Up-regulation of cryptochrome 1 gene expression in cotton bollworm (Helicoverpa armigera) during migration over the Bohai Sea

Abstract

Cryptochromes (CRYs) are flavoproteins and play a pivotal role in circadian clocks which mediate behavior of organisms such as feeding, mating and migrating navigation. Herein, we identified novel transcripts in Helicoverpa armigera of six isoforms of cry1 and seven isoforms of cry2 by Sanger sequencing. Phylogenetic analysis showed that the transcripts of cry1 and cry2 align closely with other insect crys, indicating within-species divergence of Hacry. A dn/ds analysis revealed that the encoding sequence of the cry1 was under purifying selection by a strong negative selection pressure whereas the cry2 was less constraint and showed a less strong purification selection than cry1. In general, Hacrys were more abundantly transcribed in wild migrating populations than that in laboratory maintained populations, and expression of the cry2 was lower than cry1 in all samples tested. Moreover, when compared with the migrating parental population, offspring reared in laboratory conditions showed a significant reduction on transcription of the cry1 but not cry2. These results strongly suggest that cry1 was more related to the migration behavior of H. armigera than cry2.

Keywords: Cryptochromes; Expression analysis; Helicoverpa armigera; Migration; Transcripts.

Figure 1. Transcripts of Hacry1 (A) and Hacry2 (B) in H. armigera.

“■” Stands for the open reading frame (ORF) region. “□” stands for untranslated region (UTR). “-” stands for gap. The conserved MTHF and FAD binding regions were shown on the top. The names and GenBank accession numbers of transcripts were shown on the right.

Figure 2. Phylogenetic reconstruction of cry genes in species from Lepidoptera based on maximum likelihood.

(A) Tree based analysis of cry1 genes using the ones of Drosophila melanogaster and Acyrthosiphon pisum as outgroup. (B) Tree based analysis of cry2 genes using the one of Apid mellifera as outgroup. Values on the nodes are the nonparametric bootstrap proportions (MLBPs). Moths denoted in blue and butterflies in red. Branch-specific ω values are shown on nodes of the common ancestors. The names and GenBank accession numbers of crys were shown in Table S2.

Figure 3. Relative expression level of Hacry1 and Hacry2 in different tissues (n = 3–5), day instar stage (n = 4), population (n = 4–8) and months (n = 8–11) of adults.

(A) Relative expression level of Hacry1 in different tissues of adults. (B) Relative expression level of Hacry2 in different tissues of adults. (C) Relative expression level of Hacry1 in different day instar stage of adults. (D) Relative expression level of Hacry2 in different day instar stage of adults. (E) Relative expression level of Hacry1 in different population of adults. (F) Relative expression level of Hacry2 in different population of adults. (G) Relative expression level of Hacry1 in different months of adults. (H) Relative expression level of Hacry2 in different months of adults. Mean ± SE. The “*” and “**” denote statistical significance of the expression levels in (C) and (D) (p < 0.05 and p < 0.01, respectively). The different letters were used to show significant difference in (E) and (F) (p < 0.05, by Student’s t-test).

Figure 4. The expression level of Hacrys in CD and LF population.

(A) Diel changes of relative expression level of Hacry1 in CD population. (B) Diel changes of relative expression level of Hacry2 in CD population. (C) Diel changes of relative expression level of Hacry1 in LF population with moths at 3-day after eclosion. The different letter indicates significant differences between groups (p < 0.05, by ANOVA). (D) Diel changes of relative expression level of Hacry2 in LF population with moths at 3-day after eclosion. The different letter indicates significant differences between groups (p < 0.05, by ANOVA). (E) Relative expression level of Hacry1 between CD and LF individuals. Mean ± SE. The “*” denote statistical significance of the expression levels between CD and LF individuals at the same time (p < 0.05, n = 3–4). (F) Relative expression level of Hacry2 between CD and LF individuals. Mean ± SE. The “*” denote statistical significance of the expression levels between CD and LF individuals at the same time (p < 0.05, n = 3–4).