Combinatorial expression of ebony and tan generates body color variation from nymph through adult stages in the cricket, Gryllus bimaculatus

Abstract

Insect body colors and patterns change markedly during development in some species as they adapt to their surroundings. The contribution of melanin and sclerotin pigments, both of which are synthesized from dopamine, to cuticle tanning has been well studied. Nevertheless, little is known about how insects alter their body color patterns. To investigate this mechanism, the cricket Gryllus bimaculatus, whose body color patterns change during postembryonic development, was used as a model in this study. We focused on the ebony and tan genes, which encode enzymes that catalyze the synthesis and degradation, respectively, of the precursor of yellow sclerotin N-β-alanyl dopamine (NBAD). Expression of the G. bimaculatus (Gb) ebony and tan transcripts tended to be elevated just after hatching and the molting period. We found that dynamic alterations in the combined expression levels of Gb'ebony and Gb'tan correlated with the body color transition from the nymphal stages to the adult. The body color of Gb'ebony knockout mutants generated by CRISPR/Cas9 systemically darkened. Meanwhile, Gb'tan knockout mutants displayed a yellow color in certain areas and stages. The phenotypes of the Gb'ebony and Gb'tan mutants probably result from an over-production of melanin and yellow sclerotin NBAD, respectively. Overall, stage-specific body color patterns in the postembryonic stages of the cricket are governed by the combinatorial expression of Gb'ebony and Gb'tan. Our findings provide insights into the mechanism by which insects evolve adaptive body coloration at each developmental stage.

Fig 1. Pathway of melanin and sclerotin biosynthesis in insects.

Dopamine, which is synthesized from tyrosine by the activity of TH and Ddc, becomes three pigments. Dopamine-melanin is produced by the activity of Yellow and Lac2. Excess dopamine is metabolized into NBAD and NADA by the activity of Ebony and NAT, respectively, to produce two types of sclerotins: yellow NBAD sclerotin and colorless NADA sclerotin. This pathway also includes Tan, an NBAD hydroxylase, which may coordinate the melanization level with Ebony. The metabolic pathway depicted was modified from Arakane et al. (2009) [10]. Intermediate products and pathways of DOPA-melanin biosynthesis have been omitted to demonstrate Dopamine-related pigmentation. Enzymes and metabolites are colored red and black, respectively. TH, Tyrosine hydroxylase; Ddc, DOPA decarboxylase; Ebony, NBAD synthase; Tan, NBAD hydroxylase; NAT, dopamine N-acetyltransferase; Yellow, Yellow protein.

Fig 2. Identification of Gryllus ebony and tan.

(A) Genomic structures of Gb’ebony and Gb’tan. Boxes indicate exons, and lines connecting boxes indicate introns. Black and white boxes indicate coding regions and untranslated regions, respectively. (B) The phylogenetic trees of the Ebony and Tan proteins from Gryllus and other insects. The sequences were aligned using the ClustalW program, and the phylogenetic tree was generated by a neighbor-joining method. Pa: Periplaneta americana, Bm; Bombyx mori, Hv; Henosepilachna vigintioctopunctata, Tc; Tribolium castaneum, Dm; Drosophila melanogaster, Nl; Nilaparvata lugens, Of; Oncopeltus fasciatus, Ad; Anopheles darlingi, Ld; Leptinotarsa decemlineata, Mr; Megachile rotundata, Pp; Papilio polytes, Pc; Penicillium chrysogenum.

Fig 3. Expression profiles of Gb’ebony and Gb’tan transcripts from the embryo through adult stages.

Relative expression levels of Gb’ebony (A) and Gb’tan (B) transcripts in whole embryos (D; day after egg laying) and the whole body of nymphs and adults (D; day after hatching or molting) were analyzed by RT-qPCR. Gb’β-actin was used as an internal control gene. Black, blue, and red lines indicate unsexed, male, and female crickets, respectively. Filled and unfilled points indicate pigmented and unpigmented crickets, respectively. The scale on the X-axis indicates one day. The first to third instars showed complete pigmentation within a few hours after hatching and molting. Gene expression in unpigmented and pigmented crickets on day 1 of each stage was analyzed using cDNA samples derived from crickets within 1 and 2–15 h, respectively, after hatching or molting (enclosed with a red dotted frame). Relative expression levels were calculated based on the amounts of transcripts in the first instar nymphs immediately after hatching. The data presented are the mean and SD (N ≥ 3). The lower sides of the error bars were omitted. The asterisks (*) and (**) mean P < 0.05 and P < 0.01, respectively, based on Student’s t-test. Asterisks for the first to third instars are shown for significant differences in gene expression levels between unpigmented and pigmented crickets on day 1 of each stage. Asterisks from the seventh instar onward are indicated when there was a significant difference in gene expression between males and females.

Fig 4. The generation of Gb’ebony and Gb’tan homozygous mutants using the CRISPR/Cas9 system.

(A) Guide-it Resolvase assay for CRISPR/Cas9 mutagenesis. Gel images show the results of the assay performed on eggs after injection of the Cas9-gRNA complex. Minus lanes indicate the no Guide-it Resolvase control. Digested bands were detected when the reactions were performed with the enzyme (plus lanes). L: 50 bp DNA ladder. (B) Sequences of Gb’ebony and Gb’tan mutants generated by the CRISPR/Cas9 system. (C) Immunoblot analysis confirming the knockout of Gb’ebony and Gb’tan at the protein level. Antibodies against the recombinant His-tagged Gb’Ebony and Gb’Tan proteins were used in these experiments. Crude extracts from G. bimaculatus were separated by SDS-PAGE. Gb’Ebony and Gb’Tan proteins (arrowhead) were detected only in wild-type samples. β-Actin was included as a loading control. (D) Effects of Gb’tan knockout on Gb’tan transcription. RNA was extracted from the whole body of day 1 seventh instar nymphs of the wild-type and Gb’tan mutants within 1 h of molting and subjected to RT-qPCR. Data are presented as the mean value ± SD obtained from four biological replicates and three technical replicates. The asterisks (**) mean statistical significance at P < 0.01 in a Student’s t-test.

Fig 5. Phenotype of Gb’ebony homozygous mutants.

(A) Dorsal views of wild-type and Gb’ebony ^cr1 mutant adults. (B) Effect of Gb’ebony knockout on the color of adult wings. FW: Forewing, HW: Hind wing. (C) Dorsal views of wild-type and Gb’ebony ^cr1 mutant nymph stages. (C’) Magnified image of the dorsal side of the thorax and the tail in first instar nymphs. Scale bars: 10 mm in A and B; 2 mm (1st–6th instar nymphs) and 10 mm (7th–8th instar nymphs) in C; 0.5 mm in C’.

Fig 6. Phenotype of Gb’tan homozygous mutants.

(A) Dorsal views of wild-type and Gb’tan ^cr1 mutant adults. (B) Effect of Gb’tan knockout on the color of adult wings. FW: Forewing, HW: Hindwing. (C) Dorsal views of wild-type and Gb’tan ^cr1 mutant nymph stages. (D) Quantification of grayscale intensity in wild-type and Gb’tan mutants at the seventh instar stage. The intensity of body color excluding the appendages was measured using ImageJ Fiji software (https://fiji.sc). The grayscale intensity consists of 256 tones of color gradients, with 0 and 255 indicating white and black, respectively. The asterisks (*) and (**) mean P < 0.01 and P < 0.001, respectively, based on a Mann–Whitney U test (N ≧ 12). Scale bars: 10 mm in A and B; 2 mm (1st–6th instar nymphs) and 10 mm (7th–8th instar nymphs) in C.

Fig 7. Mechanism for determining body color in G. bimaculatus.

(A) Pathway of melanin and sclerotin biosynthesis in G. bimaculatus. Knockout of Gb’ebony (encoding NBAD synthase) and Gb’tan (encoding NBAD hydroxylase) causes body color changes due to over-production of melanin and NBAD sclerotin, respectively. (B) The correlation between the expression patterns of Gb’ebony and Gb’tan and the body color transition of wild-type crickets from the nymphs through adult stages. Scales of the Y-axis in the graph differ between Gb’ebony and Gb’tan for the comparison of periodic patterns. The images of the crickets in this figure were reprinted from Figs 5 and 6. Scale bars: 2 mm in A; 1 mm (1st–4th instar nymphs) and 2 mm (5th–8th instar nymphs and adult) in B.