Effects of altered catecholamine metabolism on pigmentation and physical properties of sclerotized regions in the silkworm melanism mutant

Abstract

Catecholamine metabolism plays an important role in the determination of insect body color and cuticle sclerotization. To date, limited research has focused on these processes in silkworm. In the current study, we analyzed the interactions between catecholamines and melanin genes and their effects on the pigmentation patterns and physical properties of sclerotized regions in silkworm, using the melanic mutant melanism (mln) silkworm strain as a model. Injection of β-alanine into mln mutant silkworm induced a change in catecholamine metabolism and turned its body color yellow. Further investigation of the catecholamine content and expression levels of the corresponding melanin genes from different developmental stages of Dazao-mln (mutant) and Dazao (wild-type) silkworm revealed that at the larval and adult stages, the expression patterns of melanin genes precipitated dopamine accumulation corresponding to functional loss of Bm-iAANAT, a repressive effect of excess NBAD on ebony, and upregulation of tan in the Dazao-mln strain. During the early pupal stage, dopamine did not accumulate in Dazao-mln, since upregulation of ebony and black genes led to conversion of high amounts of dopamine into NBAD, resulting in deep yellow cuticles. Scanning electron microscope analysis of a cross-section of adult dorsal plates from both wild-type and mutant silkworm disclosed the formation of different layers in Dazao-mln owing to lack of NADA, compared to even and dense layers in Dazao. Analysis of the mechanical properties of the anterior wings revealed higher storage modulus and lower loss tangent in Dazao-mln, which was closely associated with the altered catecholamine metabolism in the mutant strain. Based on these findings, we conclude that catecholamine metabolism is crucial for the color pattern and physical properties of cuticles in silkworm. Our results should provide a significant contribution to Lepidoptera cuticle tanning research.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Phenotype of mln pupae after dopamine injection and expression levels of the corresponding melanin genes.

(A). Phenotype after dopamine injection (injected on day 6 and examined on day 7 of pupation). D represents dorsal side, V represents ventral side. bar: 1 cm. (B). Expression levels of the corresponding melanin genes. (Student's t-test, n = 3. *p

Figure 2. Phenotype of mln mutant after β-alanine injection.

(A). Body color changes of mln subjected to different doses of injection. a and b represent the phenotypes of mln male and female silkworm injected with different doses, respectively. Scale bar: 1 cm; c represents color changes of tentacle at an injection dose of 700 µg/pupa. Scale bar: 5 mm; d represents color changes of vein when the injection dose is 700 µg/pupa. Scale bar: 1 cm. (B). Expression levels of Ddc in individuals injected with different doses of β-alanine (Student's t-test, n = 3. *p<0.05, **p<0.01, data represent mean±S.D.). (C). Expression levels of ebony in individuals injected with different β-alanine doses. (Student's t-test, n = 3. **p<0.01, data are presented as mean±S.D.). (D). NBAD content in individuals injected with different doses of β-alanine (Student's t-test; *p<0.05, data are presented as mean±S.D. of three separate experiments.). (E). Schematic diagram of the mechanism by which the body color of mln is changed after β-alanine injection. The dotted blunt symbol indicates possible decreased inhibition.

Figure 3. Pigmentation of head and catecholamine metabolism in fifth instar larvae after molt.

(A). Color patterns of the heads from both wild-type and mln mutant strains immediately after the fourth molt. a and b indicate wild-type and mutant, respectively. Scale bar:1 mm. (B). Color pattern of the heads from 12 h of fifth instars of wild-type and mln mutant. c and d indicate the wild-type and mutant, respectively. Scale bar: 1 mm. (C). Differences in expression profiles of melanin metabolism genes in the fifth instars just after molt in both wild-type and mutant. (Student's t-test. n = 3. **p<0.01. Data are presented as mean±S.D.). (D). Differences in catecholamines between fifth instars just after molt of both wild-type and mutant. (Student's t-test. *p<0.05, **p<0.01. Data are presented as mean±S.D. of three separate experiments.). (E). Differences in the expression patterns of melanin genes between 12 h of fifth instars of both wild-type and mutant. (Student's t-test. n = 3. *p<0.05, **p<0.01). Data are presented as mean±S.D.). (F). Differences in catecholamine content at 12 h of fifth instars between wild-type and mutant strains. (Student's t-test. *p<0.05, **p<0.01. Data are presented as mean±S.D. of three separate experiments.). (G). Schematic diagram of the melanism process of the heads of mln fifth instar larva. Blue and red boxes indicate fifth instar immediately after molt and 12 h, respectively. The blunt symbol indicates inhibitory action, while the fork signifies dysfunction of the Bm-iAANAT gene.

Figure 4. Mechanism underlying the phenotypic similarity between the wild-type and mutant silkworm during early stages of pupation.

(A). Phenotypic similarities between the wild-type and mutant at day 2 of pupation. a and b indicate phenotype of the dorsal (D) and ventral side (V) of the wild-type and mutant strains, respectively. Scale bar: 1 cm. (B). Expression differences in melanin metabolism genes between wild-type and mutant pupae at day 2 of pupation. (Student's t-test. n = 3. **pDdc inhibition in the mutant.

Figure 5. Analysis of phenotypic differences between wild-type and mutant adult silkworm.

(A). Phenotype of mln and wild-type adults. Scale bar: 1 cm. (B). Differences in expression patterns of pigment metabolism genes between mln and wild-type adults. (Student's t-test. n = 3. *p<0.05, **p<0.01. Data are presented as mean±S.D.). (C). Differences in catecholamine content between mln and wild-type adults (Student's t-test. **p<0.01. Data are presented as mean±S.D. of three separate experiments.). (D). Schematic diagram of the melanism process in the mln adult. The Blunt symbol indicates inhibitory action, while the fork signifies dysfunction of the Bm-iAANAT gene.

Figure 6. Phenotypes and expression levels of melanin genes between mln and wild-type from pupal to moth stages.

P2, P4, P6 and P8 indicate days 2, 4, 6 and 8 of pupation, respectively. D and V represent dorsal side and ventral side of pupa. Scale bar: 1 cm. (Student's t-test. n = 3. *p

Figure 7. Scanning electron microscope analysis of a cross-section of dorsal plates from wild-type and mln strains.

A and B represent adult dorsal plates of mln and wild-type silkmoths, respectively. Scale bar: 5 mm. a and c are cross sections of the adult dorsal plate of mln. The arrow indicates stratification. b and d represents cross-sections of the adult dorsal plate of Dazao. Magnification was described as X.

Figure 8. Mechanical properties of adult wings between wild-type and mln strains.

(A). Phenotype of the anterior wing in wild-type and mln mutant. (B). Storage modulus (E′) of adult wings of wild-type and mln mutant under frequency scanning (n = 3). (C). tanδ(E″/E′) of adult wings of wild-type and mln mutant under frequency scanning (n = 3).

Figure 9. Schematic diagram of variations in dopamine and NBAD contents of mln mutant from the pupal to moth stage.

(A). Color pattern changes from pupa to moth in mln. (B). Variations in the ratios of ebony, black and tan from pupa to moth stages and the speculated NBAD content variation diagram. Ratio = (mean of quantitative PCR for Dazao-mln)/(mean of quantitative PCR for Dazao)). (C). Variations in the ratio of Ddc from pupal to moth stage and speculated dopamine content variation diagram. Ratio = (mean of quantitative PCR for Dazao-mln)/(mean of quantitative PCR for Dazao)). (D). Schematic diagram of the melanization of mln from pupa to moth stage. The circular arrow indicates that high expression of ebony and black promotes production of NBAD in the mutant. The blunt symbol indicates inhibitory action, while the fork signifies loss of Bm-iAANAT gene function.