Positional cloning of a Bombyx pink-eyed white egg locus reveals the major role of cardinal in ommochrome synthesis.
Ommochromes are major insect pigments involved in coloration of compound eyes, eggs, epidermis and wings. In the silkworm Bombyx mori, adult compound eyes and eggs contain a mixture of the ommochrome pigments such as ommin and xanthommatin. Here, we identified the gene involved in ommochrome biosynthesis by positional cloning of B. mori egg and eye color mutant pink-eyed white egg (pe). The recessive homozygote of pe has bright red eyes and white or pale pink eggs instead of a normal dark coloration due to the decrease of dark ommochrome pigments. By genetic linkage analysis, we narrowed down the pe-linked region to ~258 kb, containing 17 predicted genes. RNA sequencing analyses showed that the expression of one candidate gene, the ortholog of Drosophila haem peroxidase cardinal, coincided with egg pigmentation timing, similar to other ommochrome-related genes such as Bm-scarlet and Bm-re. In two pe strains, a common missense mutation was found within a conserved motif of B. mori cardinal homolog (Bm-cardinal). RNA interference-mediated knockdown and transcription activator-like effector nuclease (TALEN)-mediated knockout of the Bm-cardinal gene produced the same phenotype as pe in terms of egg, adult eye and larval epidermis coloration. A complementation test of the pe mutant with the TALEN-mediated Bm-cardinal-deficient strain showed that the mutant phenotype could not be rescued, indicating that Bm-cardinal is responsible for pe. Moreover, knockdown of the cardinal homolog in Tribolium castaneum also induced red compound eyes. Our results indicate that cardinal plays a major role in ommochrome synthesis of holometabolous insects.
Transgenic Silkworm Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Japan.
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M
Osanai-Futahashi
Transgenic Silkworm Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Japan.
K-I
KI
Tatematsu
Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.
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R
Futahashi
Insect Genome Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Japan.
J
J
Narukawa
Silk Materials Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Japan.
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Y
Takasu
Insect Growth Regulation Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Japan.
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T
Kayukawa
Insect Growth Regulation Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Japan.
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T
Shinoda
NODAI Genome Research Center, Tokyo University of Agriculture, Tokyo, Japan.
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T
Ishige
Department of Bioscience, Tokyo University of Agriculture, Tokyo, Japan.
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S
Yajima
Transgenic Silkworm Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Japan.
T
T
Tamura
Insect Genome Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Japan.
K
K
Yamamoto
Transgenic Silkworm Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Japan.
H
H
Sezutsu
D000818:Animals; D012831:Bombyx / Q000235:genetics*; D003001:Cloning, Molecular; D005123:Eye; D005260:Female; D055786:Gene Knockout Techniques; D017344:Genes, Insect; D005816:Genetic Complementation Test; D008040:Genetic Linkage; D019476:Insect Proteins / Q000235:genetics* / Q000378:metabolism; D007814:Larva; D008297:Male; D010063:Ovum; D010640:Phenothiazines / Q000378:metabolism*; D010641:Phenotype; D010802:Phylogeny; D010858:Pigmentation / Q000235:genetics*; D034622:RNA Interference; D014231:Tribolium / Q000235:genetics
D019476:Insect Proteins; D010640:Phenothiazines; C002772:ommochrome