Mutations in a β-group of solute carrier gene are responsible for egg and eye coloration of the brown egg 4 (b-4) mutant in the silkworm, Bombyx mori
Graphical abstract
Introduction
Ommochromes constitute a significant group of pigments that are widely distributed in the eggs, compound eyes, and epidermis of insects (Figon and Casas, 2019). Ommochrome pigments are also known to be involved in body color patterning in invertebrates, which is important for sexual dimorphism and mimicry. For example, Heliconius butterflies display mimetic wing patterning, which is regulated by several ommochrome-related genes, such as vermillion and cinnabar (Reed et al., 2008; Ferguson and Jiggins, 2009). Yellow/red color changes associated with sexual maturation in some dragonflies are regulated by redox states of epidermal ommochrome pigments (Futahashi et al., 2012). In addition, ommochromes also function as screening pigments in compound eyes (Burnet et al., 1968) and are involved in tryptophan metabolism (Linzen, 1974).
Studies on the ommochrome synthetic pathway have been developed by investigating eye color mutants of the fruit fly, Drosophila melanogaster. Most of the genes are involved in the synthetic pathway from tryptophan to 3-hydroxykynurenine (vermillion: Searles and Voelker, 1986; cinnabar: Sullivan et al., 1973), pigment granule formation (pink: Falcón-Pérez et al., 2007; garnet: Lloyd et al., 1999; deep orange: Shestopal et al., 1997), and the incorporation of 3-hydroxykynurenine into pigment granules (scarlet: Tearle et al., 1989; white: O’Hare et al., 1983). In the silkworm, Bombyx mori, ommochrome pigments accumulate in eggs, adult compound eyes, and in the reddish markings on the larval epidermis. The early steps of the ommochrome synthetic pathway are conserved between B. mori and D. melanogaster. The genes responsible for the three white egg and eye color mutants, white egg 1 (w-1), white egg 2 (w-2), and white egg 3 (w-3), in B. mori correspond to the orthologous genes of cinnabar, scarlet, and white in D. melanogaster, respectively (Quan et al., 2007; Kômoto et al., 2009; Tatematsu et al., 2011). The conservation of these genes is also reported in other insects, such as the red flour beetle, Tribolium castaneum, and the Western tarnished plant bug, Lygus hesperus (Lorenzen et al., 2002; Broehan et al., 2013; Grubbs et al., 2015; Brent and Hull, 2019). However, after 3-hydroxykynurenine incorporation into pigment granules, the ommochrome synthetic pathway begins to differ in Drosophila and Bombyx. For instance, the gene responsible for the B. mori red egg (re) mutant encodes a major facilitator superfamily transporter which is conserved in most insect species but lost in D. melanogaster (Osanai-Futahashi et al., 2012). This is possibly due to the difference of the ommochrome composition in the ommatidia; while Cyclorrhapha (including Drosophila) are reported to have only ommatins as the ommochrome pigment in the eye, other insect species, with the exception of Orthoptera, are reported to also contain ommins in the ommatidia (Figon et al., 2020; Linzen, 1974). Given that the re gene is indispensable for ommin synthesis (Kawase and Aruga, 1954), the gene loss in Dipteran insects may be the cause of lack of ommins in the compound eyes. Therefore, genetic studies based on non-Cyclorrhapha insect species are important to reveal the later steps of the ommochrome synthesis pathway.
The eggs and adult compound eyes of the B. mori contain a mixture of ommochrome pigments, such as ommin and xanthommatin, and exhibit a dark purple or black color (Kawase and Aruga, 1954; Koga and Osanai, 1967; Linzen, 1974; Sawada et al., 2000). The brown egg 4 (b-4) is a recessive mutant which causes eggs and eyes to express a reddish-brown color instead (Fig. 1; Nakaizawa and Nakajima, 1979; Nakajima, 1956). The b-4 is located at 21.9 cM in the B. mori genetic linkage group 20 (chromosome 20; Doira et al., 1974). The ommochrome precursor, 3-hydroxykynurenine, accumulates in the mother's pupal ovaries (Yamashita and Hasegawa, 1964) and is incorporated into pigment granules in eggs. The b-4 egg color phenotype is not maternally inherited, suggesting that the gene responsible for the b-4 mutation is predicted to be involved in the synthesis pathway from 3-hydroxykynurenine to ommochrome pigments. We attempted to identify the responsible gene for the mutant phenotypes for a deeper understanding of the later steps of the ommochrome biosynthetic pathway.
Here, we performed double digest restriction-site associated DNA sequencing (ddRAD-seq) and narrowed down the b-4 locus to approximately 1.1 Mb that contains 69 predicted gene models. Among these, three b-4 mutant strains investigated in this study had two independent mutations in an ortholog of the Drosophila eye color mutant gene, mahogany (mah), which encodes a putative amino acid transporter that belongs to the β-group of solute carrier (SLC) family (Grant et al., 2016). We performed CRISPR/Cas9-mediated gene knockout targeting the candidate gene and confirmed that the responsible gene for the b-4 phenotypes is B. mori mahogany (Bmmah).
Section snippets
B. mori strains
We used two wild-type B. mori strains (p50T and C108), three b-4 mutant strains (e36, e37, and No. 903), and two os mutant strains (osIn4 and osIn7) for this study. The B. mori strain, p50T, was maintained in our laboratory. Strains e36 and e37 were provided from Kyushu University (Fukuoka, Japan) with the support from the National BioResource Project (http://silkworm.nbrp.jp). Strains C108 and No. 903 were acquired from the Genetic Resource Center, National Agriculture and Food Research
Characterization of the b-4 phenotype
Eggs and adult compound eyes of the wild-type strain, p50T, exhibited a dark purple or black color, while that of the b-4 mutant strains e36, e37, and No. 903 displayed a dark reddish-brown color (Fig. 1). However, the phenotypes were slightly different between the b-4 mutant strains. Compared to e36 and No. 903, the e37 strain had darker eggs and compound eyes. We also observed a reduction of pigments in the brain and ganglia of the b-4 mutants (Fig. 1), which was not investigated in the
The functional mechanism of BmMAH predicted from the b-4 phenotypes
In this research, we performed ddRAD-seq analysis and narrowed down the b-4 candidate region to a segment that contained 69 predicted gene models (Fig. 2). RNA-seq and cDNA sequencing revealed that the gene structure is disrupted in one of the candidate genes, KWMTBOMO12119, in the b-4 mutant strains (Fig. 3, Figs. S1–2). Next, we performed CRISPR/Cas9 mediated gene knockout targeting KWMTBOMO12119 and observed reddish-brown colored eggs and adult compound eyes like in the b-4 mutants (Fig. 4B,
Acknowledgements
We are grateful to Natsuki Nakashima for technical assistance for silkworm maintenance. We also thank Seigo Kuwazaki for ddRADseq library preparation. We thank the Institute for Sustainable Agro-ecosystem Services, The University of Tokyo, for facilitating the mulberry cultivation and the Biotron Facility at the University of Tokyo for rearing the silkworms. This work was supported by JSPS KAKENHI (grant numbers 17H05047 and 20H02997) and grant from the Ministry of Agriculture, Forestry and
References (77)
- et al.
Phenogenetic studies on visual acuity in. Drosophila melanogaster
J. Insect Physiol.
(1968) - et al.
High-quality genome assembly of the silkworm, Bombyx mori
Insect Biochem. Mol. Biol.
(2019) - et al.
Mutations in an amino acid transporter gene are responsible for sex-linked translucent larval skin of the silkworm, Bombyx mori
Insect Biochem. Mol. Biol.
(2011) - et al.
A single-base deletion in an ABC transporter gene causes white eyes, white eggs, and translucent larval skin in the silkworm w-3oe mutant
Insect Biochem. Mol. Biol.
(2009) The tryptophan → omrnochrome pathway in insects
Adv. In Insect Phys.
(1974)- et al.
Identification of the Bombyx red egg gene reveals involvement of a novel transporter family gene in late steps of the insect ommochrome biosynthesis pathway
J. Biol. Chem.
(2012) - et al.
Occurrence of ommochrome-containing pigment granules in the central nervous system of the silkworm, Bombyx mori
Comp. Biochem. Physiol. B Biochem. Mol. Biol.
(2000) - et al.
Evolutionary origin of amino acid transporter families SLC32, SLC36 and SLC38 and physiological, pathological and therapeutic aspects
Mol. Aspect. Med.
(2013) - et al.
Novel non-autonomous transposable elements on W chromosome of the silkworm, Bombyx mori
J. Genet.
(2010) - et al.
Design, evaluation, and screening methods for efficient targeted mutagenesis with transcription activator-like effector nucleases in medaka
Dev. Growth Differ.
(2014)
Development of eye colors in Drosophila: the mutants bright and mahogany
Am. Nat.
A melanosomal two-pore sodium channel regulates pigmentation
Sci. Rep.
RNA interference-mediated knockdown of eye coloration genes in the western tarnished plant bug (Lygus hesperus Knight). Arch. Insect Biochem
Physiol.
Functional analysis of the ATP-binding cassette (ABC) transporter gene family of Tribolium castaneum
BMC Genom.
The vesicular GABA transporter, VGAT, localizes to synaptic vesicles in sets of glycinergic as well as GABAergic neurons
J. Neurosci.
Missense mutation in exon 2 of SLC36A1 responsible for champagne dilution in horses
PLoS Genet.
Lysosome-related organelles
Faseb. J.
A framework for variation discovery and genotyping using next-generation DNA sequencing data
Nat. Genet.
Linkage studies of Bombyx mori: discovery and establishment of the oh-Df-t-b-4 linkage group, the twenty-first chromosome
J. Seric. Sci. Jpn.
A new generation of homology search tools based on probabilistic inference
Genome Inform.
MUSCLE: multiple sequence alignment with high accuracy and high throughput
Nucleic Acids Res.
Unique pH dynamics in GABAergic synaptic vesicles illuminates the mechanism and kinetics of GABA loading
Proc. Natl. Acad. Sci. U. S. A.
The Drosophila pigmentation gene pink (p) encodes a homologue of human hermansky-pudlak syndrome 5 (HPS5)
Traffic
Single-Vesicle imaging reveals different transport mechanisms between glutamatergic and GABAergic vesicles
Science
Shared and divergent expression domains on mimetic Heliconius wings
Evol. Dev.
Pigment patterns in mutants affecting the biosynthesis of pteridines and xanthommatin in Drosophila melanogaster
Biochem. Genet.
Ommochromes in invertebrates: biochemistry and cell biology
Biol. Rev.
Uncyclized xanthommatin is a key ommochrome intermediate in invertebrate coloration
Insect Biochem. Mol. Biol.
Redox alters yellow dragonflies into red
Proc. Natl. Acad. Sci. U. S. A.
ggsashimi: Sashimi plot revised for browser- and annotation-independent splicing visualization
PLoS Comput. Biol.
Full-length transcriptome assembly from RNA-Seq data without a reference genome
Nat. Biotechnol.
An eye on trafficking genes: identification of four eye color mutations in Drosophila
G3 (Bethesda)
The ABCs of eye color in Tribolium castaneum: orthologs of the Drosophila white, scarlet, and brown genes
Genetics
Cargo sorting to lysosome-related organelles regulatessiRNA-mediated gene silencing
J. Cell Biol.
Developmental patterns of 3-hydroxykynurenine accumulation in white and various other eye color mutants of Drosophila melanogaster
Biochem. Genet.
Biochemical genetics on the pigment in eggs and compound eyes in the silkworm. Japanese J
For. Genet.
Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat
Biotechnol.
Der gehalt an tryptophan, kynurenin, 3-hydroxy-kynurenin und ommochromen bei den überwinternden eiern des seidenspinners Bombyx mori während der entwicklung
Hoppe. Seylers. Z. Physiol. Chem.
Cited by (2)
Reduced-representation libraries in insect genetics
2023, Current Opinion in Insect ScienceDevelopment of an Injection Method for the Genetic Engineering of Diapause Silkworm Egg Using Dimethyl Sulfoxide
2023, Japan Agricultural Research Quarterly