Mutations of an arylalkylamine-N-acetyltransferase, Bm-iAANAT, are responsible for silkworm melanism mutant

Abstract

Coloration is one of the most variable characters in animals and provides rich material for studying the developmental genetic basis of pigment patterns. In the silkworm, more than 100 gene mutation systems are related to aberrant color patterns. The melanism (mln) is a rare body color mutant that exhibits an easily distinguishable phenotype in both larval and adult silkworms. By positional cloning, we identified the candidate gene of the mln locus, Bm-iAANAT, whose homologous gene (Dat) converts dopamine into N-acetyldopamine, a precursor for N-acetyldopamine sclerotin in Drosophila. In the mln mutant, two types of abnormal Bm-iAANAT transcripts were identified, whose expression levels are markedly lower than the wild type (WT). Moreover, dopamine content was approximately twice as high in the sclerified tissues (head, thoracic legs, and anal plate) of the mutant as in WT, resulting in phenotypic differences between the two. Quantitative reverse transcription PCR analyses showed that other genes involved in the melanin metabolism pathway were regulated by the aberrant Bm-iAANAT activity in mln mutant in different ways and degrees. We therefore propose that greater accumulation of dopamine results from the functional deficiency of Bm-iAANAT in the mutant, causing a darker pattern in the sclerified regions than in the WT. In summary, our results indicate that Bm-iAANAT is responsible for the color pattern of the silkworm mutant, mln. To our knowledge, this is the first report showing a role for arylalkylamine-N-acetyltransferases in color pattern mutation in Lepidoptera.

FIGURE 1.

Phenotype of WT and mln mutant strain. A, WT (Dazao, +^mln/+^mln) and mln mutant strain (Dazao-mln, mln/mln) larvae are shown on day 1 of the 5th instar. In the mln mutant, the head, anal plate, thoracic legs, sieve plates of the spiracle, and claw hook of prolegs of larvae are blacker or more buffy compared with the WT. B, body, scales, dorsal plate, vein, thoracic legs, and tentacle of the mln adult are also blacker than the WT. Scale bar, 1 cm. Magnification, indicated in the panels.

FIGURE 2.

Preliminary and fine mapping of the mln locus. A, marker S1807 was tightly linked with the mln locus. The upstream and downstream sequences (∼300 kb on each side) of S1807 were analyzed. BGIBMGA008538 (Bm-iAANAT) and BGIBMGA008539 were selected as the candidate genes. B, marker of Bm-iAANAT, P3C, shows polymorphism between Dazao-mln and C108. Short lines indicate the markers. C, fine mapping of the mln locus was done using P3C. There is no recombination between P3C and the mln locus. The results indicate that P3C was also tightly linked with the mln locus.

FIGURE 3.

Schematic diagram of Bm-iAANAT sequences of WT (Dazao) and mutant (Dazao-mln). A, amplification of genome using P3C primers between Dazao and Dazao-mln. Double horizontal lines indicate the last 67 bp of exon 4 of Bm-iAANAT in Dazao. Arrows indicate splicing sites of intron 4 in Dazao and Dazao-mln, respectively. The box indicates a 29-bp-long insertion, the first 15 bp being included in the aberrant open reading frame of exon 4 in Dazao-mln. B, identification of the full-length cDNA (no poly(A) tag) of Bm-iAANAT in Dazao-mln (mutant) and Dazao (WT). Two abnormal transcripts were detected in the mutant strain. C, comparison of the structure of Bm-iAANAT between Dazao and Dazao-mln. White boxes indicate open reading frames. Coarse horizontal lines indicate untranslated regions (3′-UTR). Diagonal lines indicate introns. Gray region indicates the deletion. Gray solid box indicates the insertion of 15 bp. Black box indicates abnormal exon 5. Vertical arrows indicate the location of the termination codon. The termination codon of type 1 is located at +34 bp of exon 5. The termination codon of type 2 is located at +74 bp of 3′ UTR. D, alignments of the speculative Bm-iAANAT amino acid sequences among Dazao, Dazao-mln type 1 and type 2. Horizontal line indicates the acetyltransferase domain of the WT strain, Dazao.

FIGURE 4.

Expression patterns of Bm-iAANAT in Dazao. A, expression profile of the Bm-iAANAT gene during the molting stage of the 4th instar larvae. B, temporal expression of Bm-iAANAT in the silkworm from 5th instar to the moth stage. V1–V7 represent corresponding days of the 5th instar larva; W₀–W_2.5 represent day 0, day 0.5, day 1, day 1.5, day 2, and day 2.5 of the wandering stage. P1–P8 represent day 1 to day 8 of the pupation. M0 represents the 1st day of the moth stage. BmActin3 gene was internal control. C, spatial expression of Bm-iAANAT on the 4th day of the 5th instar larvae in Dazao strain. Total RNA samples of 10 silkworm tissues were isolated and analyzed by RT-PCR, including the ovary, testis, head, silk gland, integument, midgut, malpighian tubule, fat body, hemocyte, and trachea. D, relative expression levels of Bm-iAANAT between the sclerified (anal plate, thoracic legs, and head) and less sclerified tissues (epidermis) of the larva. The data show the mean ± S.D. (error bars) (n = 3).

FIGURE 5.

Comparison of the expression levels of Bm-iAANAT between Dazao and Dazao-mln in the several developmental stages and strain-specific phenotype tissues (head, thoracic legs, and anal plate). A, semiquantitative RT-PCR detection, BmActin3 as inner control. Lane 1, Dazao-mln. Lane 2, Dazao. B, quantitative PCR analysis. Expression levels were normalized to the expression of the eukaryotic translation initiation factor 4A gene (microarray probe ID, sw22934). C, relative expression levels of Bm-iAANAT in three strain-specific phenotype tissues of the larva in Dazao and Dazao-mln. The data indicate the mean ± S.D. (error bars) (n = 3).

FIGURE 6.

Investigation of the expression differences of key melanin metabolism genes between Dazao and Dazao-mln. A, quantitative RT-PCR detection of melanin metabolism key genes in three larva strain-specific phenotype tissues between Dazao and Dazao-mln on V1 stage. Error bars, S.D. B, result of investigations on M0 stage. **, p < 0.002, Student's t test. n = 3. Ratio = (mean of quantitative PCR for Dazao-mln)/(mean of quantitative PCR for Dazao). C, diagram of the melanin metabolism pathway (see Ref. ref. 3). Black arrows indicate the expression pattern in WT. Green color indicates expression pattern in mln mutant. Solid green arrows indicate the up-regulated expression of ebony in thoracic legs (TL) and anal plate (AP) of the larva compared with WT. Dotted line arrows indicate the down-regulated expression of the moth compared with WT. Frame indicates the accumulation of dopamine in mln mutant. Fork indicates the loss of function of Bm-iAANAT in mln mutant. POs, phenol oxidases. The genes are shown in blue. The pigment precursors are shown in red.

FIGURE 7.

Quantity of dopamine analyzed by reversed phase HPLC in the strain-specific phenotype tissues between the WT and mutant on day 2 of 4th instar. The column elution was monitored at 280 nm. A, arrows indicate the peak of dopamine. Scale bar, 5 milliarbitrary units (mAU). B, data indicate the mean ± S.D. (error bars) (n = 3). *, p < 0.05, Student's t test (n = 3).