Molecular Evolution Of Melanin Synthesis Pathway Genes In Silkworm, Bombyx Mori

The domesticated silkworm (Bombyx mori) was domesticated from wild silkworm (Bombyx mandarina) about 5,000 years ago. During domestication, body color between B. mandarina and B. mori changed dramatically. However, the molecular mechanism of the silkworm body color transition is not known. The completion of silkworm genome project and availability of over tenthounds of ESTs and complete cDNA sequences provide a good opportunity for studying the effect of artificial selection on the special biological character of B. mori and the genetic basis of differences in morphological traits between B. mandarina and B. mori. In this study, we compared the differences of body color and of the integument ultrastructure between B. mandarina and B. mori firstly. Next, because melanin is produced by epidermal cells through the melanin synthesis pathway in insects, we identified the melanin synthesis pathway genes in B. mori by bioinformatics methods and investigated the expression profiles both in tissues and at different development stages; then, we examined within-and between-species nucleotide diversity for silkworm melanin synthesis pathway genes, and the association between genetic diversity and the position in the pathway. Lastly, neutrality test, coalescent simulation, linkage disequilibrium, haplotype network analysis, phylogenetic analyses and real-time PCR were used to investigate whether some of the melanin synthesis pathway genes experienced selection during domestication and the possible molecular mechanism of selection. The main results are as follows:1. Comparison of body color and integument ultrastructure between B. mori and B. mandarinaThere are significant differences of body color at three developmental stages between B. mori and B. mandarina, including larva, pupa and moth. Domesticated silkworm larva has white body color except for eye spot, crescent-shaped spot and star spot, whereas wild silkworm has black body color with brown eye spot, demilune spot and star spot. The color of domesticated silkworm pupa is lightyellow, but wild silkworm pupa shows brown body color. B. mori adults have white body color, whereas B. mandarina adults have pitchy body color with black wing spot.There are two aspects of differences in integument ultrastructure between B. mori and B. mandarina revealed by electron microscopy:the first is the quantity of white uric acid granules, epidermis of wild silkworm has little white uric acid granules, whereas epidermis of domesticated silkworm has a great quantity of white uric acid granules; the other is whether exocuticle has melanin granules or not, exocuticle of wild silkworm has a lot of melanin granules, whereas exocuticle of domesticated silkworm has no melanin granules.In total, the results showed that B. mandarina have black and brown body color, whereas B. mori has white and lightyellow body color. The analysis of integument ultrastructure elucidated the material basis of different body color between B. mori and B.mandarina, and white body color of B. mori is due to a great quantity of white uric acid granules in epidermis, whereas black body color of B. mandarina is because of a lot of black melanin granules in exocuticle. Therefore, the change of body color during silkworn domestication may be associated with melanin synthesis, and melanin is produced by epidermal cells through the melanin synthesis pathway in insects.2. Identification and expression profiles of melanin synthesis pathway genes in B. moriAll melanin synthesis pathway genes in D. melanogaster have orthologs in B. mori genome, which are located on chromosomes 1,4,8.11,13 and 26. respectively. However, yellow-f and yellow-f2 have the same orthologous gene (annotated gene BGIBMGA014224) in the B. mori genome. Further analysis showed that all the melanin synthesis pathway genes in B. mori have more than one exon.Expression patterns of melanin synthesis pathway genes in various tissues on day 3 of the fifth instar showed that, TH gene was expressed in head, integument, fat body and trachea; Ddc gene was expressed in head, integument, fat body, trachea and gonads; yellow and yellow-f genes were expressed in head, integument, fat body and trachea; black and ebony genes were expressed in head mainly; Datl gene was expressed mainly in head, integument, silk gland and fat body and also slightly in midgut and trachea; tan gene was expressed in all 8 tissues analazed in this study and weakly in fat body.Based on RT-PCR. we analyzed the expression patterns of melanin synthesis pathway genes at different development stages in B. mori. The results showed that, TH gene was expressed in all development stages, and the highest expression exists in ecdysis period; the expression level of Ddc gene was low, and the peak of expression also appeared in ecdysis period; the highest expression of yellow gene existed in 1st instar molting,2nd instar molting,3rd instar molting and 4th instar molting; the expression pattern of yellow-f gene was similar to that of TH gene, and the highest expression existed in ecdysis period, and the expression level was low from full appetite period to the next molting; the expression level of Datl gene was low in full appetite period from 1 st instar to 4th instar, and the higher expression level of Datl gene existed from day 4 of 5th instar to the moth, and the peak of expression also appeared in ecdysis period; the expression of ebony gene was mainly in molting period, the initial stage after ecdysis, pupa and moth; tan gene was expressed in all development stages, and there was no significant difference among stages.To sum up, we identified the melanin synthesis pathway genes in B. mori successfully, and analyzed their spatial and temporal expression patterns. The tissue expression profiles showed that the melain synthesis pathway genes were expressed mainly in head and integument, which were consistent with the melanin distribution in B. mori. Development expression profiles showed that, the peaks of expression for melanin synthesis pathway genes existed in ecdysis period, which suggests that melanin synthesis pathway genes are associated with cuticle tanning. The results also implied that the functions of melanin sythesis pathway genes were not lost during silkworm domestication.3. Nucleotide diversity of melanin synthesis pathway genes in B. mori and B. mandarinaSequences of the 8 melanin synthesis pathway loci were obtained from 30 samples, including domesticated silkworm and wild silkworm, respectively. Estimates of total nucleotide diversity (πtotal) ranged from 0.00031(black) to 0.02891(tan) with an average value of 0.01340 in B. mori, whereasπtotal of B. mandarina varied from 0.00977 (TH) to 0.03496 (tan) with an average value of 0.02261. Similarly, Watterson's 0 (θW) of B. mori ranged from 0.00069 (black) to 0.02885 (tan) with a mean value of 0.01337, whereasθw of B. mandarina varied from 0.01245 (TH) to 0.04157 (tan) with an average value of 0.02633. Theπtotal of B. mandarina was significantly higher than that of B. mori (Wilcoxon signed rank test, P=0.01, df=7), as well asθw(Wilcoxon signed rank test, P=0.01, df=7), indicating that about 40.7% or 49.2% of the genetic diversity of wild silkworm was lost in domesticated silkworm measured byπtotalθw.In the melanin synthesis pathway, the 8 core structural genes can be divided into two groups. TH and black encode the two earliest-acting enzymes in the pathway, processing tyrosine and uracil into the melanin precursors, respectively, whereas Ddc, yellow, yellow-f, Datl, ebony and tan are the downstream genes. To study the relationship between nucleotide polymorphism and the position of the melanin synthesis pathway genes, we evaluated whether nucleotide polymorphisms are affected by the position of the genes in the pathway. An average value of nucleotide diversity of upstream genes(black and TH) in B. mori is 0.00064, whereas an average value of diversity of downstream genes is 0.01765. The upstream genes have significantly lower nucleotide polymorphisms than the downstream genes in B. mori (Student's t-test, P=0.0310, df=6). Similarly, an average value of nucleotide diversity of the upstream genes in B. mandarina is 0.01273 and significantly smaller than the corresponding average value of 0.02590 at the downstream loci (Student's t-test,P=0.0207, df=6). This suggested that the position of melanin synthesis pathway does affect the distribution of selective constraint along it. More specifically, the upstream genes have higher levels of selective constraint than the downstream genes.Neutrality test showed that,4 of 8 loci in B. mori and 7 of 8 loci in B. mandarina had negative Tajima's D values, and 3 of 8 loci in B. mori and 7 of 8 loci in B. mandarina had negative Fu and Li's D values, suggesting that both domesticated and wild silkworms were under directional selection. We found that 4 loci (Ddc. Datl, yellow-f and tan) in B. mori and 1 locus (ebony) in B. mandarina had positive Tajima's D and Fu and Li's D values, and another locus (ebony) in B. mori also had positive Fu and Li's D values, which indicated balancing selection or a population subdivision event because the long-term maintenance of distinct haplotypes can lead to an excess of intermediate frequency variants. Fay and Wu's H-statistic was calculated for the whole sequence of all loci in both B. mori and B. mandarina population. The H-statistic determines the level of high frequency variants to detect hitchhiking. However, none of the H-statistics was significant.The results of the linkage disequilibrium (LD) with nucleotide pairwise distance in B. mori and B. mandarina showed that, both curves of LD decayed rapidly within 200 bp and dropped slowly when distance is> 200 bp. The average r2 in B. mandarina dropped rapidly for the first 200 bp to a value of 0.22 and a value of 0.10 within 1600 bp, whereas the value in the domesticated silkworm declined much slowlv to a value of only 0.54 within 200 bp and remained a value of >0.40 by 1600 bp. The higher level of LD in B. mori was consistent with an increase in LD due to domestication.4. Evidence of artificial selection at TH gene during silkworm domesticationFor TH locus, there was a lot of evidence for artificial selection. Firstly,90% of nucleotide diversity for TH in wild silkworm lost in domesticated silkworm measured byπtotal, and the standing variation in B. mori was mainly part of natural variation B. mandarina had. More definitely, among parsimony informative sites at TH locus in B. mori, there was just one new variation which was not from B. mandarina and accumulated after domestication. There were 126 parsimony informative sites for TH in B. mandarina, but 118 of which fixed in B. mori in a haplotype pattern. At other 8 sites, B. mori showed singleton variable sites, and all 8 mutations were from B. mandarina. These results reflect the signature of selection during the domestication. Moreover, LD cannot be analyzed for TH in B. mori because there are no pairwise comparisons.Secondly, significant negative Tajima's D and Fu and Li's D value was only observed at the TH locus for B. mori, where the neutral model was rejected at P<0.001. A significant negative Tajima's D and Fu and Li's D test statistic indicates an excess of low frequency of polymorphism, which is consistent with directional selection or population expansion. Thirdly, coalescent simulation test showed that TH deviated from the bottleneck model and was selection target during the domestication. Fourthly, haplotype structure and phylogenetic analyses of TH sequences suggested that all domesticated silkworm strains fell predominantly into one clade and were subsets of wild silkworm. Lastly, both demographic effects from a bottleneck and the hitchhiking effect were excluded. Taken together, TH is a likely candidate domestication locus in silkworm.Moreover, non-coding region of TH may have been a target of selection. There were 4 fixed sites between B. mori and B. mandarina. The former two fixed sites are located in the first intron, and the third one is located in the second intron. Although the last fixed site is located in the exon, that codon (CCA) mutated to codon (CCG) does not cause an amino acid change. To investigate the contents of surrounding regions of the former 3 fixed sites between B. mori and B. mandarina, the MatInspector professional programwas used to predict transcription factor binding sites. Interestingly, we found that there was a five-base-pair motif TTTAT around the first fixed site in all B. mandarina strains, whereas the five base pair motif TTTAT mutated to TTTAA and fixed in all B. mori samples. Abd-B and paralogous HOX proteins have been shown to preferentially bind to the five base pair motif TTTAT or TTTAC, and with some affinity to the shorter motif TTAT in Drosophila. In the second fixed site, core sequence GGTC for Retinoid X Receptor (RXR) heterodimer binding sites mutated to GGCC in B. mori. In the third fixed site, there was no gain or loss of transcription factor binding sites for the mutation from C to G in B. mori. To date, there has not been any report to show association between RXR heterodimer binding sites and insect pigmentation.The loss of a putative Abd-B binding site (motif TTTAT vs TTTAA) observed in B. mori may be the result of a small sample size. To further confirm this result in a larger sample, we sequenced the region of a putative Abd-B binding site for another 60 domesticated silkworm strains and 7 wild silkworm samples. The result indeed showed that all domesticated silkworm strains had TTTAA haplotype, whereas TTTAT existed in all wild silkworms.Abd-B and paralogous HOX proteins have been shown to preferentially bind to the five base pair motif TTTAT or TTTAC in Drosophila. Abd-B directly activates the expression of yellow gene in the male D. melanogaster A5 and A6 segments through binding sites (TTTAT or TTTAC) in a specific cis-regulatory element, and mutational inactivation of a key Abd-B binding site could cause male-specific pigmentation to be lost. Real-time PCR indeed showed that the relative expression levels of TH gene in B. mandarina were significantly higher than that in B. mori at three developmental stages (Student's t-test, P<0.0001),which is consistent with light body color of domesticated silkworm relative to wild silkworm. Tyrosine hydroxylase (TH) is the rate-limiting enzyme of melanin synthesis pathway in insects.and the activity of TH directly affected insect body color.Taken together, we speculated that the loss of a key Abd-B binding site and significant difference of expression level of TH gene may be a reason for body color change between B. mori and B. mandarina during silkworm domestication. Thus, our results emphasize the exceptional role of gene expression regulation in morphological transition of domesticated animals.