Positional Cloning And Molecular Mechanism Of Sex-Controlled Melanism(sml) Mutant In Silkworm, Bombyx Mori

Insects are the most diverse and abundant animal group. The variable color patterns have fascinated biologists all over the world for more than a century, and have always been a hot research topic in the field of biology. One of the most common primary pigmentation processes is melanism, which occurs throughout Insecta. In addition to an important source of insect polymorphisms, melanism also plays an important part in ecological adaption. It is caused by the accumulation of melanin, and the genes, proteins and metabolites pertaining to melanin metabolism pathway in insects have been extensively studied. However, more and more research showed that insect melanism was not only closely related to the genes of melanin metabolism pathway, but also regulated by other genes or genetic loci, suggesting a knowledge gap in the genetic basis and regulatory mechanism of insect melanism.The silkworm, Bombyx mori, is not only an important agricultural economic insect, but also the most advanced lepidopteran model. It has important roles in pest control and biological research, with abundant body-color mutant materials(over 100 body color mutants described thus far) accumulated for more than one hundred years. Previous studies of body color mutants have enhanced our fundamental understanding of the silkworm pigmentation processes and the genetic mechanisms underlying insect melanism, and provided the theoretical basis and technical support for further elucidation of body-color regulation mechanism.Recently, a new melanic mutant in the silk moths termed sex-controlled melanism(sml) was discovered by our group. It is a spontaneous autosomal recessive mutant, and only the sml male moths exhibit the melanic phenotype. Our hereditary analysis demonstrated that this melanic phenotype was independent of the genetic variations, which were reported to be associated with melanism. This suggests that an unknown novel gene likely plays a role in creating this melanic morph. We selected sml mutant for our study. Positional cloning and other experiments were performed to find the mutant site of this mutant, and studies on functional verification and regulation mechanism were also carried out. The main findings are as follows:1. Phenotype observation and analysis of the sex-controlled melanism(sml) In order to ascertain the specific phenotypic characteristics of the sex-controlled melanism(sml), we observed and analyzed the phenotypes continuously. Its general characteristics are as follows: sml males and females exhibit ordinary body-color at larval and prepupal stages; sml male moths exhibit the melanic phenotype, while females exhibit the white appearance of typical wild-types. Under the standard conditions, visible differences were not observed before P8(the 8th day of pupae). Pigmentation in sml males were initially observed by the naked eye in P8. The whole bodies of sml males are black in P9 and adult stage. Investigation in multiple tissues revealed that in sml males the colors of other sclerotic tissues such as pupal cases, moth’s dorsal plate and tentacles were not visibly different from wild-type individuals. Experiment of removal of gonad or genital appendage anlage revealed that body-colors of sml males and females did not change after organ removal, suggesting that these organs couldn’t affect the body colors of sml imagoes. Investigation of adult longevity revealed that the adult longevity of wild-type female silkworms was conspicuously longer than the males. And there were no significant differences between sml and wild-type females in the adult longevity. In contrast, the adult longevity of sml male moths was significantly longer than wild-type males, and even longer than the females. These findings revealed that the melanic individuals in sml strain were more viable, suggesting that melanism could extend the adult longevity of insect under some conditions.2. Transcriptomes analysis of the sml To investigate global transcriptional changes associated with the mutant phenotypes of sml male, both male and female samples from sml and Dazao strains at the initial stage of melanin formation and deposition(P8) were selected for comparative analysis. The serial numbers were as follows: sml males(with the serial number as sml_M), sml females(with the serial number as sml_F), Dazao males(with the serial number as DZ_M) and Dazao females(with the serial number as DZ_F). There were 168 DEGs(differentially expressed genes), 503 DEGs and 623 DEGs detected in the three pairwise melanic-white comparisons; sml_M vs sml_F, sml_M vsDZ_M and sml_M vs DZ_F, respectively. Venn diagrams were constructed to eliminate the interference from genetic background and sex differences, and the major 59 DEGs associated with the melanic-morph were identified. Based on functional annotation and transcriptome expression data,we found that among these DEGs, 49 were up-regulated and 10 were down-regulated in the melanic sml male sample compared to the other three typical samples. Intriguingly, up to 24 genes(about 41% of the 59 candidate DEGs) were cuticular protein encoding genes, including 7 CPR(cuticular protein with the R&R Consensus), 7 CPG(glycine-rich cuticular protein), 1 CPT(cuticular protein with a Tweedle motif) and 9 CPH(hypothetical cuticular protein) genes, and 22 genes were uniquely up-regulated in the sml male sample, suggesting that these cuticular protein genes may be related to melanism(with a role in transporting and/or maintaining melanin). Two genes with key roles in insect melanism, i.e. Bm-laccase2(BGIBMGA006745) and Bm-yellow(BGIBMGA001149), were significantly up-regulated in the sml male, suggesting that sml melanic alleles likely functioned via the melanin metabolism pathway by up-regulating the expression of yellow and laccase2 genes. Meanwhile, the laccase2 B-type isoform transcript, BGIBMGA006746, was also significantly up-regulated in sml male suggesting that this isoform could also play a role in the melanism process at late pupal stage. Moreover, BGIBMGA011430 and BGIBMGA000667, encoding a catalase and an insulin-like peptide(Bombyxin E-1) respectively, were also found among the candidate DEGs. It’s report that these two genes might be related to organism survival and life span. This finding in our work may provide an important clue about the extended life span of mutant male.3. Identification of mutant site of the sml Morphological and simple sequence repeat(SSR) molecular markers on silkworm linkage groups(chromosomes) were used to perform linkage analysis of sml locus.The results showed that sml locus was linked with molecular markers on the 17 th linkage group(chromosome 17), but was independent of morphological markers or molecular markers on the other linkage groups. Therefore,sml mutant locus is located in the 17 th chromosome of silkworm. We performed primary mapping of the 159 BC1 M progeny using the molecular markers. First, we roughly mapped the sml locus between the markers, M3 and M11, within scaffold nscaf2829 on the 17 th chromosome. The distances from sml locus to these markers were 1.9c M and 3.1c M, respectively. We then designed new primer sets of molecular marker in the region of primary mapping based on the silkworm genome database. Genotyping using 1573 BC1 M individuals, further delimited the sml locus between markers M5 and M9, and markers M6、M7 and M8 was tightly linked to the sml locus. The length of this region was about 403 kb. Body-color of moth is a domestication trait of silkworm. In order to identify domestication selection sites in domesticated silkworm genome, we investigated the SNP variation of genome data from multiple strains of Bombyx mori and Bombyx Mandarina by ROD method, and performed Tajima’D and other tests to verify the reliability of these sites. The results revealed the strong domestication selection of the genomic region of sml, and suggested this region was closely related to the regulation and domestication of moth body-color.4. Candidate gene screening, cloning and functional verification 23 genes were predicted to be present within sml region. In order to screen candidate genes, we performed q RT-PCR analysis to investigate the expression pattern of these genes,and found that the expression levels of 4 genes(BGIBMGA005552, BGIBMGA005557, BGIBMGA005652 and BGIBMGA005662) in the sml mutant were significantly lower than that of the Dazao(wild type). BGIBMGA005652 is a member of the conserved fizzy family of cell cycle regulators,whose homologous gene(poik) regulates colour pattern switches in the mimetic Heliconius butterflies. Gene function analysis showed that BGIBMGA005557 encodes an ATP binding protein with transferase activity and nucleotide phosphatase activity, BGIBMGA005552 encodes a putative smooth muscle caldesmon and BGIBMGA005662 encodes a Putative protein with WD40 repeat domain 85. Further sequences analysis of these genes revealed that compared to the sequences in the WT, there were a number of single nucleotide substitutions and short fragment insertions or deletions in gene BGIBMGA005557 of the sml which destroied its ORF, thus this gene could not be translated translate into functional protein in sml; there was a large fragment(about 20kb) deletion located 5 kb upstream of BGIBMGA005652 in the sml mutant; there were a short fragment insertion and a number of single nucleotide substitutions at the upstream of BGIBMGA005552; there also were single nucleotide substitutions and short fragment insertions in the introns and downstream of BGIBMGA005662. To verify whether the deficiency of these candidate genes could cause the melanic morphs, we performed RNAi experiments to knockdown the expression of these genes in WT. BGIBMGA005552, BGIBMGA005557 and BGIBMGA005652 RNAi leaded to melanization of different degrees in moth, while BGIBMGA005662 RNAi didn’t. Considering the results in RNAi experiments and melanic morphs of the sml mutant, we speculate that BGIBMGA005552, BGIBMGA005557 and BGIBMGA005652 are involved in the regulation of adult body color of silkworm, and the melanic phenotype of sml may be caused by abnormal expression of multiple genes. The sml male moth exhibits melanic phenotype, and the results of transcriptome analysis showed that the gene expressions of the melanin metabolism pathway were abnormal in the mutant. So we performed q RT-PCR analysis to investigate the expression pattern of the key genes in the melanin metabolism pathway. In wild type(WT), one expression peak of yellow and laccase2(which were thought to play major roles in the process of the formation of melanin and its deposition in the cuticle) was observed after the compound eye darkened stage. In the sml, the fluctuating expression patterns of both the yellow and laccase2 genes were similar to that in the WT, but the expression levels were significantly higher than that of the WT, especially, the relative expression level of yellow gene in P7 was extremely high. While other genes involved in insect melanization pathway didn’t show obvious expression differences in the melanic sample compared to the WT. Based on the above results, we presume that sml alleles likely function via the melanin metabolism pathway by regulating the expression of yellow and laccase2 genes(especially yellow gene) to control the moth body color.5. Biological information analysis of sml homology region in lepidopteran insects In multiple lepidopteran insects, the homology alignment and biological information analysis were carried out on the genomic region of the sml candidate region. The candidate region of sml is homologous to the regulatory regions(Hm Yb, P, Cr and carbonaria) related to wing- or body-color variations in multiple lepidopteran insects. This indicates that the region is conserved regulatory sites of the adult body color in Lepidoptera. We also found that 2 candidate genes BGIBMGA005557 and BGIBMGA005652 of sml were also in the homologous region of the industrial melanism core regulatory region. The industrial melanism in the peppered moth(Biston betularia) is a classic textbook example of rapid evolution. However, only the chromosomal region(carbonaria) responsible for melanism has been identified in previous studies. It is worth noting that, phenotype of the sml melanistic moths is very similar to that of industrial melanism moths, and these melanistic phenotypes are also regulated by homologous loci. So we have reason to speculate that the orthologous genes of the body-color regulatory genes(BGIBMGA005557 and BGIBMGA005652) in Biston betularia are possibly the candidate genes of industrial melanism. In conclusion, our findings not only provide important experimental data supports for elucidation of the molecular regulating mechanisms of lepidopteran body color, but also provide an important reference for the study on the molecular mechanism of industrial melanism.