Comprehensive silk gland multi-omics comparison illuminates two alternative mechanisms in silkworm heterosis

Heterosis is a common phenomenon in plants and animals with diverse underlying mechanisms.Here,we applied two widely used silkworm hybrid systems and performed multi-omics analysis to identify possible intrinsic associations between different hybrid strategies and epigenetic mechanisms with silkworm heterosis.We found significant differences in the silk gland transcriptomic landscape between the two systems,including differentially expressed genes and expression patterns in the hybrid offspring compared to their parents.In the quaternary hybrid system,hybrid vigor was primarily due to upregulated genes and the parent-dominant upregulated expression pattern,involving multiple transport processes,cellular nitrogen compound catabolism,glucose metabolism,and tricarboxylic acid cycle.In the binary system,hybrid vigor was mainly due to the down-regulated genes and transgressively down-regulated expression pattern,mainly involving basic nitrogen synthesis metabolism and body function.We also demonstrated that DNA methylation may affect hybrid vigor by regulating the expression of several heterosis-related genes.Thus,this study revealed two alternative mechanisms that may contribute to silkworm heterosis,both of which facilitate the efficient utilization of energy and nitrogen for silk production.

Efficient strategies for changing the diapause character of silkworm eggs and for the germline transformation of diapause silkworm strains

To overcome the disadvantages of current silkworm Bombyx mori transgenic technology, such as costly and time-consuming to maintain non-diapause transgenic silkworms, we report here on the development of treatments for the germline transformation of diapause silkworm strains. Our results showed that HC1 treatment within 3 h of oviposition was able to prevent the diapause of eggs from Japanese lineage diapause silkworm strains and was also suitable for germline transformation of the same strains. By incu- bating developing mother eggs from Chinese lineage diapause silkworm strains at 15℃ （15℃-IME）, we were able to prevent the diapause of their daughter eggs; a similar strategy （15℃-IMES） for the germline transformation of the same strains was that the mother eggs were incubated at 15℃, and the daughter eggs were then microinjected according to the conventional microinjection methods used for non-diapause eggs. By combining tempera- ture and light controls, the improved 15℃-IMES strategy prevented diapause in daughter eggs, and also enabled the germline transformation of both Japanese and Chinese lineage diapause silkworm strains. Although each of the strategies developed here has advantages and disadvantages, we suggest that the 15℃-IMES strategy is a good reference for the establishment of germline transformation technologies of other egg diapause insects. These new strategies for the efficient germline transformation of diapause silkworm strains are likely to improve the practical use of silkworm transgenic lines in sericulture and also highlight silkworm functional genomics research and its modeling.

Expression pattern and tissue localization of the class B scavenger receptor BmSCRBQ4 in Bombyx moil

Class B scavenger receptors （SR-Bs） are cell surface glycoproteins involved in various physiological processes in vivo, including the transport and metabolism of lipids, binding and phagoeytosis of xenobiotics, and signaling. But little information is available about silkworm SR-Bs; it is necessary to study these SR-Bs for revealing their function. In this study, we cloned the full-length coding sequence of BrnSCRBQ4, a SR-B gene from the silkworm Bombyx mori L. We found that the BmSCRBQ4 gene consists of nine exons and eight introns, with an open reading frame of 1371 bp encoding 456 amino acids. Gene expression studies determined that BmSCRBQ4 messenger RNA （mRNA） was expressed in unfertilized eggs, during embryonic development and throughout the majority of the larval period. Expression of mRNA was detected in the mid gut, middle silk gland, posterior silk gland, head, integumentum, fat body, testes and the ovaries of the larval B. mori Dazao strain, as well as in the silkworm cell lines BmN and BmE. Protein expression studies found BmSCRBQ4 protein was expressed only in the testes, fat body and middle silk gland of larvae, as well as in the silkworm cell lines BmN and BmE. The BmSCRBQ4 protein showed variability in banding patterns in different tissues and cells when analyzed by Western blotting. Immunohistochemical staining showed that the BmSCRBQ4 protein localizes to the constitutive membranes or cellular membranes of these tissues. These results indicated that BmSCRBQ4 gene may play some physiologically relevant roles at the cell surface in each tissue.

Bmmp influences wing morphology by regulating anterior-posterior and proximal-distal axes sdevelopment

Insect wings are subject to strong selective pressure,resulting in the evolution of remarkably diverse wing morphologies that largely determine flight capacity.However,the genetic basis and regulatory mechanisms underlying wing size and shape development are not well understood.The silkworm Bombyx mori micropterous(mp)mutant exhibits shortened wing length and enlarged vein spacings,albeit without changes in total wing area.Thus,the mp mutant comprises a valuable genetic resource for studying wing de-velopment.In this study,we used molecular mapping to identify the gene responsible for the mp phenotype and designated it Bmmp.Phenotype-causing mutations were identified as indels and single nucleotide polymorphisms in noncoding regions.These mutations resulted in decreased Bmmp messenger RNA levels and changes in transcript isoform composition.Bmmp null mutants were generated by clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9 and exhibited changed wing shape,similar to mp mutants,and significantly smaller total wing area.By examining the expression of genes critical to wingdevelopment inwildtype and Bmmp null mutants,we found that Bmmp exerts its function by coordinately modulating anterior-posterior and proximal-distal axes development.We also studied a Drosophila mp mutant and found that Bmmp is functionally conserved in Drosophila.The Drosophila mp mutant strain exhibits curly wings of reduced size and a complete loss of flight capacity.Our results increase our understanding of the mechanisms underpinning insect wing development and reveal potential targets for pest control.

Microarray analysis of New Green Cocoon associated genes in silkworm, Bombyx mori

Green cocoons in silkworm, Bombyx mori, are caused by flavonoids accumulation in the silk proteins, fibroin and sericin. Despite the economic value of natural green cocoon and medical value of flavonoids, there is limited understanding of the molecular mechanism regulating flavonoids uptake in silkworm, which is tightly associated with the trait of green cocoon. The purpose of this study is to perform a comprehensive analysis to understand the molecular mechanisms of flavonoids uptake in silkworm based on microarray analyses. The study subject was the New Green Cocoon from the silkworm strains, G200 and N100, a new spontaneous dominant green cocoon trait identified in the 2000s. The genes regulating this trait are independent of other green cocoon genes previously reported. Genome-wide gene expression was compared between the New Green Cocoon producing silkworm strains, G200 and NI00, and the control sample, which is the white cocoon producing strain 872B. Among these strains, N100 and 872B are near-isogenic lines. The results showed that 130 genes have consistently changing expression patterns in the green cocoon strains when compared with the white cocoon strain. Among these, we focused on the genes related to flavonoids metabolism and absorption, such as sugar trans- porter genes and UDP-glucosyltransferase genes. Based on our findings, we propose the potential mechanisms for flavonoids absorption and metabolism in silkworm. Our results imply that silkworm might be used as an underlying model for flavonoids in pharmaceutical research.

Sob gene is critical to wing development in Bombyx mori and Tribolium castaneum

The development of insect appendages requires the expression of multiple genes in a strict spatial and temporal order. The odd-skipped family genes are vital transcriptional factors involved in embryonic development. The development and morphogenesis of the insect wing requires multiple transcription factors to regulate the expression of wing patterning genes at the transcriptional level. However, the function of odd-related genes in insect wing morphogenesis and development during postembryonic stages is unclear. We focused on the roles of the sister of odd and bowl (sob) gene, a member of odd-skipped family genes, during the wing morphopoiesis in Bombyx mori using the clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 system and in Tribolium castaneum by RNA interference. The results showed that the wings were significantly smaller and degenerated, and wing veins were indistinct in the sob gene loss-of-function group in both B. mori and T. castaneum. Quantitative real-time polymerase chain reaction revealed that the Tcsob gene regulated the expression of wing development genes, such as the cht 7 and the vg gene. The findings suggest the importance of sob gene in insect wing morphology formation during postembryonic stages.

Molecular basis of the silkworm mutant re^(l) causing red egg color and embryonic death

The coloration and hatchability of insect eggs can affect individual and population survival.However,few genetic loci have been documented to affect both traits,and the genes involved in regulating these two traits are unclear.The silkworm recessive mutant re^(l)shows both red egg color and embryo mortality.We studied the molecular basis of the re^(l)phenotype formation.Through genetic analysis,gene screening and sequencing,we found that two closely linked genes,BGIBMGA003497(Bmre)and BGIBMG4003697(Bm-Semala),control egg color and embryo mortality,respectively.Six base pairs of the Bm-re gene are deleted in its open reading frame,and BmSemala is expressed at abnormally low levels in mutant re^(l).BmSemala gene function verification was performed using RNA interference and clustered randomly interspersed palindromic repeats(CRISPR)/CRISPR-associate protein 9.Deficiency of the BmSemala gene can cause the death of silkworm embryos.This study revealed the molecular basis of silkworm re^(l)mutant formation and indicated that the Semala gene is essential for insect embryo development.