Publications by authors named "Anjiang Tan"

42 Publications

Two dehydroecdysone reductases act as fat body-specific 20E catalyzers in Bombyx mori.

Insect Sci 2021 May 20. Epub 2021 May 20.

Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu Province, 212100, China.

Periodic post-embryonic changes in insects, including growth, development and metamorphosis, are strictly controlled by many compounds, including steroid hormones. The biosynthesis and clearance of 20-hydroxyecdysone (20E), the major active form of the insect steroid hormone ecdysone, result in titer fluctuations that help control insect development. The inactivation of 20E in the silkworm Bombyx mori is highly tissue-specific, with CYP18A1 and ecdysone oxidase controlling 20E inactivation specifically in the mid-silk gland and midgut, respectively. Here, we characterized silkworm 3-dehydroecdysone 3α reductase (Bm3DE3α) and 3-dehydroecdysone 3β reductase (Bm3DE3β), two enzymes involved predominantly in the C-3-mediated catalysis of 20E in fat bodies. The ubiquitous and silk gland-specific overexpression of Bm3DE3α decreased the 20E titer, resulting in larval lethality and larval-pupal transition failure, respectively. In contrast, the ubiquitous and mid-silk gland-specific overexpression of Bm3DE3β increased the 20E titer, resulting in larval growth delays and lethality at the mid-fifth larval stage, respectively. Thus, Bm3DE3α and Bm3DE3β mediate fat body-specific steroid hormone metabolism in B. mori, indicating that highly diversified 20E metabolism-related mechanisms exist in different insect species.
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http://dx.doi.org/10.1111/1744-7917.12928DOI Listing
May 2021

A CCCH zinc finger gene regulates doublesex alternative splicing and male development in Bombyx mori.

Insect Sci 2020 Oct 7. Epub 2020 Oct 7.

Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, 200032, China.

Recent identification of a Piwi-interacting RNA (piRNA)-initiated sex determination cascade in the silkworm, Bombyx mori, provides novel insights into high diversity of insect sex determination pathways. In this system, the W-chromosome-derived Fem piRNA is the primary sex determination signal. A CCCH-type zinc finger gene Masculinizer (Masc), which is targeted by Fem piRNA-PIWI complex in female animals, is indispensable for male-specific splicing of B. mori doublesex (Bmdsx). Although many genes involved in this cascade have been identified, the regulatory mechanisms of silkworm sex determination remain to be elucidated. Here we show that another CCCH-type zinc finger gene, Bmznf-2, is a masculinization factor in B. mori. Bmznf-2 shows testis-abundant expression and loss of Bmznf-2 function via clustered regularly interspaced short palindromic repeats / single-guide RNA-mediated mutagenesis results in feminized differentiation and appearance of the female-specific splicing variants of Bmdsx transcripts in males. In contrast, there is no phenotypic consequence in mutant females. In mutant males, relative messenger RNA expression levels of female-dominant genes such as vitellogenin and sex-specific storage protein 1 are significantly elevated while several male-dominant genes are significantly down-regulated. Furthermore, male mutants show delayed developmental timing, smaller body sizes of larvae and malformation of moth wings. Our data thus reveal that Bmznf-2 plays an indispensable role in silkworm male sexual differentiation.
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http://dx.doi.org/10.1111/1744-7917.12876DOI Listing
October 2020

Molecular Disruption of Ion Transport Peptide Receptor Results in Impaired Water Homeostasis and Developmental Defects in .

Front Physiol 2020 20;11:424. Epub 2020 May 20.

CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, China.

Insect ion transport peptides (ITPs) are important regulators of many physiological processes and they exert their functions by interacting with their receptors (ITPRs). In the current study, we comprehensively investigated the physiological functions of ITPR in the lepidopteran model insect, the silkworm (), using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome editing technique. Mutations in silkworm ITPR () resulted in a prolongnation of the larval stage by 3.5-day as well as failure in wing expansion of moths. The mutation accelerated food transition throughout the digestive tract, which is 1.55-fold that of wild type (WT) insects. Excretion was 1.56-fold of WT insects during the larval stage, resulting in the loss of body water content. Loss of function induced significant upregulation of nitric oxide synthase (NOS) enzyme activity and nitric oxide (NO) content, as well as downstream Ca/NO/cGMP signaling pathways. Key genes in insulin and ecdysone signaling pathways were also affected by disruption. Our data show that ITPR plays key roles in regulating insect water homeostasis and development.
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http://dx.doi.org/10.3389/fphys.2020.00424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7251169PMC
May 2020

A single ortholog of teashirt and tiptop regulates larval pigmentation and adult appendage patterning in Bombyx mori.

Insect Biochem Mol Biol 2020 06 31;121:103369. Epub 2020 Mar 31.

Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, 200032, China; CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, 100049, China. Electronic address:

Two paralogous genes, teashirt (tsh) and tiptop (tio), encode zinc-finger transcription factors and play important roles in insect growth and development. In the fruit fly, Drosophila melanogaster, tsh promotes trunk segmental identities and contributes to the patterning of other tissues during the embryonic stage. During the adult stage, tsh contributes to the specification and patterning of appendages, including the leg, wing and eye. While tio acts redundantly with tsh, flies lacking tio function are viable without deleterious phenotypes. This gene pair is present in the genomes of all Drosophila species but only as a single homologue in several other insect species. In Oncopeltus fasciatus and Tribolium castaneum, tsh/tio has been functionally characterized as specifying the identity of the leg during the adult stage. However, in lepidopteran insects which include large numbers of pests in agriculture and forestry, as well as the major silk producer silkworm Bombyx mori, the biological functions of tsh/tio are still poorly understood. In the current study, we performed functional analysis of tsh/tio by using both CRISPR/Cas9-mediated mutagenesis and transposon-mediated ectopic expression in B. mori. The results show that loss of tsh/tio function affected pigmentation during the larval stage and appendage pattering during the adult stage. RNA-seq analysis and subsequent q-RT-PCR analysis revealed that depletion of tsh/tio significantly elevated the expression of the kynurenine 3-monooxygenase gene, as well as melanin synthase-related genes during the larval stage. Furthermore, ubiquitous ectopic expression of tsh/tio induces developmental retardation and eventually larval lethality. These data reveal evolutionarily conserved functions of tsh/tio in controlling adult appendage patterning, as well as the novel function of regulating larval pigmentation in B. mori, providing novel insights into how tsh/tio regulates insect growth and development.
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http://dx.doi.org/10.1016/j.ibmb.2020.103369DOI Listing
June 2020

Identification and functional analysis of promoters of heat-shock genes from the fall armyworm, Spodoptera frugiperda.

Sci Rep 2020 02 11;10(1):2363. Epub 2020 Feb 11.

Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, 40546, United States of America.

The functional information on heat-shock proteins (Hsp) and heat-shock promoters from an important agricultural insect pest, Spodoptera frugiperda, is still lacking. We conducted a genome-wide identification of Hsp genes and identified a total of 21 genes belonging to four major insect Hsp families (small heat-shock proteins, Hsp60, Hsp70, and Hsp90) in S. frugiperda. Expression of most of S. frugiperda (SfHsp) genes could be detected in Sf9 cells, embryos and larval tissues of S. frugiperda. The heat-inducible activity of heat-shock promoters from several SfHsp genes was tested in Sf9 cells and embryos. The promoter of SfHsp70D showed the high constitutive activity in cell line and embryos, while the activity of SfHsp20.15 and SfHsp20.71 promoters was most dramatically induced in Sf9 cells and embryos. In embryos, the heat-induced activity of SfHsp20.71 and SfHsp70D promoters outperformed commercially used ie1 and ie2 promoters. The heat-induced activity of SfHsp70D and SfHsp19.07 promoters were more robust than ie2 promoter in Sf9 cells. These SfHsp promoters with high basal activity or with heat-induced activity from low basal activity, could be used in S. frugiperda or other lepidopteran insects for many applications including transgenesis and genome editing.
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http://dx.doi.org/10.1038/s41598-020-59197-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012861PMC
February 2020

Maelstrom regulates spermatogenesis of the silkworm, Bombyx mori.

Insect Biochem Mol Biol 2019 06 7;109:43-51. Epub 2019 Apr 7.

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China. Electronic address:

The spermatogenesis of animal is essential for the reproduction and a very large number of genes participate in this procession. The Maelstrom (Mael) is identified essential for spermatogenesis in both Drosophila and mouse, though the mechanisms appear to differ. It was initially found that Mael gene is necessary for axis specification of oocytes in Drosophila, and recent studies suggested that Mael participates in the piRNA pathway. In this study, we obtained Bombyx mori Mael mutants by using a binary transgenic CRISPR/Cas9 system and analyzed the function of Mael in B. mori, a model lepidopteran insect. The results showed that BmMael is not necessary for piRNA pathway in the ovary of silkworm, whereas it might be essential for transposon elements (TEs) repression in testis. The BmMael mutation resulted in male sterility, and further analysis established that BmMael was essential for spermatogenesis. The spermatogenesis defects occurred in the elongation stage and resulted in nuclei concentration arrest. RNA-seq and qRT-PCR analyses demonstrated that spermatogenesis defects were associated with tight junctions and apoptosis. We also found that BmMael was not involved in the silkworm sex determination pathway. Our data provide insights into the biological function of BmMael in male spermatogenesis and might be useful for developing novel methods to control lepidopteron pests.
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http://dx.doi.org/10.1016/j.ibmb.2019.03.012DOI Listing
June 2019

Disruption of sex-specific doublesex exons results in male- and female-specific defects in the black cutworm, Agrotis ipsilon.

Pest Manag Sci 2019 Jun 7;75(6):1697-1706. Epub 2019 Jan 7.

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.

Background: Doublesex (dsx), the downstream gene in the insect sex-determination pathway, is a key regulator of sexually dimorphic development and behavior across a variety of insects. Manipulating expression of dsx could be useful in the genetic control of insects. However, information on the sex-specific function of dsx in non-model insects is lacking.

Results: In this work, we isolated a dsx homolog, which is alternatively spliced into six female-specific and one male-specific isoforms, from an important agricultural pest, the black cutworm, Agrotis ipsilon. Studies on the expression of sex-specific Aidsx mRNA during embryonic development showed that the sixth hour post oviposition is the key stage for sex determination in A. ipsilon. Functional analysis of Aidsx was conducted using a CRISPR/Cas9 system targeting female- and male-specific Aidsx exons. Disruptions of sex-specific Aidsx exons resulted in sex-specific, sexually dimorphic defects in external genitals, gonads and antennae, and expression of sex-specific genes as well as production of offspring in both sexes.

Conclusion: Our results not only demonstrate that dsx is a key player determining A. ipsilon sexually dimorphic traits, but also provide a potential method for the genetic control of this pest. © 2018 Society of Chemical Industry.
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http://dx.doi.org/10.1002/ps.5290DOI Listing
June 2019

Silkworm genetic sexing through W chromosome-linked, targeted gene integration.

Proc Natl Acad Sci U S A 2018 08 13;115(35):8752-8756. Epub 2018 Aug 13.

Key Laboratory of Insect Developmental and Evolutionary Biology, Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, 200032 Shanghai, China;

Sex separation methods are critical for genetic sexing systems in commercial insect production and sterile insect techniques. Integration of selectable marker genes into a sex chromosome is particularly useful in insects with a heterogametic sex determination system. Here, we describe targeted gene integration of fluorescent marker expression cassettes into a randomly amplified polymorphic DNA (RAPD) marker region in the W chromosome of the lepidopteran model insect using transcriptional activator-like effector nuclease (TALEN)-mediated genome editing. This silkworm strain shows ubiquitous female-specific red or green fluorescence from the embryonic to adult stages. Furthermore, we developed a binary, female-specific, embryonic lethality system combining the TALEN and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology. This system includes one strain with TALEN-mediated, W-specific Cas9 expression driven by the silkworm germ cell-specific () promoter and another strain with U6-derived single-guide RNA (sgRNA) expression targeting (), an essential gene for silkworm embryonic development. Filial 1 (F1) hybrids exhibit complete female-specific lethality during embryonic stages. Our study provides a promising approach for genetic sexing and sheds light on developing sterile insect techniques in other insect species, especially in lepidopteran pests with WZ/ZZ sex chromosome systems.
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http://dx.doi.org/10.1073/pnas.1810945115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6126770PMC
August 2018

Mass spider silk production through targeted gene replacement in .

Proc Natl Acad Sci U S A 2018 08 6;115(35):8757-8762. Epub 2018 Aug 6.

Key Laboratory of Insect Developmental and Evolutionary Biology, Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, 200032 Shanghai, China;

Spider silk is one of the best natural fibers and has superior mechanical properties. However, the large-scale harvesting of spider silk by rearing spiders is not feasible, due to their territorial and cannibalistic behaviors. The silkworm, , has been the most well known silk producer for thousands of years and has been considered an ideal bioreactor for producing exogenous proteins, including spider silk. Previous attempts using transposon-mediated transgenic silkworms to produce spider silk could not achieve efficient yields, due to variable promoter activities and endogenous silk fibroin protein expression. Here, we report a massive spider silk production system in by using transcription activator-like effector nuclease-mediated homology-directed repair to replace the silkworm fibroin heavy chain gene () with the major ampullate spidroin-1 gene () in the spider We successfully replaced the ∼16-kb endogenous gene with a 1.6-kb gene fused with a 1.1-kb partial sequence and achieved up to 35.2% chimeric MaSp1 protein amounts in transformed cocoon shells. The presence of the MaSp1 peptide significantly changed the mechanical characteristics of the silk fiber, especially the extensibility. Our study provides a native promoter-driven, highly efficient system for expressing the heterologous spider silk gene instead of the transposon-based, random insertion of the spider gene into the silkworm genome. Targeted integration into silkworm silk glands provides a paradigm for the large-scale production of spider silk protein with genetically modified silkworms, and this approach will shed light on developing new biomaterials.
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http://dx.doi.org/10.1073/pnas.1806805115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6126722PMC
August 2018

Bombyx mori histone methyltransferase BmAsh2 is essential for silkworm piRNA-mediated sex determination.

PLoS Genet 2018 02 23;14(2):e1007245. Epub 2018 Feb 23.

CAS Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China.

Sex determination is a hierarchically-regulated process with high diversity in different organisms including insects. The W chromosome-derived Fem piRNA has been identified as the primary sex determination factor in the lepidopteran insect, Bombyx mori, revealing a distinctive piRNA-mediated sex determination pathway. However, the comprehensive mechanism of silkworm sex determination is still poorly understood. We show here that the silkworm PIWI protein BmSiwi, but not BmAgo3, is essential for silkworm sex determination. CRISPR/Cas9-mediated depletion of BmSiwi results in developmental arrest in oogenesis and partial female sexual reversal, while BmAgo3 depletion only affects oogenesis. We identify three histone methyltransferases (HMTs) that are significantly down-regulated in BmSiwi mutant moths. Disruption one of these, BmAsh2, causes dysregulation of piRNAs and transposable elements (TEs), supporting a role for it in the piRNA signaling pathway. More importantly, we find that BmAsh2 mutagenesis results in oogenesis arrest and partial female-to-male sexual reversal as well as dysregulation of the sex determination genes, Bmdsx and BmMasc. Mutagenesis of other two HMTs, BmSETD2 and BmEggless, does not affect piRNA-mediated sex determination. Histological analysis and immunoprecipitation results support a functional interaction between the BmAsh2 and BmSiwi proteins. Our data provide the first evidence that the HMT, BmAsh2, plays key roles in silkworm piRNA-mediated sex determination.
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http://dx.doi.org/10.1371/journal.pgen.1007245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5841826PMC
February 2018

Identification of yellow gene family in Agrotis ipsilon and functional analysis of Aiyellow-y by CRISPR/Cas9.

Insect Biochem Mol Biol 2018 03 11;94:1-9. Epub 2018 Jan 11.

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. Electronic address:

The yellow gene family has been identified in several model insects, but yellow genes were poorly identified in non-model insects and the functions of yellow genes are largely unknown. In this study, we identified seven yellow genes in an important agricultural pest Agrotis ipsilon. Each gene encodes a protein containing a major royal jelly domain. Phylogenetic analysis defined these genes as yellow-y, -b, -b2, -c, -d, -e, and -h, respectively. The A. ipsilon yellow genes yellow-b, -b2, and -c were stably expressed in all developmental stages and tissues analyzed, whereas the other four yellow genes had unique expression patterns, suggesting distinct physiological roles of each gene. Using the CRISPR/Cas9 system, we successfully disrupted yellow-y in A. ipsilon and obtained G insects with somatic mutations. Unlike the black of wild-type newly hatched larvae and of adults, the mutants were yellow, although in the pupal stage mutant coloration did not differ from wild-type coloration. This phenotype was inherited by G offspring. The G mutants did not show any growth deficiency compared with control insects; however, a dehydration-like phenotype was observed in newly hatched G larvae from sibling crossed mutants. Our results indicate that A. ipsilon yellow-y gene plays a role in body pigmentation and also might function in waterproofing.
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http://dx.doi.org/10.1016/j.ibmb.2018.01.002DOI Listing
March 2018

MicroRNA-14 regulates larval development time in Bombyx mori.

Insect Biochem Mol Biol 2018 02 27;93:57-65. Epub 2017 Dec 27.

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. Electronic address:

MicroRNAs (miRNA) regulate multiple physiological processes including development and metamorphosis in insects. In the current study, we demonstrate that a conserved invertebrate miRNA-14 (miR-14) plays an important role in ecdysteroid regulated development in the silkworm Bombyx mori, a lepidopteran model insect. Ubiquitous transgenic overexpression of miR-14 using the GAL4/UAS system resulted in delayed silkworm larval development and smaller body size of larva and pupa with decrease in ecdysteriod titers. On the contrary, miR-14 disruption using the transgenic CRISPR/Cas9 system led to a precocious wandering stage with increase in ecdysteriod titers. We identified that the hormone receptor E75 (E75) and the ecdysone receptor isoform B (ECR-B), which both serve as essential mediators in the ecdysone signaling pathway, as putative target genes of miR-14 by in silico target prediction. Dual-luciferase reporter assays confirmed the binding of miR-14 to the 3'UTRs of E75 and ECR-B in a mammalian HEK293T cell line. Furthermore, transcription levels of E75 and ECR-B were significantly affected in both miR-14 overexpression and knockout transgenic animals. Taken together, our data suggested that the canonical invertebrate miR-14 is a general regulator in maintaining ecdysone homeostasis for normal development and metamorphosis in B. mori.
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http://dx.doi.org/10.1016/j.ibmb.2017.12.009DOI Listing
February 2018

The FOXO transcription factor controls insect growth and development by regulating juvenile hormone degradation in the silkworm, .

J Biol Chem 2017 07 10;292(28):11659-11669. Epub 2017 May 10.

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. Electronic address:

Forkhead box O (FOXO) functions as the terminal transcription factor of the insulin signaling pathway and regulates multiple physiological processes in many organisms, including lifespan in insects. However, how FOXO interacts with hormone signaling to modulate insect growth and development is largely unknown. Here, using the transgene-based CRISPR/Cas9 system, we generated and characterized mutants of the silkworm FOXO () to elucidate its physiological functions during development of this lepidopteran insect. The mutant (FOXO-M) exhibited growth delays from the first larval stage and showed precocious metamorphosis, pupating at the end of the fourth instar (trimolter) rather than at the end of the fifth instar as in the wild-type (WT) animals. However, different from previous reports on precocious metamorphosis caused by juvenile hormone (JH) deficiency in silkworm mutants, the total developmental time of the larval period in the FOXO-M was comparable with that of the WT. Exogenous application of 20-hydroxyecdysone (20E) or of the JH analog rescued the trimolter phenotype. RNA-seq and gene expression analyses indicated that genes involved in JH degradation but not in JH biosynthesis were up-regulated in the FOXO-M compared with the WT animals. Moreover, we identified several FOXO-binding sites in the promoter of genes coding for JH-degradation enzymes. These results suggest that FOXO regulates JH degradation rather than its biosynthesis, which further modulates hormone homeostasis to control growth and development in In conclusion, we have uncovered a pivotal role for FOXO in regulating JH signaling to control insect development.
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http://dx.doi.org/10.1074/jbc.M117.777797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5512063PMC
July 2017

Transgenic Clustered Regularly Interspaced Short Palindromic Repeat/Cas9-Mediated Viral Gene Targeting for Antiviral Therapy of Bombyx mori Nucleopolyhedrovirus.

J Virol 2017 04 29;91(8). Epub 2017 Mar 29.

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

We developed a novel antiviral strategy by combining transposon-based transgenesis and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system for the direct cleavage of Bombyx mori nucleopolyhedrovirus (BmNPV) genome DNA to promote virus clearance in silkworms. We demonstrate that transgenic silkworms constitutively expressing Cas9 and guide RNAs targeting the BmNPV () and genes effectively induce target-specific cleavage and subsequent mutagenesis, especially large (∼7-kbp) segment deletions in BmNPV genomes, and thus exhibit robust suppression of BmNPV proliferation. Transgenic animals exhibited higher and inheritable resistance to BmNPV infection than wild-type animals. Our approach will not only contribute to modern sericulture but also shed light on future antiviral therapy. Pathogen genome targeting has shown its potential in antiviral research. However, transgenic CRISPR/Cas9 system-mediated viral genome targeting has not been reported as an antiviral strategy in a natural animal host of a virus. Our data provide an effective approach against BmNPV infection in a real-world biological system and demonstrate the potential of transgenic CRISPR/Cas9 systems in antiviral research in other species.
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http://dx.doi.org/10.1128/JVI.02465-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5375672PMC
April 2017

Bombyx mori P-element Somatic Inhibitor (BmPSI) Is a Key Auxiliary Factor for Silkworm Male Sex Determination.

PLoS Genet 2017 01 19;13(1):e1006576. Epub 2017 Jan 19.

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.

Manipulation of sex determination pathways in insects provides the basis for a wide spectrum of strategies to benefit agriculture and public health. Furthermore, insects display a remarkable diversity in the genetic pathways that lead to sex differentiation. The silkworm, Bombyx mori, has been cultivated by humans as a beneficial insect for over two millennia, and more recently as a model system for studying lepidopteran genetics and development. Previous studies have identified the B. mori Fem piRNA as the primary female determining factor and BmMasc as its downstream target, while the genetic scenario for male sex determination was still unclear. In the current study, we exploite the transgenic CRISPR/Cas9 system to generate a comprehensive set of knockout mutations in genes BmSxl, Bmtra2, BmImp, BmImpM, BmPSI and BmMasc, to investigate their roles in silkworm sex determination. Absence of Bmtra2 results in the complete depletion of Bmdsx transcripts, which is the conserved downstream factor in the sex determination pathway, and induces embryonic lethality. Loss of BmImp or BmImpM function does not affect the sexual differentiation. Mutations in BmPSI and BmMasc genes affect the splicing of Bmdsx and the female reproductive apparatus appeared in the male external genital. Intriguingly, we identify that BmPSI regulates expression of BmMasc, BmImpM and Bmdsx, supporting the conclusion that it acts as a key auxiliary factor in silkworm male sex determination.
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http://dx.doi.org/10.1371/journal.pgen.1006576DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5289617PMC
January 2017

Depletion of juvenile hormone esterase extends larval growth in Bombyx mori.

Insect Biochem Mol Biol 2017 02 3;81:72-79. Epub 2017 Jan 3.

Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. Electronic address:

Two major hormones, juvenile hormone (JH) and 20-hydroxyecdysone (20E), regulate insect growth and development according to their precisely coordinated titres, which are controlled by both biosynthesis and degradation pathways. Juvenile hormone esterase (JHE) is the primary JH-specific degradation enzyme that plays a key role in regulating JH titers, along with JH epoxide hydrolase (JHEH) and JH diol kinase (JHDK). In the current study, a loss-of-function analysis of JHE in the silkworm, Bombyx mori, was performed by targeted gene disruption using the transgenic CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases) system. Depletion of B. mori JHE (BmJHE) resulted in the extension of larval stages, especially the penultimate and ultimate larval stages, without deleterious effects to silkworm physiology. The expression of JHEH and JHDK was upregulated in mutant animals, indicating the existence of complementary routes in the JH metabolism pathway in which inactivation of one enzyme will activate other enzymes. RNA-Seq analysis of mutant animals revealed that genes involved in protein processing in the endoplasmic reticulum and in amino acid metabolism were affected by BmJHE depletion. Depletion of JHE and subsequent delayed JH metabolism activated genes in the TOR pathway, which are ultimately responsible for extending larval growth. The transgenic Cas9 system used in the current study provides a promising approach for analysing the actions of JH, especially in nondrosophilid insects. Furthermore, prolonging larval stages produced larger larvae and cocoons, which is greatly beneficial to silk production.
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http://dx.doi.org/10.1016/j.ibmb.2017.01.001DOI Listing
February 2017

Sexually dimorphic traits in the silkworm, Bombyx mori, are regulated by doublesex.

Insect Biochem Mol Biol 2017 01 17;80:42-51. Epub 2016 Nov 17.

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. Electronic address:

The DM domain genes, doublesex (dsx) in insects, or their structural homologs, male abnormal 3 (mab-3) in nematodes and Dmrt1 (doublesex and mab-3-related transcription factor 1) in mammals, are downstream regulators of the sex determination pathway that control sexually dimorphic development. Despite the functional importance of dsx and its potential applications in sterile insect technologies (SITs), the mechanisms by which it controls sexually dimorphic traits and the subsequent developmental gene networks in insects are poorly understood. Phylogenetic analyses indicate that insect dsx genes have sex-specific alternative splicing isoforms, whereas other taxa do not. We exploited genome editing and transgenesis technologies to induce mutations in either the male-specific isoform (dsx) or common region (dsx) of dsx in the somatic tissues of the lepidopteran model insect Bombyx mori. Disruptions of gene function produced either male-specific sexually-dimorphic defects or intersexual phenotypes; these results differ from those observed in other insects, including Drosophila melanogaster. Our data provide insights into the divergence of the insect sex determination pathways related to the most conserved downstream component dsx.
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http://dx.doi.org/10.1016/j.ibmb.2016.11.005DOI Listing
January 2017

Expansion of CRISPR targeting sites in Bombyx mori.

Insect Biochem Mol Biol 2016 May 24;72:31-40. Epub 2016 Mar 24.

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. Electronic address:

The CRISPR/Cas9 system has been proven as a revolutionary genome engineering tool. In most cases, single guide RNA (sgRNA) targeting sites have been designed as GN19NGG or GGN18NGG, because of restriction of the initiation nucleotide for RNA Pol III promoters. Here, we demonstrate that the U6 promoter from a lepidopteran model insect, Bombyx mori, effectively expressed the sgRNA initiated with any nucleotide bases (adenine, thymine, guanine or cytosine), which further expands the CRISPR targeting space. A detailed expansion index in the genome was analysed when N20NGG was set as the CRISPR targeting site instead of GN19NGG, and revealed a significant increase of suitable targets, with the highest increase occurring on the Z sex chromosome. Transfection of different types of N20NGG sgRNAs targeting the enhanced green fluorescent protein (EGFP) combined with Cas9, significantly reduced EGFP expression in the BmN cells. An endogenous gene, BmBLOS2, was also disrupted by using various types of N20NGG sgRNAs, and the cleavage efficiency of N20NGG sgRNAs with different initial nucleotides and GC contents was evaluated in vitro. Furthermore, transgenic silkworms expressing Cas9 and sgRNAs targeting the BmBLOS2 gene were generated with many types of mutagenesis. The typical transparent skin phenotype in knock-out silkworms was stable and inheritable, suggesting that N20NGG sgRNAs function sufficiently in vivo. Our findings represent a renewal of CRISPR/Cas9 target design and will greatly facilitate insect functional genetics research.
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http://dx.doi.org/10.1016/j.ibmb.2016.03.006DOI Listing
May 2016

Functional analysis of Bombyx Wnt1 during embryogenesis using the CRISPR/Cas9 system.

J Insect Physiol 2015 Aug 9;79:73-9. Epub 2015 Jun 9.

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. Electronic address:

Recently established, custom-designed nuclease technologies such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system provide attractive genome editing tools. Targeted gene mutagenesis using the CRISPR/Cas9 system has been achieved in several orders of insects. However, outside of studies on Drosophila melanogaster and the lepidopteron model insect Bombyx mori, little success has been reported, which is largely due to a lack of effective genetic manipulation tools that can be used in other insect orders. To create a simple and effective method of gene knockout analysis, especially for dissecting gene functioning during insect embryogenesis, we performed a functional analysis of the Bombyx Wnt1 (BmWnt1) gene using Cas9/sgRNA-mediated gene mutagenesis. The Wnt1 gene is required for embryonic patterning in various organisms, and its crucial roles during embryogenesis have been demonstrated in several insect orders. Direct injection of Cas9 mRNA and BmWnt1-specific sgRNA into Bombyx embryos induced a typical Wnt-deficient phenotype: injected embryos could not hatch and exhibited severe defects in body segmentation and pigmentation in a dose-dependent manner. Quantitative real-time PCR (qRT-PCR) analysis revealed that Hox genes were down-regulated after BmWnt1 depletion. Furthermore, large deletion, up to 18Kb, ware generated. The current study demonstrates that using the CRISPR/Cas9 system is a promising approach to achieve targeted gene mutagenesis during insect embryogenesis.
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http://dx.doi.org/10.1016/j.jinsphys.2015.06.004DOI Listing
August 2015

Ectopic expression of ecdysone oxidase impairs tissue degeneration in Bombyx mori.

Proc Biol Sci 2015 Jun;282(1809):20150513

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, People's Republic of China

Metamorphosis in insects includes a series of programmed tissue histolysis and remolding processes that are controlled by two major classes of hormones, juvenile hormones and ecdysteroids. Precise pulses of ecdysteroids (the most active ecdysteroid is 20-hydroxyecdysone, 20E), are regulated by both biosynthesis and metabolism. In this study, we show that ecdysone oxidase (EO), a 20E inactivation enzyme, expresses predominantly in the midgut during the early pupal stage in the lepidopteran model insect, Bombyx mori. Depletion of BmEO using the transgenic CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases) system extended the duration of the final instar larval stage. Ubiquitous transgenic overexpression of BmEO using the Gal4/UAS system induced lethality during the larval-pupal transition. When BmEO was specifically overexpressed in the middle silk gland (MSG), degeneration of MSG at the onset of metamorphosis was blocked. Transmission electron microscope and LysoTracker analyses showed that the autophagy pathway in MSG is inhibited by BmEO ectopic expression. Furthermore, RNA-seq analysis revealed that the genes involved in autophagic cell death and the mTOR signal pathway are affected by overexpression of BmEO. Taken together, BmEO functional studies reported here provide insights into ecdysone regulation of tissue degeneration during metamorphosis.
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http://dx.doi.org/10.1098/rspb.2015.0513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4590451PMC
June 2015

MiR-2 family targets awd and fng to regulate wing morphogenesis in Bombyx mori.

RNA Biol 2015 ;12(7):742-8

a Faculty of Life Sciences; Northwestern Polytechnical University ; Xi'an , China.

MicroRNAs (miRNAs) are post-transcriptional regulators that target specific mRNAs for repression and thus play key roles in many biological processes, including insect wing morphogenesis. miR-2 is an invertebrate-specific miRNA family that has been predicted in the fruit fly, Drosophila melanogaster, to be involved in regulating the Notch signaling pathway. We show here that miR-2 plays a critical role in wing morphogenesis in the silkworm, Bombyx mori, a lepidopteran model insect. Transgenic over-expression of a miR-2 cluster using a Gal4/UAS system results in deformed adult wings, supporting the conclusion that miR-2 regulates functions essential for normal wing morphogenesis. Two genes, abnormal wing disc (awd) and fringe (fng), which are positive regulators in Notch signaling, are identified as miR-2 targets and validated by a dual-luciferase reporter assay. The relative abundance of both awd and fng expression products was reduced significantly in transgenic animals, implicating them in the abnormal wing phenotype. Furthermore, somatic mutagenesis analysis of awd and fng using the CRISPR/Cas9 system and knock-out mutants also resulted in deformed wings similar to those observed in the miR-2 overexpression transgenic animals. The critical role of miR-2 in Bombyx wing morphogenesis may provide a potential target in future lepidopteran pest control.
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http://dx.doi.org/10.1080/15476286.2015.1048957DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4615647PMC
March 2016

Enhancement of larval RNAi efficiency by over-expressing Argonaute2 in Bombyx mori.

Int J Biol Sci 2015 5;11(2):176-85. Epub 2015 Jan 5.

1. Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.

RNA interference has been described as a powerful genetic tool for gene functional analysis and a promising approach for pest management. However, RNAi efficiency varies significantly among insect species due to distinct RNAi machineries. Lepidopteran insects include a large number of pests as well as model insects, such as the silkworm, Bombyx mori. However, only limited success of in vivo RNAi has been reported in lepidoptera, particularly during the larval stages when the worms feed the most and do the most harm to the host plant. Enhancing the efficiency of larval RNAi in lepidoptera is urgently needed to develop RNAi-based pest management strategies. In the present study, we investigate the function of the conserved RNAi core factor, Argonaute2 (Ago2), in mediating B. mori RNAi efficiency. We demonstrate that introducing BmAgo2 dsRNA inhibits the RNAi response in both BmN cells and embryos. Furthermore, we establish several transgenic silkworm lines to assess the roles of BmAgo2 in larval RNAi. Over-expressing BmAgo2 significantly facilitated both dsRNA-mediated larval RNAi when targeting DsRed using dsRNA injection and shRNA-mediated larval RNAi when targeting BmBlos2 using transgenic shRNA expression. Our results show that BmAgo2 is involved in RNAi in B. mori and provides a promising approach for improving larval RNAi efficiency in B. mori and in lepidopteran insects in general.
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http://dx.doi.org/10.7150/ijbs.10235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4279093PMC
September 2015

Site-specific, TALENs-mediated transformation of Bombyx mori.

Insect Biochem Mol Biol 2014 12 23;55:26-30. Epub 2014 Oct 23.

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.

Transposon-based genetic transformation has facilitated insect functional genomics and new strategies of pest management. However, there is a need for alternative, site-specific approaches to overcome limitations of random integration (and associated position-effects) and potential instability of inserted transgenes. Here we describe a transposon-free, site-specific genetic transformation system mediated by transcription activator-like effector nucleases (TALENs) in the silkworm, Bombyx mori, a lepidopteran model insect. We successfully established a site-specific transgenic system with comparable transformation efficiency to transposon-based genetic transformation through microinjection of TALENs mRNA targeting the BmBLOS2 locus and a linearizable donor plasmid encoding an expression cassette of the DsRed2 red fluorescent protein. This system provides a valuable approach for insect transgenesis and will enable future functional gene analysis and generate novel applications in agricultural and medical insect pest-management technologies.
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http://dx.doi.org/10.1016/j.ibmb.2014.10.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4408225PMC
December 2014

CYP18A1 regulates tissue-specific steroid hormone inactivation in Bombyx mori.

Insect Biochem Mol Biol 2014 Nov 27;54:33-41. Epub 2014 Aug 27.

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. Electronic address:

Insect development and metamorphosis are regulated by two major hormones, juvenile hormone and ecdysteroids. Despite being the key regulator of insect developmental transitions, the metabolic pathway of the primary steroid hormone, 20-hydroxyecdysone (20E), especially its inactivation pathway, is still not completely elucidated. A cytochrome P450 enzyme, CYP18A1, has been shown to play key roles in insect steroid hormone inactivation through 26-hydroxylation. Here, we identified two CYP18 (BmCYP18A1 and BmCYP18B1) orthologs in the lepidopteran model insect, Bombyx mori. Interestingly, BmCYP18A1 gene is predominantly expressed in the middle silk gland (MSG) while BmCYP18B1 expresses ubiquitously in B. mori. BmCYP18A1 is induced by 20E in vitro, suggesting its role in 20E metabolism. Using the binary Gal4/UAS transgenic system, we ectopically overexpressed BmCYP18A1 in a MSG-specific manner with a Sericin1-Gal4 (Ser-Gal4) driver or in a ubiquitous manner with an Actin3-Gal4 (A3-Gal4) driver. Ectopic overexpression of BmCYP18A1 in MSG or in all tissues resulted in developmental arrestment of transgenic animals during the final instar larval stage. The 20E titers in the transgenic animals expressing BmCYP18A1 were lower compared to the levels in the control animals. Although the biological significance of MSG-specific expression of BmCYP18A1 is unclear, our results provide the first evidence that BmCYP18A1, which is conserved in most arthropods, is involved in a tissue-specific steroid hormone inactivation in B. mori.
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http://dx.doi.org/10.1016/j.ibmb.2014.08.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4692384PMC
November 2014

Allelic-specific expression in relation to Bombyx mori resistance to Bt toxin.

Insect Biochem Mol Biol 2014 Nov 12;54:53-60. Epub 2014 Aug 12.

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. Electronic address:

Understanding the mechanism of Bt resistance is one of the key elements of the effective application of Bt in pest control. The lepidopteran model insect, the silkworm, demonstrates qualities that make it an ideal species to use in achieving this understanding. We screened 45 strains of silkworm (Bombyx mori) using a Cry1Ab toxin variant. The sensitivity levels of the strains varied over a wide range. A resistant strain (P50) and a phylogenetically related susceptible strain (Dazao) were selected to profile the expressions of 12 Bt resistance-related genes. The SNPs in these genes were detected based on EST analysis and were validated by allelic-specific PCR. A comparison of allelic-specific expression between P50 and Dazao showed that the transcript levels of heterozygous genes containing two alleles rather than an imbalanced allelic expression contribute more to the resistance of P50 against Bt. The responses of the allelic-specific expression to Bt in hybrid larvae were then investigated. The results showed that the gene expression pattern of an ATP-binding cassette transporter C2 (ABCC2) and an aminopeptidase N (APN3), changed in an allelic-specific manner, with the increase of the resistant allele expression correlated with larval survival. The results suggest that a trans-regulatory mechanism in ABCC2 and APN3 allelic-specific expression is involved in the insect's response to the Bt toxin. The potential role of allelic-specific gene regulation in insect resistance to Bt toxins is discussed.
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http://dx.doi.org/10.1016/j.ibmb.2014.07.007DOI Listing
November 2014

MicroRNA Let-7 regulates molting and metamorphosis in the silkworm, Bombyx mori.

Insect Biochem Mol Biol 2014 Oct 9;53:13-21. Epub 2014 Jul 9.

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. Electronic address:

MicroRNAs (miRNAs) are a class of endogenous, non-coding, regulatory RNA molecules that post-transcriptionally regulate gene expression by binding to the 3'UTRs of mRNA targets and thus cause their degradation or translational inhibition. In insects, important roles of miRNAs in various biological processes have been demonstrated in Drosophila melanogaster. However, biological roles of miRNAs are barely unveiled in the majority of insect species due to limited genetic tools. In the present study, we introduce the transgenic miRNA sponge (miR-SP) technology combining with the binary GAL4/UAS system in the domesticated silkworm, Bombyx mori, to exploit the biological function of an evolutionally conserved miRNA, let-7. We successfully established transgenic silkworm lines in which a miRNA sponge construct targeting BmLet-7 seed region was expressed in a ubiquitous manner directed by A3-GAL4 driver. Transgenic animals showed decreased expression of BmLet-7, leading to developmental arrestment during the larval-larval and larval-pupal transition. Simultaneously, expression levels of the predicted BmLet-7 target genes, FTZ-F1 and Eip74EF (E74), key regulatory factors in the ecdysone pathway, were elevated in transgenic animals. The current study is the first report on application of the transgenic miR-SP technology in non-drosophilid insects, which will not only contribute to better understanding of let-7 biological roles, but also greatly facilitate future miRNA functional analysis in insects.
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http://dx.doi.org/10.1016/j.ibmb.2014.06.011DOI Listing
October 2014

PDP1 regulates energy metabolism through the IIS-TOR pathway in the red flour beetle, Tribolium castaneum.

Arch Insect Biochem Physiol 2014 Mar 29;85(3):127-36. Epub 2014 Jan 29.

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.

The PAR-domain protein 1 (PDP1) is essential for locomotor activity of insects. However, its functions in insect growth and development have not been studied extensively, which prompted our hypothesis that PDP1 acts in energy metabolism. Here we report identification of TcPDP1 in the red flour beetle, Tribolium castaneum, and its functional analysis by RNAi. Treating larvae with dsTcPDP1 induced pupae developmental arrestment, accompanied by accelerated fat body degradation. dsTcPDP1 treatments in adults resulted in reduced female fecundity. Disruption of TcPDP1 expression affected the transcription of genes involved in insulin signaling transduction and mechanistic target of rapamycin (mTOR) pathway. These results support our hypothesis that TcPDP1 acts in energy metabolism in T. castaneum.
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http://dx.doi.org/10.1002/arch.21146DOI Listing
March 2014

The CRISPR/Cas system mediates efficient genome engineering in Bombyx mori.

Cell Res 2013 Dec 29;23(12):1414-6. Epub 2013 Oct 29.

1] Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China [2] University of Chinese Academy of Sciences, Beijing 100049, China.

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http://dx.doi.org/10.1038/cr.2013.146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3847576PMC
December 2013

Comparative methylomics between domesticated and wild silkworms implies possible epigenetic influences on silkworm domestication.

BMC Genomics 2013 Sep 23;14:646. Epub 2013 Sep 23.

State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, 32 East Jiaochang Road, Kunming, Yunnan Province 650223, China.

Background: In contrast to wild species, which have typically evolved phenotypes over long periods of natural selection, domesticates rapidly gained human-preferred agronomic traits in a relatively short-time frame via artificial selection. Under domesticated conditions, many traits can be observed that cannot only be due to environmental alteration. In the case of silkworms, aside from genetic divergence, whether epigenetic divergence played a role in domestication is an unanswered question. The silkworm is still an enigma in that it has two DNA methyltransferases (DNMT1 and DNMT2) but their functionality is unknown. Even in particular the functionality of the widely distributed DNMT1 remains unknown in insects in general.

Results: By embryonic RNA interference, we reveal that knockdown of silkworm Dnmt1 caused decreased hatchability, providing the first direct experimental evidence of functional significance of insect Dnmt1. In the light of this fact and those that DNA methylation is correlated with gene expression in silkworms and some agronomic traits in domesticated organisms are not stable, we comprehensively compare silk gland methylomes of 3 domesticated (Bombyx mori) and 4 wild (Bombyx mandarina) silkworms to identify differentially methylated genes between the two. We observed 2-fold more differentiated methylated cytosinces (mCs) in domesticated silkworms as compared to their wild counterparts, suggesting a trend of increasing DNA methylation during domestication. Further study of more domesticated and wild silkworms narrowed down the domesticates' epimutations, and we were able to identify a number of differential genes. One such gene showing demethyaltion in domesticates correspondently displays lower gene expression, and more interestingly, has experienced selective sweep. A methylation-increased gene seems to result in higher expression in domesticates and the function of its Drosophila homolog was previously found to be essential for cell volume regulation, indicating a possible correlation with the enlargement of silk glands in domesticated silkworms.

Conclusions: Our results imply epigenetic influences at work during domestication, which gives insight into long time historical controversies regarding acquired inheritance.
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http://dx.doi.org/10.1186/1471-2164-14-646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3852238PMC
September 2013

MicroRNA-281 regulates the expression of ecdysone receptor (EcR) isoform B in the silkworm, Bombyx mori.

Insect Biochem Mol Biol 2013 Aug 22;43(8):692-700. Epub 2013 May 22.

Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai 200032, China.

Insect development and metamorphosis are regulated by the coordination of ecdysone and juvenile hormones. Insect microRNAs (miRNAs) also act in insect development and metamorphosis by regulating genes in the ecdysone cascade. Although hundreds of insect miRNAs have been identified, the physiological functions of most remain poorly understood. Here, we report that a conserved insect miRNA, microRNA-281 (miR-281), regulates the ecdysone receptor (EcR), in an isoform-specific manner in the silkworm Bombyx mori. The B. mori EcR (BmEcR) gene encodes three isoforms: BmEcR-A, BmEcR-B1 and BmEcR-B2. The 3'UTR regions of A and B genes, which contain multiple potential microRNA targeting sites, are distinct. Target prediction revealed that miR-281 may specifically target the 3'UTR of BmEcR-B. Using a dual luciferase reporter assay in HEK293T cells, we confirmed that miR-281 suppressed transcription of BmEcR-B but not BmEcR-A. The expression of miR-281 and BmEcR-B are well coordinated in the Malpighian tubules from the fourth larval molt to pupation. In the Malpighian tubules of fifth instar larvae, BmEcR-B protein expression was down-regulated after injection of a miR-281 mimic while up-regulated after injection of a miR-281 inhibitor. miR-281 expression was suppressed by 20-hydroxyecdysone treatments but not affected by juvenile hormone treatments. Based on these findings, we propose that miR-281 participates in B. mori developmental regulation in the Malpighian tubules through suppression of BmEcR-B expression.
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http://dx.doi.org/10.1016/j.ibmb.2013.05.002DOI Listing
August 2013