Modular cis-regulatory logic of yellow gene expression in silkmoth larvae.

Colour patterns in butterflies and moths are crucial traits for adaptation. Previous investigations have highlighted genes responsible for pigmentation (ie yellow and ebony). However, the mechanisms by which these genes are regulated in lepidopteran insects remain poorly understood. To elucidate this, molecular studies involving dipterans have largely analysed the cis-regulatory regions of pigmentation genes and have revealed cis-regulatory modularity. Here, we used well-developed transgenic techniques in Bombyx mori and demonstrated that cis-regulatory modularity controls tissue-specific expression of the yellow gene. We first identified which body parts are regulated by the yellow gene via black pigmentation. We then isolated three discrete regulatory elements driving tissue-specific gene expression in three regions of B. mori larvae. Finally, we found that there is no apparent sequence conservation of cis-regulatory regions between B. mori and Drosophila melanogaster, and no expression driven by the regulatory regions of one species when introduced into the other species. Therefore, the trans-regulatory landscapes of the yellow gene differ significantly between the two taxa. The results of this study confirm that lepidopteran species use cis-regulatory modules to control gene expression related to pigmentation, and represent a powerful cadre of transgenic tools for studying evolutionary developmental mechanisms.

Sandwich ELISA Using a Mouse/Human Chimeric CSLEX-1 Antibody.

BACKGROUND: An assay using a mouse antisialyl Lewis X (sLeX) antibody (CSLEX-1) is used clinically for screening and monitoring patients with breast cancer in Japan. However, the IgM isoform of CSLEX-1 is not preferred for the assay because the bulkiness of IgM generally causes poor accessibility to the antigen. To solve this problem, we developed an antisLeX mouse/human chimeric IgG antibody, CH-CSLEX-1, using transgenic silkworms. The performance of a homologous sandwich ELISA of CH-CSLEX1 was then evaluated. METHODS: To generate CH-CSLEX-1, we used a GAL4/UAS binary gene expression system in transgenic silkworms. The reactivities of CSLEX-1 and CH-CSLEX-1 were determined in a Biacore analysis. To confirm antigen specificity, 3 antigens [sLeX, sLeA, and Lewis Y (LeY)] were used. RESULTS: CH-CSLEX-1 formed correctly as an IgG class of immunoglobulin molecule with an isoelectric point close to the predicted value. The best combination for capturing and probing in a sandwich ELISA was determined as a homologous combination of CH-CSLEX-1. The CH-CSLEX-1 assay specifically detected sLeX, but not sLeA and LeY. A correlation analysis with 107 human samples showed good concordance between the conventional CSLEX-1 assay (homologous sandwich ELISA using CSLEX-1) and the CH-CSLEX-1 assay (r = 0.98). Moreover, the CH-CSLEX-1 assay was not affected by either human antimouse IgG antibodies (HAMA IgG) or HAMA IgM. CONCLUSIONS: The mouse/human chimeric antibody CH-CSLEX-1 allowed the establishment of a highly specific sandwich ELISA for sLeX that was not affected by HAMA.

Knockout of a transgene by transcription activator-like effector nucleases (TALENs) in the sawfly, Athalia rosae (Hymenoptera) and the ladybird beetle, Harmonia axyridis (Coleoptera).

Transcription activator-like effector nucleases (TALENs) are efficient tools for targeted genome editing and have been utilized in a number of insects. Here, we demonstrate the gene disruption (knockout) caused by TALENs targeting a transgene, 3xP3-driven enhanced green fluorescence protein (EGFP), that is integrated in the genome of two species, the sawfly Athalia rosae (Hymenoptera) and the ladybird beetle Harmonia axyridis (Coleoptera). Messenger RNAs of TALENs targeting the sequences adjacent to the chromophore region were microinjected into the eggs/embryos of each species. In At. rosae, when microinjection was performed at the posterior end of eggs, 15% of G(0) individuals showed a somatic mosaic phenotype for eye EGFP fluorescence. Three-quarters of the somatic mosaics produced EGFP-negative G(1) progeny. When eggs were injected at the anterior end, 63% of the G(0) individuals showed somatic mosaicism, and 17% of them produced EGFP-negative G(1) progeny. In H. axyridis, 25% of posterior-injected and 8% of anterior-injected G(0) individuals produced EGFP-negative G(1) progeny. In both species, the EGFP-negative progeny retained the EGFP gene, and various deletions were detected in the target sequences, indicating that gene disruption was successfully induced. Finally, for both species, 18-21% of G(0) founders produced gene knockout progeny sufficient for establishing knockout strains.

Positional cloning of a Bombyx pink-eyed white egg locus reveals the major role of cardinal in ommochrome synthesis.

Ommochromes are major insect pigments involved in coloration of compound eyes, eggs, epidermis and wings. In the silkworm Bombyx mori, adult compound eyes and eggs contain a mixture of the ommochrome pigments such as ommin and xanthommatin. Here, we identified the gene involved in ommochrome biosynthesis by positional cloning of B. mori egg and eye color mutant pink-eyed white egg (pe). The recessive homozygote of pe has bright red eyes and white or pale pink eggs instead of a normal dark coloration due to the decrease of dark ommochrome pigments. By genetic linkage analysis, we narrowed down the pe-linked region to ~258 kb, containing 17 predicted genes. RNA sequencing analyses showed that the expression of one candidate gene, the ortholog of Drosophila haem peroxidase cardinal, coincided with egg pigmentation timing, similar to other ommochrome-related genes such as Bm-scarlet and Bm-re. In two pe strains, a common missense mutation was found within a conserved motif of B. mori cardinal homolog (Bm-cardinal). RNA interference-mediated knockdown and transcription activator-like effector nuclease (TALEN)-mediated knockout of the Bm-cardinal gene produced the same phenotype as pe in terms of egg, adult eye and larval epidermis coloration. A complementation test of the pe mutant with the TALEN-mediated Bm-cardinal-deficient strain showed that the mutant phenotype could not be rescued, indicating that Bm-cardinal is responsible for pe. Moreover, knockdown of the cardinal homolog in Tribolium castaneum also induced red compound eyes. Our results indicate that cardinal plays a major role in ommochrome synthesis of holometabolous insects.

Establishment of a specific cell death induction system in Bombyx mori by a transgene with the conserved apoptotic regulator, mouse Bcl-2-associated X protein (mouse Bax).

The induction of apoptosis in vivo is a useful tool for investigating the functions and importance of particular tissues. B-cell leukaemia/lymphoma 2-associated X protein (Bax) functions as a pro-apoptotic factor and induces apoptosis in several organisms. The Bax-mediated apoptotic system is widely conserved from Caenorhabditis elegans to humans. In order to establish a tissue-specific cell death system in the domestic silkworm, Bombyx mori, we constructed a transgenic silkworm that overexpressed mouse Bax (mBax) in particular tissues by the Gal4-upstream activation sequence system. We found that the expression of mBax induced specific cell death in the silk gland, fat body and sensory cells. Fragmentation of genomic DNA was observed in the fat body, which expressed mBax, thereby supporting apoptotic cell death in this tissue. Using this system, we also demonstrated that specific cell death in sensory cells attenuated the response to the sex pheromone bombykol. These results show that we successfully established a tissue-specific cell death system in vivo that enabled specific deficiencies in particular tissues. The inducible cell death system may provide useful means for industrial applications of the silkworm and possible utilization for other species.

Establishment of transgenic silkworms expressing GAL4 specifically in the haemocyte oenocytoid cells.

Insect haemocytes play significant roles in innate immunity. The silkworm, a lepidopteran species, is often selected as the model for studies into the functions of haemocytes in immunity; however, our understanding of the role of haemocytes remains limited because the lack of haemocyte promoters for transgene expression makes genetic manipulations difficult. In the present study, we aimed to establish transgenic silkworm strains expressing GAL4 in their haemocytes. First, we identified three genes with strong expression in haemocytes, namely, lp44, Haemocyte Protease 1 (HP1) and hemocytin. Transgenic silkworms expressing GAL4 under the control of the putative promoters of these genes were then established and expression was examined. Although GAL4 expression was not detected in haemocytes of HP1-GAL4 or hemocytin-GAL4 strains, lp44-GAL4 exhibited a high level of GAL4 expression, particularly in oenocytoids. GAL4 expression was also detected in the midgut but in no other tissues, indicating that GAL4 expression in this strain is mostly oenocytoid-specific. Thus, we have identified a promoter that enables oenocytoid expression of genes of interest. Additionally, the lp44-GAL4 strain could also be used for other types of research, such as the functional analysis of genes in oenocytoids, which would facilitate advances in our understanding of insect immunity.

PhiC31 integrase-mediated cassette exchange in silkworm embryos.

To construct an effective site-specific integration system in the silkworm, we examined if phiC31 integrase works in silkworm embryos. As an assay system, we constructed an extrachromosomal cassette exchange reaction system between two attP sites of an acceptor plasmid and two attB sites of a donor plasmid. To evaluate the activity, integrase mRNAs synthesized from three different plasmids were used. We injected a mixture of the acceptor and donor plasmids with the mRNA synthesized in vitro from one of the three plasmids into silkworm embryos at 4-6 h after oviposition and recovered plasmid DNAs from the embryos 3 days after injection. The resultant plasmids were transformed into Escherichia coli and spread on selection medium plates containing the appropriate antibiotics. A colony-forming assay and restriction enzyme digestion of the plasmids purified from the colonies showed that the phiC31 integrase worked very efficiently in the silkworm embryos. Notably, a phiC31 integrase mRNA synthesized from two of the plasmids produced cassette exchange plasmids at a high frequency, suggesting that the mRNA can be used to construct a targeted integration system in silkworms.

Transgenic analysis of the BmBLOS2 gene that governs the translucency of the larval integument of the silkworm, Bombyx mori.

The larval integument of the silkworm, Bombyx mori, is opaque because urate granules accumulate in the epidermis. Although the biosynthetic pathway of uric acid is well studied, little is known about how uric acid accumulates as urate granules in epidermal cells. In the distinct oily (od) mutant silkworm, the larval integument is translucent because of the inability to construct urate granules. Recently, we have found that the od mutant has a genomic deletion in the B. mori homologue of the human biogenesis of lysosome-related organelles complex1, subunit 2 (BLOS2) gene (BmBLOS2). Here, we performed a molecular and functional characterization of BmBLOS2. Northern blot analysis showed that BmBLOS2 was ubiquitously expressed in various tissues. We analysed the structure of a newly isolated mutant (od(B) ) allelic to od and found a premature stop codon in the coding sequence of BmBLOS2 in this new mutation. Moreover, the translucent phenotype was rescued by the germ-line transformation of the wild-type BmBLOS2 allele into the od mutant. Our results suggest that BmBLOS2 is responsible for the od mutant phenotype and plays a crucial role in biogenesis of urate granules in the larval epidermis of the silkworm. The relationships amongst Hermansky-Pudlak syndrome (HPS) genes in mammals, granule group genes in Drosophila and translucent mutant genes in B. mori are discussed.

Extensive synteny conservation of holocentric chromosomes in Lepidoptera despite high rates of local genome rearrangements.

The recent assembly of the silkworm Bombyx mori genome with 432 Mb on 28 holocentric chromosomes has become a reference in the genomic analysis of the very diverse Order of Lepidoptera. We sequenced BACs from two major pests, the noctuid moths Helicoverpa armigera and Spodoptera frugiperda, corresponding to 15 regions distributed on 11 B. mori chromosomes, each BAC/region being anchored by known orthologous gene(s) to analyze syntenic relationships and genome rearrangements among the three species. Nearly 300 genes and numerous transposable elements were identified, with long interspersed nuclear elements and terminal inverted repeats the most abundant transposable element classes. There was a high degree of synteny conservation between B. mori and the two noctuid species. Conserved syntenic blocks of identified genes were very small, however, approximately 1.3 genes per block between B. mori and the two noctuid species and 2.0 genes per block between S. frugiperda and H. armigera. This corresponds to approximately two chromosome breaks per Mb DNA per My. This is a much higher evolution rate than among species of the Drosophila genus and may be related to the holocentric nature of the lepidopteran genomes. We report a large cluster of eight members of the aminopeptidase N gene family that we estimate to have been present since the Jurassic. In contrast, several clusters of cytochrome P450 genes showed multiple lineage-specific duplication events, in particular in the lepidopteran CYP9A subfamily. Our study highlights the value of the silkworm genome as a reference in lepidopteran comparative genomics.

Production of an active feline interferon in the cocoon of transgenic silkworms using the fibroin H-chain expression system.

We constructed the fibroin H-chain expression system to produce recombinant proteins in the cocoon of transgenic silkworms. Feline interferon (FeIFN) was used for production and to assess the quality of the product. Two types of FeIFN fusion protein, each with N- and C-terminal sequences of the fibroin H-chain, were designed to be secreted into the lumen of the posterior silk glands. The expression of the FeIFN/H-chain fusion gene was regulated by the fibroin H-chain promoter domain. The transgenic silkworms introduced these constructs with the piggyBac transposon-derived vector, which produced the normal sized cocoons containing each FeIFN/H-chain fusion protein. Although the native-protein produced by transgenic silkworms have almost no antiviral activity, the proteins after the treatment with PreScission protease to eliminate fibroin H-chain derived N- and C-terminal sequences from the products, had very high antiviral activity. This H-chain expression system, using transgenic silkworms, could be an alternative method to produce an active recombinant protein and silk-based biomaterials.

Mutations of the silkworm molybdenum cofactor sulfurase gene, og, cause translucent larval skin.

Normal silkworms (Bombyx mori) have opaque larval skin due to uric acid accumulation in the epidermis while a mutant, og, is translucent owing to a deficiency in xanthine dehydrogenase (XDH), which synthesizes uric acid. Molybdenum cofactor (MoCo) sulfurase is responsible for XDH activation in various organisms. A silkworm MoCo sulfurase gene was cloned and found to be on the og locus, whose mutant alleles, og(k) and og(t), show premature stop codons, proving that og is the MoCo sulfurase gene. It was observed that a miniature inverted-repeat transposable element (MITE), named Organdy, when inserted in an og(t) mutant allele exon, causes unstable splicing of a downstream intron leading to incomplete open reading frames.

Characterization of the kynurenine 3-monooxygenase gene corresponding to the white egg 1 mutant in the silkworm Bombyx mori.

Kynurenine 3-monooxygenase (KMO, EC 1.14.13.9), which catalyzes the oxidation of kynurenine to 3-hydroxykynurenine, is involved in the synthesis of ommochrome pigments in insects. A silkworm mutant, white egg 1 ( w-1), has been shown to be deficient in this enzyme activity. The mutant is characterized morphologically by its white eyes and the fact that the females lay white eggs. To analyze the relationship between the KMO gene and the mutation, we first determined the entire sequence of a full-length 2.0-kb cDNA and examined its expression pattern in the wild type. The cDNA sequence contains one ORF encoding a polypeptide of 456 amino acids, and transcripts were detected in the larval Malpighian tubules and the pupal ovaries, but not in other tissues. Southern analysis and nucleotide sequencing showed that the KMO gene is present in a single copy and consists of ten exons distributed over a 16-kb region. Comparison of the transcripts between the wild type and mutant silkworms showed that the wild type expressed a single transcript, whereas the mutant exhibited markedly reduced amounts of two transcripts with sizes of 2.0 kb and 1.8 kb. Nucleotide sequence analysis of these mutant transcripts indicated that sequences corresponding to the ninth and tenth exons were missing. Inverse PCR and Southern analysis of the mutant gene demonstrated that the corresponding genomic region was deleted in the w-1 mutant.

Identification and characterisation of a silkworm ABC transporter gene homologous to Drosophila white.

In the silkworm, Bombyx mori, many eye- and egg-colour mutations affecting the synthesis and accumulation of ommochrome pigments have been described. In order to understand the pigment precursor transporters involved, ABC transporter genes homologous to the Drosophila white gene were isolated from the silkworm. Reverse transcriptase-polymerase chain reactions (RT-PCR) using embryonic mRNA amplified three cDNA fragments, named Bmwh1, Bmwh2 and Bmwh3 that showed homology to the white gene. Since Bmwh3 shows the highest degree of sequence identity and a similar expression pattern to the Drosophila homologue, we characterised this gene further. A 2667-bp Bmwh3 cDNA isolated from an embryonic library has one ORF encoding a polypeptide of 687 amino acids. The predicted protein has one ATP-binding domain, six transmembrane-spanning segments and high similarity to the Drosophila WHITE protein. Southern analysis indicates that Bmwh3 is a single-copy gene. Polyadenylated Bmwh3 transcripts about 2.7 kb long were detected in eggs, Malpighian tubules and pupal heads, but not in testes, posterior silk glands or fat body cells. The level of Bmwh3 mRNA was reduced in w3 and w3ol mutants but normal in other egg- and eye-colour mutants, suggesting that Bmwh3 correspond to the w3 locus. Genetic analysis was used to map the cloned gene to chromosome 10.

Dynamic rearrangement within the Antheraea pernyi silk fibroin gene is associated with four types of repetitive units.

We characterized a full-length gene encoding wild silkmoth Antheraea pernyi fibroin (Ap-fibroin) to clarify the conformation of repetitive sequences. The gene consisted of a first exon encoding 14 amino acid residues, a short intron (120 bp), and a long second exon encoding 2,625 amino acid residues. Three amino acids, alanine, glycine, and serine, amounted to 81% of the Ap-fibroin sequence. The Ap-fibroin, except for 155 residues of the amino terminus, was composed of 80 tandemly arranged polyalanine-containing units (motifs). A motif was a doublet of a polyalanine block (PAB) and a nonpolyalanine block (NPAB). Seventy-eight of the 80 motifs were classified into four types based on differences in the NPAB sequences. Although respective motifs were significantly conserved, many rearrangements were observed within the second exon, i.e., the triplication of a 558-bp-long sequence and other duplication events of shorter sequences. Chi-like sequences, GCTGGAG, might contribute to the rearrangement within the gene as described in human minisatellite loci, because they were found at specific sites of NPAB-encoding sequences in three of four types of motifs. The present results support the idea that the Ap-fibroin gene is unstable like minisatellite sequences and that the evolution of this gene is strongly associated with its instability.

Evolution of the LINE-like I element in the Drosophila melanogaster species subgroup.

LINE-like retrotransposons, the so-called I elements, control the system of I-R (inducer-reactive) hybrid dysgenesis in Drosophila melanogaster. I elements are present in many Drosophila species. It has been suggested that active, complete I elements, located at different sites on the chromosomes, invaded natural populations of D. melanogaster recently (1920-1970). But old strains lacking active I elements have only defective I elements located in the chromocenter. We have cloned I elements from D. melanogaster and the melanogaster subgroup. In D. melanogaster, the nucleotide sequences of chromocentral I elements differed from those on chromosome arms by as much as 7%. All the I elements of D. mauritiana and D. sechellia are more closely related to the chromosomal I elements of D. melanogaster than to the chromocentral I elements in any species. No sequence difference was observed in the surveyed region between two chromosomal I elements isolated from D. melanogaster and one from D. simulans. These findings strongly support the idea that the defective chromocentral I elements of D. melanogaster originated before the species diverged and the chromosomal I elements were eliminated. The chromosomal I elements reinvaded natural populations of D. melanogaster recently, and were possibly introduced from D. simulans by horizontal transmission.