The direction, timing and demography of Popillia japonica (Coleoptera) invasion reconstructed using complete mitochondrial genomes.

The Japanese beetle Popillia japonica is a pest insect that feeds on hundreds of species of wild and cultivated plants including important fruit, vegetable, and field crops. Native to Japan, the pest has invaded large areas of the USA, Canada, the Azores (Portugal), Italy, and Ticino (Switzerland), and it is considered a priority for control in the European Union. We determined the complete mitochondrial genome sequence in 86 individuals covering the entire distribution of the species. Phylogenetic analysis supports a major division between South Japan and Central/North Japan, with invasive samples coming from the latter. The origin of invasive USA samples is incompatible, in terms of the timing of the event, with a single introduction, with multiple Japanese lineages having been introduced and one accounting for most of the population expansion locally. The origin of the two invasive European populations is compatible with two different invasions followed by minimal differentiation locally. Population analyses provide the possibility to estimate the rate of sequence change from the data and to date major invasion events. Demographic analysis identifies a population expansion followed by a period of contraction prior to the invasion. The present study adds a time and demographic dimension to available reconstructions.

Development of a cypovirus protein microcrystal-encapsulated Bacillus thuringiensis UV-tolerant and mosquitocidal δ-endotoxin.

The δ-endotoxin Cry4Aa from Bacillus thuringiensis israelensis (Bti) has insecticidal characteristics specific to insects of the order Diptera. Although Cry4Aa has shown potential as an effective proteinaceous pesticide against mosquitoes, it has an ultraviolet (UV)-intolerant property that limits its outdoor use. Our previous research showed that protein microcrystal polyhedra from Bombyx mori cypovirus can encapsulate diverse foreign proteins and maintain long-term protein activity under hostile environmental conditions, including UV irradiation. In this study, we report the development of polyhedra encapsulating the Cry4Aa insecticidal activity domain by using a modified baculovirus expression system. We confirmed the oral intake of recombinant polyhedra introduced into the experimental environment by the larvae of a mosquito, Aedes albopictus, and delivery of encapsulated proteins into the digestive tract. The polyhedra encapsulating partial Cry4Aa showed mosquito larvicidal activity during incubation of larvae with 50% lethal-dose value of 23.717×104 cubes for 10 Aedes albopictus larvae in 1 ml water. In addition, polyhedra showed a specific property to reduce the impact of UV-C irradiation on the activity of encapsulated partial Cry4Aa, thus demonstrating the effectiveness of encapsulating Bti δ-endotoxins inside polyhedra to increase the availability of proteinaceous pesticides for outdoor use for mosquito control.

Proliferation of Bombyx mori nucleopolyhedrovirus strain H4 in BmN cells is enhanced by exchange of the F gene sequence with type strain T3.

The Bombyx mori nucleopolyhedrovirus (BmNPV)-based baculoviral expression vector system is among the most efficient expression vector systems for eukaryotic proteins especially when used in combination with silkworms as a host. We newly isolated a novel BmNPV strain (BmNPV H4) in Hokkaido, Japan that outperforms the type strain T3 in terms of both proliferation and expression of polyhedrin protein in silkworm larvae; however, it proliferates poorly in the BmN cell line. We inferred the gene responsible for the differences in proliferation between viral strains by quantifying amino acid similarity distances in protein functional domains and identifying highly divergent alleles between the H4 and T3 strains. Among proteins that differ markedly in functional domain sequence between H4 and T3, we identified the F gene, which encodes the F protein, as a putative cause of proliferative differences between the two strains. Using recombinant viruses with the F protein-coding sequence exchanged between H4 and T3, we determined that the T3 F protein increases H4 proliferation in BmN while the H4 F protein does not improve T3 proliferation in silkworm larvae. Our results suggest that the BmNPV F protein can strongly affect viral proliferation in a genetic background-specific manner and may be an important target for manipulating the proliferation characteristics of BmNPV-based expression vectors.

Characterization of a novel negevirus isolated from Aedes larvae collected in a subarctic region of Japan.

We isolated and characterized a novel positive-sense, single-stranded RNA virus from Aedes larvae collected on Okushiri Island, Hokkaido, Japan. This virus, designated Okushiri virus (OKV), replicated in the Aedes albopictus cell line C6/36 with severe cytopathic effects and produced a large number of spherical viral particles that were 50-70 nm in diameter and released into the cell culture medium. The OKV genome consisted of 9,704 nucleotides, excluding the poly(A) tail at the 3'-terminus, and contained three major open reading frames (ORF1, ORF2, and ORF3). ORF1 encoded a putative protein of approximately 268 kDa that included a methyltransferase domain, FtsJ-like methyltransferase domain, helicase domain, and RNA-dependent RNA polymerase domain. The genome organization and results of a phylogenetic analysis based on the amino acid sequence predicted from the nucleotide sequence indicated that OKV is a member of a new insect virus group of negeviruses with a possible evolutionary relationship to some plant viruses. ORF2 and ORF3 were suggested to encode hypothetical membrane-associated proteins of approximately 45 kDa and 22 kDa, respectively. This is the first study on a novel negevirus isolated from mosquito larvae in Japan.

Analysis of the Bombyx mori nucleopolyhedrovirus ie-1 promoter in insect, mammalian, plant, and bacterial cells.

The Bombyx mori nucleopolyhedrovirus (BmNPV) ie-1 promoter exhibits strong transcriptional activity and is used in transient foreign gene expression systems in insect cells. In a reporter assay experiment using the BmNPV ie-1 promoter, we found that it exhibited activity even in non-host mammalian BHK cells, plant BY-2 cells, and also bacterial Escherichia coli cells. An analysis using a deletion series of the BmNPV ie-1 promoter demonstrated that the core promoter region of this promoter was sufficient to display promoter activity in BHK cells, BY-2 cells, and E. coli cells, whereas upstream elements were required for higher activity in insect cells. Furthermore, we found that the BmNPV ie-1 promoter exhibited sufficient activity for a ß-galactosidase assay in E. coli cells. The results obtained here suggest that the BmNPV ie-1 promoter has potential as a universal promoter for transient expression systems in insect, mammalian, plant, and bacterial cells.

Characterization of KfrA proteins encoded by a plasmid of Paenibacillus popilliae ATCC 14706(T).

A scaffold obtained from whole-genome shotgun sequencing of Paenibacillus popilliae ATCC 14706(T) shares partial homology with plasmids found in other strains of P. popilliae. PCR and sequencing for gap enclosure indicated that the scaffold originated from a 15,929-bp circular DNA. The restriction patterns of a plasmid isolated from P. popilliae ATCC 14706(T) were identical to those expected from the sequence; thus, this circular DNA was identified as a plasmid of ATCC 14706(T) and designated pPOP15.9. The plasmid encodes 17 putative open reading frames. Orfs 1, 5, 7, 8, and 9 are homologous to Orfs 11, 12, 15, 16, and 17, respectively. Orf1 and Orf11 are annotated as replication initiation proteins. Orf8 and Orf16 are homologs of KfrA, a plasmid-stabilizing protein in Gram-negative bacteria. Recombinant Orf8 and Orf16 proteins were assessed for the properties of KfrA. Indeed, they formed multimers and bound to inverted repeat sequences in upstream regions of both orf8 and orf16. A phylogenetic tree based on amino acid sequences of Orf8, Orf16 and Kfr proteins did not correlate with species lineage.

Tightly regulated expression of Autographa californica multicapsid nucleopolyhedrovirus immediate early genes emerges from their interactions and possible collective behaviors.

To infect their hosts, DNA viruses must successfully initiate the expression of viral genes that control subsequent viral gene expression and manipulate the host environment. Viral genes that are immediately expressed upon infection play critical roles in the early infection process. In this study, we investigated the expression and regulation of five canonical regulatory immediate-early (IE) genes of Autographa californica multicapsid nucleopolyhedrovirus: ie0, ie1, ie2, me53, and pe38. A systematic transient gene-expression analysis revealed that these IE genes are generally transactivators, suggesting the existence of a highly interactive regulatory network. A genetic analysis using gene knockout viruses demonstrated that the expression of these IE genes was tolerant to the single deletions of activator IE genes in the early stage of infection. A network graph analysis on the regulatory relationships observed in the transient expression analysis suggested that the robustness of IE gene expression is due to the organization of the IE gene regulatory network and how each IE gene is activated. However, some regulatory relationships detected by the genetic analysis were contradictory to those observed in the transient expression analysis, especially for IE0-mediated regulation. Statistical modeling, combined with genetic analysis using knockout alleles for ie0 and ie1, showed that the repressor function of ie0 was due to the interaction between ie0 and ie1, not ie0 itself. Taken together, these systematic approaches provided insight into the topology and nature of the IE gene regulatory network.

Phylogenetic relationship of Paenibacillus species based on putative replication origin regions and analysis of an yheCD-like sequence found in this region.

To determine the phylogenetic relationship among Paenibacillus species, putative replication origin regions were compared. In the rsmG-gyrA region, gene arrangements in Paenibacillus species were identical to those of Bacillus species, with the exception of an open reading frame (orf14) positioned between gyrB and gyrA, which was observed only in Paenibacillus species. The orf14 product was homologous to the endospore-associated proteins YheC and YheD of Bacillus subtilis. Phylogenetic analysis based on the YheCD proteins suggested that Orf14 could be categorized into the YheC group. In the Paenibacillus genome, DnaA box clusters were found in rpmH-dnaA and dnaA-dnaN intergenic regions, known as box regions C and R, respectively; this localization was similar to that observed in B. halodurans. A phylogenetic tree based on the nucleotide sequences of the whole replication origin regions suggested that P. popilliae, P. thiaminolyticus, and P. dendritiformis are closely related species.

Mos1 transposon-based transformation of fish cell lines using baculoviral vectors.

Drosophila Mos1 belongs to the mariner family of transposons, which are one of the most ubiquitous transposons among eukaryotes. We first determined nuclear transportation of the Drosophila Mos1-EGFP fusion protein in fish cell lines because it is required for a function of transposons. We next constructed recombinant baculoviral vectors harboring the Drosophila Mos1 transposon or marker genes located between Mos1 inverted repeats. The infectivity of the recombinant virus to fish cells was assessed by monitoring the expression of a fluorescent protein encoded in the viral genome. We detected transgene expression in CHSE-214, HINAE, and EPC cells, but not in GF or RTG-2 cells. In the co-infection assay of the Mos1-expressing virus and reporter gene-expressing virus, we successfully transformed CHSE-214 and HINAE cells. These results suggest that the combination of a baculovirus and Mos1 transposable element may be a tool for transgenesis in fish cells.

Identification of a Bacillus thuringiensis Cry8Da toxin-binding glucosidase from the adult Japanese beetle, Popillia japonica.

Cry8Da from Bacillus thuringiensis galleriae SDS-502 has insecticidal activity against both the larvae and adult Japanese beetle (Popillia japonica Newman). The receptor determines the specificity of the insecticidal activity of Cry proteins and hence, in order to reveal the mode of action of Cry toxin, receptor identification is a necessary step. However, a receptor for Cry8-type toxin has not been identified in the Scarabaeidae family of insects. Therefore, we aimed to identify the receptor of Cry8Da toxin in adult P. japonica BBMV. A ligand blot showed the Cry8Da toxin only bound to a 150kDa protein in the BBMV of adult P. japonica. In order to identify the Cry8Da toxin binding protein, it was purified by column chromatography and three internal amino acid sequences were determined. Two of the three internal amino acid sequences shared homology with Coleopteran ß-glucosidases. In addition, the fraction containing the Cry8Da toxin binding protein had ß-glucosidase activity but no aminopeptidase N and alkaline phosphatase activity, both of which are commonly reported as receptors for Cry toxins in Lepidopteran and Dipteran insects. The ß-glucosidase homologous genes could be amplified by PCR using degenerate oligonucleotide primers designed from a conserved sequence of Coleopteran ß-glucosidases and an internal amino acid sequence of the Cry8Da toxin binding protein. Taken together, the ß-glucosidase in adult P. japonica BBMV is the receptor for B. thuringiensis Cry8Da toxin.

A 50-kilodalton Cry2A peptide is lethal to Bombyx mori and Lymantria dispar.

The Cry2Aa3 gene was introduced into asporogenic Bacillus thuringiensis, and the synthesized protoxin killed Bombyx mori and Lymantria dispar larvae. Chymotrypsin hydrolyzed the linkages between 49Tyr/Val50 and 145Lys/Ser146 in the protoxin, and 50- and 58-kDa fragments were generated, respectively. Both peptides killed the larvae of both insects.

Phenotypic grouping of 141 BmNPVs lacking viral gene sequences.

We constructed a series of gene knockout BmNPVs (KOVs) for each of 141 genes (Gomi et al., 1999; Katsuma et al., 2011) using the BmNPV T3 bacmid system (Ono et al., 2007) and lambda red recombination system (Datsenko and Wanner, 2000). In a subsequent analysis of the properties needed for infection using a marker gene, egfp (enhanced green fluorescent protein gene), inserted into the polyhedrin locus, the knockout viruses (KOVs) were subdivided into four phenotypic types, A to D. Type-A (86 KOVs) showed the ability to expand infections equivalent to the control while type-B (8 KOVs) spread infections more slowly. Type-C (37 KOVs) expressed egfp in transfected-BmN cells but the production of infectious viruses was not observed. Type-D (10 KOVs) showed no ability to express egfp even in the transfection experiments. KOVs lacking genes (pkip (Bm15), gp41 (Bm66), bro-d (Bm131), Bm20, 48, 65, 91, 93, or 101) previously identified as being essential, were placed in the viable type-A and B categories.

Intramolecular proteolytic nicking and binding of Bacillus thuringiensis Cry8Da toxin in BBMVs of Japanese beetle.

Bacillus thuringiensis (Bt) Cry8D insecticidal proteins are unique among Cry8 family proteins in terms of its insecticidal activity against adult Scarab beetles, such as Japanese beetle (Popillia japonica Newman). From the sequence homology with other Bt Cry proteins especially those active against beetles, such as Cry3Aa whose 3D structure is available, the structure of the Cry8D protein has been predicted to be a typical three-domain Cry protein type. In addition, the activation process of Cry8D in gut juice of susceptible insects is presumed to be similar to that of Cry3A (Yamaguchi et al., 2008). In this study, the activation process of Cry8Da in insect gut juice was closely examined. Japanese beetle gut juice proteases digested the 130kDa Cry8Da protein to produce a 64kDa protein. This 64kDa protein was active against both adult and larval Japanese beetle and considered to be an activated toxin. N-terminal sequencing of this 64kDa protein revealed that the Cry8Da leader sequence consisting of 63 amino acid residues from M(1) to F(63) was removed. As in the case of Cry3Aa, the proteases further digested the 64kDa protein to two 8kDa and 54kDa fragments. N-terminal amino acid analysis of these smaller fragments indicated that the proteases digested the loop between Alpha Helix (Alpha for short) 3 and Alpha 4. This means that the 8kDa fragment consists of Alpha 1-3 of Domain I and that the 54kDa fragment contains the remaining Domain I and full Domain II and Domain III. Size exclusion chromatography and anion exchange chromatography could not separate these 64, 54 and 8kDa proteins suggesting that the 54kDa and 8kDa fragments are still forming the toxin complex equivalent to the 64kDa protein by size and ionic charge. The sequencing and chromatography results suggest that the gut juice proteases merely nicked the loop between Alpha 3 and Alpha 4. This nicking process appeared to be essential for receptor binding of the Cry8Da toxin. BBMV binding assay revealed that the Cry8Da toxin bound to BBMV preparations from both adult and larval Japanese beetle only after the loop was nicked. Only the 54kDa fragment bound to the BBMV preparations but not the 64kDa protein. Ligand blot showed that the protease activated Cry8Da toxin, presumably the 54kDa fragment, bound to specific BBMV proteins, one or more of those would be receptor(s). The sizes and binding affinities of these Cry8Da-bound proteins of Japanese beetle BBMV differed between larvae and adults.

Discovery of a novel Bacillus thuringiensis Cry8D protein and the unique toxicity of the Cry8D-class proteins against scarab beetles.

A novel cry gene, cry8Db, highly toxic to scarab beetles such as the Japanese beetle, Popillia japonica Newman, was cloned from an isolate of Bacillus thuringiensis(Bt), BBT2-5. The cry8Db gene has 3525bp nucleotides and codes for a protein of 1174 amino acid residues. The protein, Cry8Db, has typical Bt characteristics such as the 8-block, conserved sequences and the three-domain 3D toxin structure as defined with Cry3Aa. When the amino acid sequence of Cry8Db was compared with that of Cry8Da whose gene was cloned and characterized in our laboratory earlier, substantial sequence diversities were found in their Domain III. The cry8Db gene was expressed in an acrystalliferous B. thuringiensis strain, BT51. BT51 expressing cry8Db formed a spherical crystal like the natural crystal of BBT2-5. The Cry8Db protein was assayed along with the other scarab active Cry8Da and Cry8Ca against the Japanese beetle. While Cry8Da and Cry8Db had toxicity against both adults and larvae of the Japanese beetle, Cry8Ca was toxic to only larvae. Cry8Ca showed no toxicity against the adult beetle up to 30 microg per 1 cm(2) of leaf discs on which the protein was applied. The activation process of Cry8Db by adult and larval gut juice was compared in vitro with the processes of Cry8Da and Cry8Ca. All three proteins, Cry8Db, Cry8Da and Cry8Ca, produced a toxic core of approximately 70kDa equally indicating that the activation process does not inactivate the adult activity of Cry8Ca. We concluded that the adult activity of Cry8D proteins is encoded in Domain II. Further tests against other beetle species showed a significant difference between Cry8D's and Cry8Ca but no difference between Cry8Da and Cry8Db. Comparison of 3D structural models of Cry8Ca, Cry8Da and Cry8Db, which were constructed by using Cry3Bb as the structural template, indicated significant structural differences, especially between Cry8Ca and Cry8D proteins, in three major surface-exposed loops of Domain II that may be involved in determining the adult beetle activity.

Cloning and expression of two crystal protein genes, cry30Ba1 and cry44Aa1, obtained from a highly mosquitocidal strain, Bacillus thuringiensis subsp. entomocidus INA288.

Two novel crystal protein genes, cry30Ba and cry44Aa, were cloned from Bacillus thuringiensis subsp. entomocidus INA288 and expressed in an acrystalliferous strain. Cry44Aa crystals were highly toxic to second-instar Culex pipiens pallens (50% mortality concentration [LC50] = 6 ng/ml) and Aedes aegypti (LC50 = 12 ng/ml); however, Cry30Ba crystals were not toxic.

Activation process of the mosquitocidal delta-endotoxin Cry39A produced by Bacillus thuringiensis subsp. aizawai BUN1-14 and binding property to Anopheles stephensi BBMV.

Most delta-endotoxins produced by Bacillus thuringiensis require proteolytic processing in order to become active. The in vitro and in vivo activation processes of Cry39A, a delta-endotoxin that is highly toxic to Anopheles stephensi, were investigated. Cry39A with a molecular mass of 72 kDa was processed in vitro into a 60 kDa fragment by trypsin and gut extract from A. stephensi larvae. N-terminal amino acid sequencing of the 60 kDa fragment revealed that trypsin and the protease(s) in the gut extract cleaved Cry39A between Arg(61) and Gly(62). In contrast, 40 and 25 kDa polypeptides were generated in vivo by intramolecular cleavage of the 60 kDa fragment in A. stephensi larvae. Further, a co-precipitation assay was used to investigate the binding property of the activated Cry39A to A. stephensi BBMV. Cry39A bound to A. stephensi BBMV specifically and did not compete with the Cry4Aa toxin. This indicated that the binding molecule(s) for Cry39A might differ from those for Cry4A. In addition, Cry39A preferentially bound to the Triton X-100-insoluble membrane fraction.

Enhancement of cauliflower mosaic virus 35S promoter in insect cells infected with baculovirus.

We happened to discover that the cauliflower mosaic virus (CaMV) 35S promoter inserted into a recombinant Autographa californica multicapsid nucleopolyhedrovirus (rAcMNPV) was strongly activated during the replication of the recombinant virus in Spodoptera frugiperda (Sf9) cells. The expression of the luciferase gene from the 35S promoter in rAcMNPV was remarkably increased late in infection and was resistant to alpha-amanitin treatment. Primer extension indicated that transcriptional initiation from the 35S promoter in Sf9 cells occurred within one of the two baculoviral late promoter TAAG motifs located in the vicinity of the transcription start site in plant cells. These observations suggested that the CaMV 35S promoter served as a transcription start site for AcMNPV-induced RNA polymerase.

W-derived BAC probes as a new tool for identification of the W chromosome and its aberrations in Bombyx mori.

We isolated four W chromosome-derived bacterial artificial chromosome (W-BAC) clones from Bombyx mori BAC libraries by the polymerase chain reaction and used them as probes for fluorescence in situ hybridization (FISH) on chromosome preparations from B. mori females. All four W-BAC probes surprisingly highlighted the whole wild-type W sex chromosome and also identified the entire original W-chromosomal region in W chromosome-autosome translocation mutants. This is the first successful identification of a single chromosome by means of BAC-FISH in species with holokinetic chromosomes. Genomic in situ hybridization (GISH) by using female-derived genomic probes highlighted the W chromosome in a similar chromosome-painting manner. Besides the W, hybridization signals of W-BAC probes also occurred in telomeric and/or subtelomeric regions of the autosomes. These signals coincided well with those of female genomic probes except one additional GISH signal that was observed in a large heterochromatin block of one autosome pair. Our results support the opinion that the B. mori W chromosome accumulated transposable elements and other repetitive sequences that also occur, but scattered, elsewhere in the respective genome.

Use of an N-terminal half truncated IE1 as an antagonist of IE1, an essential regulatory protein in baculovirus.

An immediate-early gene product of baculovirus, IE1, is essential for viral gene expression and for viral DNA replication. It has been demonstrated for Autographa californica nuclear polyhedrosis virus (AcNPV) that the C-terminal region of IE1 is required for dimerization. And the acidic N-terminal region of IE1 has been identified as the activation domain. We constructed an N-terminal 267 amino acid (a.a.) truncated mutant of Bombyx mori nuclear polyhedrosis virus (BmNPV) IE1, which was defective as a transactivator of a viral early gene (p35) promoter. We then examined possible IE1 antagonistic functions of this defective IE1, IE1TN, in BmNPV-infected cells. A transient expression experiment demonstrated that IE1TN strongly repressed the activation of the hr5-dependent p35 promoter derived from BmNPV infection. In addition, DpnI assay elucidated an inhibitory effect of IE1TN on the hr5-dependent replication of plasmid in BmN cells induced by NPV infection. A marked reduction in the production of virus was observed when the BmN cells were infected with BmNPV after transfection with IE1TN-expression plasmids. These results suggested that IE1TN could act as an IE1 antagonist in silkworm cells infected with BmNPV. We then analyzed the ability of IE1TN to inhibit the multiplication of BmNPV using transgenic silkworms. The BmNPV-resistance of the transgenic silkworms was very weak, suggesting insufficient expression of the transgene product, IE1TN.