The pear psylla, Cacopsylla pyricola Foerster (Homoptera: Psyllidae), is a serious insect pest of commercial pear crops. The species, which resides on pear trees throughout its life cycle, is rapidly spreading in some regions of the world. Given the life cycle, it is unclear how such a rapid spread has been facilitated. Presently, the population genetic structure of the species including genetic diversity and gene flow was studied to understand the nature of dispersal and field ecology of the species. Pear psylla was collected from several pear orchards in Korea. The 658-bp region of mitochondrial COI gene and the 716-bp long complete internal transcribed spacer 2 (ITS2) of the nuclear ribosomal DNA were sequenced. Unlikely other previously studied insect pests, the COI-based genetic diversity of the pear psylla was extremely low (maximum sequence divergence of 0.15%). This finding allowed us to conclude that the species may have been introduced in Korea relatively recently, possibly with the phenomenon of genetic bottlenecks. ITS2 sequence-based analyses of phylogeny, population differentiation, gene flow, and hierarchical population structure all concordantly suggested that the pear psylla populations in Korea are neither genetically isolated nor hampered for gene flow. These genetic data are concordant with the dispersal of an overwintering winterform morph outside the non-pear habitat in the fall and the possibility of subsequently longer distant dispersal.

Bee venom contains a variety of peptides and enzymes, including serine proteases. While the presence of serine proteases in bee venom has been demonstrated, the role of these proteins in bee venom has not been elucidated. Furthermore, there is currently no information available regarding the melanization response or the fibrin(ogen)olytic activity of bee venom serine protease, and the molecular mechanism of its action remains unknown. Here we show that bee venom serine protease (Bi-VSP) is a multifunctional enzyme. In insects, Bi-VSP acts as an arthropod prophenoloxidase (proPO)-activating factor (PPAF), thereby triggering the phenoloxidase (PO) cascade. Bi-VSP injected through the stinger induces a lethal melanization response in target insects by modulating the innate immune response. In mammals, Bi-VSP acts similarly to snake venom serine protease, which exhibits fibrin(ogen)olytic activity. Bi-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products, defining roles forBi-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings provide a novel view of the mechanism of bee venom in which the bee venom serine protease kills target insects via a melanization strategy and exhibits fibrin(ogen)olytic activity.

Classical swine fever virus (CSFV) envelope glycoprotein E2 is the main target for inducing neutralizing antibodies and protective immunity in swine. Here, we report a novel strategy forthe large-scale production of a CSFV E2 subunit vaccine that demonstrates a high immunogenic capability in the larvae of a baculovirus-infected silkworm, Bombyx mori. We constructed a recombinant B. mori nucleopolyhedrovirus (BmNPV) that expressed recombinant polyhedra together with the N-terminal 179 amino acids of CSFV E2 (CSFV E2ΔC). BmNPV-E2ΔC-infected silkworm larvae expressed an approximately 44-kDa fusion protein that was detected using both anti-polyhedrin and anti-CSFV E2 antibodies. Electron and confocal microscopy both demonstrated that the recombinant polyhedra were morphologically normal and contained CSFV E2ΔC. The CSFV E2ΔC antigen produced in BmNPV-E2ΔC-infected silkworm larvae reached 0.68 mg per ml of hemolymph and 0.53 mg per larva at 6 days post-infection. Mice that were immunized with the granule form of recombinant polyhedra or the soluble form of the fusion protein elicited CSFV E2 antibodies, which indicated that the recombinant polyhedra carrying CSFV E2ΔC were immunogenic. The virus neutralization test showed that the serum from mice that were treated with recombinant polyhedra or the soluble form of the fusion protein contained significant levels of virus neutralization activity. These results demonstrate that the present strategy can be used for the large-scale production of CSFV E2 antigen and that the recombinant polyhedra containing CSFV E2ΔC as a granule antigen can be used as a potential subunit vaccine against CSFV.

Pseudorabies virus (PRV), a member of the Alphaherpesviridae, is the causative agent of Aujeszky’s disease in pigs. Glycoprotein B (gB) of PRV, a major constituent of the viral envelope, consists of 916 amino acids. We continuously combined three gB epitopes, E1 (aa 62-129), E2 (aa 217-282), and E3 (aa 543-737). The DNA fragment containing the PRV gB epitopes was fused with polyhedrin gene in order to generate recombinant baculovirus that expresses the recombinant polyhedra with PRV gB epitopes under the control of the Bombyx mori nucleopolyhedrovirus polyhedrin promoter. Recombinant baculoviruses were injected into fifth-instar B. mori larvae. SDS-PAGE and Western blot analyses revealed that recombinant polyhedra constitute polyhedrin and PRV gB epitopes, and that the recombinant PRV gB epitopes showed cross-reactivity against antiserum of PRV gB produced from pig. To examine the immunogenicity of recombinant PRV gB epitopes, we injected into mice as model animals. ELISA results indicated that antibody production is increased in a similar manner in the injection of recombinant polyhedra with PRV gB epitopes, either injected recombinant polyhedra as a granule form antigen without adjuvant or injected recombinant polyhedrin as a soluble form antigen with adjuvant. Taken together, these data show that PRV gB epitopes were produced as a granule form antigen by fusing recombinant polyhedra in baculovirus-infected silkworm larvae and displayed the immunogenicity in mice, indicating the efficacy of the granule form antigen as a PRV gB vaccine.

Diamondback moth (Plutella xylostella L.) belonging to genus Lepidoptera is a notorious pest of cruciferous crops worldwide. We evaluated the bioinsecticidal activity of the liquid cultures (LB and NB) of a bacterial strain, Serratia sp. EML-SE1, isolated from a diseased diamondback moth. The pathogenicity of a bacterial strain to diamondback moth was confirmed by the following procedures: treatment of liquid culture on cabbage leaves, ingestion of inoculated cabbage and mortality response. For the test, twenty 3rd instar larvae of diamondback moth were placed on the Chinese cabbage leaf in a round plastic cage (Ø 10 × 6 cm) and sprayed with the liquid cultures. After 72 hours, insecticidal activity of LB and NB cultures of Serratia sp. against P. xylostella larvae showed 91.7% and 88.3%, respectively. In addition, the bioinsecticidal activity on potted cabbage with 14 leaves in a growth cage (165 × 83 × 124 cm) also was similar to that of plastic cage experiment. Summarized, the Serratia sp. EML-SE1 may be a potent candidate as a bioinsecticidal agent to control diamondbac kmoth.

Background: Proteolytic enzymes are involved in insect molting and metamorphosis and play a vital role in the programmed cell death of obsolete organs. Here we show the expression profile of cathepsin B in the fat body of the silkworm Bombyx mori during development. We also compared the expression profile of B. mori cathepsins B (BmCatB) and D (BmCatD) in the fat body during the larval-pupal transformation of B. mori in the BmCatB or BmCatD RNA interference (RNAi) process. Results: BmCatB is ecdysone-induced and expressed in the fat body of B. mori during the molting, and the larval-pupal and pupal-adult transformations, and its expression leads to programmed cell death. In particular, BmCatB is highly expressed in the fat body of B. mori during the larval-pupal transformation and BmCatB RNAi treatment resulted in the arrest of the larval-pupal transformation. RNAi-treated BmCatB knock-down sustained the expression of BmCatD during the larval-pupal transformation. On the other hand, BmCatD RNAi up-regulated the expression of BmCatB in the fat body of final instar larvae. Conclusion: Based on these results, we conclude that BmCatB is involved in the programmed cell death of the fat body during B. mori metamorphosis and that BmCatB and BmCatD contribute collaboratively to B. mori metamorphosis

We have determined the complete mitochondrial genome of the yellow-spotted long horned beetle, Psacothea hilaris (Coleoptera: Cerambycidae), an endangered insect species in Korea. The 15,856-bp long P. hilaris mitogenome harbors gene content typical of the animal mitogenome and a gene arrangement identical to the most common type found in insect mitogenomes. As with all other sequenced coleopteran species, the 5-bp long TAGTA motif was also detected in the intergenic space sequence located between tRNASer (UCN) and ND1 of P. hilaris. The 1,190-bp long non-coding A+T-rich region harbors an unusual series of seven identical repeat sequences of 57-bp in length and several stretches of sequences with the potential to form stem-and-loop structures. Furthermore, it contains one tRNAArg-like sequence and one tRNALys-likes equence. Phylogenetic analysis among available coleopteran mitogenomes using the concatenated amino acid sequences of PCGs appear to support the sister group relationship of the suborder Polyphaga to all remaining suborders, including Adephaga, Myxophaga, and Archostemata. Among the two available infraorders in Polyphaga, a monophyletic Cucujiformia was confirmed, with the placement of Cleroidea as the basal lineage for Cucujiformia. On the other hand, the infraorder Elateriformia was not identified as monophyletic, thereby indicating that Scirtoidea and Buprestoidea are the basal lineages for Cucujiformia and the remaining Elateriformia.

The complete nucleotide sequences of the mitochondrial genome (mitogenome) from the white-spotted flower chafer, Protaetia brevitarsis (Coleoptera: Cetoniidae) was determined. The 20,319-bp long circular genome is the longest among the completely sequenced arthropods. This extraordinary length of the genome stemmed from 5,654-bp long A+T-rich region composed of twenty-eight 117-bp tandem repeats, seven 82-bp tandem repeats, and each two 19-bp and 38-bp tandem repeats. The P. brevitarsis contains a typical gene complement, order, and arrangement identical to most common type found in insects. The P. brevitarsis COI gene does not have typical ATN codon. Thus, we also designated it as AAC (asparagine), which is found in the start context of all sequenced Polyphaga within Coleoptera. All tRNAs showed stable canonical clover-leaf structure of other mt tRNAs, except for tRNASer (AGN), DHU arm of which could not form stable stem-loop structure. The 5bp-long motif sequence (TAGTA) that has been suggested to be the possible binding site for the transcription termination peptide for the major-strand also was found betweent RNASer (UCN) and ND1, as have been detected in all sequenced coleopteran insects.

The 15,389-bp long complete mitogenome of the endangered red-spotted apollo butterfly, Parnassius bremeri (Lepidoptera: Papilionidae) was determined. This genome has a gene arrangement identical to those of all other sequenced lepidopteran insects, which have the gene order of tRNAMet, tRNAIle, and tRNAGln at the beginning. Due to the uncertainty the start codon for COI gene in insect has been discussed extensively. We propose the CGA sequence as the start codon for COI gene in lepidopteran insects, based on complete mitogenome sequences of lepidopteran insects including our P. bremerii and additional sequences of the COI start region from a diverse taxonomic range of lepidopteran species (a total of 51 species belonging to 15 families). As has been suggested in other sequenced lepidopteran insects the 18 bp-long poly-T stretch and the downstream conserved motif ATAGA that were previously suggested to serve as a structural signal for minor-strand mtDNA replication also was found at the 3’-end region of the P. bremerii A+T-rich region. In an extensive search to find out tRNA-like structure in the A+T-rich region, each one tRNATrp-like sequence and tRNALeu (UUR)-like sequence were found in the P. bremeri A+T-rich region, and most of other sequenced lepidopteran insects were shown to have tRNA-like structure within the A+T-rich region, thereby indicating that such feature is frequent in the lepidopteran A+T-rich region. Phylogenetic analysis using the concatenated 13 amino acid sequences and nucleotide sequences of PCGs of the four macrolepidopteran suferfamilies together with Tortricoidea and Pyraloidea well recovered a monophyly of Papilionoidea and a monophyly of Bombycoidea. However, Geometroidea and Noctuoidea were unexpectedly clustered as one group and placed this group to the sister group to Bombycoidea, instead of Papilionoidea in most analyses.

Invertebrate mitochondrial genome contains 13 protein-coding genes and major start codons for them are ATA (Met) and ATG (Met). However, alternative start codons such as ATT (Ile), ATC (Ile), TTG (Leu), and GTG (Val) also have been suggested from a diverse organism. Approximately 120 complete mitochondrial genome reported showed that the start codon for COI gene evidences an array of diverse designation of COI start codon such as typical ATN, tetranucleotide TTAG and ATAA, newly proposed AAT and AAC and so on. In the case of Lepidoptera, many completely sequenced species showed no typical start codon at the start context of COI and even within the neighboring tRNATyr. In order to clarify, we newly sequenced the beginning context of COI gene, encompassing the neighboring tRNATyr and start region of COI gene from 39 species belonging to eight lepidopteran families. We found the newly sequenced 39 species and 14 available complete lepidopteran mitochondrial genomes all possessed CGA (arginine), which is the first non-overlapping in-frame codon in COI gene. Furthermore, this CGA is highly well aligned in terms of both nucleotide and amin o acid sequences with neighboring region. Thus, the CGA (arginine) may be synapomorphic character for Lepidoptera, functionally constrained. We, therefore, propose the CGA sequence as the start codon for COI gene in lepidopteran insects.

We analyzed a portion of mitochondrial COI gene sequences (658 bp) to investigate the genetic diversity and geographic variation of the swallowtail butterfly, Papilioxuthus L., and the cabbage butterfly, Pieris rapae (Lepidoptera: Papilionidae). P. xuthus showed a moderate level of sequence divergence (0.91% at maximum) in 15 haplotypes, whereas P. rapae showed a moderate to high level of sequence divergence (1.67% at maximum) in 30 haplotypes, compared with other relevant studies. Analyses of population genetic structure showed that most populations are not genetically differentiated in both species. The distribution pattern of both species appears to be consistent with category IV of the phylogeographic pattern sensu Avise (Avise et al. 1987): a phylogenetic continuity, an absence of regional isolation of mtDNA clones, and extensive distribution of close clones. The observed pattern of genetic diversity and geographic variation of the two butterfly species seems to reflect the abundant habitats, abundant host plants, and flying abilities in connection with the lack of historical biogeographic barriers.

In this study, we determined the complete mitochondrial genome of the jewel beetle, Chrysochroa fulgidissima (Coleoptera: Buprestidae), from four overlapping fragments. The 15,592-bp long C. fulgidissima mitogenome exhibits a gene arrangement and content identical to the most common type in insects. The start codon of the C. fulgidissima COI gene is unusual, in that no typical ATN codon is available. The 875-bp A+T-rich region is the shortest among the coleopteran mitogenomes that have thus far been sequenced in their entirety. The most unusual feature of the genome is the presence of three tRNA-like sequences within the A+T-rich region: two tRNALeu(UUR)-like sequences and one tRNAAsnlike sequence. These sequence stretches evidence the proper anticodon sequence and the potential to form secondary structures, but also harbor many mismatches in the stems. Phylogenetic analysis using a concatenation of 13 amino acid sequences of protein-coding genes among the available sequenced species of coleopteran superfamilies (Buprestoidea and Elateroidea belonging to the infraorder Elateriformnia, and Chrysomeloidea and Tenebrioroidea belonging to the infraorder Cucujoiformia) by Bayesian inference, maximum-parsimony analyses, and maximum-likelihood analysis unexpectedly revealed a lack of support for monophyletic Elateriformia.