The UN member states have been consulting on the establishment of high seas MPAs under the BBNJ Agreement since December 2017.This issue brings about the potential conflicts between the jurisdiction of the high seas MPAs and the traditional freedom of the high seas. Although it is generally accepted that the freedom of the high seas can be reasonably restricted, it is acknowledged that there are great controversies among States on the specific forms, approaches and applicable scopes of the restrictions of the high seas freedoms by the high seas MPAs. At present, there are four recognized high seas MPAs in the world. The practice of these four MPAs contributes positively to the interpretation of the reasonable restrictions on the freedom of the high seas. Accordingly, this paper concludes that the international community should gradually carry forward future restrictions on the freedom of the high seas with some suggestions.

Since its founding in 1949, the People’s Republic of China has acceded to more than 300 multilateral treaties. Among them, China made reservations to 83 treaties, accounting for nearly 27.7 percent of the total. Evidently, for China, formulating reservations to multilateral treaties is an issue of vital importance in the process of concluding and executing multilateral treaties. This paper examines the three main reasons why China inclines to formulate reservations to the treaties, then argues that the reservations formulated by China, whether in procedure or in substance, are not only in full conformity with the 1969 Vienna Convention on the Law of Treaties, but also full of Chinese characteristics. This paper also analyzes several specific reservations that China shall withdraw, and addresses the limitations of China’s existing treaty reservation system. Accordingly, this paper concludes that the Chinese government should improve and perfect its treaty reservation system with some suggestions.

Although baculoviruses have a long history of safe use as specific, environmentally benign insect control agents, their use has been limited by several factors, especially their slow speed of action. In this study, we intended to improve the insecticidal activities of Autographa californica nucleopolyhedrovirus (AcMNPV) by expressing Kunitz-type toxin isolated from venoms of Bombus ignitus or Araneus ventricosus. For this, recombinant AcMNPVs, AcBi-KTT, AcAv-Tox1 and AcAv-Tox2 expressing Bi-KTT, Av-Tox1 and Av-Tox2, respectively, under the control of p10 gene promoter were constructed. While polyhedra produced by these recombinant viruses were identical to those of the wild-type AcMNPV in shape, their sizes were relatively smaller than those of the AcMNPV. Among recombinant viruses, AcBi-KTT and AcAv-Tox2 showed significant reduction in median lethal time (LT50) against Spodoptera exigua larvae. Especiaaly, these two viruses showed about 6.2~10-folds higher polyhedra production rate compared to that of the AcMNPV. These results suggested that Kunitz-type toxins from insect venom could be successfully applied to improve insecticidal activity of baculoviruses.

Rice stripe virus (RSV), the type member of the genus Tenuivirus, causes rice stripe disease and the viral transmission is mediated through the sucking by small brown planthopper, Laodelphax striatellus. Considerations have been mainly focused on the protection of rice from RSV and/or the planthopper, rather than the interaction between RSV and the insect. To clarify the interaction, in this work, mRNA was extracted from RSV-viruliferous planthopper with non-viruliferous control, and expressed sequence tag (EST) databases were generated based on 454 GS-FLX pyrosequencing technology for comparative analysis. RSV-viruliferous planthopper had ca. 2500 isotigs, which included genes on biological process (19%), cellular component (13%), molecular function (22%) and no hits (46%) from gene ontology (GO) analysis; this structure was similar to the control. However, in the viruliferous planthopper, 109 isotigs were up-regulated and 660 isotigs were down-regulated, compared to the non-viruliferous control. These RSV-dependently regulated genes may have important function in the behavior of planthopper or the transmission of RSV.

ORF43 (ac43) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved baculovirus gene whose function is unknown. To determine the role of ac43 in baculovirus life cycle, we used a new AcMNPV bacmid (bAc-MK) and generated ac43 deletion virus (ac43KO) by using the plasmid capture system (PCS). After transfection into Spodoptera frugiperda cells, ac43KO produced significantly different OBs which with much larger size; and especially had much single nucleocapsids compared to Ac-MK. Furthermore, ac43KO bacmid led to defect in transcription and expression of polyhedrin, which result in less OBs production. However, ac43KO didn't affect BV production since there's no remarkable difference of BV titer in both ac43KO and Ac-MK. These results demonstrate that ac43 play an important role in polyhedrin expression, OB formation, and virion assembly.

Bacillus thuringiensis (Bt) strain K4 was isolated from fallen leaves which had been collected at a forest stand in Mungyeong city, Republic of Korea. The flagellated vegetative cells of Bt K4 were agglutinated with the H3 reference antiserum among 55 reference H-antisera. In a further test to identify subfactors, 3b and 3d monospecific antisera were reactive to the cells, followed up with introducing a novel serogroup of 3a3b3d, designated as serovar mogi. The strain K4 had mosquitocidal activity against Dipteran larvae, Anopheles sinensis and Culex pipiens pallens, with no Lepidopteran toxicity observed. The SDS-PAGE profile of K4 crystal protein, ovoidal-shaped, included several bands ranging from 30-75 kDa. Four putative peptides, Cry19Ba, Cry40ORF2, Cry27Aa and Cry20Aa were detected from the bands by a nano-LC-ESI-IT MS analysis. Through a thermal asymmetric interlaced PCR, cry19Ba, cry40ORF2 and cry27Aa genes were partially cloned from K4 strain. Three cry genes were further found in the strain by a 454 pyrosequencing, ending up to showing 58%, 39% and 84% homology in amino acids with Cry56Aa, Cry8Ba and Cry39ORF2 toxins, respectively. This novel 3a3b3d type strain, B. thuringiensis subsp. mogi, can be used as a good resource for studying unknown mosquitocidal cry genes.

Rice black-streaked dwarf virus (RBSDV), a member of the genus Fijivirus within the family Reoviridae, is the causative agent of maize rough dwarf and rice black-streaked dwarf diseases, both of which can lead to severe yield losses in east Asia. Although molecular approaches such as RT-PCR have potential for detection and diagnosis of this virus infections, their impact on high throughput certification is still limited. Therefore, the development of an antibody-based assay for rapid and effective diagnosis of RBSDV is preferable. In this study, we collected RBSDV from rice with rough dwarf disease and its complete nucleotide sequences of 10 genomic segments encoding 12 non-overlapping ORFs were determined. Among 12 ORFs, ORF1, 2 and 12 showed high level of similarities with the RdRp, major core protein and major outer shell protein, respectively. These ORFs were expressed as polyhedrin fusion protein or full-length soluble protein using baculovirus expression system for the preparation of specific antibody against RBSDV, which could be useful for the detection and diagnosis of this virus.

We isolated two baculoviruses, Spodoptera litura granulovirus (SlGV) and S. litura nucleopolyhedrovirus (SlNPV) in the dead larvae of S. litura. The granule of SlGV were ovoidal shape with an approximate measure of 240-340 nm×140-180 nm, and each granule contained one single rod-shape virion with a mean size of 180-200 nm×20-40 nm. Whereas, the polyhedra of SlNPV were irregular in shape with a approximate diameter of 1.0-1.5 ㎛, and numerous virions comprised of the multinucleocapsid were contained in each polyhedra. The major component of occlusion bodies produced by SlGV and SlNPV were about 29 and 30 kDa, respectively. When the phylogenic relationship between these viruses were analyzed using the nucleotide sequences of granulin gene from SlGV and polyhedrin gene from SlNPV, they were not closely related to each other. We also found that the two viruses showed similar insecticidal activity against 2nd instar larvae of Spodotera litura in terms of dose-response, but SlGV showed much longer LT50 than that of SlNPV. The two baculoviruses might be cooperatively be applied as biological control agent for the control of S. litura

Autographa californica nucleopolyhedrovirus (AcMNPV) has a large doublestrand DNA genome of approximately 134 kbp and harbors 156 open reading frames (ORFs). To elucidate DNA replication cascade of AcMNPV, we developed a novel baculovirus genome that can be maintained in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. This genome, named bAc-MK, contains a mini-F replicon and a kanamycin resistance marker. Using a convenient Tn7 transposon-based system, pPCS-S, 55 single ORF-truncated mutants were generated by random insertion into bAc-MK genome. These single ORF-truncated mutants were independently transfected into Sf9 cells, 16 of them were found affecting viral replication since they defected in producing polyhedra. Furthermore, to verify the pathogenicity of the single ORF-truncated mutants, the remaining 39 mutants were subjected to bioassay to Spodoptera exigua 3rd instar larvae. Among them, ac9-, ac49-, ac103- and ac105-knockout mutants showed higher mortality compared to that of bAc-MK. These results suggested that these ORFs could be related to pathogenicity of AcMNPV.

The Bacillus thuringiensis strain K4 was isolated from fallen leaves, sampled in a forest region of the city of Mungyeong, Korea. The flagellated vegetative cells of B. thuringiensis strain K4 were agglutinated with the H3 reference antiserum and further, agglutinated with 3b and 3d monospecific antisera but non-reactive for 3c and 3e factor sera. These results create a new serogroup with flagellar antigenic structure of 3a3b3d, designated serovar mogi. The strain K4 showed high activity against dipteran larvae, Anopheles sinensis and Culex pipiens pallens while no lepidopteran toxicity. It produced a single ovoidal-shaped parasporal crystal whose SDS-PAGE protein profile consisted of several bands ranging from 75 to 30 kDa. Through the protein identification by nano-LC-ESI-IT MS analysis, the putative peptides of Cry19Ba, Cry40ORF2, Cry27Aa and Cry20Aa were detected. In contrast to the plasmid profile of B. thuringiensis H3 serotype strains, the strain K4 contained only a large plasmid (~100 kb) and we cloned partial cry27Aa, cry19Ba and cry40ORF2 genes from it by thermal asymmetric interlaced PCR. Sequencing analysis showed 87%, 88% and 88% homologous with known cry27Aa, cry19Ba and cry40ORF2 genes, respectively. The new type strain, B. thuringiensis subsp. mogi (H3a3b3d) will be a good resource for new mosquitocidal cry genes.

Entomopathogenic fungi are widely available as biological control agents for controlling insect pests in agriculture and forestry. The fungal culture broth contains various pathogenesis-related components such as blastospores, mycelium and insecticidal enzymes such as chitinase, Pr1- and Pr2-proteases, which have been reported to play an important role in penetrating insect cuticles. In this study, we tried to evaluate the utility of culture broth from Beauveria bassiana SFB-205 to control lepidopteran pests. High level of insecticidal activity correspond to over 90% of mortality were observed when the culture broth of B. bassiana SFB-205 was inoculated to the Spodoptera litura larvae together with the B. thuringiensis K1. The freeze-dried culture broth showed synergistic effects in insecticidal activity against larvae of S. exigua and S. litura when treated with corresponding baculoviruses, SeNPV and SlNPV. Active ingredient of the B. bassiana SFB-205 culture broth was identified to chitinase, which have truncated form by insertional mutation compared to previously reported chitinases.

Bacillus thuringiensis 1-3 (Bt 1-3) which was isolated from a Korean soil sample showed high insecticidal activity against Aedes aegypti as well as Plutella xylostella. The isolate was determined to belong to ssp. aizawai (H7) type by an H antiserum agglutination test and produced bipyramidal-shaped crystal proteins with a molecular weight of 130 kDa. PCR analysis with cry gene specific primers showed that Bt 1-3 contained cry1Aa, cry1Ab, cry1C, cry1D and cry2A gene, differing from spp. aizawai (reference strain) which contains cry1Aa, cry1Ab, cry1C and cry1D. We modified the plasmid capture system (PCS) to clone plasmid from Bt 1-3 through in vitro transposition. Fifty-three clones were acquired and their sizes were approximately 10 kb. Based on the sequence analysis, they were classified according to similarities with four known Bt plasmids, pGI3, pBMB175, pGI1 and pGI2, respectively. One of pGI3-like clones, named as pBt1-3, was fully sequenced and its 20 putative open reading frames (ORFs), Rep-protein, double-strand origin of replication (dso), single-strand origin of replication (sso), have been identified. The structure of pBt1-3 showed high similarity with pGI3 which is one of rolling-circle replication (RCR) group VI family.

Baculovirus chitinase gene (ChiA) is a late gene and is essential for liquefying host insect at the late stage of infection for its hydrolyzing chitin function. In previous report, baculovirus ChiA can offer many interseting new opportunities for pest control. Recently, a putative chitinase gene (ChiA) was identified in the Spodopter litura nucleopolyhedorvirus (SlMNPV-K1) genome. The open reading frame (ORF) contains 1,692 nucelotides (nt) and encodes a protein of 563 amino acids (aa) with a predicted molecular weight of 62.62 kDa. To conform the insecticidal activity of ChiA from SlMNPV-K1, we constructed a baculovirus transfer vector, pBac-SlChiA, and this transfer vector was co-transfected with the bApGOZA DNA into sf9 cell to generate corresponding recombinant viru which designed Ap-SlChiA. Western blot analysis indicate that SlMNPV-K1 ChiA was successfully expressed. We found the chitinase activity of recombinant virus was enhanced 53% than wide type AcMNPV by chitinase assay, and the recombinant virus showed higher evidently insecticidal activity against 3rd instar larvae of Spodotera exigua than wide type AcMNPV (4.5 time). These results suggested that the chitinase gene from SlMNPV-K1 could be successfully applied to improve pathogenicity of bauclovirus

Autographa californica nucleopolyhedrovirus (AcMNPV) has a large doublestrand DNA genome of approximately 134 kbp and comprises 156 open reading frames (ORFs). To elucidate DNA replication cascade of AcMNPV, we developed a novel baculovirus genome that can be maintained in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. This genome, named bAc-MK, contains a mini-F replicon and a kanamycin resistance marker. Using a convenient Tn7 transposon-based system, pPCS-S, which contains an ampicillin resistance gene, 56 single ORF-truncated mutants were generated by random insertion into bAc-MK genome. These single ORF-truncated mutants were independently transfected into Sf9 cells to verify viral replication. Interestingly, both lef-1 and p48 knockout mutants showed normal viral replication in infected cells, which are reported to essential for viral replication. These results suggest that these single ORF-truncated mutants are useful for elucidation of viral replication cascade.

Through an application of plasmid capture system (PCS) to Bacillus thuringiensis plasmid DNAs, we acquired 21 polymorphic clones of putative genomic DNA of bacteriophage. The genome size of phage 1-3 (PhBT1-3) was determined to be 46,517 base pairs (bp) with 35.43% G + C content and 83% coding region. Sixty-five putative open reading frames (ORFs) with more than 50 codons were found in the new phage genome. In accordance with this genome finding, the phage particles and its DNA were confirmed from the supernatant of B. thuringiensis 1-3. Morphological characterization and infectivity assay demonstrated that PhBT1-3 belongs to the family Siphoviridae and it showed infectivity to three B. thuringiensis type strains, galleriae, entomocidus, and morrisoni. Based on these results, we screened the existence of phages in B. thuringiensis type strains by PCR with terminase small subunit-specific primers. Ten of 67 type strains showed PCR products and the similarity of those putative amino acids was more than 70%. Furthermore, we verified the existence of various shaped phages from the supernatants of 10 B. thuringiensis type cultures. In conclusion, we characterized a putative genome of phage, PhBT1-3 from B. thuringiensis 1-3, and confirmed the distribution of phages in the group of 67 B. thuringiensis type strains.

A new Bacillus thuringiensis isolate 19-22 (Bt 19-22) exhibited high anti-fungal activity against barley powdery mildew (Blumeria graminis f. sp. hordei). The cry gene content of Bt 19-22 comprised cry1Aa, cry1Ab, cry1Ac and cry1D which have high insecticidal activity against lepidopteran larvae. We tried to confer a dipteran insecticidal activity to Bt 19-22 for constructing a recombinant strain which has multiple functions, anti-fungal and dual insecticidal activity. The insecticidal cry11Aa gene of B. thuringiensis was constructed under cry1Ac promoter in an E. coli-B. thuringiensis shuttle vector (pPro11A). The plasmid, pPro11A was introduced into Bt 19-22 isolate by electroporation and four transformants which had different cry gene contents were identified by PCR with cry11Aa and cry1-type specific primers. Among them, a Bt 19-22 transformant (11A/19-22 No. 7) expressed Cry11A protein (approximately 70 kDa) successfully without change of its inherent characteristics such as Cry protein expression and antifungal activity. The insecticidal activity of 11A/19-22 No. 7 was checked against Plutella xylostella and Culex pipiens. These results suggests that the recombinant strain shows dual insecticidal activity against lepidopteran and dipteran larvae as well as antifungal activity.

Bacillus thuringiensis 1-3 (Bt 1-3), belonging to subsp. aizawai (H7), showed different characteristics in plasmid profiles from type strain and had cry2A gene in addition to cry1Aa, cry1Ab, cry1C and cry1D. To clone its plasmids and construct E.coli-Bt shuttle vector, we constructed the plasmid capture system (PCS) by inserting attB sites including lacZ between transposable elements (designated as pPCS-Troy). Through in vitro transposition with total plasmids DNA of Bt 1-3, 53 clones were acquired and their sizes were approximately 10 kb. Based on the sequence analysis, they were classified in four groups showing similarities with four known Bt plasmids, pGI3, pBMB175, pGI1 and pGI2, respectively. One of pGI3-like clones, named as pBt1-3, was fully sequenced and its putative open reading frames (ORFs), Rep-protein, double-strand origin of replication (dso), single-strand origin of replication (sso), have been identified. The structure of pBt1-3 showed high similarity with pGI3 which is one of rolling-circle replication (RCR) group VI family. As a donor for construction of shuttle vector, pDonr-attPEm vector harboring erythromycin resistant gene between attP sites was constructed. Through BP recombination with pPCS-Troy-cloned Bt plasmids and pDonr-attPEm, erythromycin resistant gene was transposed to Bt plasmids. This scheme proposes that in vitro transposition using pPCS-Troy and BP recombination using pDonr-attPEm can easily clone Bt plasmids and construct novel shuttle vectors.

Recently, the genome of Spodoptera litura granulovirus (SlGV) which encodes 133 putative open reading frames (ORFs) was completely sequenced. In this study, to screen novel insecticidal genes of SlGV, we first constructed an advanced plasmid capture system, pPCS-TPI, which contains not only pUC19 ori and ampicillin resistance gene but also Autographa californica nucleopolyhedrovirus (AcMNPV) ORF603 and ORF1629 homologous region between Tn7L and Tn7R. In order to introduce genomic segments of SlGV into the genome of AcMNPV, genomic DNA of SlGV was digested with EcoRI and self-ligated. These self-ligated segments were in vitro transposed with the pPCS-TPI donor by the help of TnsABC* transposase. By this, 10 EcoRI-digested genomic segments of the SlGV were cloned, and these clones were co-transfected with the bApGOZA DNA into sf9 cells to generate corresponding recombinant virus, respectively. The resulting recombinant viruses harboring genomic segments of the SlGV could be used to investigate the insecticidal activity and/or other functions originated from the introduced genomic segments of the SlGV.

The baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV), a large circular double-stranded DNA virus whose genome encodes at least 155 open reading frames (ORFs), is highly pathogenic to a number of lepidopteran insects and widely used to transduce various cells for exogenous gene expression. Although many genes of AcMNPV have been identified, the genome-wide study related to viral replication has not been well announced. In this study, to elucidate DNA replication cascade of AcMNPV, we firstly developed a novel baculovirus genome that can be maintained in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. This genome, named bAc-MK, contains a mini-F replicon and a kanamycin resistance marker. Using a convenient Tn7 transposon-based system, pPCS-S, which contains an ampicillin resistance gene, ORF knock-out mutants were generated by random insertion into bAc-MK genome. These mutants will be suffered DNA microarray to elucidate AcMNPV replication cascade.

Plasmid capture systems (PCS) facilitate cloning and manipulation of circular double-stranded DNA. We recently developed an improved PCS (PCS-LZ) to clone relatively large DNA molecules of 30-150 kb. The PCS-LZ donor consists of a mini-F replicon and a kanamycin resistance marker between Tn7 left and Tn7 right ends. Both the replicon and marker gene of the PCS-LZ donor are transferred into target plasmid DNAs by in vitro transposition, followed by replication in E. coli. Colonies are tested for lacZ expression by blue/white screening. Circular DNAs were obtained from plasmids of Bacillus thuringiensis, genome segments of Cotesia glomerata bracovirus and polymorphic genomes of Autographa californica nucleopolyhedrovirus. PCS-LZ is a powerful tool for use in genomic analysis and mutagenesis in invertebrate pathology, and we are extending its application to include vertebrate research.