Sang Woon Shin

Isolation and characterization of immune-related genes from the fall webworm, Hyphantria cunea, using PCR-based differential display and subtractive cloning

Following injection of bacteria into the hemocoel of the fall webworm, Hyphantria cunea, several inducible genes were identified and characterized using PCR-based differential display (DD-PCR) and subtractive cloning. Ten immune-related cDNA clones (Hdd1, Hdd2, Hdd3, Hdd11, Hdd13, Hdd15, Hdd17, Hdd23, Hs106, Hs302) were isolated and characterized. The deduced amino acid sequence of Hdd2 was shown to be a member of the copper, zinc superoxide dismutase (Cu-Zn SOD) family. The H. cunea Cu-Zn SOD is novel in that it is up-regulated following a bacterial challenge and has a putative signal peptide suggesting its secretion and involvement in the insect immune response. Hdd3 was found to encode a new member of the serpin (serine protease inhibitor) family. The putative lectin corresponding to Hdd15 is of a different kind in that it has two lectin C domains in a single molecule. These two lectin C domains show significant homology to the lectin C domain of Periplaneta lipopolysaccharide binding protein (LPS-BP). Three cloned genes, Hdd17, Hs106 and Hs302, encode a homologue to Bombyx mori Gram negative binding protein, a hemolin-like protein and a attacin-like protein, respectively. The deduced amino acid sequences from Hdd11 showed weak homology with a Locusta migratoria hemolymph protein. On the contrary, Hdd1, Hdd13 and Hdd23 did not reveal any significant homology with known proteins. All of the 10 genes were clearly inducible by E. coli and M. luteus injection. Injection of distilled water only slightly induced mRNA levels. Comparison of temporal mRNA expression following E. coli injection showed three types of expression patterns.

Isolation and Characterization of the cDNA Encoding the Prophenoloxidase of Fall Webworm, Hyphantria cunea

Two kinds of cDNA clones encoding prophenoloxidases (ProPO; zymogen of phenoloxidase (monophenol, L-dopa: oxygen oxydoreductase, EC 1.14.18.1)) were isolated by polymerase chain reaction (PCR) followed by screening of cDNA library that was prepared from whole larvae of the fall webworm, Hyphantria cunea (Lepidoptera, Arctiidae). The cDNAs encode 681 and 697 amino acids with molecular masses of 78.2 and 80.2 kDa, respectively. Deduced amino acid sequence homology between the two H. cunea ProPOs are only 49% whereas the homology against other insect ProPOs ranged from about 40 to 72%. The phylogenic analysis showed that the insect ProPOs are grouped mainly into two families. A putative proteolytic cleavage site for enzyme activation was identical to other insect ProPOs. The conserved copper binding sites were 84-62% homologous to arthropod ProPOs. Two additional highly conserved regions were found in the carboxy terminal. Furthermore, like other insect prophenoloxidases, hydrophobic signal peptide sequences were absent in the deduced ProPOs from H. cunea. Southern blot analysis indicated that the H. cunea ProPO1 is present as a single copy in the genome. Northern blot analysis showed that the expression of the ProPO genes were concentrated in mid-instar larvae, but were much lower in other developmental stages.

Two carbohydrate recognition domains of Hyphantria cunea lectin bind to bacterial lipopolysaccharides through O-specific chain.

We previously identified a novel lectin cDNA from the fall webworm [Shin et al. (1998) Insect Biochem. Mol. Biol. 28, 827–837], which encodes two carbohydrate recognition domains (CRD‐N and CRD‐C) and is up‐regulated following bacterial challenge. The lipopolysaccharide (LPS) binding activities of the recombinant CRD‐N and CRD‐C (rCRD‐N and rCRD‐C) were investigated by enzyme‐linked immunosorbent assay. The LPS binding of rCRD‐N and rCRD‐C was pH‐dependent: at pH below 6.0, they show a higher binding ability to LPS. The binding of the rCRD‐N was inhibited by both D‐mannose and N‐acetyl‐D‐glucosamine, whereas the binding of the rCRD‐C was inhibited only by D‐mannose. The binding of both rCRD‐N and rCRD‐C to Escherichia coli was mainly mediated through the O‐specific chain.

Ultrastructural and molecular identification of a Wolbachia endosymbiont in a spider, Nephila clavata

Wolbachia‐like bacteria were observed in the egg cells of golden orb‐weaving spider, Nephila clavata, by means of transmission electron microscopy. The bacteria exhibited the typical morphology of Wolbachia, including three enveloping membranes. Based on the amplification and sequencing of partial 16S rDNA and ftsZ gene, the bacteria were identified as Wolbachia, intracellular, transovarially inherited α‐proteobacteria in invertebrates. Phylogenetic analysis based on 16S rDNA and ftsZ gene sequences invariably indicated that the intracellular bacteria from N. clavata belonged to group A Wolbachia, which were found only from insects. Clustering of Wolbachia from N. clavata with group A Wolbachia indicates that the bacteria were probably transferred horizontally between insects and the spider.

Protein purification and cDNA cloning of a cecropin-like peptide from the larvae of fall webworm (Hyphantria cunea)

A proteinous antimicrobial substance was purified from the bacteria-challenged larvae of the fall webworm, Hyphantria cunea. It is a cecropin-like antibacterial peptide which exhibits antibacterial activity against Gram-negative and Gram-positive bacteria, and known as Hyphantria cecropin A. The cDNA clones corresponding to this peptide were isolated from a cDNA library constructed from the bacteria-challenged larvae and obtained complete nucleotide sequences. In addition to the Hyphantria cecropin A sequence, we obtained three other cDNAs exhibiting high sequence similarity with Hyphantria cecropin A. We synthesized the C-terminally amidated peptide of 35 residues based on the deduced sequence of the isolated cDNA of Hyphantria cecropin A. The synthetic peptide exhibited strong antibacterial activity against several microbes including medically important bacteria such as Salmonella, Shigella, and fungus such as Candida. A Southern blot experiment using these cloned cDNAs as probes predicted the existence of multiple forms of Hyphantria cecropin genes.

Protein purification and nucleotide sequence of a lysozyme from the bacteria-induced larvae of the fall webworm, Hyphantria cunea

A protein with lytic activity against Micrococcus luteus was purified from the hemolymph of the fall webworm, Hyphantria cunea, larvae challenged with live E. coli. A bacteriolytic protein of about 14,000 daltons in mass was purified by cation exchange chromatography and reverse-phased HPLC. The optimum pH and optimum temperature range for activity were around pH 6.2 and 50 degrees C, respectively, in a 100 mM phosphate buffer. The amino-terminal amino acid sequence of this protein was determined and the corresponding cDNA was isolated and analyzed. The deduced protein of 142 amino acid residues was composed of a putative leader sequence of 20 residues and the mature enzyme of 122 residues. The cloned lysozyme gene was strongly induced in response to bacterial injection, implying that the enzyme is a part of the immune response of H. cunea. Comparison with other known lysozyme sequences shows that our lysozyme belongs to the chicken lysozyme.

Conifer Diterpene Resin Acids Disrupt Juvenile Hormone-Mediated Endocrine Regulation in the Indian Meal Moth Plodia interpunctella

Diterpene resin acids (DRAs) are important components of oleoresin and greatly contribute to the defense strategies of conifers against herbivorous insects. In the present study, we determined that DRAs function as insect juvenile hormone (JH) antagonists that interfere with the juvenile hormone-mediated binding of the JH receptor Methoprene-tolerant (Met) and steroid receptor coactivator (SRC). Using a yeast two-hybrid system transformed with Met and SRC from the Indian meal moth Plodia interpunctella, we tested the interfering activity of 3704 plant extracts against JH III-mediated Met-SRC binding. Plant extracts from conifers, especially members of the Pinaceae, exhibited strong interfering activity, and four active interfering DRAs (7α-dehydroabietic acid, 7-oxodehydroabietic acid, dehydroabietic acid, and sandaracopimaric acid) were isolated from roots of the Japanese pine Pinus densiflora. The four isolated DRAs, along with abietic acid, disrupted the juvenile hormone-mediated binding of P. interpunctella Met and SRC, although only 7-oxodehydroabietic acid disrupted larval development. These results demonstrate that DRAs may play a defensive role against herbivorous insects via insect endocrine-disrupting activity.

Genomic Structure of the Immune-related Serpin Gene in the Fall Webworm, Hyphantria cunea

Abstract We previously identified a novel immune-related serine type protease inhibitor cDNA from Hyphantria cunea (Drury) (Shin et al., 1998) that suggested a specific inhibitor directed at the protease involved in the activation of prophenoloxidase (proPO) (Park et al., 2000). Herein, we report complete nucleotide sequence and the exon-intron structure of the gene. The Hyphantria serpin gene spans 18 kb and is composed of eight exons and seven introns coding for an 1.8-kb mRNA. In the 5′ flanking sequence, we found several putative regulatory motifs including an enhancer, two NF-IL6 motifs, a TNF-α responsive element, an interferon-γ-responsive element, and a GATA motif element that have been found in the promoters of the acute phase protein genes of mammals and insects. In addition, a 9-bp inverted repeat sequence located from −270 to −221 region was also found. A reactive site loop sequence was located in exon 8 flanked by 3′ untranslation sequence. Phylogenetic analysis showed that this Hyphantria serpin did not cluster with either insect or mammalian serpins that are known, or suspected, to be associated with immune function. Southern blot analysis showed that the Hyphantria serpin gene is present as a single copy in the genome.

A plant diterpene counteracts juvenile hormone-mediated gene regulation during Drosophila melanogaster larval development

Many plant species possess compounds with juvenile hormone disruptor (JHD) activity. In some plant species, such activity has been attributed to diterpene secondary metabolites. Plant JHD diterpenes disrupt insect development by interfering with the juvenile hormone (JH)-mediated formation of JH receptor complexes. Here, we demonstrate that a plant extract and a diterpene from Lindera erythrocarpa (methyl lucidone) interfere with the formation of both methoprene-tolerant (Met)/Taiman and Germ cell-expressed (GCE)/Taiman heterodimer complexes in yeast two-hybrid assays in vitro. In addition to the in vitro JHD activity, the diterpene and the plant extract from L. erythrocarpa also disrupt the development of larvae and pupae in Drosophila melanogaster. Comparing the transcriptomes of juvenile hormone analog (JHA, methoprene)- and JHD (methyl lucidone)-fed wandering third-instar larvae revealed a large number of genes that were coregulated by JHA and JHD. Moreover, most (83%) of the genes that were repressed by methyl lucidone were significantly activated by methoprene, indicating that JHDs and JHAs have opposing effects on the transcriptional regulation of many JH-dependent genes. Gene ontology analysis also suggested that some of the genes activated-by-JHA/repressed-by-JHD play roles in spermatogenesis. Affymetrix microarray-based analysis indicated that the expression of genes activated-by-JHA/repressed-by-JHD was testis-specific. Together, these results suggest that JH is involved in testis-specific gene expression and that plant JHD diterpenes function as JH antagonists in such JHA-mediated gene regulation.

Species-Specific Interactions between Plant Metabolites and Insect Juvenile Hormone Receptors

Because juvenile hormone (JH) controls insect development and its analogs are used as insecticides, juvenile hormone disruptors (JHDs) represent potential sources from which novel pesticides can be developed. Many plant species harbor JHD activity, which has previously been attributed plant secondary metabolites (i.e., diterpenes) that disrupt insect development by interfering with the JH-mediated heterodimer formation of insect juvenile receptor complexes. The results of the present study indicate that plant JHD activity is also concentrated in certain plant groups and families and that plant metabolites have insect group-specific activity. These findings suggest that reciprocal diversification has occurred between plants and insects through the evolution of the plant metabolites and JH receptors, respectively, and that plant metabolites could be developed into insect group-specific pesticides with limited effects on non-target species.