Yong Min Kwon

Modulation of MnSOD protein in response to different experimental stimulation in Hyphantria cunea

A manganese superoxide dismutase (MnSOD) gene was cloned from the fall webworm, Hyphantria cunea. MnSOD cDNAs encode precursor proteins of 215 amino acid residues. H. cunea MnSOD possesses the metal binding ligands of 3 histidines and 1 aspartic acid common to MnSODs. The deduced amino acid sequences of the H. cunea MnSOD cDNA showed 76% identity to Bombyx mori MnSOD and 56-62% identity to MnSOD sequences from other species. MnSOD and copper/zinc superoxide dismutase (Cu/ZnSOD) is expressed in all tissues of H. cunea. MnSOD expression changed at a trace-level in infected larvae, while Cu/ZnSOD expression strongly changed against Gram-positive and Gram-negative bacteria, and fungi. Environmental stresses such as different artificial photoperiods (24L:0D), ultraviolet irradiation (312 nm), and starvation condition increased Cu/ZnSOD expression, MnSOD expression, on the other hand, was increased by starvation. Moreover, MnSOD and Cu/ZnSOD expression showed no significant change in the 0L:24D condition. MnSOD and Cu/ZnSOD expression in H. cunea also significantly increased at high (37°C) and low (4°C) temperature. Oxidative stress induced by 10% H(2)O(2) reduced the expression levels of MnSOD and Cu/ZnSOD. However, paraquat-induced oxidative stress reduced MnSOD expression but increased Cu/ZnSOD expression. These results suggest that Cu/ZnSOD may play a larger role than MnSOD as a superoxide anion scavenger against oxidative stress in H. cunea.

RNA interference mediated knockdown of apolipophorin-III leads to knockdown of manganese superoxide dismutase in Hyphantria cunea

Apolipophorin-III (apoLp-III), a hemolymph protein that facilitates lipid transport in aqueous media in insects was recently shown to play a role in insect immune activation. Here, we report another novel possible function of apoLp-III in insects. To identify genes affected by apoLp-III expression in larvae, we decreased endogenous apoLp-III mRNA in Hyphantria cunea (Hc) through RNA interference; subsequently, we observed lower levels of antioxidant enzymes, including manganese superoxide dismutase (MnSOD), glutathione S-transferase, and immune proteins. Knockdown of Hc apoLp-III led to decreased MnSOD expression in fat body tissues and elevated superoxide anion levels in Hc fat body cells, suggesting that Hc apoLp-III is involved in the action and/or expression of antioxidant enzymes, especially MnSOD.

Comparative analysis of two attacin genes from Hyphantria cunea.

A full-length clone corresponding to attacin was isolated from a cDNA library made from fat body of immunized Hyphantria cunea larvae. This newly isolated attacin B shows characteristics different from those previously reported for attacin A. The two attacin cDNAs encode precursor proteins of 233 and 248 amino acid residues, respectively. The two attacins show 45.9% identity at the amino acid level, and 35.2% identity at the nucleotide level. Attacins A and B of H. cunea show significant identities with the attacins of Lepidoptera. Attacin B is a typical glycine-rich protein, while attacin A is leucine-rich. Attacin B is expressed from last instar larvae to adult, while attacin A showed stage-specific expression during the prepupal and pupal stages. Attacins A and B are predicted to have different secondary structure in that attacin A has no tendency to form helices but attacin B contains a substantial number of helices. Attacin A is induced at a trace level in infected larvae, while attacin B is strongly induced against Gram-positive and negative bacteria, fungi, and viruses. The attacin B transcripts were detected in fat body, epidermis and hemocytes after injection with Escherichia coli, Citrobacter freundii, or Candida albicans, but not in the midgut and Malpighian tubule. Recombinant attacin A showed no antibacterial activity, while recombinant attacin B showed strong antibacterial activity in proportion to the amount of the protein injected.

Molecular cloning and characterization of the STAT gene in Hyphantria cunea haemocytes.

A new insect member of the signal transducer and activator of transcription (STAT) family of transcription factors, Hyphantria cunea STAT (HcSTAT), was cloned from the lepidopteran H. cunea. The domain involved in DNA interaction and the Src homology 2 (SH2) domain were well conserved. During all developmental stages, the gene was expressed at a low level in the haemocytes, fat body cells, midgut, epidermis and Malpighian tubules. The haemocytes and Malpighian tubules showed transcriptional activation of HcSTAT upon Gram‐negative and Gram‐positive bacterial challenges. These challenges increased the induction and nuclear translocation of the HcSTAT protein that recognizes a STAT target site in H. cunea haemocytes. In vivo treatment with sodium orthovanadate translocated HcSTAT to the haemocyte nucleus. This study shows the involvement of the haemocyte Janus kinase/STAT pathway after microbial infection in lepidopteran insects.

Partial sequence and characterization of nitric oxide synthase gene from Hyphantria cunea

Nitric oxide synthase (NOS) gene has been partially sequenced from Hyphantria cunea and compared with those already determined from insects. Hyphantria cunea NOS possesses putative recognition sites for co‐factors heme, BH4, CaM, FMN, FAD, and NADPH common to NOS. The deduced amino acid sequence of H. cunea NOS cDNA showed 70.3% identity to Manduca sexta NOS and 57.6–69.5% identity to NOS sequences from other insects. Nitric oxide synthase is expressed in all tissues of H. cunea, except in hemocytes. The NOS expression in midgut, fat body, epidermis, and Malpighian tubule strongly increased against Gram‐positive and Gram‐negative bacterial infection. These results suggest that NOS may play an important role in insect defense system against bacterial infection.