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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., (© 2011 The Authors. Insect Molecular Biology © 2011 The Royal Entomological Society.)

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.

An insect defensin, named Galleria defensin, was purified from the larval haemolymph of Galleria mellonella immunized against E. coli. The peptide was composed of forty-three amino acid residues containing six cysteines that might be engaged in intramolecular disulphide bridges. The primary structure of Galleria defensin shared about 90.7% identity to that of heliomicin, which was an insect defensin isolated from Heliothis virescens. The full-length cDNA encoding Galleria defensin was cloned from the fat body of the immunized G. mellonella larvae. Northern blot analysis revealed that Galleria defensin was expressed not only in the fat body but also in the midgut against invading bacteria into haemocoel. This is the first report presenting cDNA and expression of an insect defensin in the lepidopteran species.

Storage protein-1 (HcSP-1) is a major storage protein found in the hemolymph and fat body of Hyphantria cunea. HcSP-1 has a high methionine (6.0%) and low aromatic amino acid content (8.5%) (Cheon et al., 1998). In this study, the accumulation and expression of HcSP-1 in ovary was investigated using biochemical and immunocytochemical methods. HcSP-1 was detected in the ovaries in 6-day-old pupae and accumulated toward the end of pupal life, when HcSP-1 transcripts were detectable by Northern blot analysis and RT-PCR. In situ hybridization showed that the HcSP-1 mRNA was located in the nurse cells and follicular epithelial cells, but not in the oocyte. Though most of the HcSP-1 that is incorporated in the yolk bodies of the oocyte is probably sequestered from the surrounding hemolymph, HcSP-1 is an important yolk protein contributing to early yolk body formation before the development of patency by the follicular epithelium., (Copyright 2001 Wiley-Liss, Inc.)

Storage protein-1 (SP-1) is a major storage protein found in the hemolymph and fat body of Hyphantria cunea. In this study, the uptake and accumulation of SP-1 into the ovary of H. cunea was investigated using biochemical and immunocytochemical methods. SP-1 in H. cunea has a high methionine content (4.6%) but is not female-specific, like other high methionine storage proteins. In the 6-day-old pupal ovary, SP-1 was detectable in trace amounts but accumulated to significant levels toward the end of the pupal stage. After adult emergence, SP-1 rapidly decreased in the ovarian follicles and remained low in the egg. This suggest that SP-1 is either extensively modified or degraded, causing a loss of its antigenic property in the ovary after adult emergence. During vitellogenesis, SP-1 is present in the hemolymph and penetrates through the tunica propria to reach the perioocytic space. From there, SP-1 is incorporated into yolk bodies. These results clearly show that SP-1 is taken up by the developing oocyte. Its disappearance suggests that SP-1 might be an amino acid reservoir for providing precursors for egg formation, in contrast to yolk proteins, which are utilized during postembryonic development.

The synthesis of two storage protein subunits, 76,000-Mr and 82,000-Mr polypeptides, by the testes sheath has been studied in Heliothis virescens. Like fat body, which is the primary site of synthesis for the large extratesticular pool, cells of the testes sheath secrete glycosylated storage proteins assembled into hexamers. The testis sheath differed from fat body in several important respects, including the failure to synthesize an abundant (in the hemolymph) 74,000-Mr storage protein, its relatively reduced expression of the 76,000-Mr polypeptide, and the absence of resorption of storage proteins from the lumen of the testis during pupal development. Cyst cells were also shown to import actively the 82,000-Mr storage protein by pinocytosis of testicular fluid and transfer it to the developing spermatids. Unlike other cell types that sequester storage proteins in the form of cytoplasmic granules, their localization within spermatids was exclusively mitochondrial. These observations suggest that expression of the storage protein genes is regulated tissue specifically and reveal novel pathways for their transport and, perhaps, utilization and function during development.