Insects secrete antimicrobial peptides as part of the innate immunity response. These peptides are often cationic, low molecular peptides with diverse structures. Most antimicrobial peptides from insects have antibacterial but not antifungal activity. We have characterized a newly identified peptide, antifungal peptide‐1 (AFP1) from hemolymph of a lepidopteran insect, Manduca sexta (tobacco hornworm). AFP1 was isolated by size exclusion chromatography from hemolymph plasma of larvae. Fractions containing activity against the yeast, Saccharomyces cerevisiae were analyzed my SDA‐PAGE and MALDI‐TOF MS/MS and found to contain a 41 residue peptide that was encoded by sequences identified in M. sexta transcriptome and genome data bases. AFP1 is a member of the diapausin family of peptides, which includes members known to have antifungal activity. The M. sexta genome contains 14 genes with high similarity to AFP1, each with 6 conserved Cys residues. A cDNA for AFP1 was cloned from cDNA prepared from fat body RNA. AFP1 was produced as a recombinant protein in Escherichia coli. Purified natural and recombinant AFP1 were active against S. cerevisiae, with IC50 of 15 micromolar, but they had no detectable activity against bacteria. Experiments to investigate activity of AFP1 against other fungi are ongoing. AFP1 mRNA level strongly increased after larvae were injected with yeast or with Micrococcus luteus. Our results indicate that synthesis of AFP1 is part of the innate immune response to infection in M. sexta. Grant Funding Source : Supported by NIH grant GM41247.
The stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), is an economically important pest of livestock. Previous studies demonstrated lymphocyte suppression by crude salivary gland extract (SGE) of the stable fly. A dominant 27-kDa protein identified in the SGE was reported to stimulate immunodominant antibody responses in exposed cattle. The purpose of this study was to determine whether this protein, now identified as a homolog of insect proteins named antigen 5 (Ag5), was responsible for the lymphocyte suppression and whether naïve calves can mount an immune response to it. Calves raised in the winter were immunized with recombinant Ag5 (rAg5) expressed in Drosophila S2 cells or with "natural" Ag5 protein isolated by preparative gel electrophoresis of SGE. Control calves were immunized with adjuvant alone. Rising antibody concentrations to rAg5 were detected in two of three calves immunized with rAg5 and one of three calves immunized with natural Ag5. Recall lymphocyte responses to rAg5 were detected at 21 and 28 d postimmunization in calves immunized with rAg5 but not in calves immunized with the natural Ag5 or those exposed to adjuvant alone. Mitogen-stimulated bovine lymphocyte responses were not suppressed by rAg5. Further investigation using immunoblotting revealed that rAg5 binds to the Fc and F(ab′)2 portions of bovine IgG, but not to an Fab fragment. These findings suggest that Ag5 of the stable fly salivary gland is not immunosuppressive but that it has immunoglobulin binding properties and can invoke specific antibody and memory lymphocyte responses in immunized calves.
Abstract From a Locusta migratoria genomic DNA library, a gene has been isolated that codes for a previously unrecognized hemolymph protein of M r = 19,000, designated 19k protein. The gene has at least five exons, extending over about 9 kb of DNA. Its polypeptide product, obtained by cell‐free translation of mRNA selected from adult fat body RNA by hybridization with the cloned DNA, is precipitated by antiserum against a low molecular weight hemolymph protein fraction. The mature protein product has been purified from locust hemolymph, and an N‐terminal sequence of 20 amino acids has been determined. In polyacrylamide gel electrophoresis, this protein comigrates with apolipophorin III, from which it was previously not distinguished, but it is clearly distinct by amino acid composition and sequence. The genomic clone was used as a probe to isolate a fat body cDNA clone of the 19k protein mRNA. The 938‐base pair cDNA clone contains a 516‐base pair open reading frame. The deduced 172‐amino acid polypeptide includes an apparent signal peptide, a sequence of four amino acids that may represent a prosegment, and a sequence identical (with a single exception, which may reflect polymorphism) with the N‐terminal sequence of the hemolymph protein. Its mRNA occurs at a low level in late larval fat body, is abundant in the newly eclosed adult, then declines to a low level, and rises again at days 8–10; it is greatly reduced after destruction of the corpora allata with precocene and then is elevated after treatment with methoprene, suggesting stimulation by juvenile hormone. The biological role of 19k protein is unknown.
Abstract Functional analysis of the two chitin synthase genes, TcCHS1 and TcCHS2 , in the red flour beetle, Tribolium castaneum , revealed unique and complementary roles for each gene. TcCHS1‐ specific RNA interference (RNAi) disrupted all three types of moult (larval–larval, larval–pupal and pupal–adult) and greatly reduced whole‐body chitin content. Exon‐specific RNAi showed that splice variant 8a of TcCHS1 was required for both the larval‐pupal and pupal‐adult moults, whereas splice variant 8b was required only for the latter. TcCHS2 ‐specific RNAi had no effect on metamorphosis or on total body chitin content. However, RNAi‐mediated down‐regulation of TcCHS2 , but not TcCHS1 , led to cessation of feeding, a dramatic shrinkage in larval size and reduced chitin content in the midgut.
