Isolation, Identification, and Bioinformatic Analysis of Antibacterial Proteins and Peptides from Immunized Hemolymph of Red Palm Weevil Rhynchophorus ferrugineus

Abstract

Red palm weevil (Rhynchophorus ferrugineus Olivier, 1791, Coleoptera: Curculionidae) is a destructive pest of palms, rapidly extending its native geographical range and causing large economic losses worldwide. The present work describes isolation, identification, and bioinformatic analysis of antibacterial proteins and peptides from the immunized hemolymph of this beetle. In total, 17 different bactericidal or bacteriostatic compounds were isolated via a series of high-pressure liquid chromatography steps, and their partial amino acid sequences were determined by N-terminal sequencing or by mass spectrometry. The bioinformatic analysis of the results facilitated identification and description of corresponding nucleotide coding sequences for each peptide and protein, based on the recently published R. ferrugineus transcriptome database. The identified compounds are represented by several well-known bactericidal factors: two peptides similar to defensins, one cecropin-A1-like peptide, and one attacin-B-like protein. Interestingly, we have also identified some unexpected compounds comprising five isoforms of pheromone-binding proteins as well as seven isoforms of odorant-binding proteins. The particular role of these factors in insect response to bacterial infection needs further investigation.

Keywords: Rhynchophorus ferrugineus; antimicrobial proteins/peptides; attacins; bioinformatics; cecropins; defensins; odorant-binding proteins; pheromone-binding proteins.

Figure 1

Reversed-phase high pressure liquid chromatography (RP-HPLC) of the components of the hemolymph of immunized insects. Panel (A) shows the full elution profile of the hemolymph. All visible peaks form this separation were collected and tested for antibacterial activity against Escherichia coli and Staphylococcus intermedius. The dashed square indicates the region with the highest activity. Panel (B) presents further separation of this specific region into several fractions. Of these fractions, five peaks marked from 1 to 5 had bactericidal activity. Panels (C–G) show individual separations of these fractions into subfractions containing homogeneous compounds. Those exhibiting bactericidal activity are marked with double digit numbers and were used for further SDS-PAGE analysis and identification. The details of hemolymph collection and chromatography conditions are described in the Materials and Methods section.

Figure 2

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) image of immune peptides and proteins isolated from Rhynchophorus ferrugineus hemolymph. The numbers correspond to the subfractions shown in Figure 1C–G. Selected bands denoted by arrows were identified by N-terminal amino acid sequencing after excision from the polyvinylidene difluoride (PVDF) membrane. The other proteins from subfractions 3.2, 4.1, and 4.7 were identified by mass spectrometry.