Systematic Investigation of the Hemolymph Proteome ofManduca sextaat the Fifth Instar Larvae Stage Using One- and Two-Dimensional Proteomics Platforms

Abstract

Manduca sexta is an excellent insect model for studying insect physiology, including hemolymph proteins. Larvae stages of this insect are highly damaging to tobacco leaves causing a drastic decrease in crop yield. Investigation on the larval biology should help in controlling its destructive potential, thus increasing crop yields. The hemolymph is the source of its immunity to disease and environmental factors, which invariably involves protein components. To better understand the physiology of M. sexta and the protein components expressed during its life cycle, two complementary proteomics approaches, one- and two-dimensional gel electrophoresis (1-DGE and 2-DGE) in conjunction with N-terminal amino acid sequencing and liquid chromatography-mass spectrometry, were employed to analyze the fifth instar larvae hemolymph proteins. These proteomics approaches together identified 123 proteins, which constituted a total of 58 nonredundant proteins and belonged to 10 functional categories. Defense (49%), transport and metabolism (15%), storage (9%), and metamorphosis (7%) categories were highly represented accounting for 80% of the identified proteins. Besides identification of previously reported proteins, 18 novel proteins were identified, which include the lipoprotein-releasing system transmembrane protein lolC, 50S ribosomal protein L24, inducible serine protease inhibitor 1, imaginal disk growth factor, protein disulfide-isomerase-like protein ERp57, etc. The 2-DGE data were integrated to develop a 2-D gel reference map. Data obtained from 1-DGE and 2-DGE analyses are accessible through the M. sexta proteomics portal ( http://foodfunc.agr.ibaraki.ac.jp/mansehemoprot.html). Together, this study provides evidence for the presence of a large number of functionally diverse protein families in the hemolymph of M. sexta. These proteins correlate well with the fifth instar stage, the transition from larvae to pupae.

Semi-quantitative analysis of changes in the plasma peptidome of Manduca sexta larvae and their correlation with the transcriptome variations upon immune challenge

“…Due to limited sequence information and technical hindrance, high-quality proteomic investigations have been performed in only a few insects such as Drosophila melanogaster (Veraksa, 2010). In Manduca sexta , 58 non-redundant polypeptides have been identified in larval plasma using one- and two-dimensional gel electrophoresis (Furusawa et al, 2008). …”

Extensive Conserved Synteny of Genes between the Karyotypes of Manduca sexta and Bombyx mori Revealed by BAC-FISH Mapping

“…For years this species has been used as a favorite experimental animal in insect biochemistry, physiology, and especially in neurobiology [15]–[17]. Sequence and expression data for M. sexta genes has been increasing [18]–[20].…”

Identification of Natural Target Proteins Indicates Functions of a Serralysin-Type Metalloprotease, PrtA, in Anti-Immune Mechanisms

“…In the case of PAT-35a and -b, we generated all of the possible sequence variants (2 10 ϭ 1,024 sequences) with the help of a C-script and then loaded them on the BLAST server. When we restricted the search to M. sexta sequences with an upper limit of E value at 0.1, the search resulted in two proteins, scolexins (12). However, we did not find matches.…”