Changes in the Plasma Proteome of Manduca sexta Larvae in Relation to the Transcriptome Variations after an Immune Challenge: Evidence for High Molecular Weight Immune Complex Formation

Abstract

Manduca sexta is a lepidopteran model widely used to study insect physiological processes, including innate immunity. In this study, we explored the proteomes of cellfree hemolymph from larvae injected with a sterile buffer (C for control) or a mixture of bacteria (I for induced). Of the 654 proteins identified, 70 showed 1.67 to >200-fold abundance increases after the immune challenge; 51 decreased to 0 -60% of the control levels. While there was no strong parallel between plasma protein levels and their transcript levels in hemocytes or fat body, the mRNA level changes (i.e. I/C ratios of normalized read numbers) in the tissues concurred with their protein level changes (i.e. I/C ratios of normalized spectral counts) with correlation coefficients of 0.44 and 0.57, respectively. Better correlations support that fat body contributes a more significant portion of the plasma proteins involved in various aspects of innate immunity. Consistently, ratios of mRNA and protein levels were better correlated for immunity-related proteins than unrelated ones. There is a set of proteins whose apparent molecular masses differ considerably from the calculated M r 's, suggestive of posttranslational modifications. In addition, some low M r proteins were detected in the range of 80 to >300 kDa on a reducing SDS-polyacrylamide gel, indicating the existence of high M r covalent complexes. We identified 30 serine proteases and their homologs, 11 of which are known members of an extracellular immune signaling network. Along with our quantitative transcriptome data, the protein identification, inducibility, and association provide leads toward a focused exploration of humoral immunity in M. sexta. Molecular & Cellular

In search of a function of Manduca sexta hemolymph protease-1 in the innate immune system

“…The ratios of HP1a and HP1b read numbers were 6.94 and 1.31 in hemocytes from the naïve and induced larvae, respectively. There was a 57% decrease in HP1a mRNA level in hemocytes and 17% decrease ( p < 0.05) in HP1a protein level in plasma after the immune challenge (He et al, 2016). In contrast, there was a 2.3-fold increase in HP1b mRNA and 17% increase ( p > 0.05) in HP1b protein.…”

“…These data agreed well with the developmental regulation in hemocytes. While the cross-reactivity of HP1a antibodies to HP1b made it impossible to distinguish the two proenzymes, quantitative proteomic analysis by nanoLC-MS/MS (He et al, 2016) allowed us to estimate that proHP1a was 8.8- and 6.3-fold higher in abundance than proHP1b in the naïve and induced day 3, 5 th instar larvae, respectively.…”

“…The remaining 125 genes are expressed in other tissues at different life stages. In the proteomic analysis of larval hemolymph, we identified 36 SPs and SPHs, nine of which are known to be components of the SP-SPH system (Zhang et al, 2014; He et al, 2016). They are: HP14 (HP for hemolymph protease), HP21, HP6, HP8, proPO activating proteases (PAP1–3), SPH1, and SPH2 (Jiang et al, 2010; Wang et al, 2014).…”

Serpin-9 and -13 regulate hemolymph proteases during immune responses of Manduca sexta

“…Since fat body mRNA levels in general do not correlate well with their protein abundances in M. sexta plasma (He et al, 2016), we examined levels of the two serpins in hemolymph samples, each pooled from 2 or 3 insects. Serpin-9 level did not increase at 24 h after the immune challenge and decreased after M. luteus stimulation (Fig.…”

Hemolymph protein profiles of subterranean termite Reticulitermes flavipes challenged with methicillin resistant Staphylococcus aureus or Pseudomonas aeruginosa

“…Previous studies have demonstrated changes in the hemolymph proteomes of fruit fly, silkworm, white butterfly, and tobacco hornworm in response to immune challenges 6,8–10 . In termites, Liu et al .…”