To gain an insight into the effects of different diets on growth and development of the domesticated silkworm at protein level, we employed comparative proteomic approach to investigate the proteomic differences of midgut, hemolymph, fat body and posterior silk gland of the silkworms reared on fresh mulberry leaves and on artificial diet. Seventy-six differentially expressed proteins were identified by MALDI TOF/TOF MS, and among them, 41 proteins were up-regulated, and 35 proteins were downregulated. Database searches, combined with GO analysis and KEGG pathway analysis revealed that some hemolymph proteins such as Nuecin, Gloverin-like proteins, PGRP, P50 and beta/-N-acetylglucosamidase were related to innate immunity of the silkworm, and some proteins identified in silkworm midgut including Myosin 1 light chain, Tropomyosin 1, Profilin, Serpin-2 and GSH-Px were involved in digestion and nutrition absorption. Moreover, two up-regulated enzymes in fat body of larvae reared on artificial diet were identified as V-ATPase subunit B and Arginine kinase which participate in energy metabolism. Furthermore, 6 down-regulated proteins identified in posterior silk gland of silkworm larvae reared on artificial diet including Ribosomal protein SA, EF-2, EF-1gamma, AspAT, ERp57 and PHB were related to silk synthesis. Our results suggested that the different diets could alter the expression of proteins related to immune system, digestion and absorption of nutrient, energy metabolism and silk synthesis poor nutrition and absorption of nutrition in silkworm. The results also confirmed that the poor nutrient absorption, weakened innate immunity, decreased energy metabolism and reduced silk synthesis are the main reasons for low cocoons yield, inferior filament quality, low survival rate of young larvae and insufficient resistance against specific pathogens in the silkworms fed on artificial diet.

The posterior silk gland (PSG) is the most important suborgan responsible for the synthesis and secretion of silk core fibroin proteins in silkworm. Here, we performed genome-scale expression profiling analysis of silkworm PSG at the fourth molting (M4) and at day 1 (V1), day 3 (V3), day 5 (V5), and wandering stage (W) of the fifth instar by microarray analysis with 22 987 probes. We found that the five genes of silk proteins secreted from PSG including fibroin heavy (H) and light (L) chains, P25, seroin 1, and seroin 2 basically showed obvious up-regulation at V3 which lasted to V5, while slight down-regulation at W. The expression of translation-related genes including ribosomal proteins and translation initiation factors generally remained stable from M4 to V5, whereas it showed clear down-regulation at W. Clustering analysis of the 643 significantly differentially expressed transcripts revealed that 43 of the important genes including seroin 1 and sugar transporter protein had co-expression patterns which were consistent with the rate changes of fibroin synthesis and PSG growth. Pathway analysis disclosed that the genes in different clusters might have co-regulations and direct interactions. These genes were supposed to be involved in the fibroin synthesis and secretion. The differential expression of several hormone-related genes also suggested their functions on the regulation of PSG development and fibroin synthesis. 2D gel-based proteomics and phosphoproteomics profiling revealed that the phosphorylated proteins accounted for no more than one-sixth of the total proteins at each stage, which was much lower than the level in normal eukaryotic cells. Changes in the phosphorylation status and levels of several proteins such as actin-depolymerizing factor 1 and enolase might be deeply involved in fibroin secretion and tissue development. Shotgun proteomic profiling combined with label-free quantification analysis on the PSG at V3, V5, and W revealed that many small heat shock proteins (sHSP) were specially expressed at W, which was substantially consistent with the results from 2-DE analysis, and implied the close correlations of sHSP with the physiological states of PSG at W. A majority of significantly up-regulated proteins at V5 were related to ribosome pathway, which was different from the microarray results, implying that the translation-level regulation of ribosomal proteins might be critical for fibroin synthesis. In contrast, the ubiquitin-proteasome pathway related proteins appeared obviously up-regulated at W, suggesting that the programmed cell death process of PSG cells might be started before cocooning.

To investigate the molecular mechanisms underlying the low fibroin production of the ZB silkworm strain, we used both SDS-PAGE-based and gel-free-based proteomic techniques and transcriptomic sequencing technique. Combining the data from two different proteomic techniques was preferable in the characterization of the differences between the ZB silkworm strain and the original Lan10 silkworm strain. The correlation analysis showed that the individual protein and transcript were not corresponded well, however, the differentially changed proteins and transcripts showed similar regulated direction in function at the pathway level. In the ZB strain, numerous ribosomal proteins and transcripts were down-regulated, along with the transcripts of translational related elongation factors and genes of important components of fibroin. The proteasome pathway was significantly enhanced in the ZB strain, indicating that protein degradation began on the third day of fifth instar when fibroin would have been produced in the Lan10 strain normally and plentifully. From proteome and transcriptome levels of the ZB strain, the energy-metabolism-related pathways, oxidative phosphorylation, glycolysis/gluconeogenesis, and citrate cycle were enhanced, suggesting that the energy metabolism was vigorous in the ZB strain, while the silk production was low. This may due to the inefficient energy employment in fibroin synthesis in the ZB strain. These results suggest that the reason for the decreasing of the silk production might be related to the decreased ability of fibroin synthesis, the degradation of proteins, and the inefficiency of the energy exploiting.

Juvenile hormone acid methyltransferase (JHAMT) is an enzyme involved in one of the final steps of juvenile hormone biosynthesis in insects. It transfers a methyl group from S-adenosyl-L-methionine (SAM) to the carboxyl group of either farnesoic acid (FA) or JH acid (JHA). Several genes coding for JHAMT have been cloned and characterized from insects from different orders, and they have been shown to play critical roles in metamorphosis and reproduction. However, the significance of JHAMT in Hymenopteran insects is unknown. We used RACE amplification method to clone JHAMT cDNA from the honey bee, Apis mellifera (AmJHAMT). The full length cDNA of AmJHAMT that we cloned is 1253bp long and encodes a 278-aa protein that shares 32-36% identity with known JHAMTs. A SAM-binding motif, conserved in the SAM-dependent methyltransferase (SAM-MT) superfamily, is present in AmJHAMT. Its secondary structure also contains a typical SAM-MT fold. Most of the active sites bound with SAM and substrates (JHA or FA) are conserved in AmJHAMT as in other JHAMT orthologs. Phylogenetic analysis clustered AmJHAMT with the other orthologs from Hymenoptera to form a major clade in the phylogenetic tree. Purified recombinant AmJHAMT protein expressed in E. coli was used to produce polyclonal antibodies and to verify the identity of AmJHAMT by immunoblotting and mass spectrometry. Quantitative RT-PCR and immunoblotting analyses revealed that queen larvae contained significantly higher levels of AmJHAMT mRNA and protein than worker larvae during the periods of caste development. The temporal profiles of both AmJHAMT mRNA and protein in queens and workers showed a similar pattern as the JH biosynthesis. These results suggest that the gene that we cloned codes for a functional JHAMT that catalyzes the final reactions of JH biosynthesis in honey bees. In addition, AmJHAMT may play an important role in honey bee caste differentiation.