In 2009, Kajiwara investigated the proteome of silkworm hemolymph by 2D-mapping and ion-trap mass spectrometry, and identified 67 proteins in a hemolymph map of the 3rd day of fifth instar . After comparing the 56 proteins identified in this work with those 67 proteins, we found only 5 that were identical, including juvenile hormone-binding proteins (spots H36-38); aldose reductase (spots H21, H22); low molecular-weight lipid proteins (spots H27, H28 and H29); and CI8 (spot H12). Other proteins identified by Kajiwara, such as RNA-binding proteins, carboxylesterases, zinc-finger proteins, and some hypothetical proteins, were not identified in this work. Comparing the hemolymph map of Kajiwara with the hemolymph map from this work, we found the pH range of the two maps was different. We used pH 3-10 strips to separate proteins, while Kajiwara used pH 4-7 strips. For this reason, some alkaline proteins in this work may not appear in the Kajiwara map, such as imaginal disk growth factor (spot H17-19), gelsolin (spot H20), and glyoxylate reductase (spot H23). We also compared our 2D map with the 2D map of Manduca hemolymph reported by Furusawa in 2007 , and found 10 Manduca hemolymph proteins had similar expression patterns in silkworm hemolymph protein, including transferrin protein, serine proteinase-like protein, chymotrypsin inhibitor, hemolin and prophenoloxidase. Thus, our results contribute to the understanding of development and metamorphosis not only in the silkworm, but also in other lepidopteran pests.
30 K protein of silkworm hemolymph
3 K proteins, a group of lipoproteins with a molecular mass of approximately 30 kDa, accumulate in a stage-dependent fashion in the larval hemolymph of the silkworm, Bombyx mori. In previous work, we analyzed the silkworm genome and expressed sequences, and found 10 genes encoding 30 K proteins. By semi-quantitative reverse-transcription polymerase chain reaction, eight members were found to be expressed in the fatbody . In this work, we investigated the 30 K proteins of hemolymph at the protein level using 2D-PAGE and MALDI-TOF MS. Expression of hemolymph 30 K proteins demonstrated a dynamic change from the 1st day of fifth instar larvae through eclosion, with maximum expression at the spinning stage, and almost disappearance by the 1st day of moth stage. Five 30 K protein family members were identified from the 28-31 kDa area in the hemolymph of 3rd day pupa (Additional File 2). The proteins sequences of Bmlp1, Bmlp2, Bmlp3 and Bmlp4 had more than 97% similarity with PBMHP-6, PBMHP-12, PBMHPC-19 and PBMHPC-21, low molecular lipoprotein 30 K of silkworm which were reported previously by Sakai . Because of the high similarity, these might be the same proteins in silkworm, which show some genetic variation in different varieties. Bmlp7 is a novel 30 K protein in hemolymph, and shared 93% protein sequence similarity with PBMHPC-19. Other members were not detected in hemolymph because of low expression, or expression during other stages.
In the hemolymph map of the 3rd day of fifth instar, Bmlp1, Bmlp2, Bmlp3 and Bmlp7 were found by MS, but not Bmlp4. Bmlp4 increased sharply during development, and was detected in six spots (spot 1, 4, 7, 8, 9, 10) in the hemolymph map of the 3rd day of pupation. After metamorphosis from silkworm to pupa, it decreased rapidly in the hemolymph, and was not detected in the hemolymph at the 8th day of pupation. Compared to Bmlp1, Bmlp2, Bmlp3 and Bmlp7, Bmlp4 is a special 30 K protein that appears later in the fifth instar stage, is expressed strongly, and disappears rapidly. This is similar to the mRNA expression of Bmlp4 in fatbody that was reported by Sun in 2007 . These results suggested that Bmlp4 may have a different function than other silkworm 30 K proteins.
During the later pupal stages, female silkworm proteins around 30 kD were reduced compared to males. It has been reported that during this period, the 30 K proteins were transported into the embryo in female silkworms, and became important for embryonic development [17, 18], which would result in the reduction of 30 kD in female hemolymph. While in male silkworms, the 30 K proteins accumulated in the hemolymph instead of being absorbed by the embryo. Interestingly, at the 8th day of pupation, the amount of 30 K proteins decreased sharply, both in females and males. A protease for digesting 30 K proteins, named 30 kP protease A, was found in silkworm eggs, and selectively hydrolyzes the 30 kDa yolk protein of silkworm. This exhibited a high expression level in late-pupal stages, by Northern blot . We predict that during this period, some 30 K proteins in male pupa are enzymatically degraded, producing nutrition for the activities of the last stages, such as eclosion and mating.
Low molecular weight proteins change in later pupal stages
In addition to the low molecular weight lipoprotein 30 K protein, small proteins also showed remarkable variation in the silkworm hemolymph maps, especially at the 8th day of pupation. Spots H52 and H53, considered pupa-specific proteins, were detected only in the 8th day hemolymph of pupa, in the acidic area of the 2D map. These two proteins were identified as 32 kDa apolipoproteins. In 2005, Kim investigated a similar protein, in the fall webworm, Hyphantrin. The cDNA for the Hyphantrin protein was expressed only during the middle and late pupal stages, by Northern blot , and was similar to the 32 kDa apolipoprotein of silkworm in expression pattern and molecular weight. Spot H54 was more abundant at the 8th day of pupation compared to the 3rd day of the pupa and larval stages, and was barely detectable in the hemolymph at eclosion. This was identified as a diapause bioclock protein, EA4, which is an ATPase that measures time intervals as a diapause-duration clock. Its activity can be elevated transiently by dissociation of an inhibitory peptide under cold conditions . Spots H55, H56 were identified as apolipophorin III proteins, which increased greatly in silkworm moth hemolymph. Adult moths depend on an abundance of lipids as high-powered energy sources for flight. Apolipophorin III is considered to have a key role in lipid transport systems, and serves as a reusable shuttle for transporting lipids to flight muscles, for use as fuel . Although B. mori, as a domesticated insect, has almost lost flight capability over cultivation, many activities in the adult moth, such as mating or egg laying, still rely heavily on an efficient muscle system.
Proteins involved in metamorphosis from larval to pupa
During insect metamorphosis, the larval tissues are degraded, and this decomposition involves programmed cell death (PCD) triggered by ecdysteroids. Some proteins related to PCD were found by 2D-electrophoresis and MS. Beta-N-acetylglucosaminidase (spots H8, H9, H10) is a chitinolytic enzyme that hydrolyzes chitose to chitobiose, and has an important role in metamorphosis . The molecule 20-hydroxyecdysone induces expression of beta-N-acetylglucosaminidase, and strong expression of beta-N-acetylglucosaminidase in the latter stage of the fifth larval stage contributes to larval tissue degeneration and metamorphosis . Juvenile hormone regulates growth and development in insects, and is protected from hydrolysis by general esterases by combining with juvenile hormone binding protein (JHBP) . JHBP (spots H36, H37, H38) was detected in silkworm hemolymph from larval to moth stages, and showed higher expression in the larval stage. Imaginal disc growth factor (IDGF) (spots H17, H18, H19) is a polypeptide growth factor that stimulates the growth of imaginal disk cells. In the presence of 20-hydroxyecdysion, IDGF can be induced in the anterior silk gland, suggesting that IDGF is involved in PCD during silkworm metamorphosis .
Proteins involved in food digestion and substance metabolism
During the fifth instar of the larval stage, the silkworm digests an abundance of mulberry to accumulate energy for the non-feeding pupal and adult stages. Some of the enzymes involved in substance and energy metabolism were found in this stage. For instants, aldose reductase (spots H21, H22) is a member of the family of oxidoreductases that convert glucose to sorbitol in carbohydrate metabolism . Glyoxylate reductase (spot H23) and hydroxypyruvate isomerase (spot H31) participate in glyoxylate and dicarboxylate metabolism. The former can catalyze glycolate to produce glyoxylate, while the latter interconverts aldoses and ketoses [28, 29]. Aminoacylase (spots H15, H16) belongs to a family of hydrolases that catalyze the conversion of N-acyl-L-amino acids to carboxylate and L-amino acids . CI-8 (spot H12) of silkworms also showed high expression at the 6th day of fifth instar. It is secreted by the fatbody during the feeding period, and sequestered in the fatbody after the onset of spinning . It not only interacts with two polypetides, p29 and p60, in the midgut membrane, but also inhibits the 35-kDa protease in the digesting juice of the larval midgut [32, 33]. During the period of high consumption of mulberry, we hypothesize that up-regulation of CI-8 is involved in regulating protease activity in mulberry digestion.
Proteins related to immunity in silkworm hemolymph
Silkworm hemolymph is involved in initiation of the immune response. An abundance of proteins related to immunity were also identified in this work, including hemolin (spots H48, H49), prophenoloxidase (spot H11), serine proteinases (spots H24, H26), paralytic peptide-binding protein (H39), and trypsin inhibitor (spots H1, H13, H14). The prophenoloxidase activation system is an important defense system against parasite and pathogen invasion . Some proteins of this complex system were also detected in silkworm hemolymph. Hemolin is a member of the pattern recognition proteins, which are induced after bacterial infection. Although it does not have direct antibacterial activity, it can trigger the prophenoloxidase cascade against bacterial infection, in combination with bacterial lipopolysaccharides . Two serine proteases were also found in hemolymph, which shared 66% and 67% similarities to serine protease homologs (SPHs) purified from the plasma of Manduca sexta larvae. The SPHs of M. sexta are reported to bind to pattern recognition receptor and function as mediators to recruit prophenoloxidase and prophenoloxidase-activating protease to the site of infection . Prophenoloxidase has long been considered a key enzyme in melanization, which is considered to be an effective defense against microorganisms. Without prophenoloxidase, the melanization of insects is almost completely inhibited .
Some novel silkworm hemolymph proteins were identified in this work. In this work, SP1 and SP2 were found in the hemolymph of silkworm larvae, and a new hemolymph storage protein (BGIBMGA009027) was identified, which shared high similarity and a similar expression pattern to SP2. Gelsolin (spot H20) is an actin-binding protein that interacts with actin in vitro to promote actin nucleation and actin filament formation. There are two forms of gelsolin in vertebrates, one secreted, and other cytoplasmic . The secreted gelsolin of silkworm was first reported here, in the molecular range of 45 kDa, and by hemolymph map, was expressed from larvae to moth. It is presumed to be involved in the regulation of extracellular fluid viscosity, or in wound-healing . Some protease inhibitors were present in silkworm hemolymph (spot H1, H12, H13). For example, inter-alpha-trypsin inhibitors (ITIH4) are a family of structurally related serine protease inhibitors in human plasma. Recent studies suggest they may function during infection by bacteria and viruses . Finally, some proteins (H2, H3, H4, H32, H51) are not discussed here, even though they had strong expression and showed remarkable change during development and metamorphosis, because of inconclusive results from database searches. Future analysis of these proteins and their functions will contribute to our understanding of development and metamorphosis.