Peptidomic Analysis of the Brain and Corpora Cardiaca-Corpora Allata Complex in the Bombyx mori

The silkworm, Bombyx mori, is an important economic insect for silk production. However, many of the mature peptides relevant to its various life stages remain unknown. Using RP-HPLC, MALDI-TOF MS, and previously identified peptides from B. mori and other insects in the transcriptome database, we created peptide profiles showing a total of 6 ion masses that could be assigned to peptides in eggs, including one previously unidentified peptide. A further 49 peptides were assigned to larval brains. 17 new mature peptides were identified in isolated masses. 39 peptides were found in pupal brains with 8 unidentified peptides. 48 were found in adult brains with 12 unidentified peptides. These new unidentified peptides showed highly significant matches in all MS analysis. These matches were then searched against the National Center for Biotechnology Information (NCBI) database to provide new annotations for these mature peptides. In total, 59 mature peptides in 19 categories were found in the brains of silkworms at the larval, pupal, and adult stages. These results demonstrate that peptidomic variation across different developmental stages can be dramatic. Moreover, the corpora cardiaca-corpora allata (CC-CA) complex was examined during the fifth larval instar. A total of 41 ion masses were assigned to peptides.

Neuropeptides have been broadly studied in many insect species, such as Locusta migratoria, Apis mellifera, and Manduca sexta [16,17]. However, most peptide studies focus on a specific developmental stage, either at larval or adult, which limits appreciation of the peptidomic variations that take place across different growth stages. The aim of the present study is to profile peptide complements in eggs and in the brains of silkworms at larval, pupal, and adult stages.

Insects.
Silkworms from strain P50 (Dazao) were obtained from the Institute of Sericulture in Jiangsu province. They were reared on mulberry leaves at 26 • C at 80% relative humidity and a 16L:8D photoperiod. For the experiments, eggs from day 3 were analyzed, and brains on day 4-5 of larvae, day 3 of pupae, and day 3 of male and female adults were separately dissected and analyzed.

Tissue Extraction and Liquid Chromatography.
One hundred eggs were collected and incorporated into a sample, and three thus independent biological replicates (samples) were separately collected and analyzed. Similarly, one hundred of larval brains, one hundred of pupal brains, and one hundred of adult brains of B. mori, as well as one hundred pairs of the corpora cardiaca-corpora allata (CC-CA) complex from the fifth instar larvae, were dissected and incorporated into an independent sample, respectively, and three thus independent biological replicates were also collected and analyzed, respectively. All operations were performed on ice. Each sample was placed in a tube containing ice-cold extraction medium (methanol: water: acetic acid, 90 : 9 : 1, v/v/v), homogenized, and centrifuged at 12,000 ×g at 4 • C for 15 minutes. The pellet was re extracted twice. All supernatants were pooled, the organic solvent was evaporated by vacuum centrifugation, and the residue was dissolved in 0.1% (v/v) trifluoroacetic acid (TFA).
The samples were fractionated on an Agilent 1100 HPLC system (Agilent, USA) using a ZORBAX StableBond C18 column (4.6 mm × 250 mm, 5 μm, 300Å; Agilent, USA). The column was first eluted with 3% acetonitrile in 0.1% trifluoroacetic acid (TFA) for 10 minutes. Then acetonitrile was increased to 21% over 10 minutes. This was followed by a linear gradient of 21-60% acetonitrile/0.1% TFA over 30 minutes at a flow rate of 1 mL/min. Fractions were collected manually every minute and concentrated by the vacuum desiccator (LNG-T88, Huamei Biochemical Company, P.R.China) to about 5 μL for mass spectrometry analysis.

MALDI-TOF Mass Spectrometry. MALDI-TOF MS
analysis was performed on an autoflex II TOF/TOF instrument (Bruker Daltonics, Germany). The matrix used in the analysis was a saturated solution of recrystallized α-cyano-4hydroxycinnamic acid (CHCA; Bruker Daltonics) dissolved in 70% acetonitrile containing 0.1% (v/v) TFA. Samples of 0.5 μL were added to the MALDI plate, followed by 0.5 μL of matrix solution. They were mixed and left to dry at room temperature. The spectra were obtained using an accelerating voltage of 19 KV in the reflection mode with a mass range m/z 700-3000. Laser power was adjusted to provide optimal signal-to-noise ratio. The measured monoisotopic masses [M + H] + were compared to the calculated values of known or predicted peptides. Masses were calculated using Protein Prospector (University of California, San Francisco, CA, U.S.) [1]. MALDI-TOF-TOF mass spectra were acquired on an autoflex II TOF/TOF instrument (Bruker Daltonics, Germany). Ion fragmentation data were analyzed using FlexAnalysis software (version 3.0) from Bruker Daltonics. The mature peptide in B. mori from our MS/MS results were identified and confirmed by either previous publications [3-6, 8, 12-15], or the EST from the NCBI database (http://www.ncbi.nlm.nih.gov/) and silkworm database (http://silkworm.genomics.org.cn/) [18], or the NeuroPred tool SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/).

Peptidomics in Different Organs and Developmental
Stages of B. Mori. In this study, over 100 ion peaks were obtained by MS profiling from analysis of eggs and all postembryonic stages. The mature peptides were evaluated by comparison of measured monoisotopic masses [M + H] + against combination of bioinformatics and identified peptides in Lepidopteran insects and the predicted peptides from genome databases, such as calculated masses, score, queries matched, and sequence coverage. In total, 19 newfound and 40 previously reported mature brain peptides were identified in B. mori.
Both the MALDI-TOF mass spectra of HPLC separation fractions from different developmental stages of silkworm brains and CC-CA extracts of the fifth instar larvae and the MALDI-TOF-TOF fragmentation spectra of the precursor ion at m/z were analyzed and identified. A number of b-type and y-type ions were labeled (Figures 1-3). Some of them are pGluat of the N-terminal.

Discussion
Peptides in insects are very important to regulate many physiological activities involved in feeding, ecdysis and metamorphosis, reproduction, energy homeostasis, circadian rhythm, anxiety, seizure, contraction of muscle, learning and memory, and so on [19][20][21][22]. Especially, variation of peptidomics at different developmental stages is huge, in which each different stage has characteristic peptides. In this study, we mapped peptides from important neuroendocrine organs, the CC-CA complex, and the brains of silkworms at different stages of growth. The aim of this study was exploring peptidomic composition at different stages because different development stages express specific physiological requirements.
Our results demonstrate that peptidomic variations during different developmental stages are profound in silkworms. In eggs, only few mature peptides were detected; we infer that developmental regulation is operates on a different physiological basis during the egg stage. ACP and AKHs are involved in mobilization of lipids and carbohydrates from fat bodies and ovaries [23]. Besides, allatostatin is involved in inhibition of JH and SGNP in diapause [24]. Compared to larval brains, pupal and adult brains lacked 3 categories (CAPA/CAP2b, NPLP-1, and Leucokinin) and 2 categories (NPLP-1, and Leucokinin), respectively. Of these 3 categories, the CAPA/CAP2b peptides have cardioacceleratory properties and increased heart rate [25][26][27]. The NPLP-1 was found to play a role in ecdysis behavior in D. melanogaster [1,28,29], and leucokinin is a neurohormone that participates in the regulation of water and ion homeostasis, especially the control of ion transport in the stellate cells of the insect's Malpighian tubules [1].
The peptidomic variation across organs between the brain and the CC-CA complex in larvae was also different. Compared to larval brains, CC-CA complex lacked 3 categories (diapause hormone, SIFamide and leucokinin). The SIFamide has been found to be responsible for courtship. Four SIFamidergic neurons and arborizations play an important function in the neuronal circuitry controlling sexual behavior in Drosophila [28,29]. In addition, SIFamide may play a role in processing or transmitting tactile, olfactory, and visual stimuli, which is also important for courtship behavior and partner selection [30]. But the function of SIFamide in B. mori and other moths still maintains unclear.
Nowadays, SIFamide, tachykinin (TK), CCAP, CAPA-PVK, sulfakinin, and neuropeptide-like precursor 1 (NPLP 1) were predicted by the B. mori genomic database [12]. MIP is another Lepidoptera peptide previously identified from  International Journal of Peptides   Manduca sexta [31]. It was found to have the similar peptide precursor named as B. mori PTST [2][3][4][5][6][7]. Many neuropeptide precursors undergo a series of enzymatic processes, causing the production of mature, bioactive amidated neuropeptides. Each mature peptide may display a different potential function on the cellular level [32]. The AT/AST has functions in stimulating/inhibiting JH synthesis [33,34]. The sNPF and tachykinins regulate food intake and consumption but sulfakinin inhibits food intake [35][36][37]. Corazonin may be the key factor in the formation of colors during the larval stage or reduction of spinning in the pupal stage [38,39]. The orcokinins, BRFas, and PTSPs are all involved in the regulation of insect development in ecdysteroid biosynthesis, in which orcokinins have a clear prothoracicotropic activity, in contrast with BRFas [14]. While PTSPs inhibit ecdysteroid biosynthesis in the PG [15]. Again, our results indicate that peptidomics can vary greatly between different organs and developmental stages. The present study adds valuable information to the knowledge of neuropeptidomes.
In this study, all HPLC fractions were collected and analyzed by MALDI-TOF MS, and high-intensity signal peaks consistent with B. mori neuropeptides were fragmented by TOF-TOF for peptide identification. However, the apparatus has a limited ability to hit and break ions by TOF-TOF, which is weaker than the ion-trap mass spectrum for some peptides to be not fragmented.