Annotation of novel neuropeptide precursors in the migratory locust based on transcript screening of a public EST database and mass spectrometry

Abstract

Background: For holometabolous insects there has been an explosion of proteomic and peptidomic information thanks to large genome sequencing projects. Heterometabolous insects, although comprising many important species, have been far less studied. The migratory locust Locusta migratoria, a heterometabolous insect, is one of the most infamous agricultural pests. They undergo a well-known and profound phase transition from the relatively harmless solitary form to a ferocious gregarious form. The underlying regulatory mechanisms of this phase transition are not fully understood, but it is undoubtedly that neuropeptides are involved. However, neuropeptide research in locusts is hampered by the absence of genomic information.

Results: Recently, EST (Expressed Sequence Tag) databases from Locusta migratoria were constructed. Using bioinformatical tools, we searched these EST databases specifically for neuropeptide precursors. Based on known locust neuropeptide sequences, we confirmed the sequence of several previously identified neuropeptide precursors (i.e. pacifastin-related peptides), which consolidated our method. In addition, we found two novel neuroparsin precursors and annotated the hitherto unknown tachykinin precursor. Besides one of the known tachykinin peptides, this EST contained an additional tachykinin-like sequence. Using neuropeptide precursors from Drosophila melanogaster as a query, we succeeded in annotating the Locusta neuropeptide F, allatostatin-C and ecdysis-triggering hormone precursor, which until now had not been identified in locusts or in any other heterometabolous insect. For the tachykinin precursor, the ecdysis-triggering hormone precursor and the allatostatin-C precursor, translation of the predicted neuropeptides in neural tissues was confirmed with mass spectrometric techniques.

Conclusion: In this study we describe the annotation of 6 novel neuropeptide precursors and the neuropeptides they encode from the migratory locust, Locusta migratoria. By combining the manual annotation of neuropeptides with experimental evidence provided by mass spectrometry, we demonstrate that the genes are not only transcribed but also translated into precursor proteins. In addition, we show which neuropeptides are cleaved from these precursor proteins and how they are post-translationally modified.

Figure 1

Locusta migratoria Pacifastin Precursor 3. Amino acid sequence alignment of LMPP-3 and the ORF of CO827143 and CO827145 (most identical sequences). The signal peptide is indicated in italic, dibasic cleavage sites are shown in bold and pacifastin-like peptides are marked. Identical residues are marked with an asterisk.

Figure 2

Locusta migratoria Neuroparsin Precursors. Multiple sequence alignment (ClustalW 1.82) of CO849956, CO849957, CO832876, CO849958, CO821291 and the known Locusta neuroparsin precursor (Lom-NPP). Translation initiation sites are indicated in black and cysteine residues are shaded.

Figure 3

Locusta migratoria Tachykinin Precursor (top) and MS spectrum displaying the monoisotopic masses of 5 different Tachykinins (bottom). Amino acid sequence corresponding to the ORF of CO848687. The signal peptide as predicted by SignalP is shown in italic, possible amidation and dibasic cleavage sites are indicated in bold, possible tachykinin-like peptides are marked. MALDI-TOF mass spectrum of the first abdominal ganglion of Locusta migratoria. Masses corresponding to tachykinin peptides are marked (r.i. is relative intensity, m/z is mass to charge ratio).

Figure 4

Locusta migratoria Ecdysis Triggering Hormone Precursor. Amino acid sequence alignment of CG18105 (the Drosophila ETH-precursor) and the novel Locusta ETH-precursor (assembly of ORF from CO822155 and CO833211). The signal peptide is indicated in italic, amidation sites and dibasic cleavage sites are marked in bold and (putative) ETH-peptides are marked.

Figure 5

Amino acid sequence alignment of all known ETH-like peptides.

Figure 6

MSMS spectra of the two locust ETH-peptides. MSMS fragmentation spectrum of the triple charged ion at m/z 536.67, corresponding to ETH-1 (SDFFLKTAKSVPRIamide) and the double charged ion at m/z 780.45, corresponding to ETH-2 (SDLFLKSAKSVPRIamide). A-type, b-type, y-type and z-type fragment ions are shown. The theoretical fragment ion masses found in the spectrum are indicated in bold and the mass difference between the expected and observed fragment ion masses is shown below.

Figure 7

Locusta migratoria Neuropeptide F. Amino acid sequence alignment of CG10342 (the Drosophila NPF-precursor) and the ORF of CO854418. The signal peptide is indicated in italic, possible amidation and dibasic cleavage sites are marked in bold and a (putative) NPF-peptide is marked.

Figure 8

Locusta migratoria Allatostatin-C. Amino acid sequence corresponding to the ORF of CO835369. Possible dibasic cleavage sites and translation stops are indicated in bold, a possible allatostatin-C peptide is marked.