Several recent laboratory observations that bear on the origin of the Bilateria are reviewed and interpreted in light of our set-aside cell theory for bilaterian origins. We first discuss new data concerning the phylogeny of bilaterian phyla. Next, we use systematic, molecular, and paleontological lines of evidence to argue that the latest common ancestor of echinoderms plus hemichordates used a maximal indirect mode of development. Furthermore, the latest common ancestor of molluscs and annelids was also indirectly developing. Finally, we discuss new data on Hox gene expression patterns which suggest that both sea urchins and polychaete annelids use Hox genes in a very similar fashion. Neither utilizes the complete Hox complex in the development of the larva per se, while the Hox complex is expressed in the set-aside cells from which the adult body plan is formed. Our current views on the ancestry of the bilaterians are summarized in phylogenetic terms, incorporating the characters discussed in this paper.
2.1. Two-Dimensional Gel Electrophoresis and MALDI-TOF/TOF Analyses
The gel-based proteome analysis revealed 61 and 36 spots from the soluble fraction (SF) and insoluble fraction (IF), respectively. From the spots analyzed by Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF/TOF), 23 peptide sequences belonging to eight proteins were identified in the SF, approximately 38% of the total analyzed (Figure 2, Table 1). Proteins identified in the SF comprised five different enzymes: Superoxide dismutase, Triosephosphate isomerase, Ribonuclease, two Fructose-bisphosphate aldolases and Alpha-enolase. In addition, Peroxiredoxin and two Ferritins were identified. However, three of these proteins matched to “predicted protein” as best hit, but were then further annotated using blastp algorithm in the NCBI with the accession number retrieved from the custom sea anemones databases. Unlike shotgun proteomics, for gel-based analysis were used only two sea anemones databases, since additional search was carried against UniProtKB/Swiss-Prot in the Metazoa section. However, best results corresponded to local analysis. On the other hand, no proteins were identified with statistic confidence from the IF (Figure S1) and in both cases SF and IF, the use of different database like UniProtKB/Swiss-Prot did not improved the identification. The details of blast search and protein identification by MALDI-TOF/TOF mass spectrometry of the protein identified from the 2DE is shown in Table 1. It is noteworthy, that some of the proteins identified have been previously reported in other cnidarians [46,47,48], but constitute the first report in B. verrucosa.
The identification rates obtained for SF are similar to those reported in previous studies of other marine species, when comparable proteomics protocols were used [49,50,51]. On the other hand, the absence of identifications in IF is an evidence that our proteomics protocol is likely not optimized for the analysis of the type of proteins present in this fraction. Since IF may be enriched with hydrophobic membrane proteins, the lack of identifications may be related, among other possible causes, to incomplete separation of proteins and to the inefficient digestion of these proteins with trypsin; thus, hindering the generation of a sufficient number of proteolytic peptide fragments for Mass Spectrometry/Mass Spectrometry (MS/MS) sequencing analysis. This limitation of trypsin when cleaving such proteins particularly in the hydrophobic and transmembrane domains can be overcome by combining the activities of other proteases [52,53].
The identified proteins seem to play important roles related with RNA degradation, glycolysis and antioxidant pathways. Moreover, some proteins like alpha aldolase seem to play diverse molecular and physiological roles. In fact, several antibacterial, antiparasitic, antifungical and autoantigen activities have been proposed . Alpha aldolase expression and activity have been associated with the occurrence and metastasis of cancer, as well as with growth, development and reproduction of organisms . Its expression seems to be related to heat shock , but it is also probably active under anaerobic condition . In general, some of these proteins act as stress protein against environmental changes by exerting a protective effect on cells.
Ribonucleases, also known as RNases, are common and widely distributed catalytic proteins among animals, involved in the RNA degradation . Three different RNases were detected: triosephosphate isomerase, Fructose-bisphosphate aldolase and Alpha-enolase, which are involved in the glycolytic pathway. Triosephosphate isomerase is a glycolytic enzyme that catalyzes the interconversion of the three-carbon sugars such as dihydroxyacetone phosphate and d-glyceraldehyde 3-phosphate . Aldolases are stereochemistry-specific enzymes acting in a diverse variety of condensation and cleavage reactions . Specifically, fructose-1,6-bisphosphate aldolase is involved in gluconeogenesis and glycolysis, controlling the production of fructose-1,6-bisphosphate from the condensation of dihydroxyacetone phosphate with glyceraldehyde-3-phosphate [58,59]; while Alpha-enolase is a versatile metalloenzyme, that catalyzes the conversion of 2-phosphoglyceric acid to phosphoenolpyruvic acid .
On the other hand, Ferritin is one of the most important proteins in iron metabolism, acting as primary iron storage protein or iron transporter, solubilizing iron and thus regulating its homeostasis [60,61]. Peroxiredoxin, also called thioredoxin peroxidase or alkyl hydroperoxide reductase, has been proposed as antioxidant protein [62,63,64]. Both proteins, seem to play an important role by protecting the cells against reactive oxygen species , so they are likely to be natural anti-Ultraviolet (UV) radiation agents . Similarly, superoxide dismutase is another relevant antioxidant protein [65,67]. The high expression of this protein as part of the antioxidant defense system makes sense, since aerobic organisms need to deal with oxygen species produced as a consequence of aerobic respiration and substrate oxidation .
2.2. Protein Identification from Shotgun Proteomics Analysis
A methodology based on shotgun analysis was employed to investigate the whole-body proteome of B. verrucosa. This methodology has been previously reported as suitable for diverse purposes related to protein identification such as characterization of complex sample, inference of the main enzymatic pathway involved in a tissue, even to reveal venom composition [68,69,70,71]. Altogether, 688 peptide sequences were identified among the two replicates of the fractions analyzed (SF and IF), which accounted for 412 groups of non-redundant proteins (), retrieved from custom cnidarians databases. Of all protein detected, 97 were identified from two or more peptides. Only four proteins were detected as potential contaminants in the first search against custom database, while 69 sequences accounted for 35 putative proteins as contaminants against UniProtKB/Swiss-Prot database (Table S2). Of such contaminants, 10 proteins were identified from two or more peptides and were related mostly to human keratin and trypsin. In the case of contaminants, proteolytic fragments from trypsin and keratin were the most commonly found, which are difficult to avoid and thus are ubiquitous in proteomic analysis . The functional annotation of all proteins (except for contaminants) was further addressed.
The fact that several IF proteins were identified by this shotgun method shows the increased potential of this method over 2DE/MALDI-TOF/TOF for the analysis of membrane proteins, even when carried out based on the activity of a single protease (trypsin).
All proteins identified from the gel-based analysis were also found among those identified by the shotgun proteomic analysis. As an example, the shotgun analysis allowed the identification of Peroxiredoxin (XP_001640260.1, see Table 1