Protein expression during the embryonic development of a gastropod

Proteomics 2010, 10, 2701–2711

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DOI 10.1002/pmic.200900846

RESEARCH ARTICLE

Protein expression during the embryonic development of a gastropod Jin Sun1, Yu Zhang 2, Vengatesen Thiyagarajan3,4, Pei-Yuan Qian2 and Jian-Wen Qiu1 1 2 3 4

Department of Biology, Hong Kong Baptist University, Hong Kong, P. R. China Department of Biology, The Hong Kong University of Science and Technology, Hong Kong, P. R. China School of Biological Sciences, The University of Hong Kong, Hong Kong, P. R. China Swire Institute of Marine Science, The University of Hong Kong, Hong Kong, P. R. China

Despite the potential use of gastropod embryos in basic and applied research, little is known about their protein expression. We examined, for the first time, changes in proteomic profile during embryonic development of Pomacea canaliculata from an embryo without a shell (stage II) to an embryo with a fully formed shell (stage III) to understand the roles that proteins play in critical developmental events, such as the formation of shell, operculum and heart, and the differentiation of head and foot. To analyze protein expression during development, we used 2-DE to detect, MS to analyze, and de novo peptide sequencing followed by MS-BLAST to identify the proteins. The de novo cross-species protein identification method was adopted because of a lack of genomic and proteomic data in the whole class of Gastropoda. 2-DE detected approximately 700 protein spots. Among the 125 spots that were abundant, 52% were identified, a marked improvement over the conventional direct MSBLAST method. These proteins function in perivitelline fluid utilization, shell formation, protein synthesis and folding, and cell cycle and cell fate determination, providing evidence to support that this embryonic period is a period of dynamic protein synthesis and metabolism. The data shall provide a basis for further studies of how gastropod embryos respond to natural and human-induced changes in the environment.

Received: December 27, 2009 Revised: March 28, 2010 Accepted: April 27, 2010

Keywords: 2-DE / Animal proteomics / Apple snail / de novo sequencing / Embryonic development / Gastropod

1

Introduction

Embryonic development is of interest to biologists from both basic research and applied research perspectives. Embryos are widely used in basic research of cell signaling, genetic control of early development and certain diseases, and mechanisms of endocrine disruption [1–2]. In applied research, understanding animal embryology has led to great development of the aquaculture industry and embryonic Correspondence: Dr. Jian-Wen Qiu, Department of Biology, Hong Kong Baptist University, Kowloon, Hong Kong, P. R. China E-mail: [email protected] Fax: 1852-34115995 Abbreviations: FA, formic acid; NDPK, nucleotide diphosphate kinase; PVF, perivitelline fluid

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

assays for monitoring environmental pollution [3–5]. Gastropod embryonic development has several unique features including torsion, shell formation, coiling, gut formation and head–foot differentiation [6–7]. For example, torsion involves reorganization of the mantle cavity, visceral mass and pallial organs, which may confer some advantages for gastropods during the early radiation of mollusks [8]. Calcareous shell is a typical structure of mollusks including gastropods, which protects them from mechanical damage as well as predation. The shell formation involves changes in body shape and deposition of minerals and pigments in a matrix of proteins [9]. These dramatic changes must have required the action of many genes, which coordinate and modulate various developmental events [10]. For example, handedness determining genes dictate the expression of the nodal and Pitx genes of the pond snail Lymnaea stagnalis, www.proteomics-journal.com

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manipulating the layout of the early embryonic cells resulted in a change in the expression of these genes, which altered the left–right asymmetry of the shell [11]. Proteomic profiling, a direct reflection of the gene expression pattern at a given stage and time, can help us understand gene regulation during the embryonic development. This technique has been applied to studies of the embryos of several species of invertebrates, such as fruit flies [12], brine shrimp [13], honey bees [14], barnacles [15, 16], polychaetes [17] and ascidians [18]. Two proteomic studies on adult gastropods [19, 20] have identified some proteins that perform important biological functions, i.e. resistance/susceptibility of an intermediate host snail (Biomphalaria glabrata) to a parasite (Echinostoma caproni) [19] and adaptation to wave exposure environment in a marine snail (Littorina saxatilis) [20], showing the great potential of proteomics in ecological and biomedical studies of gastropods. Between these two studies, a higher proportion of the differentially expressed proteins (5 of 12 spots) was identified in the B. glabrata study, due to the presence of an ESTlibrary, compared with a low proportion (2 of the 34 spots) in L. saxatilis, which does not have extensive nucleotide or protein sequence resources [20]. These two studies thus highlight the complementary role of genomic and proteomic studies. However, proteomic studies have not been conducted in gastropod embryos. Recent development of the de novo cross-species identification method [21, 22] has offered a viable option for conducting proteomic studies in species where the genome is not sequenced or EST library is not available. This method has been successfully applied to nonmodel organisms such as the alga Dunaliella salina [23], pine Araucaria angustifolia [24] and moths Cerodirphia speciosa [25] and Helicoverpa armigera [26]. The golden apple snail Pomacea canaliculata (Lamarck) is native to South America. It invaded Asia in the 1980s and has become widely distributed in the tropical and subtropical freshwater wetlands throughout Asia [27, 28]. This snail has an extended reproductive season (up to 10 months [29]). A female lays a large number (up to 8680 [30]) of large eggs (2.5 mm in diameter, Fig. 1) during a breeding season. A juvenile grows and attains maturity quickly (2–4 months [30]). In addition, this species can be reared in laboratory to provide eggs throughout the year [31]. These characteristics make it easy to obtain sufficient material to conduct proteomic studies throughout the year. Furthermore, since morphogenesis during the embryonic development of P. canaliculata has been described [32, 33], proteomic changes can be correlated with morphological changes. In this study, we examined the changes in proteomic profile during the developmental period of P. canaliculata from an embryo with a newly formed mantle cavity to one with an operculum and a fully formed but nonpigmented shell (Fig. 1), corresponding to stage II–stage III in Heras et al. [32]. Because of the lack of genomic and proteomic work in P. canaliculata and other gastropods, we applied the & 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Proteomics 2010, 10, 2701–2711

A

B

C

D

Figure 1. Embryonic development of P. canaliculata showing the eggs (A), a stage II embryo (B), a stage III embryo (C) and newly hatched juveniles (D). Embryonic stages were determined according to the previous studies [32, 33]. Stages II and III embryos were used in proteomic analysis. In stage II, the head–foot is in the early stage of differentiation. In stage III, most of the external and internal organs are fully formed; the embryo resembles a juvenile. Scale: 1 mm for (A) and (D); 0.5 mm for (B) and (C).

de novo cross-species identification method to analyze protein spots. We also assessed the function of the identified proteins in morphological changes during the embryonic development.

2

Material and methods

2.1 Snail collection, rearing and egg incubation Approximately 60 adults of P. canaliculata (25–35 mm shell length) were collected from a freshwater pond in the Hong Kong Wetland Park (221280 21.5200 N, 1141000 07.3700 E) and kept in a laboratory aquarium containing 250-L dechlorinated tap water. A canister filter was used to clean the water and an electric air pump was used to provide aeration. Snails received Romaine lettuce (Lactuca sativa longifolia) ad libitum three times a week and commercial fish food (Tetras Bits) once a week. The filer and tank were cleaned, and one-third of the water was changed each week. Fertilized eggs laid by the snails were removed and incubated at 26711C. The egg shells of two eggs from the same clutch were broken daily and embryonic stages were determined under a dissecting microscope [32]. It took approximately 5 and 9 days of incubation for fertilized eggs to develop into stages II and III, respectively. www.proteomics-journal.com

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2.2 Sample preparation Upon reaching the desired stages, egg shells were broken gently and embryos were extracted from the perivitelline fluid (PVF). For each stage, three independent samples were prepared, with each sample containing approximately 300 embryos derived from three to four females. Each sample was rinsed in Milli-Qs water to remove the PVF on the surface of the embryos. Sample preparation protocol was identical to that in Thiyagarajan and Qian [15]. In brief, sample was placed in 500 mL of lysis buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 40 mM DTT and 2% BioLyte 3/10 ampholyte immediately. Each sample was ground in a 1.5 ml eppendorf tube, sonicated with a Branson Digital Sonicator set at 15% amplitude (ten blasts each lasting 5 s, with 10 s pauses), and centrifuged at 13 000 rpm for 20 min. The pellet was discarded and the protein supernatant was stored at 801C until electrophoresis. Protein content was determined using the modified Bradford method [34].

2.3 2-DE Protein samples were purified using a 2-D cleanup kit (BioRad, Hercules, CA, USA) and rehydrated in a buffer (7 M urea, 2 M thiourea, 2% CHAPS, 40 mM DTT, 0.2% Bio-Lyte, 3/10 ampholyte and 1% Bromophenol blue). Each sample was divided into two technical replicates. For each technical replicate, 1 mg protein (300 mL solution) was loaded onto a 17 cm IPG strip (Bio-Rad) with pH 3–10 (linear) to allow for a 12-h active rehydration, followed by IEF in a Protean IEF Cell at 201C. The electrophoresis was run at 250 V for 20 min, followed by a linear increase from 250 to 8500 V in 2.5 h, and at 8500 V for a total of 60 000 Vh. The current did not exceed 50 mA per strip. Focused IPG strips were equilibrated for 15 min in equilibration buffer I (6 M urea, 2% SDS, 0.05 M Tris-HCl (pH 8.8), 50% glycerol and 2% w/v 1,4-DTT) followed by 15 min in equilibration buffer II (same as buffer I except for replacing DTT with 2.5% iodoacetamide). To capture the second dimension, the equilibrated IPG strips were loaded on the top of 12.5% SDS-PAGE (18 cm 18 cm) and sealed with 0.5% w/v agarose. To reduce the chance of keratin contamination (the dust, hair, etc.), all solutions (acrylamide stock, Tris buffer, ddH2O and 10% SDS stock) were filtered through 0.22 mm filters before use. The running buffer system was the standard Laemmli buffer for SDS-PAGE. The gels were run for 12 h at 16 mA per gel; afterwards they were stained with G-250 Colloidal Coomassie [35].

2.4 Gel image analysis The 12 2-D gels (two stages three replicates two technical replicates) were each scanned at an optical resolution of 300 dpi using an ImageScanner II (Amersham Pharmacia & 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Biotech, Uppsala, Sweden). The images were analyzed using PDQuest version 8.0 (Bio-Rad) according to the manufacturer’s manual. All of the automatically detected spots were confirmed by visual inspection. Only dots presented in all technical and sample replicates were included in analysis. Comparison of gels between the two developmental stages was conducted assuming the protein spots to conform to a 3-D Gaussian distribution and determining the maximum absorption after raw image correction and background subtraction. Total spot intensities across gels were normalized based on the embryo origin spots. This was conducted after removing spots of nonembryo origin (i.e. those from the PVF). This removal step was essential as we were only interested in comparison of embryonic proteins between the two stages. Without normalization, the starting concentrations of embryonic proteins would have been different. Intensities for all protein spots for each stage were generated as percentage values. These values were then compared for significant differences between stages. Analysis was performed using the software’s default qualitative and quantitative modes. For qualitative analysis, spots showing at least tenfold changes were considered as absent/ present. For quantitative analysis, spots showing at least twofold changes between the two stages were compared using Student’s t-test. As the t-test was conducted for many pairs of spots, p-value was adjusted using Benjamini and Hochberg’s False Discovery Rate Correction [36]. Values that showed at least twofold difference and had a p-value of o0.02 were considered as up or downregulated (Supporting Information Table S1).

2.5 MALDI-TOF/TOF and ESI QqTOF analyses MS analyses were conducted with 125 most abundant protein spots. These included but not limited to proteins that were turned on and off, as well as proteins that were up and downregulated. Spots were cut from the gels and digested in gel according to Shevchenko et al. [37]. Briefly, protein spots were destained twice with 50 mM NH4HCO3 in 50% methanol and washed three times with Milli-Qs water. Gel pieces were dried and rehydrated twice with 100% ACN and 100 mM NH4HCO3, respectively, and then digested overnight at 371C in 10 mL of 12.5 ng/mL sequencing grade trypsin (Promega, Madison, WI, USA) in 50 mM NH4HCO3. Peptides were extracted twice using 5% formic acid (FA) in 50% ACN. Extracts were pooled, dried in a speed-vacuum (Thermo Electron, Waltham, MA, USA), redissolved in 50 mL of 0.1% v/v FA, and desalted with a C18 ZipTip (Millipore, MA, USA). Digests were mixed with 5 mg/mL CHCA in 0.1% FA and 50% ACN, spotted onto MALDI target plates, and analyzed by a MALDI TOF/TOF mass spectrometer (Autoflex III smartbeam, Bruker Daltonik, Bremen, Germany). Spectra were externally calibrated with a calibration standard mixture (P/N 206195, Bruker Daltonik). www.proteomics-journal.com

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If proteins were not positively identified by the MALDI TOF/TOF analysis, protein digests were reconstituted in 5% FA and desalted with a C18 ZipTip, and analyzed on a quadrupole TOF mass spectrometer (QSTARPulsar, Applied Biosystems/Sciex, Ont., Canada) equipped with a nanoelectrospray ion source [38]. The mass spectrometer was set to the positive ion mode, with a selected mass range of m/z 350–1600. Peptides with 12 to 14 charge states were selected for MS/MS. Top abundant peptides above the five count threshold were selected for the MS/MS. Smart information-dependent acquisition was activated with automatic collision energy and automatic MS/MS accumulation.

2.6 Mass spectrum interpretation We followed the de novo cross-species protein identification strategy [22]. For the spots subjected to MALDI TOF/TOF identification, the data were submitted through MASCOT to the NCBI nonredundant database without taxon restriction. Mass searches were performed using mass tolerance settings of 75 ppm for the precursor and 0.3 Da for the fragment masses. The following parameters were used in database searching: fixed modification: carbamidomethyl (cysteine); variable modification: oxidation (methionine). Up to one missed trypsin cleavage was allowed. Hits were considered significant when at least three peptides of a protein showing MASCOT scores of over 50 simultaneously. For spots subjected to ESI QqTOF identification, a protein was considered positively identified based on the combining criteria of the MASCOT and MS-BLAST. The ESI QqTOF data were submitted through a locally installed MASCOT to the MSDB database (3 239 079 sequences entries; updated September 2006). The following parameters were used in database search: tolerance for peptides and fragments: 0.5 and 0.2 Da respectively; fixed modifica-

Proteomics 2010, 10, 2701–2711

tion: carbamidomethyl (cysteine); variable modification: oxidation (methionine); and one tolerant missed cleavage. For the MASCOT, a protein was considered positive identification only when it had a significant score (po0.05). In the MS-BLAST, we followed the color-based criteria [39] after merging the candidate sequences from in silico de novo sequencing deduced from PepNovo. De novo sequencing was performed as described by Waridel et al. [39]. Briefly, the ESI QqTOF peak files were converted into MASCOT generic file (mgf ) files manually. Automated de novo peptides sequencing was produced via the modified version of PepNovo under default settings. The top seven-candidate sequences derived from the significant results (with a score of 2.0 or above [23], unless otherwise specified) were merged into a single query string, and submitted to MS-BLAST at http://genetics.bwh.harvard.edu/msblast/ [24] for search against a nonredundant database (nrdb95). Only hits with a total BLAST score above 100 or with at least one highscoring segment pair above 72 were considered as positive [23]. The identification results were confirmed by determining the molecular weight and pI of the matched proteins using the ExPASy Proteomics Server (www.expasy.org).

3

Results

3.1 Proteome from stages II and III embryos A reference proteome map was constructed for analyzing the proteomic profile during the embryonic development from stage II to III. Several abundant spots that had a smear streak (Fig. 2) were of PVF origin and not analyzed. An analysis of the spots of embryonic origin using the PDQuest software revealed on average 718 spots per gel in stage II and 635 spots in stage III. Most of the protein spots were

Figure 2. Representative 2-DE gel images selected from one of each three replicate samples of embryonic stages II and III, showing the spots selected for mass spectrum identification.


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Proteomics 2010, 10, 2701–2711

concentrated in the pI range of 5–8. Qualitative and quantitative analyses were performed on the most abundant 125 protein spots of these two stages. These included 18 spots that appeared only in stage II, 5 spots that appeared only in stage III, 8 spots that showed upregulation, 10 spots that showed downregulation and 84 spots that did not show significant changes between the two stages. Overall, the percentage of differentially expressed spots (32.8%), including up and downregulated and on/off spots, was about half that of the spots not showing significant changes (67.2%).

3.2 Protein identification Among the 125 protein spots submitted for MS analysis, 65 protein spots were identified (Table 1). These included 6 spots that appeared only in stage II, 1 spot that appeared only in stage III, 2 spots that showed upregulation, 6 spots that showed downregulation and 50 spots that did not show significant changes between the two stages. Overall, the percentage of differentially expressed proteins successfully identified (15/41 5 36.6%) appeared to be smaller than that of proteins not showing significant changes between the two stages (50/84 5 63.4%). Among the differentially expressed proteins, five (actin-related protein, ERp 57, G protein, ´ja ` vu housekeeping tropomyosin and annexin A11b) were De proteins [40] but ten were not. These proteins were assigned a Gene Ontology (http:// www.geneontology.org) term according to their molecular function, and were categorized into eleven functional groups using the ‘‘GO-MIPS funcat conversion table’’ at the Munich Information Center for Protein Sequences (Fig. 3) [40]. Most of the identified proteins (63%) were related to metabolism (11%), energy (11%), protein fate (29%) and protein with binding function or cofactor requirement (12%). Among the identified proteins, proliferating cell nuclear antigen, actin-related protein 3, dihydrolipoamide S-acetyltransferase, ERp 57, G protein and putative septin 10 were present in stage II but not in stage III. On the other hand, cytoplasmic intermediate filament protein was present only in stage III. Nucleoside diphosphate kinase, ubiquinol cytochrome c reductase core protein 2, poly A binding protein, ATP synthesis a-subunit and D-chain, and ribosomal P0 protein were downregulated from stage II to stage III, whereas tropomyosin and annexin A11b were upregulated.

4

Discussion

To our knowledge, this is the only proteomic study of gastropod embryos, which resulted in the identification of 65 of the 125 analyzed protein spots. This successful rate (52%) was high but was not unusual for proteomic studies & 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2705 using the de novo-identified strategy. For example, Shevchenko and colleagues were able to identify 48 of 58 (83%) proteins from the venom of the moth C. speciosa (another nonmodel organism). A comparison with proteins identified in other studies of mollusks showed that among the 65 proteins identified from apple snails in this study, none has been identified from the snail L. saxatilis [20], 1 has been identified from the snail B. glabrata [19], and 13 have been identified from the mussel Mytilus edulis [41–44]. These data again illustrate the lack of proteomic study in mollusks and the usefulness of the de novo cross-species identification method in proteomic studies of nonmodel organisms [21]. In this study, we loaded 1 mg of protein, the upper limit in Bio-Rad’s 2-D recommended procedure, onto each gel and we focused on the identification of 125 abundant proteins of embryo origin. This protein loading was essential because of the presence of a substantial proportion of proteins in the PVF. Too large a protein loading would lead to very strong smears by the PVF, which could reduce the resolution. Too small a protein loading would reduce the amount of embryonic proteins available for MS analysis. A previous review has shown that the large range of protein expression levels limits the ability of the 2-DE MS approach to identify proteins of low abundance [45]. Indeed, protein quantity is critical for the accuracy of the deduced sequences when using the de novo peptide sequencing method. A sufficient amount of protein facilitates the interpretation of MS result of peptide fragments by the software PepNovo, for example the determination of b- (N-terminus fragment) and y- (C-terminus fragment) ions. PepNovo calculated the expected confidence in the produced sequence candidates by assigning a score [23]. Based on our experience, PepNovo insignificant results (with a score o2.0) are typically associated with faint spots. Since we focused only on the most abundant proteins, one may wonder whether the identified spots correspond to highly conserved proteins that perform basic functions across taxa. A comparison with the common housekeeping ´ja ` vu proteins [46] shows that 40 of our 65 identified or De proteins do not belong to this category. Therefore, we feel that the de novo approach will not just result in the identification of housekeeping proteins, although housekeeping proteins themselves may be important as their differential expression may indicate important changes in biological functions. This study focused on a period of the embryonic development of P. canaliculata when most of the organs are formed [32, 33]. In stage II, the head–foot is in early stage of differentiation, the foregut and midgut are being connected and the primitive mantle cavity is being developed [33]. In stage III, most of the external (e.g. shell, operculum, head with tentacles, eyes, foot and associated operculum) and internal (e.g. stomach, kidney, osphradium, gill and lung) organs are fully formed and the embryo now resembles a juvenile. The 65 identified proteins are categorized into www.proteomics-journal.com

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Table 1. List of identified proteins from P. canaliculata embryos

Spot no.a)

Homologous proteinb)

Species and accession numberc)

Molecular weight (kDa)

pI

Obs./Theo.

Obs./Theo.

Metabolism 22 Aldehyde dehydrogenase 1 family, A1 63 Putative inorganic pyrophosphatase 66 Glutamine synthetase 78 Dihydropyrimidine amidohydrolase 88 Glutamate oxaloacetate transaminase 2 113 Nucleoside diphosphate kinase

Homo sapiens, AAP36480 Xenopus laevis, AAH70619 X. laevis, Q642P9 Drosophila melanogaster, AAO33382 Danio rerio, NP_956283 Trypanosoma brucei, XP_829670

55/65 37/35 41/41 65/65 40/48 17/17

6.0/6.3 5.8/5.8 5.9/6.0 6.3/6.2 7.3/8.9 8.2/8.7

Energy 21 60 70 87 89 92 97 102

D. rerio, NP_997832 Gallus gallus, XP_414404 Blattella germanica, ABC96322 Bombyx mori, NP_001040257 Schistosoma japonicum, AAW27782 G. gallus, NP_990316 Plicopurpura patula, Q86DP2 Leishmania major, CAJ06684

54/69 36/39 47/47 38/36 44/52 42/44 37/24 38/39

6.0/8.8 5.9/6.0 6.6/5.9 7.0/6.8 7.7/8.0 8.1/8.3 7.6/6.8 9.0/9.0

Sarcophaga crassipalpis, O16852 Mus musculus, P50580 Monodelphis domestica, XP_001373099

34/29 45/44 46/54

4.6/4.8 6.4/6.4 7.4/6.6

B. mori, CAD29995 Pinctada fucata, ABO10190 Culex quinquefasciatus, B0WDA9 Strongylocentrotus purpuratus, NP_001020382 Aedes aegypti, AAK01430

34/34 42/ 34 38/ 38 45/49

6.3/5.9 5.1/5.2 6.0/5.6 6.3/5.7

92/ 94

6.9/6.0

Aplysia californica, AAB24569 S. purpuratus, NP_999808 Oncorhynchus mykiss, BAD90028 D. rerio, AAH44397 S. purpuratus, XP_782717 A. californica, Q16956 Daphnia magna, ACB11340 Apis mellifera, NP_001153520 Anopheles gambiae, XP_310465

57/47 92/92 62/59 63/60 62/58 74/74 71/71 71/76 49/46

4.5/4.4 5.0/4.5 5.9/5.2 6.6/6.0 6.9/7.3 5.1/4.8 5.8/6.1 6.1/6.4 5.4/5.4

Lucilia cuprina, ABO09590 Conus marmoreus, ABF48564 M. domestica, XP_001379009 Herdmania curvata, AAB62537 D. melanogaster, XP_001360097 Canis lupus familiaris, XP_848238 Cricetulus griseus, AAL18160 D. rerio, AAQ97835 M. musculus, NP_080175

62/62 59/57 35/33 38/38 59/59 62/72 62/57 40/47 46/48

5.5/5.8 4.7/4.6 5.9/7.6 6.5/6.6 6.6/5.5 6.0/5.0 6.0/6.0 6.3/6.1 8.8/9.3

M. domestica, XP_001366685

24/24

8.8/9.4

A. mellifera, XP_393451 D. pseudoobscura, XP_001355773 S. purpuratus, XP_001177619 A. mellifera, XP_624894

38/32 38/44 18/18 54/49

8.0/6.5 8.4/6.3 3.8/4.1 5.8/5.6

Dihydrolipoamide S-acetyltransferase Pyruvate dehydrogenase b Enolase Cytosolic malate dehydrogenase Citrate synthase Phosphoglycerate kinase 1 Mitochondrial malate dehydrogenase Glyceraldehyde 3-phosphate dehydrogenase

Cell cycle and DNA processing 10 Proliferating cell nuclear antigen 68 Proliferation-associated protein 2G4 91 Putative septin 10 Protein synthesis 29 Ribosomal P0 protein 43 67 kDa laminin receptor precursor 64 Initiation factor 3 subunit H 67 Elongation factor 1 g-subunit 80

Elongation factor 2

Protein fate 3 Calreticulin 16 HSP GP96 24 TCP1 subunit 8 25 TCP1 a-subunit 26 TCP1, subunit 6A 30 78 kDa glucose-regulated protein 32 HSP 70 33 HSP cognate 5 39 ATP-dependent 26S proteasome regulatory subunit 40 HSP 60 45 Protein disulfide isomerase 61 Proteasome a1 subunit 65 Phosphatase-1 72 Putatively TCP1, e-subunit 75 Putative protein ERP-72 76 ERp57 86 Proteasome 26S subunit, non-ATPase, 13 94 Ubiquinol cytochrome c reductase core protein 2 110 Putative peptidylprolyl isomerase Protein with binding function or cofactor requirement 4 Heterogeneous nuclear ribonucleoprotein 5 Heterogeneous nuclear ribonucleoprotein 7 Calmodulin 19 RNA Helicase



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Spot no.a)

Homologous proteinb)

Species and accession numberc)

Molecular weight (kDa)

pI

Obs./Theo.

Obs./Theo.

Bos taurus, XP_594772

38/38

7.2/9.1

B. mori, ABH10797 D. rerio, NP_001025456 D. rerio, NP_861431

33/68 75/69 35/52

7.4/9.4 6.1/5.7 7.0/7.5

M. musculus, EDL32222

54/40

5.8/9.2

Cellular transport, transport facilitation and transport routes 36 ATP synthase b-subunit Tribolium castaneum, XP_972481 74 H1-exporting ATPase Heliothis virescens, S18395 90 ATP synthase a-subunit S. purpuratus, Q94760 101 Voltage-dependent anion channel 1 X. (Silurana) tropicalis, NP_001016492 109 Putative ATP synthase D-chain H. discus, B6RB53

55/55 60/55 55/60 30/31 26/20

5.2/5.2 5.9/5.3 7.4/8.2 7.0/6.9 9.7/9.0

Cellular communication/signal transduction mechanism 62 Guanine nucleotide-binding protein 3 A. aegypti, EAT47861 96 Guanine nucleotide-binding protein Myxine glutinosa, AAM88903

37/37 35/33

6.0/6.0 7.5/7.0

Biogenesis of cellular components 18 Actin T2 20 Actin-related protein 3 34 b-Tubulin 35 Tubulin 122 Cytoplasmic intermediate filament protein

Litopenaeus vannamei, Q6DTY3 C. briggsae, Q61WW9 Paracentrotus lividus, P11833 Crassostrea gigas, BAD88768 A. californica, CAA42839

42/42 50/48 57/51 58/51 65/65

5.5/5.0 5.9/5.5 5.1/4.7 5.3/5.0 5.5/5.5

Subcellular localization 42 Similar to set b

Ciona intestinalis, XP_002130376

40/30

3.9/4.3

Unknown 49 Tropomyosin-2 95 Calponin homolog

B. glabrata, P43689 S. japonicum, AAD11976

35/33 42/38

4.6/4.6 9.8/9.0

27 28 31 99

Heterogeneous nuclear ribonucleoprotein A2/B1 Poly A binding protein Villin2 Annexin A11b

Protein activity regulation 47 GDP dissociation inhibitor 2

a) Spot number corresponds to the number on the 2-DE in Fig. 2. b) Protein identified by the de novo sequencing and MASCOT (www.matrixscience.com) from the NCBI nonredundant database. c) Species and accession number from the NCBI nonredundant, except for spot 109 (EMBL database).

eleven types of cellular functions or pathways (Fig. 3), supporting that the studied embryonic stages are going through active protein synthesis and catabolism. In fact, a large proportion of the proteins identified are related to energy, metabolism, transcription, protein synthesis, translation and modification. In the following paragraphs, we present our analysis of the putative functions of some of these proteins. The PVF of P. canaliculata contains all proteins, carbohydrates and lipids needed to provide the energy and raw material for the embryonic development [32]. The transition from stage II to III coincides with a significant drop in the amount of PVF and the major proteins PV1 (mainly 300 kDa) and PV2 (mainly 400 kDa) [32, 47]. In this study, we identified several proteins that are involved in energy and metabolism in the embryos (Table 1). Among these proteins, glycolysis-related proteins (i.e. dihydrolipoamide S-acetyltransferase, pyruvate dehydrogenase b, enolase, phosphoglycerate kinase 1 and glyceraldehyde 3-phosphate dehydrogenase) have been reported to play a critical role in & 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

the embryonic development of zebrafish and ascidians [18, 48]. In addition, we found annexin, a protein mainly involved in vesicle trafficking in the cells [49], to be upregulated. This protein may be related to PVF endocytosis when midgut giant cells absorb large polar molecules including proteins [33]. Shell formation and the associated changes in body shape are important events during molluscan development. We found at least three proteins that have been reported to be involved in molluscan shell formation: calponin [50], calmodulin [10] and calreticulin [51]. From stage II to III, the embryo has transformed from one lacking a shell to one having a fully formed shell. These abundant calcium metabolism-related proteins may have been involved in the deposition of calcium into the matrix of proteins during the shell formation. Furthermore, tropomyosin, a protein that has been reported to be important in muscle rearrangement and contraction of the abalone Haliotis rufescens [52], may be involved in the shell formation. Connection of the adductor muscle with the shell during the shell formation is essential www.proteomics-journal.com

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Subcellular locolization Cellular communication/ (2%) Function signal transduction Biogenesis unknown (3%) of cellular (3%) components (8%)

Metabolism 11%

Cellular transport, transport facilitation and routes (8%)

Energy (11%)

Protein activity regulation (2%)

cell cycle and DNA processing (5%)

Protein with binding function or cofactor requirement (12%)

Protein synthesis (8%)

Protein fate (29%)

to allow the head–foot to be withdrawn into the shell. The upregulation of tropomyosin during this period may thus be related to the contraction of the adductor muscle. In addition, cytoplasmic intermediate filament, a newly appeared protein in stage III, is required for tissue integrity during the late embryogenesis of Caenorhabditis elegans [53]. We postulate that this protein may coordinate the maturation of most organs or tissue in the late embryonic development of the apple snail. Morphology alteration in animals is usually accompanied by apoptotic cell death [54]. Apoptotic cell death plays an essential part in sculpting some organs, such as the formation of digits in vertebrate and resorption of the tadpole tail [55]. Cell death is related to the elimination of abnormal cells, nuclear and chromosomes or removal of cells that fail to activate the embryonic genome [55]. From stage II to stage III, the morphology of the embryo has changed significantly to resemble a juvenile snail. It is not surprising that five cell death-related proteins were identified. Among them, voltage-dependent anion channel, major constituents of the outer mitochondrial membrane, has been reported to affect the membrane permeability and the subsequent release of apoptosis promoting factors [56]. ER stress-related mechanism may induce apoptosis during embryonic development of the central nervous system in mice [57]. After the nascent proteins translation, proteins are usually transported to ER for the following modification and folding. ER stress-related proteins, such as calreticulin, ERp57, protein disulfide isomerase, were detected in P. canaliculata, indicating their participation in the protein folding in the lumen or ER may initiate the programmed cell death. In addition, proliferating cell & 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 3. Functional grouping of the identified proteins from the embryos of P. canaliculata.

nuclear antigen protein, one of the central molecules responsible for decisions of life and cell death, was also present in the embryo. This protein is important in multicellular differentiation, senescence and apoptosis [58]. Apoptosis occurs when proliferating cell nuclear antigen is absent, present in low quantities or present but not functional in the cell [58]. Some proteins that are critical to cell-cycle regulation and cell communications or signal transduction were also identified in this study. Among these was cell proliferatingassociated protein 2G4, which has been known to control cell-cycle activities [59]. This protein was abundant in both stages, indicating it is required for the cell-cycle control during the organogenesis. Nucleotide diphosphate kinase (NDPK) that regulates cell growth and differentiation is usually abundant in rapidly dividing cells [60]. NDPK also participates in hormone-dependent signal transduction pathways by activating guanine nucleotide-binding proteins [61]. The downregulation of the NDPK in our study corresponded to the disappearance of one Guanine nucleotide-binding protein (G protein), an important protein involved in second messenger cascades. G protein has been reported to affect the larval metamorphosis of the abalone H. rufescens [62]. The decline in G protein may suggest the attenuation of the signal transduction in the latter stage of the organogenesis. This signal transduction attenuation is consistent with the size limitation on the embryo imposed by the egg shell. In addition, the disappearance of septin in stage III which plays an essential function in cytokinesis may indicate that cell division is more active in stage II, whereas cell elongation may be mainly responsible for the increment in embryo size in stage III. www.proteomics-journal.com

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In summary, this article reports the first proteomic analysis of gastropod embryos. In total, 65 proteins were identified. Identification of these proteins shall provide a basis for the application of proteomics studies of snail– parasite interaction as well as snail’s responses to environmental and human-induced stressors to gastropod embryos [19–20]. In addition, our de novo peptide sequences can be used for primer design in cloning and expression experiments (Supporting Information Table S2) for a better understanding of the functions of these novel genes. However, despite the usefulness of the cross-species strategy, 48% of the 125 spots in P. canaliculata remain unidentified. Some of those spots may be the gene products unique to Pomacea or even Gastropoda. Taxon-unique genes have been reported from many groups of animals, i.e. 20% genes in Nematoda [63] and 2.5% genes in Drosophila [64]. Alternatively, the paucity of sequence resources of the related species may have limited the chance of identifying homologues in P. canaliculata. The authors thank Professor Andrej Shevchenko for his guidance on de novo protein identification, Pak Ki Wong for collecting snails from the field, Yin Ki Tam and Jason Tam for conducting mass spectrometry analyses, and two anonymous reviewers for critical comments. This study was supported by a HKBU postgraduate scholarship to J. S., a grant (HKBU FRG/0809/II-08) to J. W. Q., and a grant (AoE/P-04/04-2-II) to P. Y. Q. The authors have declared no conflict of interest.

5

References

[1] Schoenwolf, G. C., Laboratory Studies of Vertebrate and Invertebrate Embryos: Guide and Atlas of Descriptive and Experimental Development, Prentice Hall, Upper Saddle River, NJ 2001. [2] Carlson, B. M., Human Embryology and Developmental Biology, Mosby, St. Louis 2008. [3] McVey J. P. (Ed.), CRC Handbook of Mariculture, 2nd Edn, CRC Press, Boca Raton, Florida 1993. [4] His, E., Beiras, R., Seaman, M., The assessment of aquatic contamination: bioassays with bivalve larvae. Adv. Mar. Biol. 1999, 37, 1–178. [5] USEPA, Methods for measuring the acute toxicity of effluents and receiving waters to freshwater and marine organisms, 5th Edn, EPA-821-R-02-012, 2002. [6] Ruppert, E. E., Fox, R. S., Barnes, R. B., Invertebrate Zoology: A Functional Evolutionary Approach, 7th Edn, Brooks Cole Thomson, Belmont, CA 2004.

2709 [9] Liu, H., Liu, S., Ge, Y., Liu, J. et al., Identification and characterization of a biomineralization related gene PFMG1 highly expressed in the mantle of Pinctada fucata. Biochemistry 2007, 46, 844–851. [10] Jackson, D. J., Wo¨rheide, W., Degnan, B. M. et al., Dynamic expression of ancient and novel molluscan shell genes during ecological transitions. BMC 2007, 7, 160. [11] Kuroda, R., Endo, B., Abe, M., Shimizu, M., Chiral blastomere arrangement dictates zygotic left–right asymmetry pathway in snails. Nature 2009, 462, 790–795. ¨ nlu, . M., Young, M. et al., Drosophila [12] Gong, L., Puri, M., U ventral furrow morphogenesis: a proteomic analysis. Development 2004, 131, 643–656. [13] Wang, W. W., Meng, B., Chen, W. H., Ge, X. M. et al., A proteomic study on postdiapaused embryonic development of brine shrimp (Artemia franciscana), Proteomics 2007, 7, 3580–3591. [14] Li, J., Zhang, L., Feng, M., Zhang, Z. et al., Identification of the proteome composition occurring during the course of embryonic development of bees (Apis mellifera). Insect Mol. Biol. 2009, 18, 1–9. [15] Thiyagarajan, V., Qian, P. Y., Proteomic analysis of larvae during development, attachment, and metamorphosis in the fouling barnacle, Balanus amphitrite. Proteomics 2008, 8, 3164–3172. [16] Thiyagarajan, V., Wong, T., Qian, P. Y., 2D gel-based proteome and phosphoproteome analysis during larval metamorphosis in two major marine biofouling invertebrates. J. Proteome Res. 2009, 8, 2708–2719. [17] Mok, F. S., Thiyagarajan, V., Qian, P Y., Proteomic analysis during larval development and metamorphosis of the spionid polychaete Pseudopolydora vexillosa. Proteome Sci. 2009, 7, 44. [18] Nomura, M., Nakajima, A., Inaba, K., Proteomic profiles of embryonic development in the ascidian Ciona intestinalis. Develop. Biol. 2009, 325, 468–481. [19] Bouchut, A., Sautiere, P. E., Coustau, C., Mitta, G., Compatibility in the Biomphalaria glabrata/Echinostoma caproni model: potential involvement of proteins from hemocytes revealed by a proteomic approach. Acta Trop. 2006, 98, 234–246. [20] Martıńez-Fernańdez, M., Rodrı´guez-Pin˜eiro, A. M., Oliveira, E., Paéz de la Cadena, M. et al., Proteomic comparison between two marine snail ecotypes reveals details about the biochemistry of adaptation. J. Proteome Res. 2008, 7, 4926–4934. [21] Habermann, B., Oegema, J., Sunyaev, S., Shevchenko, A., The power and the limitations of cross-species protein identification by mass spectrometry-driven sequence similarity searches. Mol. Cell. Proteomics 2004, 3, 238–249.

[7] Pechenik, J. A., Biology of the Invertebrates, McGraw Hill, NY 2005.

[22] Junqueira, M., Spirin, V., Balbuena, T. S., Thomas, H. et al., Protein identification pipeline for the homology driven proteomics. J. Proteomics 2008, 71, 346–356.

[8] Bondar, C. A., Page, L. R., Development of asymmetry in the caenogastropods Amphissa columbiana and Euspira lewisii. Invertebr. Biol. 2003, 122, 28–41.

[23] Katz, A., Waridel, P., Shevchenko, A., Pick, U., Salt-induced changes in the plasma membrane proteome of the halotolerant alga Dunaliella salina as revealed by blue native gel



2710

J. Sun et al.

electrophoresis and nano-LC-MS/MS analysis. Mol. Cell. Proteomics 2007, 6, 1459–1472. [24] Balbuena, T. S., Silveira, V., Junqueira, M., Dias, L. L. C. et al., Changes in the 2-DE protein profile during zygotic embryogenesis in the Brazilian pine (Araucaria angustifolia). J. Proteomics 2009, 72, 337–352. [25] Shevchenko, A., de Sousa, M. M., Waridel, P., Bittencourt, S. T. et al., Sequence similarity-based proteomics in insects: characterization of the larvae venom of the Brazilian moth Cerodirphia speciosa. J. Proteome Res. 2005, 4, 862–869. [26] Pauchet, Y., Muck, A., Svatos, A., Heckel, D. G. et al., Mapping the larval midgut lumen proteome of Helicoverpa armigera, a generalist herbivorous insect. J. Proteome Res. 2008, 7, 1629–1639. [27] Hayes, K. A., Joshi, R. C., Thiengo, S. C., Cowie, R. H. et al., Out of South America: multiple origins of non-native apple snails in Asia. Divers. Distrib. 2008, 14, 701–712. [28] Kwong, K. L., Wong, P. K., Lau, S. S. S., Qiu, J. W., Determinants of the distribution of apple snails in Hong Kong two decades after their initial invasion. Malacologia 2008, 50, 293–302. [29] Kwong, K. L., Dudgeon, D., Wong, P. K., Qiu, J. W., Secondary production and diet of an invasive snail in freshwater wetlands: implications for resource utilization and competition. Biol. Invasion 2010, 12, 1153–1164. [30] Cowie, R. H., Apple snails (Ampullariidae) as agricultural pests: their biology, impacts and management, in: Barker, G. M. (Ed.), Molluscs as Crop Pests, CABI Publishing, Wallingford 2002, pp. 145–192. [31] Qiu, J. W., Kwong, K. L., Effects of macrophytes on feeding and life-history traits of the invasive apple snail Pomacea canaliculata. Freshw. Biol. 2009, 54, 1720–1730. [32] Heras, H., Garin, C. F., Pollero, R. J., Biochemical composition and energy sources during embryo development and in early juveniles of the snail Pomacea canaliculata (Mollusca: Gastropoda). J. Exp. Zool. 1998, 280, 375–383. [33] Koch, E., Winik, B. C., Castro-Vazquez, A., Development beyond the gastrula stage and digestive organogenesis in the apple-snail Pomacea canaliculata (Architaenioglossa, Ampullariidae). Biocell 2009, 33, 49–65. [34] Ramagli, L. S., Rodriguez, L. V., Quantitation of microgram amounts of protein in two-dimensional polyacrylamide gel electrophoresis sample buffer. Electrophoresis 1985, 6, 559–563. [35] Neuhoff, V., Arold, N., Taube, D., Ehrhardt, W., Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 1988, 9, 255–262. [36] Benjamini, Y., Hochberg, Y., Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. Roy. Stat. Soc. B 1995, 57, 289–300. [37] Shevchenko, A., Wilm, M., Vorm, O., Mann, M., Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal. Chem. 1996, 68, 850–858.


Proteomics 2010, 10, 2701–2711 [38] Shevchenko, A., Chernushevich, I., Shevchenko, A., Wilm, M. et al., ‘‘De novo’’ sequencing of peptides recovered from in-gel digested proteins by nanoelectrospray tandem mass spectrometry. Mol. Biotechnol. 2002, 20, 107–118. [39] Waridel, P., Frank, A., Thomas, H., Surendranath, V. et al., Sequence similarity-driven proteomics in organisms with unknown genomes by LC-MS/MS and automated de novo sequencing. Proteomics 2007, 7, 2318–2329. [40] Ruepp, A., Zollner, A., Maier, D., Albermann, K. et al., The FunCat, a functional annotation scheme for systematic classification of proteins from whole genomes. Nucleic Acids Res. 2004, 32, 5539–5545. [41] Amelina, H., Apraiz, I., Sun, W., Cristobal, S., Proteomicsbased method for the assessment of marine pollution using liquid chromatography coupled with twodimensional electrophoresis. J. Proteome Res. 2007, 6, 2094–2104. [42] Apraiz, I., Mi, J., Cristoba, S., Identification of proteomic signatures of exposure to marine pollutants in mussels (Mytilus edulis). Mol. Cell. Proteomics 2006, 5, 1274–1285. [43] McDonagh, B., Sheehan, D., Effect of oxidative stress on protein thiols in the blue mussel Mytilus edulis: proteomic identification of target proteins. Proteomics 2007, 7, 3395–3403. [44] Diz, A. P., Dudley, E., MacDonald, B. W., Pinã, B. et al., Genetic variation underlying protein expression in eggs of the marine mussel Mytilus edulis. Mol. Cell. Proteomics 2009, 8, 132–144. [45] Gygi, S. P., Corthals, G. L., Zhang, Y., Rochon, Y. et al., Evaluation of two-dimensional gel electrophoresis based proteome analysis technology. Proc. Natl. Acad. Sci. USA 2000, 97, 9390–9395. [46] Petrak, J., Ivanek, R., Toman, O., Cmejla, R. et al., De´ja` vu in proteomics. A hit parade of repeatedly identified differentially expressed proteins. Proteomics 2008, 8, 1744–1749. [47] Garin, C. F., Heras, H., Pollero, R. J., Lipoproteins of the egg perivitelline fluid of Pomacea canaliculata snails (Mollusca: Gastropoda). J. Exp. Zool. 1996, 276, 307–314. [48] Lucitt, M. B., Price, T. S., Pizarro, A., Wu, W. et al., Analysis of the zebrafish proteome during embryonic development. Mol. Cell. Proteomics 2008, 7, 981–994. [49] Gerke, V., Creutz, C. E., Moss, S. E., Annexins: linking Ca21 signalling to membrane dynamics. Nat. Rev. Mol. Cell. Biol. 2005, 6, 449–461. [50] Miyamoto, H., Hamaguchi, M., Okoshi, K., Analysis of genes expressed in the mantle of oyster Crassostrea gigas. Fisheries Sci. 2002, 68, 651–658. [51] Fan, W., Hu, Y., Li, C., Xie, L. et al., Cloning, characterization, and expression analysis of calreticulin from pearl oyster Pinctada fucata. Tsinghua Sci. Technol. 2008, 13, 466–473. [52] Degnan, B. M., Degnan, S. M., Morse, D. E., Muscle-specific regulation of tropomyosin gene expression and myofibrillogenesis differs among muscle systems examined at metamorphosis of the gastropod Haliotis rufescens. Dev. Genes Evol. 1997, 206, 464–471.


Proteomics 2010, 10, 2701–2711 . [53] Karabinos, A., Schunemann, J., Weber, K., Most genes encoding cytoplasmic intermediate filament (IF) proteins of the nematode Caenorhabditis elegans are required in late embryogenesis. Eur. J. Cell Biol. 2004, 83, 457–468. . [54] Hacker, G., The morphology of apoptosis. Cell Tissue Res. 2000, 301, 5–17. [55] Jacobson, M. D., Weil, M., Raff, M. C., Programmed cell death review in animal development. Cell 1997, 88, 347–354. [56] Granville, D. J., Gottlieb, R. A., The mitochondrial voltagedependent anion channel (VDAC) as a therapeutic target for initiating cell death. Curr. Med. Chem. 2003, 10, 1527–1533. [57] Zhang, X., Szabo, E., Michalak, M., Opas, M., Endoplasmic reticulum stress during the embryonic development of the central nervous system in the mouse. Int. J. Dev. Neurosci. 2007, 25, 455–463. [58] Paunesku, T., Mittal, S., Protic´, M., Oryhon, J. et al., Proliferating cell nuclear antigen (PCNA): ringmaster of the genome. Int. J. Radiat. Biol. 2001, 77, 1007–1021.


2711 [59] Radomski, N., Jost, E., Molecular cloning of a murine cDNA encoding a novel protein, p38-2G4, which varies with the cell cycle. Exp. Cell Res. 1995, 220, 434–445. [60] Ouatas, T., Hourdry, J., Mazabraud, A., Functional role of nucleotide diphosphate kinase during the development and oncogenesis. Biol. Cell 1996, 88, 83–83. [61] Bominaar, A. A., Molijn, A. C., Pestel, M., Veron, M. et al., Activation of G-protein by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium. EMBO J. 1993, 12, 2275–2279. [62] Baxter, G., Morse, D. E., G protein and diacylglycerol regulate metamorphosis of planktonic molluscan larvae. Proc. Natl. Acad. Sci. USA 1987, 84, 1867–1870. [63] Parkinson, J., Mitreva, M., Whitton, C., Thomson, M. et al., A transcriptomic analysis of the phylum Nematoda. Nat. Genet. 2004, 36, 1259–1267. [64] Clark, A. G., Eisen, M. B., Smith, D. R., Bergman, C. M. et al., Evolution of genes and genomes on the Drosophila phylogeny. Nature 2007, 450, 203–218.
