Di et al. BMC Genomics (2017) 18:809 DOI 10.1186/s12864-017-4203-7 RESEARCHARTICLE Open Access Proteomic analysis of trochophore and veliger larvae development in the small abalone Haliotis diversicolor Guilan Di1,2, Xianghui Kong1, Xiulian Miao3, Yifang Zhang2, Miaoqin Huang2, Yuting Gu2, Weiwei You2*, Jianxin Zhang1 and Caihuan Ke2* Abstract Background: Haliotis diversicolor is commercially important species. The trochophore and veliger are distinct larval stages in gastropod development. Their development involves complex morphological and physiological changes. We studied protein changes during the embryonic development of H. diversicolor using two dimensional electrophoresis (2-DE) and label-free methods, tandem mass spectrometry (MS/ MS), and Mascot for protein identification. Results: A total of 150 2-DE gel spots were identified. Protein spots showed upregulation of 15 proteins and downregulation of 28 proteins as H. diversicolor developed from trochophore to veliger larvae. Trochophore and veliger larvae were compared using a label-free quantitative proteomic approach. A total of 526 proteins were identified from both samples, and 104 proteins were differentially expressed (> 1.5 fold). Compared with trochophore larvae, veliger larvae had 55 proteins upregulated and 49 proteins downregulated. These differentially expressed proteins were involved in shell formation, energy metabolism, cellular and stress response processes, protein synthesis and folding, cell cycle, and cell fate determination. Compared with the 5 protein (fructose- bisphosphate aldolase, 14–3-3ε, profilin, actin-depolymerizing factor (ADF)/cofilin) and calreticulin) expression patterns, the mRNA expression exhibited similar patterns except gene of fructose-bisphosphate aldolase. Conclusion: Our results provide insight into novel aspects of protein function in shell formation, torsion, and nervous system development, and muscle system differentiation in H. diversicolor larvae. “Quality control” proteins were identified to be involved in abalone larval development. Keywords: Haliotis Diversicolor, Embryonic development, 2-de, Label-free Background contributes to the development of the aquaculture in- Some of gastropod larval development includes a pelagic dustry and environmental pollution monitoring [4]. The phase (trochophore and veliger) and a benthonic phase. embryonic development of gastropods includes several The trochophore and veliger are morphologically and unique features including shell formation and head–foot behaviorally distinct developmental stages. Complex differentiation [5]. Mollusk development involves shell morphological and physiological processes occur during formation or shell reduction, during which changes in the transition between these stages. Morphological dif- body shape, deposition of minerals, and pigment depos- ferences in the development of gastropod larvae have ition in a protein matrix occur [6]. The molecular mech- been reported [1–3]. Animal embryology research anisms underlying these changes are unclear. Abalones are marine gastropods distributed worldwide * Correspondence: [email protected]; [email protected] along coastal waters in tropical and temperate areas [7]. 2State Key Laboratory of Marine Environmental Science, Fujian Collaborative The small abalone Haliotis diversicolor (Mollusca, Gastro- Innovation Center for Exploitation and Utilization of Marine Biological poda, Archaeogastropoda) is a commercially important Resources, Xiamen University, Xiamen, Fujian Province 361005, People’s Republic of China Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Di et al. BMC Genomics (2017) 18:809 Page 2 of 15 species cultured along coastal waters [8]. In 2010, 50,000 differentiation of the larval retractor muscle, foot tons of abalone were harvested in China [9]. mass, mantle, and the onset of shell mineralization Jackson et al. [1] observed significant differences [10, 11]. in developmental events of the abalone Haliotis as- Sequencing techniques, genomic data, and transcrip- inine. These included hatching from the vitelline en- tomic analysis have been used to study larval develop- velope, variation in larval shell development, and ment, the transcriptome of the early life history stages of metamorphosis-inducing cues in older larvae. While the California Sea Hare Aplysia californica has been re- many proteins are involved in abalone shell forma- ported [12]. However, current genomic data are insuffi- tion, their presence and roles in early developmental cient to reveal the molecular mechanisms underlying the stages of larval shell formation are not well under- complex cellular processes of embryonic development stood [2, 3]. Larval torsion of the shell is also im- [13]. The process by which shell matrix proteins are se- portant in the development of gastropods. The creted and organized into larval shell architecture is abalone trochophore and veliger larvae possess shell largely unknown. Proteins determine phenotypes, which at the different stages of growth. Larvae hatch as can be considered snapshots of genome expression [14]. trochophores, at approximately 19 h post- Phenotypes are more complex than mere genomic ex- fertilization (Fig. 1a), and the trochophore is the ini- pression, due to lack of a direct correlation between tial shell stage. The trochophore then undergoes a gene expression intensity and protein abundance, prote- series of morphological changes, including velum omic studies are powerful tools for the discovery of new acquisition, at which pointitbecomesaswimming proteins involved in developmental processes. Proteomic larva in the pre-veliger phase (at 30 h). The trocho- profiling has been applied to embryo studies of several phore then transforms into a swimming veliger invertebrates, including fruit flies (Drosophila melanoga- larva, and the late calcified protoconch forms ster) [15], brine shrimps (Artemia franciscana) [16], (Fig. 1b). The veliger stage is characterized by honey bees (Apis mellifera) [17], fouling barnacles Fig. 1 Embryonic development of Haliotis diversicolor and 2-D gel images of silver-stained proteins (120 μg). a a trochophore larvae stage, and (b) veliger larvae. Larvae hatch as trochophores at about 19 h post-fertilization. The trochophore larvae transform into swimming veliger larvae (at 30 h). Images of 2-DE from (c) the trochophore larval proteins; (d) the veliger larval stage proteins. The spot-numbering scheme is based on the description in Additional file 2: Table S2 Di et al. BMC Genomics (2017) 18:809 Page 3 of 15 (Balanus amphitrite) [18, 19], polychaetes (Pseudopoly- 2-de dora vexillosa) [20], ascidians (Ciona intestinalis) [21], A 2-DE was conducted as described previously [26]. Total golden apple snail (Pomacea canaliculata) [22], and Pa- protein (120 μg) was used for each test. The first dimension cific oyster (Crassostrea gigas) [13]. (IEF) of gel electrophoresis was conducted on 18 cm immo- Two-dimensional electrophoresis (2-DE) permits bilized pH gradient (IPG) strips using a nonlinear pH gradi- quantitative examination within a single gel electrophor- ent (pH 3–10, Amersham Pharmacia Biotech) with a esis experiment and it is a powerful tool for total protein horizontal electrophoresis apparatus (Bio-Rad). The sample, separation. Matrix-assisted laser desorption/ionization included in the rehydration solution was loaded on the IPG (MALDI) mass spectrometry is a promising technique (Immobilized pH Gradient) strip holder. IPG dry strips [23]. Label-free-based proteomics is broadly applied to were rehydrated with a rehydration buffer (8 M urea, 2% biomarker discovery and proteomic profiling [24]. This (w/v) CHAPS, 20 mM DTT, 0.5% (v/v) IPG buffer 3–10, technique is rapid, clean and simple. It can directly and and0.01%(w/v)bromophenolblue,pH7.4)at50V.IEF precisely quantify protein expression without using la- was then initiated at 100 V for 2 h, 200 V for 2 h, 500 V for beling. Proteomic studies of abalone embryos have not 1 h, 1000 V for 2 h, 4000 V for 2 h, and finally 8000 V until been previous reported. To thoroughly evaluate larval 50,000 Vh. Before the second dimension, the IPG strips proteome changes, we investigated the differential pro- were soaked for 17 min in equilibration solution (6 M urea, tein abundances during the different development stages 50 mM Tris-HCI buffer, pH 8.8, 30% v/v glycerol, 2% of H. diversicolor using a label-free, coupled 2-DE prote- w/v SDS, and a trace of bromophenol blue) contain- omic approach. The results provide insights into the dif- ing 1% w/v DTT. Incubation was continued with ferent protein groupings during shell formation and the equilibration solution containing 2.5% (w/v) iodoace- differentiation of the larval retractor muscle, foot mass, tamide for an additional 17 min. The second dimension and mantle. of gel electrophoresis was conducted on 12.5% polyacryl- amide gels (20 cm × 20 cm × 1.5