Research Article Differential Gene Expression …...Research Article Differential Gene Expression during Larval Metamorphic Development in the Pearl Oyster, Pinctada fucata ,Basedon

Research ArticleDifferential Gene Expression during Larval MetamorphicDevelopment in the Pearl Oyster Pinctada fucata Based onTranscriptome Analysis

Haimei Li12 Bo Zhang1 Guiju Huang1 Baosuo Liu1 Sigang Fan1

Dongling Zhang3 and Dahui Yu1

1Key Laboratory of South China Sea Fishery Resources Exploitation amp Utilization of Ministry of AgricultureSouth China Sea Fisheries Research Institute Chinese Academy of Fishery Sciences Guangzhou Guangdong 510300 China2Shanghai Ocean University Shanghai 201306 China3Jimei University Xiamen Fujian 361021 China

Correspondence should be addressed to Dahui Yu pearlydh163com

Received 28 June 2016 Revised 26 August 2016 Accepted 20 September 2016

Academic Editor Quanhu Sheng

Copyright copy 2016 Haimei Li et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

P fucata experiences a series of transformations in appearance from swimming larvae to sessile juveniles during which significantchanges in gene expression likely occur Thus P fucata could be an ideal model in which to study the molecular mechanisms oflarvalmetamorphosis during development in invertebrates To study themolecular driving force behindmetamorphic developmentin larvae of P fucata transcriptomes of five larval stages (trochophore D-shape umbonal eyespots and spats) were sequencedusing an Illumina HiSeq 2000 system and assembled and characterized with the transcripts of six tissues As a result a totalof 174126 unique transcripts were assembled and 60999 were annotated The number of unigenes varied among the five larvalstages Expression profiles were distinctly different between trochophore D-shape umbonal eyespots and spats larvae As aresult 29 expression trends were sorted of which eight were significant Among others 80 development-related differentiallyexpressed unigenes (DEGs) were identified of which the majority were homeobox-containing genes Most DEGs occurred amongtrochophore D-shaped and UES (umbonal eyespots and spats) larvae as verified by qPCR Principal component analysis (PCA)also revealed significant differences in expression among trochophore D-shaped and UES larvae with ten transcripts identifiedbut no matching annotations

1 Introduction

Metamorphosis is a series of key steps in the process of larvaldevelopment the success of which affect the survival of theorganism Metamorphosis is prevalent in insects amphib-ians some fishes and many marine invertebrates such asbarnacles sponges shellfishes shrimps and echinodermsSimilar to most benthic marine invertebrates the pearloyster (Pinctada fucata) has a microscopic free-swimminglarval phase in their complex life cycle [1] Oyster larvaespend several weeks in the water column before attainingcompetency to attach andmetamorphose commencing theirsessile life The developmental processes of P fucata fromswimming larvae to sessile spats have been classified into

six stages fertilized egg trochophore D-shaped umbonaljuvenile and adult stages [2] In oysters the transition fromfree-swimming larvae to the attached juvenile form oftenrequires morphological physiological structural and func-tional changes which are under genetic regulatory control[3] Therefore the identification of key developmental genesinvolved in the metamorphosis of P fucata larvae as wellas characterizing their expression patterns is importantto understand the molecular mechanism of metamorphicdevelopment of this economically important species

Numerous studies have been conducted to explorethe mechanisms of hormones neurotransmitters genesand signaling pathways that regulate larval metamorphicdevelopment Some studies have demonstrated that eight

Hindawi Publishing CorporationInternational Journal of GenomicsVolume 2016 Article ID 2895303 15 pageshttpdxdoiorg10115520162895303

2 International Journal of Genomics

superfamily genes showed differential expression during themetamorphosis ofCiona intestinalis [4ndash7] Several homeobox-containing genes were found to be responsible for larvalmetamorphic development in Haliotis rufescens [8ndash10] Inaddition abnormal dopamine and adrenaline were observedin the larval attaching stage of the Pacific oyster Crassostreagigas [11] while a different study observed increased expressionof a molluscan growth and differentiation factor (mGDF) inthe metamorphosing stage of the same organism [12] Thesefindings indicate the diversity of genes involved in the transi-tions of larval forms

However previous studies have focused on changes in asmall number of genes and have provided a fragmented viewof the genetic modulation of larval metamorphosis Recentdevelopments in sequencing technology have allowed for thedevelopment of new genomic tools which can provide amoreglobal view of changes in gene expression over the courseof larval developmental stages [13ndash16] In terms of genome-wide studies transcriptome analyses are considered to bean ideal choice for obtaining comprehensive informationregarding animal development and growth [17 18] For Pfucata the draft genome [19] and tissue transcriptomes [20ndash23] have been recently reported Based on the transcriptomicsequences from a mixture of nine developmental stagesof P fucata [19] biomineralization-related gene expressionprofiles during larval development have been investigated[24 25] and genes involved in body patterning [26] tran-scription factors [27] and homeobox genes [28] have beenidentified Nonetheless developmentally important genesand their expression patterns during the larval stages ofdeveloping P fucata have not been systematically studiedat the transcript level to date In the present study thetranscriptomes of five larval stages (trochophore D-shapeumbonal eyespots and spats) and six tissues (gill adductormuscle hepatopancreas mantle hemocytes and pearl sac)from P fucata were sequenced using Illumina HiSeq 2000with an emphasis on the molecular mechanisms underlyinglarval metamorphic developmentThis study aims to providea valuable insight into themechanisms of genetic modulationover the course of larval metamorphic development for Pfucata as well as for other molluscan species

2 Materials and Methods

21 Larval Culture and Sample Collection Larvae of P fucatawere bred (using several females and males of a selectivelybred F3 generation as parents) through artificial insemina-tion on March 10 2013 in Sanya Hainan Island Chinaas described by Fujimura et al [2] Fertilized eggs wereincubated in a 1000 L tank at 24∘C After removing nonde-veloping embryos and dead larvae trochophore D-shapedumbonal eyespots and spats larvae stages were harvestedwith filtering net at 12 h 36 h 115 d 185 d and 235 d afterfertilization respectively and immediately preserved in RNAlater (TaKaRa Bio Inc) until RNA extraction MeanwhileRNAs of six tissues (gill adductor muscle hepatopancreasmantle hemocytes andpearl sac) of three other adult animalswere sequenced for a more robust assembly

22 RNA Extraction and cDNA Library Preparation Follow-ing the manufacturerrsquos instructions total RNA was extractedfrom five developmental stages (each stage with thousands oflarvae) and six tissues using Trizol and RNAs of each type oftissues of the three individuals were mixed by equal weightRNA integrity and quantity were confirmed by lab-on-chipanalysis using a 2100 Bioanalyzer (Agilent TechnologiesSanta Clara CA USA) and visualized on a 1 agarose gelThen cDNA was synthesized using the mRNA fragments astemplates as usual and was sequenced by the BGI (ShenzhenChina) using the Illumina HiSeq 2000 system (San DiegoCA USA) (PE100)

23 Sequence Assembly and Annotation After filtering outlow quality sequences (containing more than 5 ambiguousldquoNrdquo nucleotides or gt20 119876 ≦ 10 reads) and the removal ofadapters from raw data clean sequence data was assembledinto unigenes usingTrinity software and subsequently clusteredby TGICL v21 (-l 40 -c 10 -v 20) [29] Phrap (-repeatstringency 095 -minmatch 35 -minscore 35) (Release 230)was used to produce the longest sequence possible (httpwwwphraporg) Assembled unigenes were annotated based onthe Nr Swissprot KEGG and COG databases The sequencedirection and amino sequence of the predicted coding region(CDS) of unannotated unigenes were determined usingESTScan with default settings [30] Functional annotationsand classifications were performed by using Blast2GO [31]and WEGO [32] (119864 value threshold 1 times 10minus5) respectively

24 Normalization and Quantification of Gene ExpressionSequencing reads were mapped to the assembled referencesequence using SOAP alignersoap2 (-m 0 -x 500 -s 40 -l 35-v 5 -r 2) [33] a tool designed specifically to assemble shortsequence alignmentsThe coverage of reads from a given genewas used to calculate the expression level of that gene whichwas measured by fragments per kilobase exon per millionfragments (FPKM) [34] with the following formula

FPKM = 106119862

119873119871103 (1)

where FPKM is the expression level of a unigene 119862 is thenumber of fragments that uniquely aligned to the unigene119873 is the total number of fragments that uniquely alignedto all unigenes and 119871 is the number of bases in the CDSof the unigene The FPKM method eliminates the influenceof sequences of differing lengths and coverage level on thecalculation of gene expressionTherefore the calculated geneexpression can be directly used for comparing the differencein gene expression between samples

25 Differential Gene Expression (DEGs) across Develop-mental Stages Differential gene expression among differentlarval stages was carried out via principal component anal-ysis (PCA) using the R package (httpwwwr-projectorg)according to the manualThe pairwise differential expressionconducted by edgeR with a threshold of the false discoveryrate (FDR) le 0001 and an absolute value of log 2 Ratioge 1 was used to judge the significance of differences ingene expression Trends in the expression of all differentially

International Journal of Genomics 3

Table 1 Primers for genes used for qPCR verification

Trend Unigene code Annotated gene Primer

0 Unigene27615 All Brachyury qBran-S 51015840 GCCAAAGAAAGACCAGAAGG 31015840

qBran-A 51015840 TCAGGCTAAGGCGATCACAA 31015840

0 Unigene27517 All Six3 qSix3-S 51015840 ATACAGGGTGAGGAAGAAGT 31015840

qSix3-A 51015840 TTATCTCCGCCTTGCTGTTG 31015840

3 Unigene23318 All Engrailed qEng-S 51015840 TAGACAGAGCATCGCCTTTA 31015840

qEng-A 51015840 TTGTGATTTAACTGCCTGCT 31015840

3 Unigene41009 All Pax-7 qPax-S 51015840 GCGGAAACAGATGGGAAGCA 31015840

qPax-A 51015840 ACCGAATGACGGAAACGACT 31015840

26 CL664Crontig4 All MAPK qMAPK-S 51015840 TTTACTCCAAACAGCCCTAC 31015840

qMAPK-A 51015840 TTGCTATCTGGTCCACTTCA 31015840

29 CL7953Contig2 All Notch qNotch-S 51015840 CCAGCCACGGTATCCAAGTA 31015840

qNotch-A 51015840AGCCTCGAACAGAATATCCACT 31015840

29 CL1306Contig2 All Wnt 1 qWnt1-S 51015840 TGATGCCTACGGTAAATACG 31015840

qWnt1-A 51015840 TAACCTTGAGGTGGGAGAAC 31015840

29 Unigene27337 All Lox2 qLox2-S 51015840 CTACCCGAGTTGAATGTGGG 31015840

qLox2-A 51015840 GAAAGTAAGACGGACGAGCC 31015840

expressed genes were sorted using STEM (Short Time-Series Expression Miner v138) [35] Functional annotationand classification of genes involved in significant trendswere performed by using Blast2GO [31] and WEGO [32]respectively The enriched metabolic pathways or signaltransduction pathways of genes were identified based on theKEGG database [36]

26 Identification and Expression Profile of Genes Involved inthe Larval Metamorphic Development of P fucata Accordingto annotations by Nr and Swissprot development-relatedgenes were identified with those that had been previouslyidentified as keywords in the significant trends from the priorstep If several unigenes were assigned to the same referencegene the sequence with the lowest 119864 value (Nr and Swissprotannotation119864 value)was selected as a representativeThen theheatmap 2 module of the gplots package in R (httpscranr-projectorgwebpackagesgplotsindexhtml) was used toperform the clustering analysis of gene expression on thenormalized filtered sequences to identify genes that weresignificantly different among the five developmental stages

27 qPCR Verification of Expression Trends of Development-Related Genes In order to verify the integrity of the tran-scriptome sequences and the expression levels as revealedby RNA-Seq eight development-related genes were selectedrandomly for qPCR verification The genes and respectiveprimers are given in Table 1 qPCR was performed usingan Eppendorf real-time- (RT-) PCR system (EppendorfHamburg Germany) using a SYBR(R) Premix Ex TaqTMkit (TaKaRa) according to the manufacturerrsquos protocol Tran-script levels of target genes were normalized against the levelof a reference gene (18S rRNA) The qRT-PCR reactionswere performed under the following conditions 94∘C for5min (one cycle) 94∘C for 20 s 50∘C to 60∘C for 20 sand 72∘C for 20 s (50 cycles) The comparative CT method

UnigeneCDS

0

20000

40000

60000

80000

100000

120000

Num

ber

500ndash1000 1000ndash1500 1500ndash2000100ndash500 ge2000

Length distribution (nt)

Figure 1 Length distribution of unigenes and CDS

(2minusΔΔCT method) was used to determine the relative mRNAabundance [37]

3 Results

31 Sequence Assembly and Annotation Over 55 millionreads per sample were generated with a base call accuracy(Q20) of over 97 The number of contigs varied from118010 to 215808 with a median length (N50) of 352 to582 bp (Table 2) The number of unigenes varied from 51102to 113516 among samples with the mean length rangingfrom 536 to 689 bp while N50 ranged from 495 to 1025respectively In total 174126 unigenes were assembled witha mean length of 866 bp and an N50 of 1 569 (based on 11samples)Most unigeneswere 100ndash500 bp long and 26weregreater than 1000 bp (Figure 1)


Table 2 Summary statistics of sequence assembly from 11 samples of the pearl oyster Pinctada fucata

Sample Total raw reads Total clean reads Q20 Number of contigs ContigN50 Number of unigenes Mean length N50Gill 59492360 53662442 9767 215808 380 113516 592 977Adductor muscle 58602062 53889264 9742 127634 347 76240 415 495Hepatopancreas 57854582 52672774 9775 163089 398 85839 544 811Pearl sac 56926624 52155950 9749 149236 582 97501 545 827Mantle 58610258 52433102 9772 183633 384 100679 567 900Hemocytes 54707500 51751784 9854 178460 509 96469 599 1025Trochophore 59887806 54413910 9796 175174 399 75400 584 785D-shaped 58511662 51746334 9798 190135 485 88830 626 886Umbonal 66858916 55046652 9788 118010 352 51102 536 683Eyespots 58534394 52943288 9788 182671 504 84045 649 937Spats 60561378 54999258 9785 180265 521 82133 689 1022All 174126 866 1569

Table 3 Summarized statistics of the functional annotation in 11samples of the pearl oyster Pinctada fucata

Database Number of annotated genesNr annotation 38857Swissprot annotation 49580Annotation unigenes for KEGG 43753Annotation unigenes for GO 13465Annotation unigenes for COG 23754CDS 60946CDS by ESTScan 13966Total 60999

In total 60999 unigenes were annotated (Table 3) and74912 CDSs (4302) were predicted (13966 predicted byESTScan) (Figure 1 Table 3) Different databases annotateddifferent numbers of unigenes (Table 3) where the mostunigenes (49580) were annotated by Swissprot database andthe least (13465) by the GO database (Table 3) Numbers ofspecific and shared unigenes annotated by COG KEGG Nrand Swissprot terms can be visualized in Figure 2 Amongthem 12582 unigenes were annotated by the four databasesand 8784 were annotated specifically by Nr (Figure 2) BothKEGG and Swissprot analyses shared the most unigenes(41605) and COG and Nr shared the least (13385)

The 23754 COG-annotated unigenes can be furtherclassified into 25 functional groups half of which weresorted into the ldquogeneral functionrdquo group (Figure 3) TheGO analysis revealed that 10165 unigenes were attributed tobiological process 8442 unigenes to cell components and10588 unigenes to molecular function (Figure 4) The top 26KEGG pathways are summarized in Table 4 Most unigenes(5184 out of 43753) were involved inmetabolic pathways and1401 unigenes were involved in calcium signaling pathwayssome of which may be involved in shell formation Finallymany unigenes in the top 26 KEGG pathways were involvedin immune pathways

32 Differential Gene Expression (DEGs) and ExpressionTrends during Developmental Stages Principal component

COG

KEGG Nr

Swissprot

219

637 8784

3193

92 1311 3971

108

12582

11406

268

427

7943

3849674

Figure 2 Number of genes annotated by COG KEGG Nr andSwissprot terms based on five larval stages and six tissues ofPinctadafucata

analysis (PCA) revealed that differences in the expressionof unigenes were vast among trochophore D-shaped andUES (umbonal eyespots and spats) larvae but small withinUES stages (Figure 5(a)) Based on gene effects measured bythe first principal component value a total of 10 transcriptswith unknown functions were identified to be key factorsinvolved in the larval development of P fucata Differencesin the gene expression of these transcripts were the great-est in trochophore D-shaped and UES larvae They wererelatively highly expressed in trochophore larvae and thendownregulated during the D-shaped stage and some weresubsequently upregulated during the UES stage includingUnigens23340 All Unigene8217 All Unigene50061 All andCl616 All (Figure 5(b))

The numbers of up- and downregulated unigenes werealso much greater during early stage transitions (Figure 6)consistent with the results of the PCA From trochophoreto D-shape larvae there were 18725 unigenes upregulatedand 13162 downregulated In total there were 57228 DEGsamong the five developmental stages (Figure 7) Additionally17609 genes were preferentially expressed at a single devel-opmental stage which indicates that they play an importantrole in the corresponding developmental stage while 39619were expressed preferentially during more than two stages


Table 4 Top 26 KEGG pathways

Pathway Count (43753) Pathway IDMetabolic pathways 5184 ko01100Regulation of actincytoskeleton 2302 ko04810

Vascular smooth musclecontraction 2176 ko04270

Focal adhesion 2142 ko04510Pathways in cancer 1607 ko05200Tight junction 1544 ko04530Hypertrophiccardiomyopathy (HCM) 1426 ko05410

Dilated cardiomyopathy 1402 ko05414Calcium signaling pathway 1401 ko04020Amoebiasis 1384 ko05146Tuberculosis 1379 ko05152RNA transport 1336 ko03013Salmonella infection 1333 ko05132Neuroactiveligand-receptor interaction 1304 ko04080

Epstein-Barr virus infection 1240 ko05169Phagosome 1239 ko04145Spliceosome 1231 ko03040Purine metabolism 1191 ko00230Vibrio cholera infection 1157 ko05110Endocytosis 1139 ko04144Huntingtonrsquos disease 1121 ko05016Viral myocarditis 1090 ko05416MAPK signaling pathway 1046 ko04010Cardiac muscle contraction 1044 ko04260Gastric acid secretion 1019 ko04971Ubiquitin mediatedproteolysis 1014 ko04120

A total of 20518 genes were differentially expressed in all fiveof the development stages All differentially expressed geneswere sorted into 29 expression trends (Figure 8) of whicheight trendswere significant comprising over 45of the totalDEGs Furthermore 6653 unigenes were expressed highlyonly during the trochophore stage Across the five stages3340 unigenes were expressed in an increasing pattern while2631 unigenes were expressed in a decreasing pattern

33 Functional Enrichment Analysis A functional enrich-ment analysis of the unigenes from the eight significant trendsshowed that there were 104 54 and 46 GO terms for biologi-cal processes molecular functions and cellular componentsrespectively identified for GO function enrichment (seeSupplementary Table 1 in Supplementary Material availableonline at httpdxdoiorg10115520162895303) and 272pathways identified for KEGG pathway enrichment (Supple-mentary Table 2) For GO enrichment data trends 0 2 3 2426 28 and 29 were involved in biological processes where

5911

269

4518

5055

802

4286

11336

3121

4357

1429

2108

158

28933199

905

3442

1915

404866 913

1252

1893

12069

4511

A RNA processing and modification K transcription L replication recombination and repair B chromatin structure and dynamics D cell cycle control cell division and chromosome partitioning Y nuclear structure V defense mechanisms T signal transduction mechanisms M cell wallmembraneenvelope biogenesis N cell motility Z cytoskeleton W extracellular structures U intracellular trafficking secretion and vesicular transport O posttranslational modification protein turnover and chaperones C energy production and conversion G carbohydrate transport and metabolism E amino acid transport and metabolism F nucleotide transport and metabolism H coenzyme transport and metabolism I lipid transport and metabolism P inorganic ion transport and metabolism Q secondary metabolites biosynthesis transport and catabolism R general function prediction only S function unknown

COG function classification

0

2500

5000

7500

10000

12500

Num

ber o

f gen

es

A K L B D Y V T M N Z W U O C G E F H I P Q R SJ

J translation ribosomal structure and biogenesis

Function class

Figure 3 COG function classification of all unigenes

most unigenes belonged to trend 3 andwere related to variousmetabolic processes Trends 0 3 24 28 and 29 were involvedinmolecular function wheremost unigenes fell within trends0 3 and 28 and were related to binding and catalyticactivity Only trends 0 3 28 and 29 were involved in cellularcomponents and most unigenes fell within trend 3 and wereinvolved in processes related to membranes and organelles

Trends 0 2 3 24 26 27 28 and 29 were implicatedin KEGG pathway enrichment For 272 significant enrichedpathways 81 pathways were observed in trend 29 77 in trend28 30 in trend 27 and 27 in trend 26 (Supplementary Table 2)In trends 28 and 29 most unigenes were involved in immuneresponses


Biological processCellular componentMolecular function

0

1000

2000

3000

4000

5000

6000

7000

8000

Num

bers

Biol

ogic

al ad

hesio

n

Cel

lC

ell j

unct

ion

Mac

rom

olec

ular

com

plex

Cel

l par

t

Mem

bran

e-en

close

d lu

men

Extr

acel

lula

r mat

rix

Extr

acel

lula

r reg

ion

part

Mem

bran

eM

embr

ane p

art

Extr

acel

lula

r mat

rix p

art

Extr

acel

lula

r reg

ion

Met

abol

ic p

roce

ss

Regu

latio

n of

bio

logi

cal p

roce

ss

Mul

tiorg

anism

pro

cess

Mul

ticel

lula

r org

anism

al p

roce

ssN

egat

ive r

egul

atio

n of

bio

logi

cal p

roce

ss

Cel

lula

r com

pone

nt o

rgan

izat

ion

or b

ioge

nesis

Imm

une s

yste

m p

roce

ss

Repr

oduc

tion

Repr

oduc

tive p

roce

ssRe

spon

se to

stim

ulus

Esta

blish

men

t of l

ocal

izat

ion

Sing

le-o

rgan

ism p

roce

ss

Cel

l pro

lifer

atio

nC

ell k

illin

gCa

rbon

util

izat

ion

Dev

elopm

enta

l pro

cess

Dea

th

Loca

lizat

ion

Cel

lula

r pro

cess

Biol

ogic

al re

gula

tion

Posit

ive r

egul

atio

n of

bio

logi

cal p

roce

ss

Loco

mot

ion

Gro

wth

Sign

alin

g

Prot

ein

bind

ing

tran

scrip

tion

fact

or ac

tivity

Mol

ecul

ar tr

ansd

ucer

activ

ityM

orph

ogen

activ

ity

Met

allo

chap

eron

e act

ivity

Bind

ing

Syna

pse

Syna

pse p

art

Stru

ctur

al m

olec

ule a

ctiv

ity

Enzy

me r

egul

ator

activ

ityEl

ectro

n ca

rrie

r act

ivity

Nuc

leic

acid

bin

ding

tran

scrip

tion

fact

or ac

tivity

Nuc

leoi

d

Virio

n pa

rtVi

rion

Tran

slatio

n re

gulat

or ac

tivity

Tran

spor

ter a

ctiv

ity

Cata

lytic

activ

ity

Org

anel

leO

rgan

elle

par

t

Chem

oattr

acta

nt ac

tivity

Chan

nel r

egul

ator

activ

ity

Ant

ioxi

dant

activ

ity

Rece

ptor

activ

ityRe

cept

or re

gula

tor a

ctiv

ity

Class

Figure 4 GO categories of unigenes 13465 of 174126 unigenes were assigned to GO annotation and divided into three categories biologicalprocesses cellular components and molecular functions

In trend 0 GO enrichment showed that macromoleculemetabolic processes were the dominant groups in biologicalprocess followed by positive regulation of biological pro-cess For pathways involved in molecular function DNAbinding was the most representative category while forcellular component pathways all of the genes participate inprocesses integral to the inner mitochondrial membrane andare intrinsic to the inner mitochondrial membrane In theKEGG category spliceosomes were prevalent followed bygenes involved in the cell cycle

In trend 3 GO enrichment data showed that 6653 DEGunigenes were further categorized into 43 functional groupsamong them macromolecule metabolic processes were thedominant groups in biological process followed by cellu-lar macromolecule metabolic processes In the molecularfunction category a high percentage of genes came from

the binding and protein binding groups Spliceosomes werethe most representative followed by RNA transport andregulation of the actin cytoskeleton

In trends 27 and 28 GO enrichment reveled that therewere no significant categories However the calcium sig-naling pathway hedgehog signaling pathway and insulinsignaling pathway were significantly enriched in the KEGGdatabase as they are all involved in early development Intrend 28 small molecule metabolic processes were the dom-inant group in biological process followed by ion transportCatalytic activity was the most prevalent in the molecu-lar function category followed by transporter activity andtransmembrane transporter activity In cellular componentpathways membrane was the most representative followedby plasma membrane In KEGG enrichment categories wealso found genes related to the calcium signaling pathway intrend 27


PCA

TrochophoreD-shapeUmbonal

EyespotsSpats

CL616Contig4_All

Unigene23340_All

Unigene32284_All

Unigene36784_All

Unigene37237_AllUnigene37601_All

Unigene45746_All

Unigene46572_All


minus002 000 002 004minus004PC1(656)

minus003

minus002

minus001

000

001

002

PC2(

228

)

(a)

Unigene23340_AllCL616Contig4_AllUnigene8217_AllUnigene50061_AllUnigene32284_All

Unigene37237_AllUnigene37601_AllUnigene36784_AllUnigene45746_AllUnigene46572_All

D-shaped Umbonal SpatsTrochophore EyespotsStages

0

10000

20000

30000

40000

50000

60000

70000

FPKM

(b)

Figure 5 The distinction between five developmental stages as indicated by (a) principal component analysis and (b) expression levels of 10representative genes identified to be responsible for the distinction among the developmental stages

18725

8539

977 327

minus13162

minus804000

minus4622minus21800

DownUp

DCBA

minus15000

minus10000

minus5000

0

5000

10000

15000

20000

25000

Num

ber o

f DEG

s

Figure 6 Numbers of up- (red) and downregulated (blue) unigenesThe numbers on column indicate the quantity of up- (red) anddownregulated (blue) genes The results of four comparisons areshownThe signal intensities of each feature of the DEGs are plottedon a logarithmic scale Statistical criteria for designation of genes asup- or downregulated are outlined in the methods

In trend 29 translational elongation was the onlyenriched category for biological processes while three cate-gories were enriched in molecular function including genesinvolved in oxidoreductase activity catalytic activity and

D-shape

Umbonal Eyespots

Spats

5973

6309 2314

3013

1085 1258 2966

725

20518

8237

639

1398

1244

776773

Figure 7 Specific and shared genes in five developmental stages ofthe pearl oyster Pinctada fucata

lyase activity In cellular component pathways five categorieswere enriched while vacuole was the dominant group fol-lowed by lytic vacuole and lysosome groups Genes in trend29 were enriched in only one KEGG pathway translationalelongation with a significant 119864 value

34 Identification and Expression Profiling of Genes Involvedin the Larval Metamorphic Development of P fucata In total80 development-related candidate DEGs were identified andsummarized in Table 5 which can be mapped to knowndevelopmentally important genes including several home-obox genes and can be sorted into 10 trends trend 0 (25unigenes) trend 28 (16) trend 3 (15) trend 29 (9) andsix other trends (1ndash5) Cluster analyses suggested that mostdevelopment-related candidate genes were highly expressed


Profile 3 6653 genes Profile 28 4444 genes Profile 29 3340 genes Profile 0 2631 genes Profile 21 1759 genes Profile 24 1173 genes





Figure 8 Expression trends of unigenes across trochophore D-shaped umbonal eyespots and spats larval stages of Pinctada fucata Theprofiles were ordered based on the 119875 value of the number (at bottom-left corner) of genes assigned versus expected Color square framesdenote significant profiles (119875 le 001) Each graph displays the mean pattern of expression (black lines) of the profiled genes The numberof profiles in each cluster is indicated in the top left corner The 119909-axis represents stages and the 119910-axis represents log 2-fold change of geneexpression

in the early developmental stages (Figure 9) includingengrailed-2-B pax family fox family members e1 and p1Wnt-4 and BMP33B upregulated in the trochophore stageand LIM foxg1 Hox3 bicaudal hedgehog EGFR foxl2 andbmp 2b genes upregulated from the D-shaped stage until theeyespots stage In the spats stagewnt1 and notch-like protein 2gene were upregulated The qPCR showed that the trends inthe expression of selected genes (Figure 10) were consistentwith the expression trends indicated by the trend analysis ofRNA-Seq data (Figure 9) indicating that the sequence data inour study are reliable

4 DiscussionNot only does the pearl oyster P fucata make an ideal modelorganism for studies of biomineralization but also it is a goodmodel to study the early stage metamorphic development ofinvertebrates In this study we sequenced the transcriptomes

of five developmental stages inP fucata with the aimof devel-oping a better understanding of the molecular mechanismsdriving the change of one larval stage to the next during earlylife history In our study the de novo assembly was performedwith six tissue transcriptomes as the draft genome of Pfucata is not complete [19] As a result we obtained 174126unigenes with a mean length of 866 bp A total of 60999unigenes (35) were annotated a value slightly higher thanprevious reports [21ndash23 38] Poor annotation efficiencieshave been widely prevalent in many marine organisms likelyowing limited genomic resources from aquaculture speciesin public databases to date [21ndash23 38] Alternatively poorannotation efficiencies could be the result of the short lengthof the assembled unigene sequences [22] and great divergenceamong the genomes of marine organism Similar scenarioshave been reported in other marine organisms [39 40] Inthe KEGG annotation we observed that many pathways were


Table 5 Early development-related DEGs and their expression trends in Pinctada fucata (80)

Unigene ID Annotated gene Reference species 119864 value TrendUnigene41154 All Nanos-like protein 1 Crassostrea gigas 500119864 minus 73 0Unigene40485 All EGF-like 4 Crassostrea gigas 900119864 minus 41 0Unigene27615 All brachyury Saccostrea kegaki 0 0Unigene40515 All TBX6 Crassostrea gigas 200119864 minus 49 0CL19363Contig1 All foxn1 Homo sapiens 200119864 minus 48 0Unigene36457 All foxb1 Crassostrea gigas 600119864 minus 129 0Unigene47210 All foxi1c Caenorhabditis brenneri 400119864 minus 16 0Unigene49637 All OAR Crassostrea gigas 3119864 minus 96 0Unigene49559 All notch-like protein 1 Crassostrea gigas 300119864 minus 67 0CL26075Contig2 All Cbx1 Mus musculus 500119864 minus 60 0CL4780Contig2 All XHOX-3 Crassostrea gigas 1119864 minus 124 0Unigene23054 All ZF E-box-binding 400119864 minus 60 0Unigene30958 All ZF protein 64 600119864 minus 08 0Unigene18460 All aristaless-like 4 1119864 minus 120 0Unigene22708 All ARX 400119864 minus 87 0Unigene22940 All IRX-6 2119864 minus 101 0Unigene27129 All engrailed 200119864 minus 56 0Unigene27119 All homeobox-like protein 200119864 minus 45 0Unigene27517 All SIX3 4119864 minus 129 0Unigene31497 All ceh-9 400119864 minus 64 0Unigene31654 All Slou 1119864 minus 103 0Unigene40727 All unc-4-like protein 7119864 minus 139 0Unigene40815 All homeobox 2 7119864 minus 120 0Unigene49664 All even-skipped-like protein 1 2119864 minus 107 0Unigene50280 All not2 9119864 minus 88 0Unigene23318 All engrailed-2-B 8119864 minus 61 3Unigene51601 All BarH-like 1 6119864 minus 31 3Unigene31090 All PKNOX2 3119864 minus 156 3Unigene49991 All HMX1 2119864 minus 27 3Unigene41009 All Pax-7 4119864 minus 128 3Unigene17191 All Pax-2-A 5119864 minus 102 3CL11027Contig2 All Polycomb BMI-1-A Danio rerio 300119864 minus 78 3CL13897Contig2 All Polycomb suz12 Xenopus tropicalis 200119864 minus 149 3CL20556Contig2 All pcgf3 Xenopus tropicalis 100119864 minus 77 3CL26777Contig2 All EPC1 Homo sapiens 400119864 minus 147 3CL15780Contig2 All Wnt-4 Homo sapiens 300119864 minus 93 3Unigene44847 All BMP 33b Branchiostoma japonicus 200119864 minus 59 3CL11806Contig6 All FOXP1 Homo sapiens 500119864 minus 104 3Unigene32767 All foxe1 Crassostrea gigas 300119864 minus 81 3CL4477Contig6 All Msx2 Crassostrea gigas 100119864 minus 22 3Unigene45481 All ceh-37 2119864 minus 59 12Unigene55326 All Pax-8 7119864 minus 59 20Unigene18153 All corepressor 1-like 0 21Unigene45344 All SIX4 7119864 minus 84 21Unigene45422 All aristaless 5119864 minus 72 21Unigene45788 All HMX3-B 1119864 minus 55 21Unigene40909 All odd-skipped-related 1 Crassostrea gigas 700119864 minus 74 21Unigene22645 All Hox5 Haliotis rufescens 300119864 minus 85 24Unigene35405 All EGF-like 1 Crassostrea gigas 600119864 minus 66 24Unigene17944 All foxc2 Crassostrea gigas 800119864 minus 174 24


Table 5 Continued

Unigene ID Annotated gene Reference species 119864 value TrendUnigene45321 All Dorsal root ganglia 2119864 minus 123 26CL664Contig4 All MAPK Homo sapiens 700119864 minus 127 26Unigene23113 All LIM 7119864 minus 122 27Unigene44988 All DBX1-A 3119864 minus 93 27CL19911Contig2 All foxg1 Xenopus laevis 500119864 minus 25 27Unigene17797 All Nkx-22a 2119864 minus 137 28Unigene44641 All Nkx-62 1119864 minus 125 28Unigene22323 All hhex 3119864 minus 92 28Unigene22601 All Xlox Euprymna scolopes 500119864 minus 55 28Unigene33207 All GBX-1 300119864 minus 07 28Unigene36590 All HOX3 8119864 minus 89 28Unigene45891 All MOX-2 9119864 minus 82 28CL20549Contig2 All bicaudal Xenopus laevis 100119864 minus 45 28Unigene23139 All hedgehog Crassostrea gigas 500119864 minus 99 28CL13232Contig2 All EGF-like D10442 Caenorhabditis elegans 900119864 minus 06 28CL178Contig1 All EGFR Apis mellifera 0 28CL5633Contig3 All MAPKAPK5 Homo sapiens 500119864 minus 132 28Unigene22098 All O-fut1 Crassostrea gigas 100119864 minus 131 28Unigene31699 All foxl2 Crassostrea gigas 100119864 minus 119 28Unigene36180 All foxl1 Crassostrea gigas 100119864 minus 119 28Unigene40504 All foxslp2 Crassostrea gigas 800119864 minus 116 28Unigene31565 All bmp 2b Crassostrea gigas 600119864 minus 96 29CL7953Contig2 All Notch Crassostrea gigas 100119864 minus 139 29Unigene36286 All Notch-like protein 2 Crassostrea gigas 500119864 minus 65 29Unigene27672 All ZF C2H2 Brugia malayi 400119864 minus 07 29Unigene27337 All LOX2 800119864 minus 98 29Unigene27686 All BarH-like 1-like Oreochromis niloticus 300119864 minus 36 29CL12322Contig2 All ALDH16A1 Bos taurus 100119864 minus 179 29CL3565Contig3 All ALDH2 Crassostrea gigas 0 29CL1306Contig2 All Wnt 1 Homo sapiens 700119864 minus 13 29

related to immunity indicating that innate protection is vitalin the early developmental stages

Differential gene expressions (DEGs) occurred mainlyduring early stage transitions (Figure 6) Most genes wereup- or downregulated from trochophore to D-shaped andfrom D-shaped to umbonal stages indicating that pro-cesses associated with these transitions are very complicatedPrincipal component analyses yielded consistent resultswhere we identified 10 unigenes attributed to the divergenceamong trochophore D-shaped andUES (umbonal eyespotsand spats) stages in P fucata being highly expressed inthe trochophore stage However no functional annotationsmatch these functionally important sequences indicatingthat further research would help to elucidate the molecularmechanism of metamorphosis in this species in the future

The analysis of expression trends indicated that 12009 of13277 unigenes are sorted into eight significant expressiontrend groups Among the significant trends there were 10031(trends 3 0 and 2) 11978 (trends 28 29 21 24 26 and27) 9046 (trend 28 29 26 and 27) 9518 (trend 28 29 24and 27) and 5214 (trend 29 24 and 26) unigenes displaying

increased expression in trochophore D-shaped umbonaleyespots and spats stages respectively This conveys thatmore genes are expressed in the early stages consistentwith the DEG and PCA analyses in our study Particularly6653 unigenes (trend 3) were highly expressed only in thetrochophore stage 3340 unigenes (trend 29) expressed in anincreasing pattern over the course of development and 2631unigenes (trend 0) expressed in a decreasing pattern Thesegenes are worth further investigation

The KEGG pathway enrichment analysis indicated thatmost unigenes in trend 3 were involved in pathways ofspliceosome or RNA transport indicating that in the earlystage of P fucata RNA synthesis is more predominantOn the contrary genes in trend 29 showed significantenrichment in translational elongation pathways suggestingthat protein synthesis is more and more prevalent duringlarval development In trends 27 and 28 a large number ofunigenes were involved in the calcium signaling pathwaysynchronizing with the shell formation of prodissoconchs Iand II in D-shaped and umbonal stages [24 41] In additionimmune pathways were also enriched indicating that innate


Trochophore D-shape Umbonal Eyespots Spats

ZF C2H2ceh-37aristaless-like 4TBX6unc-4-like proteinNotch-like protein 1OARceh-9Sloufoxi1ceven-skipped-like protein 1ARXSIX3XHOX-3foxb1EGF-like 4homeobox-like proteinfoxn1homeobox 2IRX-6EngrailedZF protein 64pcgf3EPC1PKNOX2 BarH-like 1Cbx1foxe1Engrailed-2-BPolycomb suz12Polycomb BMI-1-AFOXP1Nanos-like protein 1BMP 33bWnt-4ZF E-box-bindingHMX1Msx2Pax-7Pax-2-AEGF-like 1DBX1-ALIMHOX3MAPKfoxl2foxg1odd-skipped-related 1Dorsal root ganglianot2BrachyuryaristalessPax-8corepressor 1-likeSIX4HMX3-BBarH-like 1-likeLOX2Wnt 1bmp 2bNotch-like protein 2EGFRO-fut1EGF-like D10442NotchALDH16A1bicaudalXloxhhexhedgehogHox5foxc2GBX-1Nkx-22aALDH2Nkx-62MOX-2MAPKAPK5foxl1foxslp2

minus15

minus1

minus05

0

05

1

15

Figure 9 Clusters of expression patterns of development-related genes across five larval stages in Pinctada fucata

protection is important during the entire course of larvaldevelopment [3 42 43]

In our study 80 known development-related differen-tially expressed unigenes were identified throughout thefive larval stages (Table 5) Half of them were homeobox-containing genes including genes known to be involved inthe development of body patterning (engrailed SIX3 Pax-7LIM and Hox family members) suggesting that these genesplay important roles in the metamorphic changes of P fucatalarvae Nearly half of the homeobox-containing genes wereupregulated in trochophore and D-shaped stages (Figure 9)We identified two Hox genes Hox5 and Hox3 (Figure 9)which are highly expressed in D-shaped veliger indicatingthat they are involved in the growth of D-shaped larvaeWe also found early developmentally relevant signaling

molecules such as Hedghog TGF120573 and Wnt family whichare known to play important roles in axis formation muscledifferentiation andnervous systemdevelopment [26] Recentevidence has suggested that classic morphogens such asWnts TGF120573BMP family members and Hedgehogs may allserve as axon guidance cues for a variety of axons in differentorganisms [44] Several studies have provided increasingevidence that Sonic hedgehog (Shh) is an important axonguidance cue throughout vertebrate neural development [4546] In our study one hedgehog gene (Unigene23139 All)was identified and highly expressed in umbonal and eyespotsstages (Figure 9) suggesting that increased neural develop-ment was likely taking place during those stages

The Wnt signal pathway has been shown to play animportant role in the segmentation of the marine polychaete


Wnt1

05

101520253035

Expr

essio

n le

vel

UmbonalTrochophore D-shape Eyespots SpatsStages

RELFPKM

(a)

Six3

0102030405060708090

100

Expr

essio

n le

vel

RELFPKM

D-shape Umbonal Eyespots SpatsTrochophoreStages

(b)

Notch


005

115

225

335

445

5

Expr

essio

n le

vel

RELFPKM

(c)

Lox2


0123456789

Expr

essio

n le

vel

RELFPKM

(d)

Engrailed


01020304050607080

Expr

essio

n le

vel

RELFPKM

(e)

Branchyury

05

1015202530354045

Expr

essio

n le

vel


RELFPKM

(f)

MAPK


0123456789

Expr

essio

n le

vel

RELFPKM

(g)

pax7

02468

10121416

Expr

essio

n le

vel

D-shape Umbonal Eyespots MetamorphosisTrochophoreStages

RELFPKM

(h)

Figure 10 Expression changes in (a)Wnt1 (b) Six3 (c) notch (d) lox2 (e) engrailed (f) branchyury (g)MAPK and (h) pax7 in trochophoreD-shaped umbonal eyespots and spats larval stages by qPCR


Capitella capitata [47 48] while themaintenance of primitivehematopoiesis has been attributed to Wnt4 in the vertebrateembryo [49] Both Wnt4 and Wnt1 were observed in thisstudy Wnt4 was highly expressed in the trochophore stagewhile Wnt1 was expressed in an increasing pattern over thecourse of larval development suggesting possible involve-ment in blood formation from the beginning of developmentand in body transformation during all stages Ten classes offox genes were also found in our study comprising the largestnumber of genes identified in the DEGs and displaying dif-ferent trends in expression FoxL2 XX-dominantly expressedin the differentiating ovaries of mammals [50] birds [51]and fish [52ndash54] was expressed highly in the D-shaped stagesuggesting the possible beginning of sexual developmentSome important growth-related genes were also identifiedand differentially expressed among the five developmentstages including EGF-likeMAPK andMAPKK genes whichwere actively expressed during the five developmental stagesand may contribute significantly to the transitions betweendevelopmental stages in P fucata larvae

Nonetheless the body form transformations that takeplace during larval development involve a series of morpho-logical and physiological changes and corresponding molec-ular changes which have not been systematically studied andremain unclearTherefore amore broad understanding of themolecular underpinnings of important biological processesstill merits further investigation

5 Conclusions

In this study a total of 174126 unique transcripts were assem-bled and 60999 were annotated The number of unigenesvaried between the five larval stages The expression profilesof trochophore D-shaped and UES (umbonal eyespots andspats) larvaewere distinctly differentMost unigeneswere up-or downregulated in early stage transitions and 29 expressiontrendswere sorted eight of whichwere significant In total 80development-related differentially expressed unigenes wereidentified and eight were verified by qPCR These obser-vations should be helpful in understanding the molecularmechanisms of the larval metamorphic development of Pfucata

Additional Points

Highlights(i) A large number of assembled transcripts fromPinctada fucata are reported for the first time (ii) Largevariations in expression ofDEGs related to development wereobserved in early larval stages (iii) Twenty-nine expressiontrends were identified for the first time

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Haimei Li and Dahui Yu conceived and designed the projectHaimei Li Bo Zhang and Baosuo Liu cultured and collected

the P fucata larval samples and the adult samples Haimei LiGuiju Huang and Sigang Fan carried out the transcriptomeanalysis and qPCR experiments Dongling Zhang providedtechnical assistance Haimei Li and Dahui Yu wrote themanuscript All listed authors have read edited and approvedthe final manuscript

Acknowledgments

This work was supported by The National Natural ScienceFoundation of China (NSFC) (31372525) the EarmarkedFund for China Agriculture Research System (Grant noCARS-48) and Special Fund for Marine Fisheries Researchand Extension of Guangdong Province (A201301A02A201301A08 Z2014003 Z2014006 and Z2015009) Theauthors are also grateful to the Guangzhou Gene denovoBiotechnology Co Ltd for assisting in the data analysis

References

[1] P-Y Qian ldquoLarval settlement of polychaetesrdquo Hydrobiologiavol 402 pp 239ndash253 1999

[2] T Fujimura K Wada and T Iwaki ldquoDevelopment and mor-phology of the pearl oyster larvae Pinctada fucatardquo Venus vol54 pp 25ndash48 1995

[3] AHeyland and L LMoroz ldquoSignalingmechanisms underlyingmetamorphic transitions in animalsrdquo Integrative and Compara-tive Biology vol 46 no 6 pp 743ndash759 2006

[4] C Devine V F Hinman and B M Degnan ldquoEvolution anddevelopmental expression of nuclear receptor genes in theascidian HerdmaniardquoThe International Journal of Developmen-tal Biology vol 46 no 4 pp 687ndash692 2002

[5] J M Arnold R Eri B M Degnan and M F Lavin ldquoNovelgene containing multiple epidermal growth factor-like motifstransiently expressed in the papillae of the ascidian tadpolelarvaerdquo Developmental Dynamics vol 210 no 3 pp 264ndash2731997

[6] A Nakayama Y Satou and N Satoh ldquoIsolation and charac-terization of genes that are expressed during Ciona intestinalismetamorphosisrdquoDevelopment Genes and Evolution vol 211 no4 pp 184ndash189 2001

[7] R Eri J M Arnold and V F Hinman ldquoPatterning of dopamin-ergic neurotransmitter identity among Caenorhabditis elegansray sensory neurons by a TGF family signaling pathway and aHox generdquo Development vol 126 no 24 pp 5819ndash5831 1999

[8] B M Degnan and D E Morse ldquoIdentification of eighthomeobox-containing transcripts expressed during larvaldevelopment and at metamorphosis in the gastropod molluscHaliotis rufescensrdquoMolecularMarine Biology and Biotechnologyvol 2 no 1 pp 1ndash9 1993

[9] B M Degnan S M Degnan G Fentenany and D E Morse ldquoAMoxhomeobox gene in the gastropodmolluscHaliotis rufescensis differentially expressed during larval morphogenesis andmetamorphosisrdquo FEBS Letters vol 411 no 1 pp 119ndash122 1997

[10] A F Giusti V F Hinman S M Degnan B M Degnan and DE Morse ldquoExpression of a ScrHox5 gene in the larval centralnervous system of the gastropod Haliotis a non-segmentedspiralian lophotrochozoanrdquo Evolution and Development vol 2no 5 pp 294ndash302 2000


[11] S L Coon W K Fitt and D B Bonar ldquoCompetence anddelay of metamorphosis in the Pacific oyster Crassostrea gigasrdquoMarine Biology vol 106 no 3 pp 379ndash387 1990

[12] C Lelong M Mathieu and P Favrel ldquoStructure and expressionof mGDF a new member of the transforming growth factor-120573superfamily in the bivalve mollusc Crassostrea gigasrdquo EuropeanJournal of Biochemistry vol 267 no 13 pp 3986ndash3993 2000

[13] T J Fiedler A Hudder S J McKay et al ldquoThe transcriptomeof the early life history stages of the California sea hare Aplysiacalifornicardquo Comparative Biochemistry and Physiology Part DGenomics and Proteomics vol 5 no 2 pp 165ndash170 2010

[14] J D Lambert X Y Chan B Spiecker and H C Sweet ldquoChar-acterizing the embryonic transcriptome of the snail IlyanassardquoIntegrative and Comparative Biology vol 50 no 5 pp 768ndash7772010

[15] P Huan H Wang and B Liu ldquoTranscriptomic analysis ofthe clam Meretrix meretrix on different larval stagesrdquo MarineBiotechnology vol 14 no 1 pp 69ndash78 2012

[16] Z-X Huang Z-S Chen C-H Ke et al ldquoPyrosequencing ofHaliotis diversicolor transcriptomes insights into early develop-mental molluscan gene expressionrdquo PLoS ONE vol 7 no 12Article ID e51279 2012

[17] J Qin Z Huang J Chen Q ZouW You and C Ke ldquoSequenc-ing and de novo analysis ofCrassostrea angulata (FujianOyster)from 8 different developing phases using 454 GSFLxrdquo PLoSONE vol 7 no 8 Article ID e43653 2012

[18] S Bassim A Tanguy B Genard D Moraga and R TremblayldquoIdentification ofMytilus edulis genetic regulators during earlydevelopmentrdquo Gene vol 551 no 1 pp 65ndash78 2014

[19] T Takeuchi T Kawashima R Koyanagi et al ldquoDraft genome ofthe pearl oyster Pinctada fucata a platform for understandingbivalve biologyrdquo DNA Research vol 19 no 2 pp 117ndash130 2012

[20] Y Shi C Yu Z Gu X Zhan Y Wang and A WangldquoCharacterization of the pearl oyster (Pinctada martensii) man-tle transcriptome unravels biomineralization genesrdquo MarineBiotechnology vol 15 no 2 pp 175ndash187 2013

[21] A Wang Y Wang Z Gu S Li Y Shi and X Guo ldquoDevelop-ment of expressed sequence tags from the pearl oyster PinctadamartensiiDunkerrdquoMarine Biotechnology vol 13 no 2 pp 275ndash283 2011

[22] X Zhao Q Wang Y Jiao et al ldquoIdentification of genes poten-tially related to biomineralization and immunity by transcrip-tome analysis of pearl sac in pearl oyster Pinctada martensiirdquoMarine Biotechnology vol 14 no 6 pp 730ndash739 2012

[23] S Kinoshita N Wang H Inoue et al ldquoDeep sequencing ofESTs from nacreous and prismatic layer producing tissues anda screen for novel shell formation-related genes in the pearloysterrdquo PLoS ONE vol 6 no 6 Article ID e21238 2011

[24] Y Miyazaki T Nishida H Aoki and T Samata ldquoExpression ofgenes responsible for biomineralization of Pinctada fucata dur-ing developmentrdquo Comparative Biochemistry and PhysiologymdashB Biochemistry and Molecular Biology vol 155 no 3 pp 241ndash248 2010

[25] J Liu D Yang S Liu et al ldquoMicroarray a global analysisof biomineralization-related gene expression profiles duringlarval development in the pearl oyster Pinctada fucatardquo BMCGenomics vol 16 no 1 article 325 2015

[26] D H E Setiamarga K Shimizu J Kuroda et al ldquoAn in-silico genomic survey to annotate genes coding for earlydevelopment-relevant signaling molecules in the pearl oysterPinctada fucatardquo Zoological Science vol 30 no 10 pp 877ndash8882013

[27] H Koga N Hashimoto D G Suzuki et al ldquoA genome-widesurvey of genes encoding transcription factors in Japanese pearloyster Pinctada fucata II Tbx Fox Ets HMGNF120581B bZIP andC2H2 zinc fingersrdquo Zoological Science vol 30 no 10 pp 858ndash867 2013

[28] Y Morino K Okada M Niikura M Honda N Satoh and HWada ldquoA genome-wide survey of genes encoding transcriptionfactors in the Japanese pearl oyster Pinctada fucata I Home-obox genesrdquo Zoological Science vol 30 no 10 pp 851ndash857 2013

[29] G Pertea X Huang F Liang et al ldquoTIGR gene indicesclustering tools (TGICL) a software system for fast clusteringof large EST datasetsrdquo Bioinformatics vol 19 no 5 pp 651ndash6522003

[30] C Iseli C V Jongeneel and P Bucher ldquoESTScan a programfor detecting evaluating and reconstructing potential codingregions in EST sequences rdquo in Proceedings of the InternationalConference on Intelligent Systems for Molecular Biology (ISMBrsquo99) pp 138ndash148 Heidelberg Germany 1999

[31] A Conesa S Gotz J M Garcıa-Gomez J Terol M Talonand M Robles ldquoBlast2GO a universal tool for annotationvisualization and analysis in functional genomics researchrdquoBioinformatics vol 21 no 18 pp 3674ndash3676 2005

[32] J Ye L Fang H Zheng et al ldquoWEGO a web tool for plottingGO annotationsrdquo Nucleic Acids Research vol 34 pp W293ndashW297 2006

[33] R Li C Yu Y Li et al ldquoSOAP2 an improved ultrafast tool forshort read alignmentrdquo Bioinformatics vol 25 no 15 pp 1966ndash1967 2009

[34] A Mortazavi B A Williams K McCue L Schaeffer and BWold ldquoMapping and quantifying mammalian transcriptomesby RNA-Seqrdquo Nature Methods vol 5 no 7 pp 621ndash628 2008

[35] J Ernst and Z Bar-Joseph ldquoSTEM a tool for the analysis ofshort time series gene expression datardquo BMC Bioinformaticsvol 7 article 191 2006

[36] M Kanehisa M Araki S Goto et al ldquoKEGG for linkinggenomes to life and the environmentrdquo Nucleic Acids Researchvol 36 no 1 pp D480ndashD484 2008

[37] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCRand the 2minusΔΔ119862TmethodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[38] X-D Huang M Zhao W-G Liu et al ldquoGigabase-scale tran-scriptome analysis on four species of pearl oystersrdquo MarineBiotechnology vol 15 no 3 pp 253ndash264 2013

[39] E Meyer G V Aglyamova S Wang et al ldquoSequencing and denovo analysis of a coral larval transcriptome using 454 GSFlxrdquoBMC Genomics vol 10 article 219 pp 1ndash8 2009

[40] R Bettencourt M Pinheiro C Egas et al ldquoHigh-throughputsequencing and analysis of the gill tissue transcriptome from thedeep-sea hydrothermal vent mussel Bathymodiolus azoricusrdquoBMC Genomics vol 11 article 559 2010

[41] D Fang G Xu Y Hu C Pan L Xie and R Zhang ldquoIdenti-fication of genes directly involved in shell formation and theirfunctions in pearl oyster Pinctada fucatardquo PLoSONE vol 6 no7 Article ID e21860 2011

[42] E A Williams B M Degnan H Gunter D J Jackson BJ Woodcroft and S M Degnan ldquoWidespread transcriptionalchanges pre-empt the critical pelagic-benthic transition in thevetigastropodHaliotis asininardquoMolecular Ecology vol 18 no 5pp 1006ndash1025 2009

[43] D E Clapham ldquoCalcium signalingrdquo Cell vol 131 no 6 pp1047ndash1058 2007


[44] F Charron and M Tessier-Lavigne ldquoNovel brain wiring func-tions for classical morphogens a role as graded positional cuesin axon guidancerdquo Development vol 132 no 10 pp 2251ndash22622005

[45] F Charron E Stein J Jeong A P McMahon and MTessier-Lavigne ldquoThe morphogen sonic hedgehog is an axonalchemoattractant that collaborates with netrin-1 inmidline axonguidancerdquo Cell vol 113 no 1 pp 11ndash23 2003

[46] J K Chen J TaipaleMKCooper andPA Beachy ldquoInhibitionof Hedgehog signaling by direct binding of cyclopamine toSmoothenedrdquo Genes amp Development vol 16 no 21 pp 2743ndash2748 2002

[47] E C Seaver and LM Kaneshige ldquoExpression of lsquosegmentationrsquogenes during larval and juvenile development in the polychaetesCapitella sp I and H elegansrdquo Developmental Biology vol 289no 1 pp 179ndash194 2006

[48] J L Christian B J Gavin A P McMahon and R T MoonldquoIsolation of cDNAs partially encoding four Xenopus Wnt-1int-1-related proteins and characterization of their transientexpression during embryonic developmentrdquo DevelopmentalBiology vol 143 no 2 pp 230ndash234 1991

[49] H T Tran B Sekkali G Van Imschoot S Janssens andK Vleminckx ldquoWnt120573-catenin signaling is involved in theinduction and maintenance of primitive hematopoiesis in thevertebrate embryordquo Proceedings of the National Academy ofSciences of the United States of America vol 107 no 37 pp16160ndash16165 2010

[50] D Baron F Batista J S Chaffaux Cocquet et al ldquoFoxl2 geneand the development of the ovary a story about goat mousefish and womanrdquo Reproduction Nutrition Development vol 45no 6 pp 729ndash729 2005

[51] M S Govoroun M Pannetier E Pailhoux et al ldquoIsolationof chicken homolog of the FOXL2 gene and comparison ofits expression patterns with those of aromatase during ovariandevelopmentrdquoDevelopmental Dynamics vol 231 no 4 pp 859ndash870 2004

[52] D Baron J Cocquet X Xia M Fellous Y Guiguen and RA Veitia ldquoAn evolutionary and functional analysis of FoxL2in rainbow trout gonad differentiationrdquo Journal of MolecularEndocrinology vol 33 no 3 pp 705ndash715 2004

[53] M Nakamoto M Matsuda D-S Wang Y Nagahama and NShibata ldquoMolecular cloning and analysis of gonadal expressionof Foxl2 in the medaka Oryzias latipesrdquo Biochemical andBiophysical Research Communications vol 344 no 1 pp 353ndash361 2006

[54] D-S Wang T Kobayashi L-Y Zhou et al ldquoFoxl2 up-regulatesaromatase gene transcription in a female-specific manner bybinding to the promoter as well as interacting with Ad4 bindingproteinsteroidogenic factor 1rdquoMolecular Endocrinology vol 21no 3 pp 712ndash725 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


superfamily genes showed differential expression during themetamorphosis ofCiona intestinalis [4ndash7] Several homeobox-containing genes were found to be responsible for larvalmetamorphic development in Haliotis rufescens [8ndash10] Inaddition abnormal dopamine and adrenaline were observedin the larval attaching stage of the Pacific oyster Crassostreagigas [11] while a different study observed increased expressionof a molluscan growth and differentiation factor (mGDF) inthe metamorphosing stage of the same organism [12] Thesefindings indicate the diversity of genes involved in the transi-tions of larval forms

However previous studies have focused on changes in asmall number of genes and have provided a fragmented viewof the genetic modulation of larval metamorphosis Recentdevelopments in sequencing technology have allowed for thedevelopment of new genomic tools which can provide amoreglobal view of changes in gene expression over the courseof larval developmental stages [13ndash16] In terms of genome-wide studies transcriptome analyses are considered to bean ideal choice for obtaining comprehensive informationregarding animal development and growth [17 18] For Pfucata the draft genome [19] and tissue transcriptomes [20ndash23] have been recently reported Based on the transcriptomicsequences from a mixture of nine developmental stagesof P fucata [19] biomineralization-related gene expressionprofiles during larval development have been investigated[24 25] and genes involved in body patterning [26] tran-scription factors [27] and homeobox genes [28] have beenidentified Nonetheless developmentally important genesand their expression patterns during the larval stages ofdeveloping P fucata have not been systematically studiedat the transcript level to date In the present study thetranscriptomes of five larval stages (trochophore D-shapeumbonal eyespots and spats) and six tissues (gill adductormuscle hepatopancreas mantle hemocytes and pearl sac)from P fucata were sequenced using Illumina HiSeq 2000with an emphasis on the molecular mechanisms underlyinglarval metamorphic developmentThis study aims to providea valuable insight into themechanisms of genetic modulationover the course of larval metamorphic development for Pfucata as well as for other molluscan species

2 Materials and Methods

21 Larval Culture and Sample Collection Larvae of P fucatawere bred (using several females and males of a selectivelybred F3 generation as parents) through artificial insemina-tion on March 10 2013 in Sanya Hainan Island Chinaas described by Fujimura et al [2] Fertilized eggs wereincubated in a 1000 L tank at 24∘C After removing nonde-veloping embryos and dead larvae trochophore D-shapedumbonal eyespots and spats larvae stages were harvestedwith filtering net at 12 h 36 h 115 d 185 d and 235 d afterfertilization respectively and immediately preserved in RNAlater (TaKaRa Bio Inc) until RNA extraction MeanwhileRNAs of six tissues (gill adductor muscle hepatopancreasmantle hemocytes andpearl sac) of three other adult animalswere sequenced for a more robust assembly

22 RNA Extraction and cDNA Library Preparation Follow-ing the manufacturerrsquos instructions total RNA was extractedfrom five developmental stages (each stage with thousands oflarvae) and six tissues using Trizol and RNAs of each type oftissues of the three individuals were mixed by equal weightRNA integrity and quantity were confirmed by lab-on-chipanalysis using a 2100 Bioanalyzer (Agilent TechnologiesSanta Clara CA USA) and visualized on a 1 agarose gelThen cDNA was synthesized using the mRNA fragments astemplates as usual and was sequenced by the BGI (ShenzhenChina) using the Illumina HiSeq 2000 system (San DiegoCA USA) (PE100)

23 Sequence Assembly and Annotation After filtering outlow quality sequences (containing more than 5 ambiguousldquoNrdquo nucleotides or gt20 119876 ≦ 10 reads) and the removal ofadapters from raw data clean sequence data was assembledinto unigenes usingTrinity software and subsequently clusteredby TGICL v21 (-l 40 -c 10 -v 20) [29] Phrap (-repeatstringency 095 -minmatch 35 -minscore 35) (Release 230)was used to produce the longest sequence possible (httpwwwphraporg) Assembled unigenes were annotated based onthe Nr Swissprot KEGG and COG databases The sequencedirection and amino sequence of the predicted coding region(CDS) of unannotated unigenes were determined usingESTScan with default settings [30] Functional annotationsand classifications were performed by using Blast2GO [31]and WEGO [32] (119864 value threshold 1 times 10minus5) respectively

24 Normalization and Quantification of Gene ExpressionSequencing reads were mapped to the assembled referencesequence using SOAP alignersoap2 (-m 0 -x 500 -s 40 -l 35-v 5 -r 2) [33] a tool designed specifically to assemble shortsequence alignmentsThe coverage of reads from a given genewas used to calculate the expression level of that gene whichwas measured by fragments per kilobase exon per millionfragments (FPKM) [34] with the following formula

FPKM = 106119862

119873119871103 (1)

where FPKM is the expression level of a unigene 119862 is thenumber of fragments that uniquely aligned to the unigene119873 is the total number of fragments that uniquely alignedto all unigenes and 119871 is the number of bases in the CDSof the unigene The FPKM method eliminates the influenceof sequences of differing lengths and coverage level on thecalculation of gene expressionTherefore the calculated geneexpression can be directly used for comparing the differencein gene expression between samples

25 Differential Gene Expression (DEGs) across Develop-mental Stages Differential gene expression among differentlarval stages was carried out via principal component anal-ysis (PCA) using the R package (httpwwwr-projectorg)according to the manualThe pairwise differential expressionconducted by edgeR with a threshold of the false discoveryrate (FDR) le 0001 and an absolute value of log 2 Ratioge 1 was used to judge the significance of differences ingene expression Trends in the expression of all differentially























UnigeneCDS

0

20000

40000

60000

80000

100000

120000

Num

ber





3 Results










COG

KEGG Nr

Swissprot

219

637 8784

3193

92 1311 3971

108

12582

11406

268

427

7943

3849674













5911

269

4518

5055

802

4286

11336

3121

4357

1429

2108

158

28933199

905

3442

1915

404866 913

1252

1893

12069

4511



0

2500

5000

7500

10000

12500

Num

ber o

f gen

es



Function class






0

1000

2000

3000

4000

5000

6000

7000

8000

Num

bers

Biol

ogic

al ad

hesio

n

Cel

lC

ell j

unct

ion

Mac

rom

olec

ular

com

plex

Cel

l par

t

Mem

bran

e-en

close

d lu

men

Extr

acel

lula

r mat

rix

Extr

acel

lula

r reg

ion

part

Mem

bran

eM

embr

ane p

art

Extr

acel

lula

r mat

rix p

art

Extr

acel

lula

r reg

ion

Met

abol

ic p

roce

ss

Regu

latio

n of

bio

logi

cal p

roce

ss

Mul

tiorg

anism

pro

cess

Mul

ticel

lula

r org

anism

al p

roce

ssN

egat

ive r

egul

atio

n of

bio

logi

cal p

roce

ss

Cel

lula

r com

pone

nt o

rgan

izat

ion

or b

ioge

nesis

Imm

une s

yste

m p

roce

ss

Repr

oduc

tion

Repr

oduc

tive p

roce

ssRe

spon

se to

stim

ulus

Esta

blish

men

t of l

ocal

izat

ion

Sing

le-o

rgan

ism p

roce

ss

Cel

l pro

lifer

atio

nC

ell k

illin

gCa

rbon

util

izat

ion

Dev

elopm

enta

l pro

cess

Dea

th

Loca

lizat

ion

Cel

lula

r pro

cess

Biol

ogic

al re

gula

tion

Posit

ive r

egul

atio

n of

bio

logi

cal p

roce

ss

Loco

mot

ion

Gro

wth

Sign

alin

g

Prot

ein

bind

ing

tran

scrip

tion

fact

or ac

tivity

Mol

ecul

ar tr

ansd

ucer

activ

ityM

orph

ogen

activ

ity

Met

allo

chap

eron

e act

ivity

Bind

ing

Syna

pse

Syna

pse p

art

Stru

ctur

al m

olec

ule a

ctiv

ity

Enzy

me r

egul

ator

activ

ityEl

ectro

n ca

rrie

r act

ivity

Nuc

leic

acid

bin

ding

tran

scrip

tion

fact

or ac

tivity

Nuc

leoi

d

Virio

n pa

rtVi

rion

Tran

slatio

n re

gulat

or ac

tivity

Tran

spor

ter a

ctiv

ity

Cata

lytic

activ

ity

Org

anel

leO

rgan

elle

par

t

Chem

oattr

acta

nt ac

tivity

Chan

nel r

egul

ator

activ

ity

Ant

ioxi

dant

activ

ity

Rece

ptor

activ

ityRe

cept

or re

gula

tor a

ctiv

ity

Class







PCA


EyespotsSpats

CL616Contig4_All

Unigene23340_All

Unigene32284_All

Unigene36784_All


Unigene45746_All

Unigene46572_All


minus002 000 002 004minus004PC1(656)

minus003

minus002

minus001

000

001

002

PC2(

228

)

(a)




0

10000

20000

30000

40000

50000

60000

70000

FPKM

(b)


18725

8539

977 327

minus13162

minus804000

minus4622minus21800

DownUp

DCBA

minus15000

minus10000

minus5000

0

5000

10000

15000

20000

25000

Num

ber o

f DEG

s



D-shape

Umbonal Eyespots

Spats

5973

6309 2314

3013

1085 1258 2966

725

20518

8237

639

1398

1244

776773


















Table 5 Continued










minus15

minus1

minus05

0

05

1

15







Wnt1

05

101520253035

Expr

essio

n le

vel


RELFPKM

(a)

Six3

0102030405060708090

100

Expr

essio

n le

vel

RELFPKM


(b)

Notch


005

115

225

335

445

5

Expr

essio

n le

vel

RELFPKM

(c)

Lox2


0123456789

Expr

essio

n le

vel

RELFPKM

(d)

Engrailed


01020304050607080

Expr

essio

n le

vel

RELFPKM

(e)

Branchyury

05

1015202530354045

Expr

essio

n le

vel


RELFPKM

(f)

MAPK


0123456789

Expr

essio

n le

vel

RELFPKM

(g)

pax7

02468

10121416

Expr

essio

n le

vel


RELFPKM

(h)





5 Conclusions


Additional Points


Competing Interests





Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology























UnigeneCDS

0

20000

40000

60000

80000

100000

120000

Num

ber





3 Results










COG

KEGG Nr

Swissprot

219

637 8784

3193

92 1311 3971

108

12582

11406

268

427

7943

3849674













5911

269

4518

5055

802

4286

11336

3121

4357

1429

2108

158

28933199

905

3442

1915

404866 913

1252

1893

12069

4511



0

2500

5000

7500

10000

12500

Num

ber o

f gen

es



Function class






0

1000

2000

3000

4000

5000

6000

7000

8000

Num

bers

Biol

ogic

al ad

hesio

n

Cel

lC

ell j

unct

ion

Mac

rom

olec

ular

com

plex

Cel

l par

t

Mem

bran

e-en

close

d lu

men

Extr

acel

lula

r mat

rix

Extr

acel

lula

r reg

ion

part

Mem

bran

eM

embr

ane p

art

Extr

acel

lula

r mat

rix p

art

Extr

acel

lula

r reg

ion

Met

abol

ic p

roce

ss

Regu

latio

n of

bio

logi

cal p

roce

ss

Mul

tiorg

anism

pro

cess

Mul

ticel

lula

r org

anism

al p

roce

ssN

egat

ive r

egul

atio

n of

bio

logi

cal p

roce

ss

Cel

lula

r com

pone

nt o

rgan

izat

ion

or b

ioge

nesis

Imm

une s

yste

m p

roce

ss

Repr

oduc

tion

Repr

oduc

tive p

roce

ssRe

spon

se to

stim

ulus

Esta

blish

men

t of l

ocal

izat

ion

Sing

le-o

rgan

ism p

roce

ss

Cel

l pro

lifer

atio

nC

ell k

illin

gCa

rbon

util

izat

ion

Dev

elopm

enta

l pro

cess

Dea

th

Loca

lizat

ion

Cel

lula

r pro

cess

Biol

ogic

al re

gula

tion

Posit

ive r

egul

atio

n of

bio

logi

cal p

roce

ss

Loco

mot

ion

Gro

wth

Sign

alin

g

Prot

ein

bind

ing

tran

scrip

tion

fact

or ac

tivity

Mol

ecul

ar tr

ansd

ucer

activ

ityM

orph

ogen

activ

ity

Met

allo

chap

eron

e act

ivity

Bind

ing

Syna

pse

Syna

pse p

art

Stru

ctur

al m

olec

ule a

ctiv

ity

Enzy

me r

egul

ator

activ

ityEl

ectro

n ca

rrie

r act

ivity

Nuc

leic

acid

bin

ding

tran

scrip

tion

fact

or ac

tivity

Nuc

leoi

d

Virio

n pa

rtVi

rion

Tran

slatio

n re

gulat

or ac

tivity

Tran

spor

ter a

ctiv

ity

Cata

lytic

activ

ity

Org

anel

leO

rgan

elle

par

t

Chem

oattr

acta

nt ac

tivity

Chan

nel r

egul

ator

activ

ity

Ant

ioxi

dant

activ

ity

Rece

ptor

activ

ityRe

cept

or re

gula

tor a

ctiv

ity

Class







PCA


EyespotsSpats

CL616Contig4_All

Unigene23340_All

Unigene32284_All

Unigene36784_All


Unigene45746_All

Unigene46572_All


minus002 000 002 004minus004PC1(656)

minus003

minus002

minus001

000

001

002

PC2(

228

)

(a)




0

10000

20000

30000

40000

50000

60000

70000

FPKM

(b)


18725

8539

977 327

minus13162

minus804000

minus4622minus21800

DownUp

DCBA

minus15000

minus10000

minus5000

0

5000

10000

15000

20000

25000

Num

ber o

f DEG

s



D-shape

Umbonal Eyespots

Spats

5973

6309 2314

3013

1085 1258 2966

725

20518

8237

639

1398

1244

776773


















Table 5 Continued










minus15

minus1

minus05

0

05

1

15







Wnt1

05

101520253035

Expr

essio

n le

vel


RELFPKM

(a)

Six3

0102030405060708090

100

Expr

essio

n le

vel

RELFPKM


(b)

Notch


005

115

225

335

445

5

Expr

essio

n le

vel

RELFPKM

(c)

Lox2


0123456789

Expr

essio

n le

vel

RELFPKM

(d)

Engrailed


01020304050607080

Expr

essio

n le

vel

RELFPKM

(e)

Branchyury

05

1015202530354045

Expr

essio

n le

vel


RELFPKM

(f)

MAPK


0123456789

Expr

essio

n le

vel

RELFPKM

(g)

pax7

02468

10121416

Expr

essio

n le

vel


RELFPKM

(h)





5 Conclusions


Additional Points


Competing Interests





Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









COG

KEGG Nr

Swissprot

219

637 8784

3193

92 1311 3971

108

12582

11406

268

427

7943

3849674













5911

269

4518

5055

802

4286

11336

3121

4357

1429

2108

158

28933199

905

3442

1915

404866 913

1252

1893

12069

4511



0

2500

5000

7500

10000

12500

Num

ber o

f gen

es



Function class






0

1000

2000

3000

4000

5000

6000

7000

8000

Num

bers

Biol

ogic

al ad

hesio

n

Cel

lC

ell j

unct

ion

Mac

rom

olec

ular

com

plex

Cel

l par

t

Mem

bran

e-en

close

d lu

men

Extr

acel

lula

r mat

rix

Extr

acel

lula

r reg

ion

part

Mem

bran

eM

embr

ane p

art

Extr

acel

lula

r mat

rix p

art

Extr

acel

lula

r reg

ion

Met

abol

ic p

roce

ss

Regu

latio

n of

bio

logi

cal p

roce

ss

Mul

tiorg

anism

pro

cess

Mul

ticel

lula

r org

anism

al p

roce

ssN

egat

ive r

egul

atio

n of

bio

logi

cal p

roce

ss

Cel

lula

r com

pone

nt o

rgan

izat

ion

or b

ioge

nesis

Imm

une s

yste

m p

roce

ss

Repr

oduc

tion

Repr

oduc

tive p

roce

ssRe

spon

se to

stim

ulus

Esta

blish

men

t of l

ocal

izat

ion

Sing

le-o

rgan

ism p

roce

ss

Cel

l pro

lifer

atio

nC

ell k

illin

gCa

rbon

util

izat

ion

Dev

elopm

enta

l pro

cess

Dea

th

Loca

lizat

ion

Cel

lula

r pro

cess

Biol

ogic

al re

gula

tion

Posit

ive r

egul

atio

n of

bio

logi

cal p

roce

ss

Loco

mot

ion

Gro

wth

Sign

alin

g

Prot

ein

bind

ing

tran

scrip

tion

fact

or ac

tivity

Mol

ecul

ar tr

ansd

ucer

activ

ityM

orph

ogen

activ

ity

Met

allo

chap

eron

e act

ivity

Bind

ing

Syna

pse

Syna

pse p

art

Stru

ctur

al m

olec

ule a

ctiv

ity

Enzy

me r

egul

ator

activ

ityEl

ectro

n ca

rrie

r act

ivity

Nuc

leic

acid

bin

ding

tran

scrip

tion

fact

or ac

tivity

Nuc

leoi

d

Virio

n pa

rtVi

rion

Tran

slatio

n re

gulat

or ac

tivity

Tran

spor

ter a

ctiv

ity

Cata

lytic

activ

ity

Org

anel

leO

rgan

elle

par

t

Chem

oattr

acta

nt ac

tivity

Chan

nel r

egul

ator

activ

ity

Ant

ioxi

dant

activ

ity

Rece

ptor

activ

ityRe

cept

or re

gula

tor a

ctiv

ity

Class







PCA


EyespotsSpats

CL616Contig4_All

Unigene23340_All

Unigene32284_All

Unigene36784_All


Unigene45746_All

Unigene46572_All


minus002 000 002 004minus004PC1(656)

minus003

minus002

minus001

000

001

002

PC2(

228

)

(a)




0

10000

20000

30000

40000

50000

60000

70000

FPKM

(b)


18725

8539

977 327

minus13162

minus804000

minus4622minus21800

DownUp

DCBA

minus15000

minus10000

minus5000

0

5000

10000

15000

20000

25000

Num

ber o

f DEG

s



D-shape

Umbonal Eyespots

Spats

5973

6309 2314

3013

1085 1258 2966

725

20518

8237

639

1398

1244

776773


















Table 5 Continued










minus15

minus1

minus05

0

05

1

15







Wnt1

05

101520253035

Expr

essio

n le

vel


RELFPKM

(a)

Six3

0102030405060708090

100

Expr

essio

n le

vel

RELFPKM


(b)

Notch


005

115

225

335

445

5

Expr

essio

n le

vel

RELFPKM

(c)

Lox2


0123456789

Expr

essio

n le

vel

RELFPKM

(d)

Engrailed


01020304050607080

Expr

essio

n le

vel

RELFPKM

(e)

Branchyury

05

1015202530354045

Expr

essio

n le

vel


RELFPKM

(f)

MAPK


0123456789

Expr

essio

n le

vel

RELFPKM

(g)

pax7

02468

10121416

Expr

essio

n le

vel


RELFPKM

(h)





5 Conclusions


Additional Points


Competing Interests





Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










5911

269

4518

5055

802

4286

11336

3121

4357

1429

2108

158

28933199

905

3442

1915

404866 913

1252

1893

12069

4511



0

2500

5000

7500

10000

12500

Num

ber o

f gen

es



Function class






0

1000

2000

3000

4000

5000

6000

7000

8000

Num

bers

Biol

ogic

al ad

hesio

n

Cel

lC

ell j

unct

ion

Mac

rom

olec

ular

com

plex

Cel

l par

t

Mem

bran

e-en

close

d lu

men

Extr

acel

lula

r mat

rix

Extr

acel

lula

r reg

ion

part

Mem

bran

eM

embr

ane p

art

Extr

acel

lula

r mat

rix p

art

Extr

acel

lula

r reg

ion

Met

abol

ic p

roce

ss

Regu

latio

n of

bio

logi

cal p

roce

ss

Mul

tiorg

anism

pro

cess

Mul

ticel

lula

r org

anism

al p

roce

ssN

egat

ive r

egul

atio

n of

bio

logi

cal p

roce

ss

Cel

lula

r com

pone

nt o

rgan

izat

ion

or b

ioge

nesis

Imm

une s

yste

m p

roce

ss

Repr

oduc

tion

Repr

oduc

tive p

roce

ssRe

spon

se to

stim

ulus

Esta

blish

men

t of l

ocal

izat

ion

Sing

le-o

rgan

ism p

roce

ss

Cel

l pro

lifer

atio

nC

ell k

illin

gCa

rbon

util

izat

ion

Dev

elopm

enta

l pro

cess

Dea

th

Loca

lizat

ion

Cel

lula

r pro

cess

Biol

ogic

al re

gula

tion

Posit

ive r

egul

atio

n of

bio

logi

cal p

roce

ss

Loco

mot

ion

Gro

wth

Sign

alin

g

Prot

ein

bind

ing

tran

scrip

tion

fact

or ac

tivity

Mol

ecul

ar tr

ansd

ucer

activ

ityM

orph

ogen

activ

ity

Met

allo

chap

eron

e act

ivity

Bind

ing

Syna

pse

Syna

pse p

art

Stru

ctur

al m

olec

ule a

ctiv

ity

Enzy

me r

egul

ator

activ

ityEl

ectro

n ca

rrie

r act

ivity

Nuc

leic

acid

bin

ding

tran

scrip

tion

fact

or ac

tivity

Nuc

leoi

d

Virio

n pa

rtVi

rion

Tran

slatio

n re

gulat

or ac

tivity

Tran

spor

ter a

ctiv

ity

Cata

lytic

activ

ity

Org

anel

leO

rgan

elle

par

t

Chem

oattr

acta

nt ac

tivity

Chan

nel r

egul

ator

activ

ity

Ant

ioxi

dant

activ

ity

Rece

ptor

activ

ityRe

cept

or re

gula

tor a

ctiv

ity

Class







PCA


EyespotsSpats

CL616Contig4_All

Unigene23340_All

Unigene32284_All

Unigene36784_All


Unigene45746_All

Unigene46572_All


minus002 000 002 004minus004PC1(656)

minus003

minus002

minus001

000

001

002

PC2(

228

)

(a)




0

10000

20000

30000

40000

50000

60000

70000

FPKM

(b)


18725

8539

977 327

minus13162

minus804000

minus4622minus21800

DownUp

DCBA

minus15000

minus10000

minus5000

0

5000

10000

15000

20000

25000

Num

ber o

f DEG

s



D-shape

Umbonal Eyespots

Spats

5973

6309 2314

3013

1085 1258 2966

725

20518

8237

639

1398

1244

776773


















Table 5 Continued










minus15

minus1

minus05

0

05

1

15







Wnt1

05

101520253035

Expr

essio

n le

vel


RELFPKM

(a)

Six3

0102030405060708090

100

Expr

essio

n le

vel

RELFPKM


(b)

Notch


005

115

225

335

445

5

Expr

essio

n le

vel

RELFPKM

(c)

Lox2


0123456789

Expr

essio

n le

vel

RELFPKM

(d)

Engrailed


01020304050607080

Expr

essio

n le

vel

RELFPKM

(e)

Branchyury

05

1015202530354045

Expr

essio

n le

vel


RELFPKM

(f)

MAPK


0123456789

Expr

essio

n le

vel

RELFPKM

(g)

pax7

02468

10121416

Expr

essio

n le

vel


RELFPKM

(h)





5 Conclusions


Additional Points


Competing Interests





Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



0

1000

2000

3000

4000

5000

6000

7000

8000

Num

bers

Biol

ogic

al ad

hesio

n

Cel

lC

ell j

unct

ion

Mac

rom

olec

ular

com

plex

Cel

l par

t

Mem

bran

e-en

close

d lu

men

Extr

acel

lula

r mat

rix

Extr

acel

lula

r reg

ion

part

Mem

bran

eM

embr

ane p

art

Extr

acel

lula

r mat

rix p

art

Extr

acel

lula

r reg

ion

Met

abol

ic p

roce

ss

Regu

latio

n of

bio

logi

cal p

roce

ss

Mul

tiorg

anism

pro

cess

Mul

ticel

lula

r org

anism

al p

roce

ssN

egat

ive r

egul

atio

n of

bio

logi

cal p

roce

ss

Cel

lula

r com

pone

nt o

rgan

izat

ion

or b

ioge

nesis

Imm

une s

yste

m p

roce

ss

Repr

oduc

tion

Repr

oduc

tive p

roce

ssRe

spon

se to

stim

ulus

Esta

blish

men

t of l

ocal

izat

ion

Sing

le-o

rgan

ism p

roce

ss

Cel

l pro

lifer

atio

nC

ell k

illin

gCa

rbon

util

izat

ion

Dev

elopm

enta

l pro

cess

Dea

th

Loca

lizat

ion

Cel

lula

r pro

cess

Biol

ogic

al re

gula

tion

Posit

ive r

egul

atio

n of

bio

logi

cal p

roce

ss

Loco

mot

ion

Gro

wth

Sign

alin

g

Prot

ein

bind

ing

tran

scrip

tion

fact

or ac

tivity

Mol

ecul

ar tr

ansd

ucer

activ

ityM

orph

ogen

activ

ity

Met

allo

chap

eron

e act

ivity

Bind

ing

Syna

pse

Syna

pse p

art

Stru

ctur

al m

olec

ule a

ctiv

ity

Enzy

me r

egul

ator

activ

ityEl

ectro

n ca

rrie

r act

ivity

Nuc

leic

acid

bin

ding

tran

scrip

tion

fact

or ac

tivity

Nuc

leoi

d

Virio

n pa

rtVi

rion

Tran

slatio

n re

gulat

or ac

tivity

Tran

spor

ter a

ctiv

ity

Cata

lytic

activ

ity

Org

anel

leO

rgan

elle

par

t

Chem

oattr

acta

nt ac

tivity

Chan

nel r

egul

ator

activ

ity

Ant

ioxi

dant

activ

ity

Rece

ptor

activ

ityRe

cept

or re

gula

tor a

ctiv

ity

Class







PCA


EyespotsSpats

CL616Contig4_All

Unigene23340_All

Unigene32284_All

Unigene36784_All


Unigene45746_All

Unigene46572_All


minus002 000 002 004minus004PC1(656)

minus003

minus002

minus001

000

001

002

PC2(

228

)

(a)




0

10000

20000

30000

40000

50000

60000

70000

FPKM

(b)


18725

8539

977 327

minus13162

minus804000

minus4622minus21800

DownUp

DCBA

minus15000

minus10000

minus5000

0

5000

10000

15000

20000

25000

Num

ber o

f DEG

s



D-shape

Umbonal Eyespots

Spats

5973

6309 2314

3013

1085 1258 2966

725

20518

8237

639

1398

1244

776773


















Table 5 Continued










minus15

minus1

minus05

0

05

1

15







Wnt1

05

101520253035

Expr

essio

n le

vel


RELFPKM

(a)

Six3

0102030405060708090

100

Expr

essio

n le

vel

RELFPKM


(b)

Notch


005

115

225

335

445

5

Expr

essio

n le

vel

RELFPKM

(c)

Lox2


0123456789

Expr

essio

n le

vel

RELFPKM

(d)

Engrailed


01020304050607080

Expr

essio

n le

vel

RELFPKM

(e)

Branchyury

05

1015202530354045

Expr

essio

n le

vel


RELFPKM

(f)

MAPK


0123456789

Expr

essio

n le

vel

RELFPKM

(g)

pax7

02468

10121416

Expr

essio

n le

vel


RELFPKM

(h)





5 Conclusions


Additional Points


Competing Interests





Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


PCA


EyespotsSpats

CL616Contig4_All

Unigene23340_All

Unigene32284_All

Unigene36784_All


Unigene45746_All

Unigene46572_All


minus002 000 002 004minus004PC1(656)

minus003

minus002

minus001

000

001

002

PC2(

228

)

(a)




0

10000

20000

30000

40000

50000

60000

70000

FPKM

(b)


18725

8539

977 327

minus13162

minus804000

minus4622minus21800

DownUp

DCBA

minus15000

minus10000

minus5000

0

5000

10000

15000

20000

25000

Num

ber o

f DEG

s



D-shape

Umbonal Eyespots

Spats

5973

6309 2314

3013

1085 1258 2966

725

20518

8237

639

1398

1244

776773


















Table 5 Continued










minus15

minus1

minus05

0

05

1

15







Wnt1

05

101520253035

Expr

essio

n le

vel


RELFPKM

(a)

Six3

0102030405060708090

100

Expr

essio

n le

vel

RELFPKM


(b)

Notch


005

115

225

335

445

5

Expr

essio

n le

vel

RELFPKM

(c)

Lox2


0123456789

Expr

essio

n le

vel

RELFPKM

(d)

Engrailed


01020304050607080

Expr

essio

n le

vel

RELFPKM

(e)

Branchyury

05

1015202530354045

Expr

essio

n le

vel


RELFPKM

(f)

MAPK


0123456789

Expr

essio

n le

vel

RELFPKM

(g)

pax7

02468

10121416

Expr

essio

n le

vel


RELFPKM

(h)





5 Conclusions


Additional Points


Competing Interests





Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















Table 5 Continued










minus15

minus1

minus05

0

05

1

15







Wnt1

05

101520253035

Expr

essio

n le

vel


RELFPKM

(a)

Six3

0102030405060708090

100

Expr

essio

n le

vel

RELFPKM


(b)

Notch


005

115

225

335

445

5

Expr

essio

n le

vel

RELFPKM

(c)

Lox2


0123456789

Expr

essio

n le

vel

RELFPKM

(d)

Engrailed


01020304050607080

Expr

essio

n le

vel

RELFPKM

(e)

Branchyury

05

1015202530354045

Expr

essio

n le

vel


RELFPKM

(f)

MAPK


0123456789

Expr

essio

n le

vel

RELFPKM

(g)

pax7

02468

10121416

Expr

essio

n le

vel


RELFPKM

(h)





5 Conclusions


Additional Points


Competing Interests





Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Table 5 Continued










minus15

minus1

minus05

0

05

1

15







Wnt1

05

101520253035

Expr

essio

n le

vel


RELFPKM

(a)

Six3

0102030405060708090

100

Expr

essio

n le

vel

RELFPKM


(b)

Notch


005

115

225

335

445

5

Expr

essio

n le

vel

RELFPKM

(c)

Lox2


0123456789

Expr

essio

n le

vel

RELFPKM

(d)

Engrailed


01020304050607080

Expr

essio

n le

vel

RELFPKM

(e)

Branchyury

05

1015202530354045

Expr

essio

n le

vel


RELFPKM

(f)

MAPK


0123456789

Expr

essio

n le

vel

RELFPKM

(g)

pax7

02468

10121416

Expr

essio

n le

vel


RELFPKM

(h)





5 Conclusions


Additional Points


Competing Interests





Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 5 Continued










minus15

minus1

minus05

0

05

1

15







Wnt1

05

101520253035

Expr

essio

n le

vel


RELFPKM

(a)

Six3

0102030405060708090

100

Expr

essio

n le

vel

RELFPKM


(b)

Notch


005

115

225

335

445

5

Expr

essio

n le

vel

RELFPKM

(c)

Lox2


0123456789

Expr

essio

n le

vel

RELFPKM

(d)

Engrailed


01020304050607080

Expr

essio

n le

vel

RELFPKM

(e)

Branchyury

05

1015202530354045

Expr

essio

n le

vel


RELFPKM

(f)

MAPK


0123456789

Expr

essio

n le

vel

RELFPKM

(g)

pax7

02468

10121416

Expr

essio

n le

vel


RELFPKM

(h)





5 Conclusions


Additional Points


Competing Interests





Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




minus15

minus1

minus05

0

05

1

15







Wnt1

05

101520253035

Expr

essio

n le

vel


RELFPKM

(a)

Six3

0102030405060708090

100

Expr

essio

n le

vel

RELFPKM


(b)

Notch


005

115

225

335

445

5

Expr

essio

n le

vel

RELFPKM

(c)

Lox2


0123456789

Expr

essio

n le

vel

RELFPKM

(d)

Engrailed


01020304050607080

Expr

essio

n le

vel

RELFPKM

(e)

Branchyury

05

1015202530354045

Expr

essio

n le

vel


RELFPKM

(f)

MAPK


0123456789

Expr

essio

n le

vel

RELFPKM

(g)

pax7

02468

10121416

Expr

essio

n le

vel


RELFPKM

(h)





5 Conclusions


Additional Points


Competing Interests





Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Wnt1

05

101520253035

Expr

essio

n le

vel


RELFPKM

(a)

Six3

0102030405060708090

100

Expr

essio

n le

vel

RELFPKM


(b)

Notch


005

115

225

335

445

5

Expr

essio

n le

vel

RELFPKM

(c)

Lox2


0123456789

Expr

essio

n le

vel

RELFPKM

(d)

Engrailed


01020304050607080

Expr

essio

n le

vel

RELFPKM

(e)

Branchyury

05

1015202530354045

Expr

essio

n le

vel


RELFPKM

(f)

MAPK


0123456789

Expr

essio

n le

vel

RELFPKM

(g)

pax7

02468

10121416

Expr

essio

n le

vel


RELFPKM

(h)





5 Conclusions


Additional Points


Competing Interests





Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




5 Conclusions


Additional Points


Competing Interests





Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Differential Gene Expression …...Research Article Differential Gene Expression during Larval Metamorphic Development in the Pearl Oyster, Pinctada fucata ,Basedon