research article transcriptome analysis of the carmine...

Research ArticleTranscriptome Analysis of the Carmine Spider MiteTetranychus cinnabarinus (Boisduval 1867)(Acari Tetranychidae) and Its Response to 120573-Sitosterol

Chunya Bu12 Jinling Li3 Xiao-Qin Wang12 Guanglu Shi12 Bo Peng3

Jingyu Han3 Pin Gao3 and Younian Wang12

1College of Biological Science and Engineering Beijing University of Agriculture Beijing 102206 China2Key Laboratory of Urban Agriculture (North) Ministry of Agriculture Beijing 102206 China3Plant Science and Technology College Beijing University of Agriculture Beijing 102206 China

Correspondence should be addressed to Younian Wang chunyagmailcom

Received 5 January 2015 Accepted 12 February 2015

Academic Editor Marina Sokovic

Copyright copy 2015 Chunya Bu 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

Tetranychus cinnabarinus (Acari Tetranychidae) is a worldwide polyphagous agricultural pest that has the title of resistancechampion among arthropods We reported previously the identification of the acaricidal compound 120573-sitosterol from Menthapiperita and Inula japonica However the acaricidal mechanism of 120573-sitosterol is unclear Due to the limited genetic researchcarried out we de novo assembled the transcriptome of T cinnabarinus using Illumina sequencing and conducted a differentialexpression analysis of control and 120573-sitosterol-treated mites In total we obtained gt54G high-quality bases for each sample withunprecedented sequencing depth and assembled them into 22941 unigenes We identified 617 xenobiotic metabolism-relatedgenes involved in detoxification binding and transporting of xenobiotics A highly expanded xenobiotic metabolic system wasfound in mites T cinnabarinus detoxification genesmdashincluding carboxylcholinesterase and ABC transporter class Cmdashwereupregulated after 120573-sitosterol treatment Defense-related proteins such as Toll-like receptor legumain and serine proteaseswere also activated Furthermore other important genesmdashsuch as the chloride channel protein cytochrome b carboxypeptidaseperitrophic membrane chitin binding protein and calphostinmdashmay also play important roles in mitesrsquo response to 120573-sitosterolOur results demonstrate that high-throughput-omics tool facilitates identification of xenobiotic metabolism-related genes andillustration of the acaricidal mechanisms of 120573-sitosterol

1 Introduction

The carmine spider mite Tetranychus cinnabarinus (Bois-duval 1867) (Acari Tetranychidae) is one of the mostsignificant species of the genus Tetranychus which includesover 140 species [1 2] It belongs to the class Arachnidainfraclass Acari order Prostigmata and family Tetranychi-dae T cinnabarinus is an economically important pest thatinfests greenhouse and field crops such as tomatoes pepperscucumbers strawberries maize soy apples grapes andcitrus [1] T cinnabarinus is one of the most polyphagousarthropod herbivores known It has been documented to feedon more than 1100 plant species belonging to gt140 plant

families [1] This mite has a worldwide distribution and hasan extensive host plant range throughout ChinaThese spidermites are the ldquoresistance championrdquo among arthropods asthey have an extreme record of resistance to almost all aca-ricides [1 3 4] Unreasonable and intensive use of acaricideshas accelerated the development of resistance by mites inChina Rapid development high fecundity and haplodiploidsex determination may contribute to the rapid evolution ofpesticide resistance in this mite [1]

Considering the seriousness of themite problem it is nec-essary to develop alternative acaricides to overcome the resis-tance of T cinnabarinus Botanical pesticides are desirable inpest management as they are often environmentally benign

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 794718 12 pageshttpdxdoiorg1011552015794718

2 BioMed Research International

and induce resistance less frequently 120573-Sitosterol one ofthree common phytosterols was isolated from a petroleum-ether extract of Sapium baccatum (Roxb) leaves and showshigh activity in a brine shrimp bioassay with an LC

50of

158mgmL [5] In addition 120573-sitosterol has been identifiedin Dunalia spinosa (Meyen) extracts where it exhibits strongantimicrobial activity [6]120573-Sitosterol is present in a variety ofplants and is an antitumor antimicrobial chemopreventiveand immunomodulatory compound [7 8]120573-Sitosterol is alsothe active compound of the mulberry plant that induces bit-ing behavior from Bombyx mori (Linnaeus) in a low concen-tration [9] However 120573-sitosterol alone has greatly reducedeffects on biting behavior of Antheraea assamensis (Helfer)[10] Antifungal and antibacterial activities of 120573-sitosterolhave been detected in a methanol extract of dried-groundaerial parts of Senecio lyratus (Bullock) [11] We previouslyisolated 120573-sitosterol from Mentha piperita (Linnaeus) andreported high acaricidal activity (LC

50 05mgmL) [12] 120573-

Sitosterol may thus be a candidate lead botanical compoundHowever the acaricidal mechanism of 120573-sitosterol is unclear

The availability of genome and transcriptome sequencesprovides unique information and enables investigation of themode of action of an acaricidal compound and the rolesof the gene families involved in pesticide metabolism inspider mites [13ndash15] Characterizing the spider mite genefamilies associated with 120573-sitosterol metabolism is the firststep towards a better understanding of the effects of 120573-sitosterol on spider mites In this study we performed adetailed analysis of the expression of120573-sitosterolmetabolism-associated gene families in T cinnabarinus

2 Materials and Methods

21 Carmine Spider Mite Collection and Handling A colonyof T cinnabarinus (Boisduval 1867) was originally collectedfrom a field in Qingxu County Taiyuan Municipality ShanxiProvince China in the spring of 2004 and had been freeof pesticides for gt10 years This insecticide-susceptible strainof T cinnabarinus was maintained at the Key Laboratory ofUrban Agriculture (North) Ministry of Agriculture (BeijingChina) at 26 plusmn 2∘C 60 relative humidity (RH) and a 16 hlight 8 h dark photoperiod under pesticide-free conditionsThe mites were reared on Vigna unguiculata (L) leaves atthe sixth true-leaf growth stage in a greenhouse The miteswere tested annually for high sensitivity to organophosphateinsecticides using the glass slide-dipping method accordingto FAO standards

22 120573-Sitosterol Treatment Our laboratory determined pre-viously that 120573-sitosterol has high acaricidal activity against TcinnabarinusThe changes in T cinnabarinus gene expressionwere evaluated after exposure to a sublethal concentration(LC30) of 120573-sitosterol according to the procedure described

by [16 17] The LC30is the concentration required to kill 30

of the members of a tested population after a specified testduration and is commonly used in mite pesticide exposureresearch [18] The mites were treated with 120573-sitosterol usingthe glass slide-dipping method according to the FAO [19]120573-sitosterol (Sigma-Aldrich St Louis MO USA) was diluted

in sterile distilled water containing 1 Tween 80 (Sigma-Aldrich) and 06 DMSO (Sigma-Aldrich) Sterile distilledwater with 1 Tween 80 and 06 DMSO was used as thecontrol Killing of the female adults is critical for controllingspider mites Thus female adults were used in our experi-ment Female adult mites were affixed onto one end of a 10 times2 cm glass slide using double-sided adhesive tapeThe affixedmites were checked under a stereomicroscope and inactivemites were removedThemites were dipped individually into120573-sitosterol solutions for 5 s Following removal of the slidethe excess solution was absorbed with filter paper All treatedmites were maintained at 26 plusmn 2∘C 60 RH and a 16 h light8 h dark photoperiod The dead mites were removed 24 hafter treatment and the live mites were frozen immediatelyin liquid nitrogen and stored for later extraction of RNA [19]This experiment was repeated twice

23 RNA Extraction cDNA Library Construction and Illu-mina Sequencing Total RNAs were extracted using TRIzolreagent (Invitrogen Beijing China) according to the man-ufacturerrsquos instructions Genomic DNA was removed usingRNase-free DNase (Takara Kyoto Japan) The quantity andquality of total RNA were determined using a NanoDrop2000 UV-Vis Spectrophotometer (Thermo Scientific Wilm-ington DE USA) Ultrasec 2100 pro UVVisible Spectropho-tometer (Amersham Biosciences Uppsala Sweden) and gelelectrophoresis

Poly-(A) mRNA was enriched and isolated from thehigh-quality total RNA samples using Magnetic Oligo(dT)Beads followed by fragmentation of the mRNA with aRNA fragmentation kit (Ambion Austin TX USA) Reversetranscription into cDNA was performed using random hex-amers and reverse transcriptase (Promega Madison WIUSA) and then second-strand cDNA was synthesized usingDNA polymerase I (Invitrogen) and RNaseH (New EnglandBiolabs Ipswich MA USA) After purification with theQIAquick PCRPurificationKit (Qiagen Valencia CA USA)the cDNA fragments were end-repaired and ligated to aSolexa adaptor after adding a single ldquoArdquo at the 31015840 end Thedesired size of adaptor-ligated fragment was selected byagarose gel electrophoresis Polymerase chain reaction (PCR)was performed to selectively enrich and amplify the selectedcDNA fragments to construct cDNA libraries for paired-end sequencing using the Illumina HiSeq 2000 (BiomarkerTechnologies Co Ltd Beijing China)

24 De Novo Assembly and Clustering Analysis De novoassembly of the mRNA-seq reads was performed using theTrinity method (httptrinityrnaseqsourceforgenet) afterthe raw reads were filtered by discarding the adaptorsequences low-quality sequences (reads with ambiguous ldquoNrdquobases) and reads in which gt10 of the bases had 119876 lt 20An optimized k-mer length of 25 was used to assemble thetranscriptome A clustering analysis was then conducted toconstruct the unigene database

25 Functional Annotation Open reading frames (ORF) ofthe unigenes were predicted using the ldquogetorf rdquo program in

BioMed Research International 3

the EMBOSS software package (httpembosssourceforgenetappscvsembossappsgetorfhtml) and the longestORFwas selected for each unigene Unigene functions wereannotated by a BLASTX search against the nonredundantprotein database (httpwwwncbinlmnihgov) the Swiss-Prot database (httpwwwuniprotorg) the TrEMBL data-base (httpwwwuniprotorg) the clusters of orthologousgroups (COG) database (httpwwwncbinlmnihgovCOG) and the Kyoto Encyclopedia of Genes and Genomes(KEGG database httpwwwgenomejpkegg) using aBLASTN search against the nonredundant nucleotide data-base (Nt database httpwwwncbinlmnihgov) and by aBlast2GO search against the Gene Ontology (GO) database(httpwwwgeneontologyorg) with an 119864-value le 1119890minus5

26 Simple Sequence Repeat (SSR) Analysis The MISA soft-ware (MIcroSAtellite identification tool httppgrcipk-gate-rslebendemisa) was used to identify potential SSRs fromthe unigene database The parameters were set as followsa minimum of 10 repeats for mononucleotides six fordinucleotides and five for tri- penta- and hexanucleotidemotifs

27 Differential Expression of Unigenes Differential expres-sion of the unigenes between control mites and the 120573-sitosterol-treated mites was analyzed using the IDEG6 soft-ware (httptelethonbiounipditbioinfoIDEG6) In thisstudy false discovery rate (FDR)le 001 a reads per kilobase ofexonmodel per millionmapped reads (PRKM) value ge2 andthe absolute value of log

2ratio ge1 were used as the thresholds

of significant differences in gene expression between controland 120573-sitosterol treated mites

28 Reverse Transcription Quantitative Real-Time PCR (RT-qPCR) A subset of highly differentially expressed genes wasvalidated by RT-qPCR Total RNA from the 120573-sitosterol-treated female adult mites and the control female adult miteswas extracted as described above and total RNA qualitywas high The genomic DNA was removed and cDNA wassynthesized using a PrimeScript RT reagent kit with thegDNA Eraser (Takara Dalian China) qPCR was performedon an ABI 7300 Real-Time PCR System (ABI) using SYBRPremix Ex Taq II (Tli RNaseH Plus) (Takara Kyoto Japan)The qPCR was performed as follows 95∘C for 30 s followedby 40 cycles of 5 s at 95∘C and 31 s at 60∘C and a meltingcurve analysis was conducted from 60 to 95∘C The qPCRprimers are listed in Table S1 in Supplementary Materialavailable online at httpdxdoiorg1011552015794718 No-reverse-transcription controls and nontemplate controls wereincluded on each plate Each of the two primer pairs for thetested genes and endogenous references produced a singlepeak in melting curve analyses and gel electrophoresis andhad PCR efficiency close to 100 as determined using acDNA dilution series obtained from a single sample

Transcript levels of the tested genes were determinedusing the 120573-actin and a-tub genes as endogenous referencesfor normalization by the 2minusΔΔCt method Experiments wererepeated three times Differences in mRNA levels between

Table 1 Summary of Illumina transcriptome sequencing forTetran-ychus cinnabarinus

Control 120573-Sitosterol-exposedTotal number ofreads 27495256 26870180

Total number ofnucleotides (bp) 5553615807 5427335287

11987620percentage 9888 9888

GC percentage 3887 3910

the 120573-sitosterol-treated and control mites were statisticallyanalyzed using equal or unequal variance 119905-tests dependingon the results of 119865-tests for homoscedasticity

3 Results

31 Sequence Analysis and De Novo Assembly The transcrip-tome of 120573-sitosterol-treated female adult and control miteswere sequenced using the Illumina HiSeq 2000 We obtainedgt54G high-quality bases with 9888 119876

20bases for each T

cinnabarinus sample after removing the dirty reads from theraw reads an unprecedented sequencing depth (Table 1)TheT cinnabarinus contigs had an average GC content of 3898

These high-quality reads were de novo assembled usingthe Trinity method and 22941 unigenes with a meanlength of 1468 bp were obtained (Table 2) All unigenes weregt200 bp with ge2000 bp sequences representing the highestproportion followed by 1000ndash2000 bp sequences A total of11114 (4845) unigenes were longer than 1000 bp and 5960(2598) were longer than 2000 bp In total 14831 (6465)unigenes were longer than 500 bp These results show thatIllumina sequencing enables rapid capture of a large portionof a transcriptome

32 Functional Annotation All unique unigenes were anno-tated by a BLAST search against the sequences in the nonre-dundant database SwissProt database TrEMBLdatabaseGOdatabase COG database KEGG database and Nt databasewith a cut-off E-value of 1 times 10minus5 A total of 12049 of all(5252) unigenes were annotated in our database (Table 3)A wide array of genes was homologous to genes in differentclasses and different organisms A total of 11453 unigenes hadmatches in the Nr database and 4754 had matches in the Ntdatabase whereas 9259 unigenes yielded a significant hit inthe SwissProt database

The unigenes for the GO analysis were divided intothree categories and 53 subcategories 2918 unigenes forcellular components (16 subcategories) 1476 for molecularfunctions (15 subcategories) and 5605 for biological pro-cesses (22 subcategories) (Figure 1) Some of these unigenesparticipated in multiple GO terms The three major subcate-gories were cellular and metabolic processes in the biologicalprocesses category and cell parts in the cellular componentcategory The smallest subcategories were metallochaperoneactivity (molecular functions) viral reproduction (biological


7

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olec

ular

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Mem

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embr

ane-

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e par

tC

ell j

unct

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r reg

ion

part

Extr

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rixEx

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lar m

atrix

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tN

ucle

oid

Bind

ing

Cata

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ityTr

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orte

r act

ivity

Mol

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ar tr

ansd

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ityRe

cept

or ac

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leic

acid

bin

ding

tran

scrip

tion

fact

or ac

tivity

Stru

ctur

al m

olec

ule a

ctiv

ityEn

zym

e reg

ulat

or ac

tivity

Prot

ein

bind

ing

tran

scrip

tion

fact

or ac

tivity

Ant

ioxi

dant

activ

ityEl

ectro

n ca

rrie

r act

ivity

Tran

slatio

n re

gula

tor a

ctiv

ityCh

anne

l reg

ulat

or ac

tivity

Mor

phog

en ac

tivity

Met

allo

chap

eron

e act

ivity

Cel

lula

r pro

cess

Met

abol

ic p

roce

ssD

evelo

pmen

tal p

roce

ssBi

olog

ical

regu

latio

nM

ultic

ellu

lar o

rgan

ismal

pro

cess

Resp

onse

to st

imul

usLo

caliz

atio

nRe

prod

uctio

nC

ellul

ar co

mpo

nent

org

aniz

atio

n or

bio

gene

sisRe

prod

uctiv

e pro

cess

Esta

blish

men

t of l

ocal

izat

ion

Sign

alin

gLo

com

otio

nM

ulti-

orga

nism

pro

cess

Gro

wth

deat

hIm

mun

e sys

tem

pro

cess

Biol

ogic

al ad

hesio

nC

ell p

rolif

erat

ion

Rhyt

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pro

cess

Pigm

enta

tion

Vira

l rep

rodu

ctio

n

Num

ber o

f gen

es

Gen

es (

)

Cellular component Molecular function Biological process

Tetranychus

GO classification

Figure 1 Functional annotation of assembled sequences based on the Gene Ontology (GO) categorization The 119909-axis indicates the GOcategories which are grouped into three main ontologies cellular components molecular functions and biological processes The left 119910-axisindicates the percentage of genes in the category whereas the right 119910-axis indicates the number of genes in each category ldquoTetranychusrdquoindicates that the unigenes were assembled using the reads from control and 120573-sitosterol-exposed Tetranychus cinnabarinus

Table 2 The length distribution of unigenes after de novo assemblyusing the reads from control and 120573-sitosterol-exposed Tetranychuscinnabarinus

Length range The unigene number Percentage200ndash300 4495 1959300ndash500 3615 1576500ndash1000 3717 16201000ndash2000 5154 22472000+ 5960 2598Total number of unigenes 22941Total length of unigenes 3369694911987350length of unigenes 2534

Mean length of unigenes 146885

processes) morphogen activity (molecular functions) andnucleoid (cellular components)

In addition 4283 unigenes had significantmatches in theCOG database Among the 25 COG categories the ldquogeneralfunction predictionrdquo cluster occupied the highest propor-tion (1273 2166) followed by ldquoreplication recombination

Table 3 Function annotation of theTetranychus cinnabarinus trans-criptome

Annotateddatabases All sequences ge300 bp ge1000 bp

COG 4283 552 3626GO 7180 1184 5702KEGG 4455 633 3676Swissprot 9259 1563 7370TrEMBL 11442 2140 8783nr 11453 2151 8788nt 4754 805 3768All 12049 2415 9001

and repairrdquo (463 788) and ldquotranscriptionrdquo (438 745)(Figure 2) The three smallest groups were ldquoextracellularstructuresrdquo ldquocell cycle controlrdquo ldquocell division and chromo-some partitioningrdquo and ldquocell motilityrdquo

33 SSR Discovery SSRs are widely used for evolution andgenetics studies [20 21] The spider mite had a relatively


Table 4 Summary of simple sequence repeat (SSR) types in the Tetranychus cinnabarinus transcriptome

Repeat motif Numbera Percentage () Repeat motif Numbera Percentage ()b

Dinucleotide Tetra-nucleotideACGT 143 AAACGTTT 3AGCT 468 AAAGCTTT 5ATAT 85 AAATATTT 6Total 696 3017 AAGCCTTG 2Trinucleotide AATCATTG 2AACGTT 310 AATGATTC 2AAGCTT 182 ACTCAGTG 1AATATT 182 Total 21 091ACCGGT 75 PentanucleotideACGCGT 2 AAAACGTTTT 1ACTAGT 8 AAGAGCTCTT 1AGCCTG 59 AATATATATT 1AGGCCT 48 Total 3 013ATCATG 715 Compound SSR 4 017CCGCGG 2Total 1583 6862aNumber of the specified SSRs detected in unigenesbThe relative percentage of the specified SSRs among the total SSRs

low density of microsatellites (Table 4) Even dinucleotiderepeats (696 3017) which are typically the most abundantmicrosatellite type were markedly less frequent than trin-ucleotides (1583 6862) Tetranucleotide pentanucleotideand compound SSRs comprised lt1 of all SSRs

34 Analysis of Genes Involved in Insecticide Metabolism Tounderstand how T cinnabarinus metabolizes xenobioticssuch as insecticides and secondary plant chemicals we identi-fied known genes implicated in digestion detoxification andxenobiotic transport (Table 5) The xenobiotic metabolic sys-tem of the spider mite was expanded Eighty-one cytochromeP450 (CYP) genes were detected in the T cinnabarinustranscriptome (Tables S2 and S3) Thirty-five glutathione S-transferases (GSTs) and 28 carboxylcholinesterases (CCEs)were identified (Tables S4ndashS6) In addition a group of11 Mu-class GSTs were detected within the family of 35GSTs There were also a large number of genes involved ingeneral pesticide detoxification six catalases 12 superoxidedismutases and 28 NADH dehydrogenases

In addition to metabolizing enzymes a large numberof transporters were also identified We identified 49 ATP-binding cassette transporters (class C) The MFS family isone of the largest membrane transporter families along withABC transporters Seventeen major facilitator superfamilygenes were annotated in T cinnabarinus The transporterswere expanded in T cinnabarinus

The main insecticide targets for commonly used insec-ticides were identified in the T cinnabarinus transcriptome(Table 5) Nine gamma-aminobutyric acid (GABA) recep-tors 15 glutamate-gated chloride channels (GluCls) and 25nicotinic acetylcholine receptors (nAChR) were identified

One acetylcholinesterase (AChE) 10 voltage-gated sodiumchannels and nine cytochromes b were also identified Inaddition 14 GABA-gated chloride channels and two ryan-odine receptors were identified These target sequences willfacilitate understanding of the development of resistance inthese mites and assist in development of new pesticides

35 Differential Expression Analysis The differences in geneexpression levels between control and 120573-sitosterol-treatedmites after 120573-sitosterol exposure were analyzed using theIDEG6 software FDR le 001 PRKM value ge 2 and theabsolute value of the log

2ratio ge 1 were used as the threshold

to determine significant differences in gene expression Overhalf of all differentially expressed genes were assigned to aGO category (Figure 3) We found 34 genes upregulated and57 downregulated in the 120573-sitosterol-treated mites comparedto in the control mites (Figure 4 Table S7) The highlydifferentially expressed genes are listed in Table 6 Nine ofthe differentially expressed genes were annotated using theKEGGdatabase and eight pathwayswere involved Pathwayssuch as glycan degradation fatty acid biosynthesis antigenprocessing and presentation extracellular matrix- (ECM-)receptor interactions and oxidative phosphorylation playroles in detoxifying 120573-sitosterol

The expression profiles of some insecticide metabolicgenes changed such as CCE ABC transporter class Ccarboxypeptidase fatty acid synthase cytochrome b Toll-like receptor chloride channel proteins and serine pro-teases (Table 6) Among these differentially expressed genesdetoxification enzymes transporters small secreted proteinsmembrane-binding proteins and chitin proteins featuredprominently


J Translation ribosomal structure and biogenesisA RNA processing and modificationK TranscriptionL Replication recombination and repairB Chromatin structure and dynamicsD Cell cycle control cell division and chromosome partitioningY Nuclear structureV Defense mechanismsT Signal transduction mechanismsM Cell wallmembraneenvelope biogenesisN Cell motilityZ CytoskeletonW Extracellular structuresU Intracellular trafficking secretion and vesicular transportO Posttranslational modification protein turnover and chaperones C Energy production and conversionG Carbohydrate transport and metabolismE Amino acid transport and metabolismF Nucleotide transport and metabolismH Coenzyme transport and metabolismI Lipid transport and metabolismP Inorganic ion transport and metabolismQ Secondary metabolites biosynthesis transport and catabolism R General function prediction onlyS Function unknown

0

300

600

900

1200

1500

1800

J A K L B D Y V T MN Z WU O C G E F H I P Q R S

Freq

uenc

y

Function class

COG function classification of consensus sequence

Figure 2 Clusters of orthologous (COG) classification A total of4283 unigenes were grouped into 25 COG classifications The 119910-axis indicates the number of genes in a specific function cluster Thelegend shows the 25 function categories

36 RT-qPCR Validation Study A subset of 40 highly dif-ferentially expressed genes between the 120573-sitosterol-treatedand control mites identified by transcriptomic analyses werevalidated by RT-qPCR (Table 6)Thirty-six of the forty testedgenes showed agreement with the transcriptomic resultsTheexpression profiles of these 40 genes are shown in Table 6including CCE ABC transporter class C peritrophic mem-brane chitin binding protein calphostin fatty acid synthasechloride channel protein cytochrome b zinc carboxypepti-dase putative transcription factor and Toll-like receptor

Table 5 Genes involved in insecticide metabolism

Gene name Number of sequenceswith TrEMBL hit

Cytochrome P450 81Carboxylesterase 28Glutathione S-transferase 35Catalase 6Superoxide dismutase 12NADH dehydrogenase 28GABA receptor 9Cytochrome b 9Nicotinic acetylcholine receptor 25Voltage-gated sodium channel 10Glutamate-gated chloride channel 15GABA-gated chloride channel 3Ryanodine receptor 2Acetylcholinesterase 1MFS-type transporter 17ABC transporter 130Intradiol dioxygenase-like protein 17Nuclear receptor 15Major facilitator superfamily 26Kelch-like protein 33TWiK family of potassium channels 5Inward rectifier potassium channel 4Two pore potassium channel 3Calcium-activated potassium channel 9Potassium voltage-gated channel 29Voltage-dependent calcium channel 11Calcium release-activated calcium channel 3

4 Discussion

41 Exploring the SpiderMite Transcriptome TheTetranychusurticae (Koch 1836) genome (strain London) has beensequenced and the size of the genome was sim90Mb Thusthe T urticae genome is the smallest arthropod genomesequenced to date [1] However the T cinnabarinus genomeremains unknown According to available T cinnabarinusgene sequence information there are interesting differencesbetween T cinnabarinus and T urticae Here we performedde novo assembly of the T cinnabarinus transcriptome usingIllumina paired-end sequencing technology In total weobtained gt54G high-quality bases for each T cinnabarinussample with unprecedented sequencing depth (Table 1)These high-quality reads were de novo assembled and 22941unigenes were obtained with a mean length of 1468 bp(Table 2) The insecticide resistance-related genes of T cin-nabarinus were recently analyzed [22] Here we focused onthe differential expression of T cinnabarinus genes inducedby 120573-sitosterol treatment We explored genes involved in


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art

Cel

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egio

n pa

rtEx

trac

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atrix

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ansp

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ansd

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or ac

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olec

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ulat

or ac

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ioxi

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ectro

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rrie

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anne

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allo

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abol

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ssD

evelo

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tal p

roce

ssBi

olog

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regu

latio

nM

ultic

ellu

lar o

rgan

ismal

pro

cess

Resp

onse

to st

imul

usLo

caliz

atio

nRe

prod

uctiv

e pro

cess

Repr

oduc

tion

Esta

blish

men

t of l

ocal

izat

ion

Sign

alin

gLo

com

otio

nM

ulti-

orga

nism

pro

cess

Gro

wth

Dea

thIm

mun

e sys

tem

pro

cess

Biol

ogic

al ad

hesio

nC

ell p

rolif

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enta

tion

Vira

l rep

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ctio

n

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ber o

f gen

es

Gen

es (

)35

3

1

0


GO classification

All unigenesDEG unigenes

Cell

ular

com

pone

nt o

rgan

izat

ion

or b

ioge

nesis

Nuc

leic

acid

bin

ding

tran

scrip

tion

fact

or ac

tivity

Prot

ein

bind

ing

tran

scrip

tion

fact

or ac

tivity

Figure 3 Gene Ontology (GO) functional classification of all and differentially expressed unigenes ldquoAll unigenesrdquo indicates unigenes thatwere assembled using the reads from control and 120573-sitosterol-exposed mites ldquoDEG unigenerdquo indicates the unigenes differentially expressedbetween control and 120573-sitosterol-exposed mites

0

10

20

30

40

50

60

Upregulated Downregulated

Num

ber o

f uni

gene

s

Figure 4 Gene expression profiles of control and 120573-sitosterol-exposed mites

xenobiotic metabolism and 120573-sitosterol detoxification inparticular

The T cinnabarinus sequences we obtained were similarto those of other arthropods some species of insects andother organisms [17 23 24] The most related organisms areT urticae and Panonychus citri (McGregor 1916) [25 26]particularly T urticae More than 70 of the unigenes in ourdataset significantly matched the T urticae genome databasefollowing alignment of T cinnabarinus transcriptome tothe T urticae genome As mites have some of the smallestgenomes in the animal kingdom [23] T cinnabarinus has arelatively smallmicrosatellite density which is correlatedwith

its compact size [1 22] A significant portion of the unigeneshad novel sequences with unknown function These resultsshow that our knowledge of mite genes is limited and furtherresearch is needed to characterize mite genes and exploretheir functions [23]

42 Spider Mite Metabolism of Xenobiotics T cinnabarinushas an extensive host plant range and an extraordinary abilityto develop resistance to pesticides A link between the broadresponse to diverse synthetic and natural xenobiotics andthe evolution of pesticide resistance has been proposed [1327 28] However the specific metabolic processes involvedin digesting detoxifying and transporting xenobiotics havebeen unclear to date Increasing the metabolic capabilityof detoxifying systems and reducing xenobiotic target sitesensitivity are thought to be major mechanisms of devel-opment of xenobiotic resistance [27] Amplification overex-pression and variations in the coding sequences of the threemajor metabolic enzymesmdashCYP CCEs and GSTsmdashhavebeen reported [27] This spider mite has a unique xenobioticmetabolic system and a considerably greater number ofmajormetabolic enzymes compared to insects [1 29] This wasalso evident in our transcriptome data Eighty-one CYPstwenty-eight CCEs and thirty-five GSTs were identified inthe T cinnabarinus transcriptome Six major GST subclassesoccur in insects the sigma omega theta and zeta and


Table 6 The significantly differentially expressed unigenes between control and 120573-sitosterol-exposed mites

Number ID Annotation Differentially expressed genes KEGG annotationTranscriptome qPCR

T2 Unigene BMK2524 Carboxylcholinesterase Up UpT2 Unigene BMK7122 Carboxypeptidase Up Up mdashCL121Contig1 ABC transporter class C Up Up mdashT2 Unigene BMK2982 Chloride channel protein 2 Up Up K12880CL5336Contig1 Cytochrome b Up Up K00412CL1378Contig1 Calphostin Up Up mdashT2 Unigene BMK8426 Calphostin Up Up mdashT1 Unigene BMK15610 fatty acid synthase Down Down K00665CL8369Contig1 Peritrophic membrane chitin binding protein Up Up mdashCL8324Contig1 Fibroin Down Unchanged mdashT1 Unigene BMK9681 Fibroin Down Unchanged mdashT3 Unigene BMK5391 Fibroin Down Unchanged mdashT2 Unigene BMK8596 Fibroin Down Unchanged mdashCL8591Contig1 Fibroin heavy chain (Precursor) Down Down mdashT3 Unigene BMK5709 Fibroin heavy chain (Precursor) Down Down mdashT1 Unigene BMK7441 Putative transcription factor Up Up mdashCL9149Contig1 Toll-like receptor 8 Up Up mdashCL9567Contig1 legumain Up Up mdashT1 Unigene BMK2690 legumain Up Up K01369CL2236Contig1 Legumain Up Up K01369CL710Contig1 beta-mannosidase Down Down K01192T2 Unigene BMK6324 beta-mannosidase Down Down K01192CL7517Contig1 serine protease Up Up mdashT2 Unigene BMK3603 serine protease Up Up mdashCL1681Contig1 serine proteinase Up Up mdashCL7152Contig1 serine protease homologue Down Down mdashT2 Unigene BMK7468 serine protease homologue Up Up mdashT2 Unigene BMK3702 serine protease polymerase Up Up mdashT2 Unigene BMK6592 serine protease Up Up mdashT2 Unigene BMK1689 Probable serinethreonine-protein kinase Up Up mdashCL2365Contig1 Probable serinethreonine-protein kinase Up Up mdashCL5455Contig1 Fibrillar collagen precursor Up Up K06236T2 Unigene BMK12938 Histidine-rich glycoprotein Down Down mdashCL10295Contig1 Uncharacterized histidine-rich protein Down Down mdashCL3394Contig1 Endonuclease Down Down mdashCL7961Contig1 Ribonuclease Up Up mdashCL8740Contig1 RNA helicase Down Down mdashT2 Unigene BMK4212 Ras guanine nucleotide exchange factor Up Up mdashT2 Unigene BMK17224 helicase Up Up mdashCL8128Contig1 deoxyribonuclease Up Up K01158

the insect-specific delta and epsilon subclasses [30 31] Thedelta and epsilonGST classes seem to be involved in detoxify-ing xenobiotics However a group of Mu-class GSTs but notepsilon-class GSTs was found in T cinnabarinus Mu-classGSTs are believed to be vertebrate-specific [1] and may per-form the role of epsilon-class GSTs in Tetranychus taxa [32]CYP genes are assigned to four clades the CYP2 CYP3 andCYP4 clades and themitochondrial CYP clade (CYPM) [33]Expansion of clade 2 and contraction of clade 3were observed

in Tetranychus taxa compared to that in insects The CYP3and CYP4 clades in most insect species are involved indetoxifying xenobiotics and phytotoxins [34] whereas thesefunctions might be fulfilled by the CYP2 and CYP4 clades inTetranychus taxa [22]

The insect metabolic detoxification system typicallyincludes phase I and phase II metabolizing enzymes as wellas phase III transporters which are induced after xenobioticexposure Expansion of the transporters was also found in


T cinnabarinus Thirty-nine multidrug resistance proteinsbelonged to the ATP-binding cassette transporters (class C)[1] In this study 49 ATP-binding cassette transporters (classC) were found in T cinnabarinus which far exceeds thenumber (9ndash14) found in crustaceans insects vertebratesand nematodes [1] In brief many gene families from tran-scription factors to effector genes involved in detoxificationbinding and transport of xenobiotics were characterized inthis study The expansion of the xenobiotic metabolic systemsuggests amechanism for the high level of resistance of spidermites to almost all acaricides

The main targets of commonly used insecticides wereidentified in T cinnabarinus (Table 5) GABA receptorsGluCls and nAChR are important ligand-gated ion channels[35]TheGABA receptor is themain target of phenylpyrazoleinsecticides such as fipronil abamectin and cyclopentyldiene insecticides [22 36] We identified nine GABA recep-tors GluCls are the ideal targets for highly selective insecti-cides as they are found only in invertebrates [22] Insecticidestargeting GluCls include abamectin ivermectin fipronil andthe indole diterpenoid compound nodulisporic acid [22]FifteenGluCls were identified inT cinnabarinus in this studynAChR is another important selective insecticide targetNewly developed insecticides including nereistoxin neoni-cotinoid and the biological insecticide spinosad specificallytarget the insect nAChR [35] AChE is the major targetfor organophosphates and carbamates Insecticides targetingAChE account for more than one-third of insecticide salesworldwide [37] AChEmutations and their combinations canresult in varying levels of resistance to many organophos-phate and carbamate insecticides [38] In this study a singleAChE gene was identified in T cinnabarinus Voltage-gatedsodium channels are frequently studied voltage-gated ionchannels and are targets of pyrethroids and fenpropathrin[39] Here 10 sodium channel genes were identified Full-length target gene sequences can be amplified based on thepartial sequence obtained from the transcriptome Abundantinformation regarding the sequences of these pesticide targetswill improve our understanding of the molecular mecha-nisms of xenobiotic resistance It may be possible to designnew effective pesticides in the near future

43 Analysis of Differentially Expressed Genes Related to 120573-Sitosterol Metabolism 120573-Sitosterol is present in a variety ofplants and has antitumor antimicrobial chemopreventiveand immunomodulatory activities [7 8] We reported pre-viously identification of 120573-sitosterol in acaricidal fractionsof Mentha piperita [12] 120573-Sitosterol has also been detectedin Inula japonica Thunberg and had high acaricidal activityagainst T cinnabarinus [40] 120573-Sitosterol may be a candidatenovel botanical pesticideHowever the acaricidalmechanismof action of 120573-sitosterol is unclear

Large-scale sequencing technologies such as IlluminaSolexa enable evaluation of transcriptome dynamics undervarious conditions without sophisticated normalization ofdatasets [41] Investigating fine transcriptome variations aftershort-term exposure of insects to xenobiotics could provideinformation useful for identifying novel transcripts involved

in the response to xenobiotics and insecticide tolerance[42] We identified genes of many pathways involved inthe 120573-sitosterol-related response such as glycan degradationfatty acid biosynthesis antigen processing and presentationECM-receptor interactions and oxidative phosphorylationAmong these differentially expressed genes those encodingdetoxification enzymes defense-related factors silk proteinsand several of unknown function featured prominently

Exposing living organisms to xenobiotics induces a coor-dinated transcriptional response that regulates the expressionof enzymes and transporters to facilitate detoxification [1743 44] Some acaricidal components alter the activities ofmite detoxification enzymes [22] In this study expressionof the CCE and transporter ATP-binding cassette class Cgene detoxification enzymes was upregulated after exposureof T cinnabarinus to 120573-sitosterol It was demonstrated thatmany ATP-binding cassette genes respond transcriptionallyto xenobiotic exposure [4] Moreover the broad plant hostrange and high pesticide resistance levels of T urticae areassociated with lineage-specific expansion of detoxificationgenes [4] Exposingmites to120573-sitosterol resulted in upregula-tion of the expression of CCE and transporters that facilitatedetoxification

Defense-related proteins such as Toll-like receptor legu-main and serine proteases responded significantly to the 120573-sitosterol treatment After pathogen infection the expressionlevels of a coordinated set ofCaenorhabditis elegans (Maupas1900) defense genes involving proteolysis cell death andthe stress response was altered [45 46] The recognitionof different pathogen classes via germline-encoded proteinssuch as the Toll-like receptors is the first line of defensein vertebrates [47] Endosomelysosome-located proteasesplay key roles in defense and these proteases may beattractive drug targets Asparagine endopeptidase (AEP) orlegumain is an unusual lysosomal cysteine protease [48]This enzyme has been linked to a diverse range of biologicalphenomena including plant toxic protein processing antigenprocessing and presentation [49] neuronal cell death [50]and tumor invasion [51] Some endosomelysosome-locatedserine proteases have important roles in the defense system[52] However further work is needed to determine the rolesof Toll-like receptors legumain and serine proteases in theresponse of T cinnabarinus to 120573-sitosterol

Silk webs protect spider mites against predators rain andwind [53] Silk also plays an important role during collectivemigration by aerial dispersal or by walking [53] Seventeenfibroin genes have been identified in the T urticae genome[1] Our results demonstrate that fibroin gene expression wasdownregulated during the 120573-sitosterol response Our previ-ous studies also show that exposure of mites to xenobioticscan decrease silk production Environmental stressors suchas pesticides can be detrimental to organisms and reducetheir activities [54]

The expression profiles of several potential pesticidetarget genes such as cytochrome b and the chloride channelprotein were altered Cytochrome b is a target of bifenazateand acequinocyl [55] Moreover a proportion of the dif-ferentially expressed genes has not been annotated Future


research involving cloning and functionally analyzing ofthese significantly altered genes could facilitate discovery ofadditional factors involved in pesticide metabolism

5 Conclusion

We identified 22941 unigenes by Illumina-sequencing thetranscriptome of T cinnabarinus of which 12049 were anno-tated Abundant xenobiotic metabolism-related genes wereidentified in T cinnabarinus In total 81 P450-related genes28 CCEs 35 GSTs nine GABA receptors 15 GluCls oneAChE 10 voltage-gated sodium channels nine cytochromesb 25 nAChRs six catalases 12 superoxide dismutases and 28NADH dehydrogenases were identified from the T cinnabar-inus transcriptome Expansion of the xenobiotic metabolismsystem was found which may be responsible for the highlevel of resistance to almost all acaricides of T cinnabarinusThe T cinnabarinus detoxification systemmdashincluding CCEand ABC transporter class Cmdashwas upregulated after 120573-sitosterol treatment Defense-related proteins such as Toll-like receptors legumain and serine proteases were alsoactivated Furthermore other important proteinsmdashsuch aschloride channel protein cytochrome b carboxypeptidaseperitrophic membrane chitin-binding protein calphostinand transcription factorsmdashmay also play important roles inthe interaction between120573-sitosterol and spidermites Furtherwork is needed to determine their roles in the response of Tcinnabarinus to 120573-sitosterol treatment

Data Deposition

Our sequencing data has been deposited in SRA (SRP043517)

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Chunya Bu and Jinling Li conceived and designed theexperiments Jinling Li Chunya Bu Xiao-Qin Wang BoPeng Jingyu Han and Pin Gao performed the experimentsChunya Bu and Jinling Li analyzed the data Chunya BuGuanglu Shia andYounianWang contributed reagentsmate-rialsanalysis tools Chunya Bu wrote the paper

Acknowledgments

This work was financially supported by the National NaturalScience Foundation of China (no 31200483) Beijing NaturalScience Foundation (no 6122005) the Key Project of ChineseMinistry of Education (no 212001) the Program of BeijingMunicipal Commission of Education (KM201210020001)and the PlatformConstruction Program of BeijingMunicipalCommission of Education (KCT2014001) The authors thankallmembers of Key Laboratory ofUrbanAgriculture (North)

Ministry of Agriculture (Beijing China) for their adviceassistance and technical help

References

[1] M Grbic T van Leeuwen R M Clark et al ldquoThe genomeof Tetranychus urticae reveals herbivorous pest adaptationsrdquoNature vol 479 no 7374 pp 487ndash492 2011

[2] M S Lababidi Biologische Bekampfungsmoglichkeiten der Bau-mwollspinnmilbe Tetranychus cinnabarinus (Boisduval) (AcariTetranychidae) [PhD thesis] Rheinische Friedrich-Wilhelms-Universitat zu Bonn Bonn Germany 1988

[3] T Van Leeuwen J Vontas A Tsagkarakou W Dermauw andL Tirry ldquoAcaricide resistance mechanisms in the two-spottedspider mite Tetranychus urticae and other important Acari areviewrdquo Insect Biochemistry and Molecular Biology vol 40 no8 pp 563ndash572 2010

[4] W Dermauw E J Osborne RM Clark M Grbic L Tirry andT Van Leeuwen ldquoA burst of ABC genes in the genome of thepolyphagous spider mite Tetranychus urticaerdquo BMC Genomicsvol 14 no 1 article 317 2013

[5] Y Ahmed M H Sohrab S M Al-Reza F S Tareq C MHasan and M A Sattar ldquoAntimicrobial and cytotoxic con-stituents from leaves of Sapium baccatumrdquo Food and ChemicalToxicology vol 48 no 2 pp 549ndash552 2010

[6] S Erazo G Rocco M Zaldivar et al ldquoActive metabo-lites from Dunalia spinosa resinous exudatesrdquo Zeitschrift furNaturforschungmdashSection C Journal of Biosciences vol 63 no 7-8 pp 492ndash496 2008

[7] J E Yuk J S Woo C-Y Yun et al ldquoEffects of lactose-120573-sitosterol and 120573-sitosterol on ovalbumin-induced lung inflam-mation in actively sensitized micerdquo International Immunophar-macology vol 7 no 12 pp 1517ndash1527 2007

[8] Z Ovesna A Vachalkova and K Horvathova ldquoTaraxasteroland 120573-sitosterol new naturally compounds with chemoprotec-tivechemopreventive effectsrdquoNeoplasma vol 51 no 6 pp 407ndash414 2004

[9] Y Hamamura K Hayashiya andK-I Naito ldquoFood selection bysilkworm larvaelig Bombyx mori 120573-sitosterol as one of the bitingfactorsrdquo Nature vol 190 no 4779 pp 880ndash881 1961

[10] K Neog B Unni and G Ahmed ldquoStudies on the influenceof host plants and effect of chemical stimulants on the feedingbehavior in themuga silkworm Antheraea assamensisrdquo Journalof Insect Science vol 11 article 133 2011

[11] P C Kiprono F Kaberia J M Keriko and J N KaranjaldquoThe in vitro anti-fungal and anti-bacterial activities of 120573-sitosterol from Senecio lyratus (Asteraceae)rdquo Zeitschrift furNaturforschungmdashSection C Journal of Biosciences vol 55 no 5-6 pp 485ndash488 2000

[12] J Ren G Shi J Wang J Gu and Y Wang ldquoIsolation andidentification of the principal acaricidal components extractedfromMentha piperitardquo Scientia Silvae Sinicae vol 45 no 6 pp77ndash82 2009

[13] W Dermauw N Wybouw S Rombauts et al ldquoA link betweenhost plant adaptation and pesticide resistance in the polyphag-ous spider mite Tetranychus urticaerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 110 no2 pp E113ndashE122 2013

[14] T van Leeuwen P Demaeght E J Osborne et al ldquoPopulationbulk segregant mapping uncovers resistance mutations and themode of action of a chitin synthesis inhibitor in arthropodsrdquo


Proceedings of the National Academy of Sciences of the UnitedStates of America vol 109 no 12 pp 4407ndash4412 2012

[15] T van Leeuwen W Dermauw M Grbic L Tirry and RFeyereisen ldquoSpider mite control and resistance managementdoes a genome helprdquo Pest Management Science vol 69 no 2pp 156ndash159 2013

[16] J-Z Niu W Dou T-B Ding et al ldquoTranscriptome analysis ofthe citrus red mite Panonychus citri and its gene expression byexposure to insecticideacariciderdquo Insect Molecular Biology vol21 no 4 pp 422ndash436 2012

[17] N Yang W Xie X Yang et al ldquoTranscriptomic and pro-teomic responses of sweetpotato whitefly Bemisia tabaci tothiamethoxamrdquo PLoSONE vol 8 no 5 Article ID e61820 2013

[18] Y N Feng J Yan W Sun et al ldquoTranscription and inductionprofiles of two esterase genes in susceptible and acaricide-resistant Tetranychus cinnabarinusrdquo Pesticide Biochemistry andPhysiology vol 100 no 1 pp 70ndash73 2011

[19] J R Busvine ldquoRecommendedmethods formeasurement of pestresistance to pesticiderdquo in Towards Integrated Commodity andPest Management in Grain Storage R L Semple P A Hicks JV Lozare andA Castermans Eds FAOPlant Protection Paper21 p 526 UN Food and Agricultural Organization Rome Italy1992

[20] E-H Chen D-D Wei G-M Shen G-R Yuan P-P Bai andJ-J Wang ldquoDe novo characterization of the Dialeurodes citritranscriptome mining genes involved in stress resistance andsimple sequence repeats (SSRs) discoveryrdquo Insect MolecularBiology vol 23 no 1 pp 52ndash66 2014

[21] E Stolle JHKidner andR FAMoritz ldquoPatterns of evolution-ary conservation of microsatellites (SSRs) suggest a faster rateof genome evolution in hymenoptera than in Dipterardquo GenomeBiology and Evolution vol 5 no 1 pp 151ndash162 2013

[22] Z Xu W Zhu Y Liu et al ldquoAnalysis of insecticide resistance-related genes of the carmine spider mite Tetranychus cinnabar-inus based on a de novo assembled transcriptomerdquo PLoS ONEvol 9 no 5 Article ID e94779 2014

[23] A R Cabrera K V Donohue S M S Khalil et al ldquoNewapproach for the study of mite reproduction the first tran-scriptome analysis of a mite Phytoseiulus persimilis (AcariPhytoseiidae)rdquo Journal of Insect Physiology vol 57 no 1 pp 52ndash61 2011

[24] Q Lin F Jin Z Hu et al ldquoTranscriptome analysis of chlo-rantraniliprole resistance development in the diamondbackmoth Plutella xylostellardquo PLoS ONE vol 8 no 8 Article IDe72314 2013

[25] B Liu G Jiang Y Zhang et al ldquoAnalysis of transcriptomedifferences between resistant and susceptible strains of thecitrus red mite Panonychus citri (Acari Tetranychidae)rdquo PLoSONE vol 6 no 12 Article ID e28516 2011

[26] B Liu W Dou T-B Ding et al ldquoAn analysis of the small RNAtranscriptome of four developmental stages of the citrus redmite (Panonychus citri)rdquo Insect Molecular Biology vol 23 no2 pp 216ndash229 2014

[27] X Li M A Schuler and M R Berenbaum ldquoMolecularmechanisms of metabolic resistance to synthetic and naturalxenobioticsrdquoAnnual Review of Entomology vol 52 pp 231ndash2532007

[28] R Ozawa O Nishimura S Yazawa A Muroi J Takabayashiand G-I Arimura ldquoTemperature-dependent behavioural andtranscriptional variability of a tritrophic interaction consistingof bean herbivorousmite and predatorrdquoMolecular Ecology vol21 no 22 pp 5624ndash5635 2012

[29] A Bryon N Wybouw W Dermauw L Tirry and T vanLeeuwen ldquoGenomewide gene-expression analysis of facultativereproductive diapause in the two-spotted spider mite Tetrany-chus urticaerdquo BMC Genomics vol 14 no 1 article 815 2013

[30] J D Hayes J U Flanagan and I R Jowsey ldquoGlutathionetransferasesrdquo Annual Review of Pharmacology and Toxicologyvol 45 pp 51ndash88 2005

[31] C-P D Tu and B Akgul ldquoDrosophila glutathione S-trans-ferasesrdquoMethods in Enzymology vol 401 pp 204ndash226 2005

[32] H Ranson C Claudianos F Ortelli et al ldquoEvolution of super-gene families associated with insecticide resistancerdquo Sciencevol 298 no 5591 pp 179ndash181 2002

[33] N Tijet C Helvig and R Feyereisen ldquoThe cytochromeP450 gene superfamily inDrosophila melanogaster annotationintron-exon organization and phylogenyrdquo Gene vol 262 no 1-2 pp 189ndash198 2001

[34] D Werck-Reichhart and R Feyereisen ldquoCytochromes P450 asuccess storyrdquo Genome Biology vol 1 no 6 pp 30031ndash300392000

[35] W Dermauw A Ilias M Riga et al ldquoThe cys-loop ligand-gatedion channel gene family of Tetranychus urticae implicationsfor acaricide toxicology and a novel mutation associated withabamectin resistancerdquo Insect Biochemistry and Molecular Biol-ogy vol 42 no 7 pp 455ndash465 2012

[36] S D Buckingham P C Biggin B M Sattelle L A Brownand D B Sattelle ldquoInsect GABA receptors splicing editingand targeting by antiparasitics and insecticidesrdquo MolecularPharmacology vol 68 no 4 pp 942ndash951 2005

[37] G-J Lang K Y Zhu and C-X Zhang ldquoCan acetylcholi-nesterase serve as a target for developing more selective insec-ticidesrdquo Current Drug Targets vol 13 no 4 pp 495ndash501 2012

[38] J Khajehali T van Leeuwen M Grispou et al ldquoAcetyl-cholinesterase point mutations in European strains of Tetrany-chus urticae (Acari Tetranychidae) resistant to organophos-phatesrdquo Pest Management Science vol 66 no 2 pp 220ndash2282010

[39] X H Qiu ldquoInsecticide resistance genetics genomics andimplications for pest controlrdquoActa Entomologica Sinica vol 48no 6 pp 960ndash967 2005

[40] D D Duan C Y Bu J Cheng Y N Wang and G L ShildquoIsolation and identification of acaricidal compounds in Inulajaponica (Asteraceae)rdquo Journal of Economic Entomology vol104 no 2 pp 375ndash378 2011

[41] Z Wang M Gerstein and M Snyder ldquoRNA-Seq a revolution-ary tool for transcriptomicsrdquo Nature Reviews Genetics vol 10no 1 pp 57ndash63 2009

[42] J-P David E Coissac C Melodelima et al ldquoTranscriptomeresponse to pollutants and insecticides in the dengue vectorAedes aegypti using next-generation sequencing technologyrdquoBMC Genomics vol 11 no 1 article 216 2010

[43] J R Misra M A Horner G Lam and C S Thummel ldquoTrans-criptional regulation of xenobiotic detoxification inDrosophilardquoGenes and Development vol 25 no 17 pp 1796ndash1806 2011

[44] C Xu C Y T Li and A N T Kong ldquoInduction of phase I IIand III drug metabolismtransport by xenobioticsrdquo Archives ofPharmacal Research vol 28 no 3 pp 249ndash268 2005

[45] D OrsquoRourke D Baban M Demidova R Mott and J HodgkinldquoGenomic clusters putative pathogen recognition moleculesand antimicrobial genes are induced by infection of C eleganswith M nematophilumrdquo Genome Research vol 16 no 8 pp1005ndash1016 2006


[46] D Wong D Bazopoulou N Pujol N Tavernarakis andJ J Ewbank ldquoGenome-wide investigation reveals pathogen-specific and shared signatures in the response ofCaenorhabditiselegans to infectionrdquo Genome Biology vol 8 no 9 article R1942007

[47] S Akira S Uematsu and O Takeuchi ldquoPathogen recognitionand innate immunityrdquo Cell vol 124 no 4 pp 783ndash801 2006

[48] G Miller S P Matthews T Reinheckel S Fleming and CWatts ldquoAsparagine endopeptidase is required for normal kidneyphysiology and homeostasisrdquoThe FASEB Journal vol 25 no 5pp 1606ndash1617 2011

[49] C Watts S P Matthews D Mazzeo B Manoury and C XMoss ldquoAsparaginyl endopeptidase case history of a class IIMHC compartment proteaserdquo Immunological Reviews vol 207pp 218ndash228 2005

[50] Z Liu S-W Jang X Liu et al ldquoNeuroprotective actionsof PIKE-L by inhibition of SET proteolytic degradation byasparagine endopeptidaserdquo Molecular Cell vol 29 no 6 pp665ndash678 2008

[51] C Liu C Sun H Huang K Janda and T Edgington ldquoOver-expression of legumain in tumors is significant for inva-sionmetastasis and a candidate enzymatic target for prodrugtherapyrdquo Cancer Research vol 63 no 11 pp 2957ndash2964 2003

[52] J D Colbert S P Matthews G Miller and C WattsldquoDiverse regulatory roles for lysosomal proteases in the immuneresponserdquo European Journal of Immunology vol 39 no 11 pp2955ndash2965 2009

[53] G J Le Goff T Hance C Detrain J-L Deneubourg and A-C Mailleux ldquoThe locomotor activities on sites covered by silkproduced by related and unrelated spider mites in Tetranychusurticae (Acari Tetranychidae)rdquoComptes RendusmdashBiologies vol335 no 3 pp 226ndash231 2012

[54] W Aktar D Sengupta and A Chowdhury ldquoImpact of pesti-cides use in agriculture their benefits and hazardsrdquo Interdisci-plinary Toxicology vol 2 no 1 pp 1ndash12 2009

[55] T van Leeuwen P van Nieuwenhuyse B Vanholme W Der-mauw RNauen and L Tirry ldquoParallel evolution of cytochromeb mediated bifenazate resistance in the citrus red mite Panony-chus citrirdquo Insect Molecular Biology vol 20 no 1 pp 135ndash1402011

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Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


and induce resistance less frequently 120573-Sitosterol one ofthree common phytosterols was isolated from a petroleum-ether extract of Sapium baccatum (Roxb) leaves and showshigh activity in a brine shrimp bioassay with an LC

50of

158mgmL [5] In addition 120573-sitosterol has been identifiedin Dunalia spinosa (Meyen) extracts where it exhibits strongantimicrobial activity [6]120573-Sitosterol is present in a variety ofplants and is an antitumor antimicrobial chemopreventiveand immunomodulatory compound [7 8]120573-Sitosterol is alsothe active compound of the mulberry plant that induces bit-ing behavior from Bombyx mori (Linnaeus) in a low concen-tration [9] However 120573-sitosterol alone has greatly reducedeffects on biting behavior of Antheraea assamensis (Helfer)[10] Antifungal and antibacterial activities of 120573-sitosterolhave been detected in a methanol extract of dried-groundaerial parts of Senecio lyratus (Bullock) [11] We previouslyisolated 120573-sitosterol from Mentha piperita (Linnaeus) andreported high acaricidal activity (LC

50 05mgmL) [12] 120573-

Sitosterol may thus be a candidate lead botanical compoundHowever the acaricidal mechanism of 120573-sitosterol is unclear

The availability of genome and transcriptome sequencesprovides unique information and enables investigation of themode of action of an acaricidal compound and the rolesof the gene families involved in pesticide metabolism inspider mites [13ndash15] Characterizing the spider mite genefamilies associated with 120573-sitosterol metabolism is the firststep towards a better understanding of the effects of 120573-sitosterol on spider mites In this study we performed adetailed analysis of the expression of120573-sitosterolmetabolism-associated gene families in T cinnabarinus

2 Materials and Methods

21 Carmine Spider Mite Collection and Handling A colonyof T cinnabarinus (Boisduval 1867) was originally collectedfrom a field in Qingxu County Taiyuan Municipality ShanxiProvince China in the spring of 2004 and had been freeof pesticides for gt10 years This insecticide-susceptible strainof T cinnabarinus was maintained at the Key Laboratory ofUrban Agriculture (North) Ministry of Agriculture (BeijingChina) at 26 plusmn 2∘C 60 relative humidity (RH) and a 16 hlight 8 h dark photoperiod under pesticide-free conditionsThe mites were reared on Vigna unguiculata (L) leaves atthe sixth true-leaf growth stage in a greenhouse The miteswere tested annually for high sensitivity to organophosphateinsecticides using the glass slide-dipping method accordingto FAO standards

22 120573-Sitosterol Treatment Our laboratory determined pre-viously that 120573-sitosterol has high acaricidal activity against TcinnabarinusThe changes in T cinnabarinus gene expressionwere evaluated after exposure to a sublethal concentration(LC30) of 120573-sitosterol according to the procedure described

by [16 17] The LC30is the concentration required to kill 30

of the members of a tested population after a specified testduration and is commonly used in mite pesticide exposureresearch [18] The mites were treated with 120573-sitosterol usingthe glass slide-dipping method according to the FAO [19]120573-sitosterol (Sigma-Aldrich St Louis MO USA) was diluted

in sterile distilled water containing 1 Tween 80 (Sigma-Aldrich) and 06 DMSO (Sigma-Aldrich) Sterile distilledwater with 1 Tween 80 and 06 DMSO was used as thecontrol Killing of the female adults is critical for controllingspider mites Thus female adults were used in our experi-ment Female adult mites were affixed onto one end of a 10 times2 cm glass slide using double-sided adhesive tapeThe affixedmites were checked under a stereomicroscope and inactivemites were removedThemites were dipped individually into120573-sitosterol solutions for 5 s Following removal of the slidethe excess solution was absorbed with filter paper All treatedmites were maintained at 26 plusmn 2∘C 60 RH and a 16 h light8 h dark photoperiod The dead mites were removed 24 hafter treatment and the live mites were frozen immediatelyin liquid nitrogen and stored for later extraction of RNA [19]This experiment was repeated twice

23 RNA Extraction cDNA Library Construction and Illu-mina Sequencing Total RNAs were extracted using TRIzolreagent (Invitrogen Beijing China) according to the man-ufacturerrsquos instructions Genomic DNA was removed usingRNase-free DNase (Takara Kyoto Japan) The quantity andquality of total RNA were determined using a NanoDrop2000 UV-Vis Spectrophotometer (Thermo Scientific Wilm-ington DE USA) Ultrasec 2100 pro UVVisible Spectropho-tometer (Amersham Biosciences Uppsala Sweden) and gelelectrophoresis

Poly-(A) mRNA was enriched and isolated from thehigh-quality total RNA samples using Magnetic Oligo(dT)Beads followed by fragmentation of the mRNA with aRNA fragmentation kit (Ambion Austin TX USA) Reversetranscription into cDNA was performed using random hex-amers and reverse transcriptase (Promega Madison WIUSA) and then second-strand cDNA was synthesized usingDNA polymerase I (Invitrogen) and RNaseH (New EnglandBiolabs Ipswich MA USA) After purification with theQIAquick PCRPurificationKit (Qiagen Valencia CA USA)the cDNA fragments were end-repaired and ligated to aSolexa adaptor after adding a single ldquoArdquo at the 31015840 end Thedesired size of adaptor-ligated fragment was selected byagarose gel electrophoresis Polymerase chain reaction (PCR)was performed to selectively enrich and amplify the selectedcDNA fragments to construct cDNA libraries for paired-end sequencing using the Illumina HiSeq 2000 (BiomarkerTechnologies Co Ltd Beijing China)

24 De Novo Assembly and Clustering Analysis De novoassembly of the mRNA-seq reads was performed using theTrinity method (httptrinityrnaseqsourceforgenet) afterthe raw reads were filtered by discarding the adaptorsequences low-quality sequences (reads with ambiguous ldquoNrdquobases) and reads in which gt10 of the bases had 119876 lt 20An optimized k-mer length of 25 was used to assemble thetranscriptome A clustering analysis was then conducted toconstruct the unigene database

25 Functional Annotation Open reading frames (ORF) ofthe unigenes were predicted using the ldquogetorf rdquo program in












11987620percentage 9888 9888



3 Results


20bases for each T






7

70

700

7000

0

1

10

100

Cel

l par

tC

ell

Org

anel

leM

embr

ane

Org

anel

le p

art

Mac

rom

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ular

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plex

Mem

bran

e par

tM

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ane-

enclo

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trac

ellu

lar r

egio

nSy

naps

eSy

naps

e par

tC

ell j

unct

ion

Extr

acel

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ion

part

Extr

acel

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r mat

rixEx

trac

ellu

lar m

atrix

par

tN

ucle

oid

Bind

ing

Cata

lytic

activ

ityTr

ansp

orte

r act

ivity

Mol

ecul

ar tr

ansd

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ityRe

cept

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tivity

Nuc

leic

acid

bin

ding

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scrip

tion

fact

or ac

tivity

Stru

ctur

al m

olec

ule a

ctiv

ityEn

zym

e reg

ulat

or ac

tivity

Prot

ein

bind

ing

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scrip

tion

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or ac

tivity

Ant

ioxi

dant

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ityEl

ectro

n ca

rrie

r act

ivity

Tran

slatio

n re

gula

tor a

ctiv

ityCh

anne

l reg

ulat

or ac

tivity

Mor

phog

en ac

tivity

Met

allo

chap

eron

e act

ivity

Cel

lula

r pro

cess

Met

abol

ic p

roce

ssD

evelo

pmen

tal p

roce

ssBi

olog

ical

regu

latio

nM

ultic

ellu

lar o

rgan

ismal

pro

cess

Resp

onse

to st

imul

usLo

caliz

atio

nRe

prod

uctio

nC

ellul

ar co

mpo

nent

org

aniz

atio

n or

bio

gene

sisRe

prod

uctiv

e pro

cess

Esta

blish

men

t of l

ocal

izat

ion

Sign

alin

gLo

com

otio

nM

ulti-

orga

nism

pro

cess

Gro

wth

deat

hIm

mun

e sys

tem

pro

cess

Biol

ogic

al ad

hesio

nC

ell p

rolif

erat

ion

Rhyt

hmic

pro

cess

Pigm

enta

tion

Vira

l rep

rodu

ctio

n

Num

ber o

f gen

es

Gen

es (

)


Tetranychus

GO classification



























0

300

600

900

1200

1500

1800


Freq

uenc

y

Function class







4 Discussion



7

70

700

7000

0

1

10

100

Cel

l par

tC

ell

Org

anel

leM

embr

ane

Org

anel

le pa

rtM

acro

mol

ecul

ar co

mpl

exM

embr

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art

Mem

bran

e-en

close

d lu

men

Extr

acel

lula

r reg

ion

Syna

pse

Syna

pse p

art

Cel

l jun

ctio

nEx

trac

ellu

lar r

egio

n pa

rtEx

trac

ellu

lar m

atrix

Extr

acel

lula

r mat

rix p

art

Nuc

leoi

d

Bind

ing

Cata

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ityTr

ansp

orte

r act

ivity

Mol

ecul

ar tr

ansd

ucer

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ityRe

cept

or ac

tivity

Stru

ctur

al m

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ule a

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ulat

or ac

tivity

Ant

ioxi

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ectro

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rrie

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ivity

Tran

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ulat

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Mor

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Met

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ivity

Cel

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cess

Met

abol

ic p

roce

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pmen

tal p

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ellu

lar o

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Resp

onse

to st

imul

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nRe

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uctiv

e pro

cess

Repr

oduc

tion

Esta

blish

men

t of l

ocal

izat

ion

Sign

alin

gLo

com

otio

nM

ulti-

orga

nism

pro

cess

Gro

wth

Dea

thIm

mun

e sys

tem

pro

cess

Biol

ogic

al ad

hesio

nC

ell p

rolif

erat

ion

Rhyt

hmic

pro

cess

Pigm

enta

tion

Vira

l rep

rodu

ctio

n

Num

ber o

f gen

es

Gen

es (

)35

3

1

0


GO classification


Cell

ular

com

pone

nt o

rgan

izat

ion

or b

ioge

nesis

Nuc

leic

acid

bin

ding

tran

scrip

tion

fact

or ac

tivity

Prot

ein

bind

ing

tran

scrip

tion

fact

or ac

tivity


0

10

20

30

40

50

60


Num

ber o

f uni

gene

s

























5 Conclusion


Data Deposition






Acknowledgments



References































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of












11987620percentage 9888 9888



3 Results


20bases for each T






7

70

700

7000

0

1

10

100

Cel

l par

tC

ell

Org

anel

leM

embr

ane

Org

anel

le p

art

Mac

rom

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ular

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plex

Mem

bran

e par

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enEx

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ellu

lar r

egio

nSy

naps

eSy

naps

e par

tC

ell j

unct

ion

Extr

acel

lula

r reg

ion

part

Extr

acel

lula

r mat

rixEx

trac

ellu

lar m

atrix

par

tN

ucle

oid

Bind

ing

Cata

lytic

activ

ityTr

ansp

orte

r act

ivity

Mol

ecul

ar tr

ansd

ucer

activ

ityRe

cept

or ac

tivity

Nuc

leic

acid

bin

ding

tran

scrip

tion

fact

or ac

tivity

Stru

ctur

al m

olec

ule a

ctiv

ityEn

zym

e reg

ulat

or ac

tivity

Prot

ein

bind

ing

tran

scrip

tion

fact

or ac

tivity

Ant

ioxi

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activ

ityEl

ectro

n ca

rrie

r act

ivity

Tran

slatio

n re

gula

tor a

ctiv

ityCh

anne

l reg

ulat

or ac

tivity

Mor

phog

en ac

tivity

Met

allo

chap

eron

e act

ivity

Cel

lula

r pro

cess

Met

abol

ic p

roce

ssD

evelo

pmen

tal p

roce

ssBi

olog

ical

regu

latio

nM

ultic

ellu

lar o

rgan

ismal

pro

cess

Resp

onse

to st

imul

usLo

caliz

atio

nRe

prod

uctio

nC

ellul

ar co

mpo

nent

org

aniz

atio

n or

bio

gene

sisRe

prod

uctiv

e pro

cess

Esta

blish

men

t of l

ocal

izat

ion

Sign

alin

gLo

com

otio

nM

ulti-

orga

nism

pro

cess

Gro

wth

deat

hIm

mun

e sys

tem

pro

cess

Biol

ogic

al ad

hesio

nC

ell p

rolif

erat

ion

Rhyt

hmic

pro

cess

Pigm

enta

tion

Vira

l rep

rodu

ctio

n

Num

ber o

f gen

es

Gen

es (

)


Tetranychus

GO classification



























0

300

600

900

1200

1500

1800


Freq

uenc

y

Function class







4 Discussion



7

70

700

7000

0

1

10

100

Cel

l par

tC

ell

Org

anel

leM

embr

ane

Org

anel

le pa

rtM

acro

mol

ecul

ar co

mpl

exM

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art

Mem

bran

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d lu

men

Extr

acel

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ion

Syna

pse

Syna

pse p

art

Cel

l jun

ctio

nEx

trac

ellu

lar r

egio

n pa

rtEx

trac

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atrix

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rix p

art

Nuc

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Bind

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ansp

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r act

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Mol

ecul

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ansd

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ityRe

cept

or ac

tivity

Stru

ctur

al m

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ule a

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zym

e reg

ulat

or ac

tivity

Ant

ioxi

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ityEl

ectro

n ca

rrie

r act

ivity

Tran

slatio

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gula

tor a

ctiv

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anne

l reg

ulat

or ac

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Mor

phog

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Met

allo

chap

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e act

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Cel

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cess

Met

abol

ic p

roce

ssD

evelo

pmen

tal p

roce

ssBi

olog

ical

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latio

nM

ultic

ellu

lar o

rgan

ismal

pro

cess

Resp

onse

to st

imul

usLo

caliz

atio

nRe

prod

uctiv

e pro

cess

Repr

oduc

tion

Esta

blish

men

t of l

ocal

izat

ion

Sign

alin

gLo

com

otio

nM

ulti-

orga

nism

pro

cess

Gro

wth

Dea

thIm

mun

e sys

tem

pro

cess

Biol

ogic

al ad

hesio

nC

ell p

rolif

erat

ion

Rhyt

hmic

pro

cess

Pigm

enta

tion

Vira

l rep

rodu

ctio

n

Num

ber o

f gen

es

Gen

es (

)35

3

1

0


GO classification


Cell

ular

com

pone

nt o

rgan

izat

ion

or b

ioge

nesis

Nuc

leic

acid

bin

ding

tran

scrip

tion

fact

or ac

tivity

Prot

ein

bind

ing

tran

scrip

tion

fact

or ac

tivity


0

10

20

30

40

50

60


Num

ber o

f uni

gene

s

























5 Conclusion


Data Deposition






Acknowledgments



References































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


7

70

700

7000

0

1

10

100

Cel

l par

tC

ell

Org

anel

leM

embr

ane

Org

anel

le p

art

Mac

rom

olec

ular

com

plex

Mem

bran

e par

tM

embr

ane-

enclo

sed

lum

enEx

trac

ellu

lar r

egio

nSy

naps

eSy

naps

e par

tC

ell j

unct

ion

Extr

acel

lula

r reg

ion

part

Extr

acel

lula

r mat

rixEx

trac

ellu

lar m

atrix

par

tN

ucle

oid

Bind

ing

Cata

lytic

activ

ityTr

ansp

orte

r act

ivity

Mol

ecul

ar tr

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activ

ityRe

cept

or ac

tivity

Nuc

leic

acid

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ding

tran

scrip

tion

fact

or ac

tivity

Stru

ctur

al m

olec

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ctiv

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zym

e reg

ulat

or ac

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ein

bind

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slatio

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cess

Met

abol

ic p

roce

ssD

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pmen

tal p

roce

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olog

ical

regu

latio

nM

ultic

ellu

lar o

rgan

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pro

cess

Resp

onse

to st

imul

usLo

caliz

atio

nRe

prod

uctio

nC

ellul

ar co

mpo

nent

org

aniz

atio

n or

bio

gene

sisRe

prod

uctiv

e pro

cess

Esta

blish

men

t of l

ocal

izat

ion

Sign

alin

gLo

com

otio

nM

ulti-

orga

nism

pro

cess

Gro

wth

deat

hIm

mun

e sys

tem

pro

cess

Biol

ogic

al ad

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rolif

erat

ion

Rhyt

hmic

pro

cess

Pigm

enta

tion

Vira

l rep

rodu

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n

Num

ber o

f gen

es

Gen

es (

)


Tetranychus

GO classification



























0

300

600

900

1200

1500

1800


Freq

uenc

y

Function class







4 Discussion



7

70

700

7000

0

1

10

100

Cel

l par

tC

ell

Org

anel

leM

embr

ane

Org

anel

le pa

rtM

acro

mol

ecul

ar co

mpl

exM

embr

ane p

art

Mem

bran

e-en

close

d lu

men

Extr

acel

lula

r reg

ion

Syna

pse

Syna

pse p

art

Cel

l jun

ctio

nEx

trac

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lar r

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n pa

rtEx

trac

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lar m

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acel

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r mat

rix p

art

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leoi

d

Bind

ing

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r act

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ctur

al m

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e reg

ulat

or ac

tivity

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ioxi

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ectro

n ca

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r act

ivity

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slatio

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anne

l reg

ulat

or ac

tivity

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phog

en ac

tivity

Met

allo

chap

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e act

ivity

Cel

lula

r pro

cess

Met

abol

ic p

roce

ssD

evelo

pmen

tal p

roce

ssBi

olog

ical

regu

latio

nM

ultic

ellu

lar o

rgan

ismal

pro

cess

Resp

onse

to st

imul

usLo

caliz

atio

nRe

prod

uctiv

e pro

cess

Repr

oduc

tion

Esta

blish

men

t of l

ocal

izat

ion

Sign

alin

gLo

com

otio

nM

ulti-

orga

nism

pro

cess

Gro

wth

Dea

thIm

mun

e sys

tem

pro

cess

Biol

ogic

al ad

hesio

nC

ell p

rolif

erat

ion

Rhyt

hmic

pro

cess

Pigm

enta

tion

Vira

l rep

rodu

ctio

n

Num

ber o

f gen

es

Gen

es (

)35

3

1

0


GO classification


Cell

ular

com

pone

nt o

rgan

izat

ion

or b

ioge

nesis

Nuc

leic

acid

bin

ding

tran

scrip

tion

fact

or ac

tivity

Prot

ein

bind

ing

tran

scrip

tion

fact

or ac

tivity


0

10

20

30

40

50

60


Num

ber o

f uni

gene

s

























5 Conclusion


Data Deposition






Acknowledgments



References































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of
















0

300

600

900

1200

1500

1800


Freq

uenc

y

Function class







4 Discussion



7

70

700

7000

0

1

10

100

Cel

l par

tC

ell

Org

anel

leM

embr

ane

Org

anel

le pa

rtM

acro

mol

ecul

ar co

mpl

exM

embr

ane p

art

Mem

bran

e-en

close

d lu

men

Extr

acel

lula

r reg

ion

Syna

pse

Syna

pse p

art

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l jun

ctio

nEx

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n pa

rtEx

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lar m

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acel

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r mat

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leoi

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r act

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e reg

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phog

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Met

allo

chap

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lula

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cess

Met

abol

ic p

roce

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pmen

tal p

roce

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olog

ical

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latio

nM

ultic

ellu

lar o

rgan

ismal

pro

cess

Resp

onse

to st

imul

usLo

caliz

atio

nRe

prod

uctiv

e pro

cess

Repr

oduc

tion

Esta

blish

men

t of l

ocal

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ion

Sign

alin

gLo

com

otio

nM

ulti-

orga

nism

pro

cess

Gro

wth

Dea

thIm

mun

e sys

tem

pro

cess

Biol

ogic

al ad

hesio

nC

ell p

rolif

erat

ion

Rhyt

hmic

pro

cess

Pigm

enta

tion

Vira

l rep

rodu

ctio

n

Num

ber o

f gen

es

Gen

es (

)35

3

1

0


GO classification


Cell

ular

com

pone

nt o

rgan

izat

ion

or b

ioge

nesis

Nuc

leic

acid

bin

ding

tran

scrip

tion

fact

or ac

tivity

Prot

ein

bind

ing

tran

scrip

tion

fact

or ac

tivity


0

10

20

30

40

50

60


Num

ber o

f uni

gene

s

























5 Conclusion


Data Deposition






Acknowledgments



References































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


7

70

700

7000

0

1

10

100

Cel

l par

tC

ell

Org

anel

leM

embr

ane

Org

anel

le pa

rtM

acro

mol

ecul

ar co

mpl

exM

embr

ane p

art

Mem

bran

e-en

close

d lu

men

Extr

acel

lula

r reg

ion

Syna

pse

Syna

pse p

art

Cel

l jun

ctio

nEx

trac

ellu

lar r

egio

n pa

rtEx

trac

ellu

lar m

atrix

Extr

acel

lula

r mat

rix p

art

Nuc

leoi

d

Bind

ing

Cata

lytic

activ

ityTr

ansp

orte

r act

ivity

Mol

ecul

ar tr

ansd

ucer

activ

ityRe

cept

or ac

tivity

Stru

ctur

al m

olec

ule a

ctiv

ityEn

zym

e reg

ulat

or ac

tivity

Ant

ioxi

dant

activ

ityEl

ectro

n ca

rrie

r act

ivity

Tran

slatio

n re

gula

tor a

ctiv

ityCh

anne

l reg

ulat

or ac

tivity

Mor

phog

en ac

tivity

Met

allo

chap

eron

e act

ivity

Cel

lula

r pro

cess

Met

abol

ic p

roce

ssD

evelo

pmen

tal p

roce

ssBi

olog

ical

regu

latio

nM

ultic

ellu

lar o

rgan

ismal

pro

cess

Resp

onse

to st

imul

usLo

caliz

atio

nRe

prod

uctiv

e pro

cess

Repr

oduc

tion

Esta

blish

men

t of l

ocal

izat

ion

Sign

alin

gLo

com

otio

nM

ulti-

orga

nism

pro

cess

Gro

wth

Dea

thIm

mun

e sys

tem

pro

cess

Biol

ogic

al ad

hesio

nC

ell p

rolif

erat

ion

Rhyt

hmic

pro

cess

Pigm

enta

tion

Vira

l rep

rodu

ctio

n

Num

ber o

f gen

es

Gen

es (

)35

3

1

0


GO classification


Cell

ular

com

pone

nt o

rgan

izat

ion

or b

ioge

nesis

Nuc

leic

acid

bin

ding

tran

scrip

tion

fact

or ac

tivity

Prot

ein

bind

ing

tran

scrip

tion

fact

or ac

tivity


0

10

20

30

40

50

60


Num

ber o

f uni

gene

s

























5 Conclusion


Data Deposition






Acknowledgments



References































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




















5 Conclusion


Data Deposition






Acknowledgments



References































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

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