Evolution of Soldier-Specific Venomous Protease in Social Aphids

Evolution of Soldier-Specific Venomous Protease in Social Aphids

Mayako Kutsukake,* Naruo Nikoh,� Harunobu Shibao,� Claude Rispe,§ Jean-Christophe Simon,§and Takema Fukatsu*

*Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST),Tsukuba, Japan; �Division of Natural Sciences, The Open University of Japan, Chiba, Japan; �Department of System Sciences(Biology), University of Tokyo, Tokyo, Japan; and §Institut National de la Recherche Agronomique (INRA), Unite Mixte deRecherche (UMR)1099 BIO3P, Le Rheu, France

In social aphids of the genus Tuberaphis a cysteine protease gene of the family cathepsin B exhibits soldier-specificexpression and intestinal protease production. The product is orally excreted and injected by soldier nymphs into naturalenemies, thereby exerting an insecticidal activity. In an attempt to gain insights into when and how the novel venomousprotease for the altruistic caste has evolved, we investigated the soldier-specific type (S-type) and nonspecific type(N-type) cathepsin B genes from social and nonsocial aphids. All the social aphids examined, representing the generaTuberaphis, Astegopteryx, and Cerataphis, possessed both the S-type and N-type genes. Phylogenetically distantnonsocial aphids also possessed cathepsin B genes allied to the S-type and the N-type, indicating the evolutionary originof these genes in the common ancestor of extant aphids. In Tuberaphis species the S-type genes exhibited significantsoldier-specific expression and accelerated molecular evolution whereas the N-type genes did not. In Astegopteryx andCerataphis species, meanwhile, both the S-type and N-type genes exhibited neither remarkable soldier-specificexpression nor accelerated molecular evolution. These results suggest that the S-type gene acquired the soldier-specificexpression and the venom function after divergence of the genus Tuberaphis. On the structural model of the S-typeprotease of Tuberaphis styraci the accelerated molecular evolution was associated with the molecular surface rather thanthe catalytic cleft, suggesting that the venom activity was probably acquired by relatively minor modifications on themolecular surface rather than by generation of a novel active site. In Cerataphis jamuritsu the S-type gene was, althoughcontaining a stop codon, structurally almost intact and still transcribed, suggesting recent pseudogenization of the genecopy and possible relevance to relaxed functional constraint in the highly multiplied protease gene family. On the basis ofthese results we suggest that the massive amplification in aphid cathepsin B genes might have predisposed the evolutionof venomous protease in the social aphid lineage and argue that gene duplication, accelerated molecular evolution, andacquisition of novel gene function must have played considerable roles in the evolution of complex biological systemsincluding insect sociality.

Introduction

In colonies of social insects, hundreds, thousands, ormillions of individuals are integrated into a highly orga-nized and homeostatic system, comprising one of the mostimpressive biological entities in nature. Some individualsare engaged in reproduction, whereas others produce fewor no offspring and constitute specialized castes that arecharacterized by distinct morphological, physiological,and behavioral traits for their altruistic functions. Morpho-logically and reproductively differentiated castes (e.g.,queens, workers, soldiers, etc.) are well known from socialinsect groups such as bees, ants, wasps, and termites(Wilson 1971), but such castes have also been found in lessstudied groups like aphids (Stern and Foster 1996).

Some 50 species of aphids, representing two subfami-lies Hormaphidinae and Eriosomatinae, are known to pro-duce nymphs that altruistically sacrifice their ownreproduction for the benefit of their colony mates. Suchnymphs are called ‘‘soldiers’’ because their primary socialrole is defense, whereas this caste may also play a nonde-fensive altruistic role such as gall cleaning. In highly socialaphids, soldier nymphs are morphologically differentiatedfrom normal nymphs and unable to grow, constituting a ster-ile caste (Aoki 1987; Ito 1989; Stern and Foster 1996).

The establishment and maintenance of the insect soci-ality must entail a number of evolutionary novelties that are

involved in caste differentiation, division of labor, socialcommunication, colony regulation, nest building, and manyother social traits. It is of great interest what molecularmechanisms have been acquired and/or recruited in the evo-lutionary course of the insect sociality. Despite technicaldifficulties with the nonmodel insects, recent developmentin molecular genetics and genomics has unveiled some in-triguing molecular aspects relevant to social traits in bees,ants, and termites (Abouheif and Wray 2002; Ben-Shaharet al. 2002; Krieger and Ross 2002, 2005; Bulmer andCrozier 2004; Robinson et al. 2005; Drapeau et al. 2006;Foret and Maleszka 2006; The Honeybee Genome Se-quencing Consortium 2006).

Members of the aphid tribe Cerataphidini, represent-ing the subfamily Hormaphidinae and embracing the generaTuberaphis, Astegopteryx, Cerataphis, and others, areknown as social aphids with soldier caste. They form con-spicuous galls on trees of the genus Styrax (Styracaceae),wherein adult females parthenogenetically produce mono-morphic 1st instar nymphs. When they molt into 2nd instar,two distinct morphs, normal nymphs and soldier nymphs,appear. Normal nymphs reach adulthood and reproduce,whereas soldier nymphs neither grow nor reproduce butare specialized for altruistic social tasks, colony defenseand gall cleaning. At ordinary times, soldier nymphs gatheraround exit holes of the gall, guarding the openings and dis-carding the wastes (e.g., honeydew globules, shed skins,corpses) from the holes. Encountering intruders, soldiernymphs aggressively exhibit attacking behavior by stingingwith their stylet. Aphid predators such as hoverfly maggotsand lacewing larvae are tormented, paralyzed, or killed bythe attack and usually drop off the gall surface together

Key words: social aphid, soldier caste, venom protein, cathepsin Bprotease, accelerated evolution, gene duplication.

E-mail: [email protected].

Mol. Biol. Evol. 25(12):2627–2641. 2008doi:10.1093/molbev/msn203Advance Access publication September 26, 2008

� The Author 2008. Published by Oxford University Press on behalf ofthe Society for Molecular Biology and Evolution. All rights reserved.For permissions, please e-mail: [email protected]

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with attacking soldier nymphs. Accidental bite of soldiernymphs on human skin causes itch, indicating that they pos-sess some toxic substance and inject it into enemies throughtheir stylet. These remarkable social traits associated withthe 2nd instar soldier caste are commonly found in the gall-ing generation of all cerataphidine aphids, suggesting thatthe soldier caste had already evolved in the common ances-tor of the aphid group (Aoki 1987; Ito 1989; Stern andFoster 1996).

Tuberaphis styraci is a representative of cerataphidinesocial aphids, forming large coral-shaped galls on the treeStyrax obassia and producing 2nd instar soldier nymphs(Aoki and Kurosu 1989, 1990). Owing to the developmentof an artificial diet rearing system for the species (Shibaoet al. 2002), T. styraci has recently been investigated inten-sively as experimental model of social aphid, which has un-veiled the density-mediated control mechanisms overinduction and suppression of soldier differentiation (Shibaoet al. 2003, 2004a, 2004b, 2004c).

In this and other social aphids, normal nymphs andsoldier nymphs are clonal offspring of the same motherand thus genetically identical to each other. Notwithstand-ing this, they are strikingly different in morphology, phys-iology, behavior, and reproduction, which must beattributed to differential gene expression between thecastes. In this context, a subtraction screening of soldier-specific gene expression was performed with T. styraci,which led to the discovery of an interesting cysteine pro-tease gene of the family cathepsin B (Kutsukake et al.2004). The soldier-specific type (S-type) cathepsin B geneis expressed about 2,000 times higher in soldier nymphsthan in normal nymphs, exhibiting specific localizationin the midgut epithelium. The S-type cathepsin B proteaseis secreted into the midgut cavity, vomited out through thestylet, and injected into the victims, thereby exerting an in-secticidal activity. Namely, the S-type cathepsin B is an ac-tive component of the aphid venom. Certainly, the S-typecathepsin B gene shows accelerated molecular evolutiondue to positive selection acting on the molecule, as has beenreported for other venomous proteins of snakes, scorpions,and cone snails. Meanwhile, another copy of cathepsin Bgene, which is expressed irrespective of the castes andshows no accelerated molecular evolution, was also de-tected from T. styraci. Both the nonspecific type (N-type)gene and the S-type gene were identified from several othersocial aphids of the genus Tuberaphis, forming distinctclades in the cathepsin B gene phylogeny, respectively.These results suggest that the S-type gene and the N-typegene had already been duplicated in the common ancestorof the Tuberaphis species (Kutsukake et al. 2004). Whenand how the cathepsin B genes have been duplicated andspecialized for the novel venom function are intriguingfor understanding of the evolutionary process of the soci-ality in the aphid group Cerataphidini.

Another recent study also highlighted the relevance ofcathepsin B proteases to the aphid biology and evolution.From the expressed sequence tag (EST) collections and thegenome analysis of the pea aphid Acyrthosiphon pisum,a total of 28 cathepsin B gene copies, which were classifiedinto 17 clusters, were identified (Rispe et al. 2008). Itshould be noted that of six representative gene copies sub-

jected to quantitative reverse transcriptase–polymerasechain reaction (RT-PCR) analysis, five genes exhibitedgut-specific expression patterns (Rispe et al. 2008), whichis reminiscent of the expression pattern of the S-type ca-thepsin B in T. styraci. Hence, the relationship of the S-typeand N-type cathepsin B genes of the social aphid T. styracito the diverse cathepsin B genes of the nonsocial aphid A.pisum is evolutionarily quite interesting.

To gain insights into the evolutionary origin and pro-cess of the soldier-specific venomous cathepsin B proteasegene, we identified here the S-type and N-type cathepsin Bgenes from diverse cerataphidine social aphids and ana-lyzed their molecular phylogenetic and evolutionary as-pects together with members of cathepsin B genesfamily from nonsocial aphids.

Materials and MethodsInsect Materials

Table 1 shows the social aphids examined in thisstudy. Galls of the aphids were collected from the hostplants, and insects were harvested from the galls and imme-diately preserved in acetone or 99% ethanol (Fukatsu1999).

Cathepsin B Genes

Soldier nymphs were subjected to total RNA extrac-tion using RNeasy mini kit (Qiagen GmbH, Hilden, Ger-many). RNA samples were treated with RQ1 RNase-freeDNase (Promega, Madison, WI), reverse transcribed usingSuperScript II (Invitrogen, Carlsbad, CA) with oligo d(T)16

primer, and treated with RNase H (TaKaRa, Shiga, Japan).From T. styraci, Tuberaphis coreana, Tuberaphis taiwana,and Tuberaphis sumatrana, around 1.1-kb cDNA region ofS-type cathepsin B gene, which contained a complete openreading frame (ORF), was amplified by RT-PCR with pri-mers TUB1-F (5#-GGA CTC CTG TAG ATT TAT TTACGC GA-3#) and TUB1-R (5#-GAT AAA AGC CGCGCA AAA ACT A-3#) and was cloned and sequenced.Polymerase chain reaction (PCR) was conducted using Ad-vantage 2 cDNA polymerase (Clontech, Shiga, Japan) un-der a temperature profile of 94 �C for 2 min, followed by35 cycles of 94 �C for 1 min, 55 �C for 1 min, and 72 �C for3 min, and final extension at 72 �C for 5 min. From Tuber-aphis takenouchii, around 0.5-kb cDNA region of S-typecathepsin B gene was amplified by RT-PCR with degener-ate primers DEG-F (5#-TGY GGN WSN TGY TGG GCNKT-3#) and DEG-R (5#-YKN AYN GCR TGN CCN CC-3#) and then the full length of cDNA sequence was obtainedby 5#- and 3#-rapid amplification of cDNA ends (RACE)procedures using Marathon cDNA Amplification Kit(Clontech). Then, around 1.1-kb cDNA region of S-typecathepsin B gene from T. takenouchii was amplifiedby RT-PCR with primers TKNU1-F (5#-CGG CTCCTG TAG ATT AAT TAA CGC GA-3#) and TKNU1-R(5#-GCC ACG CAT AAA AGG CAC ACG AAA A-3#)and was cloned and sequenced. Around 1.0-kb cDNA re-gion of N-type cathepsin B gene, containing an almost fulllength of ORF but three amino acid residues at each of

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Table 1Social Aphids Examined in This Study

Species Collection Locality Collection Date Collector Host PlantDDBJ/EMBL/GenBank Accession Number

Referenced

S-type CatBa N-type CatBb MtrDNAc

Tuberaphis styraci Minakami, Gunma, Japan 28 July 2002 H. Shibao Styrax obassia AB371608 AB162623 (N1) AB371629 Aoki and Kurosu (1989)AB371618 (N2)AB371619 (N3)

Tuberaphis coreana Kiso-Fukushima, Nagano,Japan

October 2002 Y. Tohsaka Styrax japonicus AB167466 (S1)AB371609 (S2)

AB167467 (N1)AB371620 (N2)

AB371630 Kutsukake et al. (2004)

Tuberaphis taiwana Habon, Nantou, Taiwan 4 November 2003 U. Kurosu Styrax formosanus AB371610 (S1) AB371621 AB371631 Aoki and Kurosu (1993)AB371611 (S2)

Tuberaphis sumatrana Tarutung, Sumatra,Indonesia

18 February 1996 T. Fukatsu Styrax subpaniculatus AB371612 (S1)AB371613 (S2)

AB371622 (N1)AB371623 (N2)

AB371632 Fukatsu et al. (1994)

Tuberaphis takenouchii Habon, Nantou, Taiwan 4 November 2003 U. Kurosu Styrax formosanus AB371614 AB371624 (N1) AB371633 Aoki and Usuba (1989)AB371625 (N2)

Tuberaphis leeuweni Tarutung, Sumatra,Indonesia

19 February 1996 T. Fukatsu Styrax subpaniculatus AB371634 Aoki et al. (1998)

Astegopteryx styracophila Lubuk-Linggau,Sumatra,Indonesia

27 January 1997 T. Fukatsu Styrax benzoin AB371615 AB371626 Kurosu et al. (1998)

Astegopteryx spinocephala Doi Suthep, Chiang Mai,Thailand

29 March 2003 U. Kurosu Styrax benzoides AB371616 AB371627 Kurosu et al. (2006)

Cerataphis jamuritsu Sun Moon Lake, Taiwan 24 April 2005 M. Kutsukake Styrax suberifolius AB371617 AB371628 Aoki et al. (1998)

a Soldier-specific type cathepsin B gene.b Nonspecific type cathepsin B gene.c Mitochondrial rRNA gene.d Literatures describing biological information of the social aphids.

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5# and 3# ends, was amplified by RT-PCR from Tuberaphisspecies with primers TUB2-F (5#-TTA TTA AAA ACGTCG ACA TGA TTC G-3#) and TUB2-R (5#-TTT TCTTCG GTG TTT AAT AGG TCA C-3#) and was clonedand sequenced. From Astegopteryx styracophila, Astegop-teryx spinocephala, and Cerataphis jamuritsu, around 0.2-kb cDNA region of S-type cathepsin B gene was amplifiedby RT-PCR with primers KT47-F (5#-AAG CCA ATGGAA CAC AAT CAC A-3#) and KT47-R (5#-CCCCAT CCG ATG AGC TTY AC-3#) and then the full lengthof cDNA sequence was obtained by 5#- and 3#-RACE pro-cedures. Around 1.0-kb cDNA region of N-type cathepsinB gene was obtained from A. styracophila, A. spinocephala,and C. jamuritsu as described above for N-type cathepsin Bgene of Tuberaphis species.

Mitochondrial rRNA Genes

Total DNA was extracted from an individual insectusing QIAamp tissue kit (Qiagen). Around 1.6-kb mito-chondrial DNA segment, containing small subunit rRNAgene, tRNA-Val gene, and large subunit rRNA gene,was amplified by PCR using primers MtrA1 (5#-AAWAAA CTA GGA TTA GAT ACC CTA-3#) and MtrB1(5#-TCT TAA TYC AAC AYC GAG GTC GCA A-3#) un-der the temperature profile of 94 �C for 2 min followed by30 cycles of 94 �C for 1 min, 48 �C for 1 min, and 65 �C for3 min. The PCR product was cloned and sequenced as de-scribed previously (Fukatsu et al. 2001).

Molecular Phylogenetic Analysis

Multiple alignment was performed using the programMAFFT 5.8 (Katoh et al. 2005), followed by manual refine-ment. Aligned sites that included alignment gaps wereomitted from the analysis. Molecular phylogenetic analyseswere conducted by three methods, Neighbor-Joining (NJ),maximum likelihood (ML), and Bayesian (BA). NJ trees(Saitou and Nei 1987) were constructed using the programClustalW (Thompson et al. 1994). Bootstrap values wereobtained by generating 1,000 bootstrap replications. MLtrees were estimated using the program RAxML-VI-HPCVersion 2.2.3 (Stamatakis 2006). In the ML analysis, thePROTCATWAG option and the GTRMIX option wereused as substitution models for amino acid sequencesand nucleotide sequences, respectively. Bootstrap valueswere obtained by generating 1,000 bootstrap replications.In the BA analysis, we used the program MrBayes 3.1.2(Ronquist and Huelsenbeck 2003). The WAG þ C þInv model and GTR þ C þ Inv model were used as sub-stitution models for amino acid sequences and nucleotidesequences, respectively. In total, 4,100 trees were obtained(ngen 410,000, samplefreq 100), and the first 2,100 of thesewere considered as ‘‘burn-in’’ and discarded. We used theprogram ProtTest v1.4 (Abascal et al. 2005) and Modelgen-erator v0.84 (Keane et al. 2006) for the selection of the sub-stitution models of amino acid sequences and nucleotidesequences, respectively. We confirmed that the potentialscale reduction factor was around 1.00 for all parameters

and that the average standard deviation of split frequenciesconverged toward zero.

Quantitative RT-PCR

Semiquantitative RT-PCR was conducted as describedpreviously (Kutsukake et al. 2004). For each of the socialaphid species, soldier nymphs, normal 2nd instar nymphs,and adult insects were subjected to RNA extraction and re-verse transcription as described above, respectively. ThecDNA samples were adjusted to the same concentrationand subjected to PCR amplification by using primersCATB1-F (5#-TAC GAC GAA CAG GGA AAA AACACG-3#) and CATB1-R (5#-TCC CCA GAA CTT ACTCCA CGA ATT-3#) for S-type cathepsin B gene of Tuber-aphis species; primers KT47-F and KT47-R for S-type ca-thepsin B gene of Astegopteryx and Cerataphis species;primers CATB2-F (5#-ATA AAT GCG GGT TCG GATGTT CTG-3#) and CATB2-R (5#-AAC GCC TGA TTTGTA ACT CGG GAA-3#) for N-type cathepsin B geneof Tuberaphis species; and primers F4K1-F (5#-TTCTGY TGT CAC HHG TGC GGA T-3#) and F4K1-R(5#-TCA WAC GAT GCT TCG ATA GGT CC-3#) forN-type cathepsin B gene of Astegopteryx and Cerataphisspecies. PCR was conducted with Advantage 2 cDNA poly-merase (Clontech) under a temperature profile of 94 �C for2 min, followed by 34 cycles of 94 �C for 1 min, 55 �C for 1min, and 72 �C for 1 min, and final extension at 72 �C for 5min. Aliquots of the PCR solution were sampled every twocycles from 24 cycles to 34 cycles, the samples were elec-trophoresed on agarose gels, and quantity of the PCR prod-ucts was evaluated densitometrically after ethidiumbromide staining. By identifying PCR cycles that gave al-most the same levels of amplified product in soldiernymphs, normal nymphs, and adult insects, relative expres-sion levels of S-type and N-type cathepsin B genes wereevaluated across the soldiers and the nonsoldiers.

Estimation of Solvent Accessibility of Amino AcidResidues in Cathepsin B Proteins

Three-dimensional models of S-type and N-type ca-thepsin B mature proteins of T. styraci were inferred bySWISS-Model (Schwede et al. 2003; http://swissmodel.ex-pasy.org/) using the crystal structure of bovine cathepsin Bmature protein as template (Yamamoto et al. 2000; PDBaccession 1qdq). Solvent-accessible surface area (SAS)for each of amino acid residues was computed from the pre-dicted structure models using the program GETAREA1.1(Fraczkiewicz and Braun 1998; http://www.scsb.utmb.edu/cgi-bin/get_a_form.tcl). Each of the amino acid resi-dues was classified into ‘‘exposed,’’ ‘‘partially exposed,’’or ‘‘buried’’ class, according to SAS proportion of the sidechain, which was formulated as follows: [SAS of the sidechain of the residue calculated from the predicted model]/[standard SAS of the side chain of the residue] � 100. Stan-dard SAS of residue X was defined as the average SAS of Xin the tripeptide Gly-X-Gly in an ensemble of 30 randomconformations. Each of the residues was regarded as




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exposed when the SAS proportion exceeded 50%, as par-tially exposed when the proportion was 20–50%, and asburied when the ratio was less than 20%.

Estimation of Proximity of Amino Acid Residues toCatalytic Cleft of Cathepsin B Proteins

For estimating amino acid residues forming the cata-lytic center, the distances between each of the atoms of thebovine cathepsin B protein and the inhibitor CA074, whichwas cocrystallized with the protein and occupied the cata-lytic cleft (Yamamoto et al. 2000), were calculated on thebasis of the three-dimensional positions of the atoms. Eachof the amino acid residues was categorized into either ‘‘nearcatalytic’’ when the residue had atoms within 10 A distancefrom the inhibitor or ‘‘far catalytic’’ when the residue had nosuch atoms. In reference to the categorization, correspond-ing amino acid residues in the S-type and N-type cathepsinB mature proteins of T. styraci were assigned on the basis ofthe alignment of the amino acid sequences for the phyloge-netic inference.

Synonymous and Nonsynonymous Substitution Rates

Synonymous substitutions per site (KS) and nonsynon-ymous substitutions per site (KA) were calculated as de-scribed (Miyata and Yasunaga 1980). Multiplesubstitutions were corrected by Kimura’s two-parametermethod (Kimura 1980). For each of the species, the pre-dominant sequence type among the 10 clones sequencedwas subjected to the analysis. To define clusters used forthe comparison, the phylogenetic relationship of Tubera-phis species inferred from mitochondrial rRNA gene se-quences was used. For comparison between the clusters,mean evolutionary distances between the gene clusterswere computed according to the phylogenetic informationbased on mitochondrial rRNA gene sequences. For exam-ple, we calculated KS[T. sumatrana vs. (T. styraci, T. coreana, T. tai-

wana)], which means KS between T. sumatrana and (T.styraci, T. coreana, T. taiwana), by (KS[T. sumatrana vs.

T. styraci] þ KS [T. sumatrana vs. T. coreana] þ KS [T. sumatrana

vs. T. taiwana]) � 1/3. We calculated KA in a similar wayand estimated the ratio KA/KS, defining the ratio for eachof the clusters. In the estimation of KA/KS ratios for eachof the partitions defined on the three-dimensional modelsof cathepsin B mature proteins, we assumed that synony-mous rates are constant among codon sites. Thus, KS valuescalculated from the full-coding regions of the cathepsin Bmature proteins were used for calculation of KA/KS ratiosfor each of the partitions. Statistical significance of the ob-tained KA/KS value was tested against a bootstrap distribu-tion of KA/KS values, which was generated by 10,000bootstrap resamplings of codons from the original alignment.

Synonymous and Nonsynonymous SubstitutionsEstimated on Tree Branches

The phylogenetic relationship inferred from mitochon-drial rRNA gene sequences was adopted as the backbone

phylogeny of the Tuberaphis species (cf. supplementaryfig. S1, Supplementary Material online). To reconstructthe ancestral nucleotide sequences on each of the internalnodes and to compute the numbers of synonymous and non-synonymous nucleotide differences for each of the branchesof the tree, the codeml program of the PAML softwarepackage (Yang 1997) was used under the following set-tings: the average nucleotide frequencies at the three codonpositions (CodonFreq 5 2) were used as a model of codonfrequency; equal amino acid distance (aaDist 5 0) was as-sumed as a codon substitution model; and one nonsynon-ymous/synonymous substitution rate ratio (x) was appliedto all the branches (model 5 0). Model selection did notaffect our results (data not shown). The statistical analysiswas conducted by the method of Zhang et al. (1998), whichis based on the Fisher’s exact test in a 2 � 2 contingencytable with the numbers of nonsynonymous sites and synon-ymous sites as rows and the numbers of changed sites andthe numbers of unchanged sites as columns.

Accession Numbers

The nucleotide sequences determined in this studywere deposited in the DDBJ/EMBL/GenBank nucleotidesequence databases (for accession numbers, see table 1).

ResultsSoldier-Specific and Nonspecific Cathepsin B Genes inSocial Aphids

From each of T. styraci, T. coreana, T. taiwana,T. sumatrana, and T. takenouchii, S-type cathepsin B genewas amplified by RT-PCR and cloned. For each of the spe-cies, 10 clones were sequenced. In T. styraci and T. taken-ouchii, all 10 sequences were identical. In T. coreana,T. taiwana, and T. sumatrana, two types of sequences wereidentified, wherein numbers of nucleotide differences were25, 6, and 1, respectively.

Similarly, N-type cathepsin B gene from each of theTuberaphis species was analyzed. Of 10 sequenced clones,a single sequence was identified in T. taiwana, two types ofsequences were obtained from T. coreana, T. sumatrana,and T. takenouchii (differences in 2, 13, and 39 nt sites,respectively), and three types of sequences were detectedfrom T. styraci (differences in 16 sites between N1 andN2, 21 sites between N1 and N3, and 5 sites betweenN2 and N3).

The slightly different S-type and N-type gene sequen-ces from the same Tuberaphis species are plausibly due toallelic polymorphisms, although the possibilities of recentgene duplications and/or gene conversions cannot be ruledout.

From A. styracophila, A. spinocephala, and C. jamur-itsu, S-type cathepsin B gene was obtained by 5#- and 3#-RACE methods. It should be noted that the S-type genesequence from C. jamuritsu was disrupted by a nonsensemutation located at a 5# region. All six clones determinedby 5# RACE method exhibited the same sequence, andthree clones of the full-length cDNA we inspected also

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represented the same sequence. Hence, it is unlikely that thenonsense mutation is due to an experimental artifact likePCR error. Except for the stop codon mutation, the S-typegene was structurally intact. The S-type gene sequencesfrom A. styracophila and A. spinocephala contained a com-plete ORF.

From the Astegopteryx and Cerataphis species, N-typecathepsin B gene was amplified and cloned. For A. styra-cophila, A. spinocephala, and C. jamuritsu, 3, 5, and 9clones were sequenced, respectively. All the sequenceswere identical within the aphid species.

Phylogenetic Relationship of the Cathepsin B Genesfrom the Social Aphids to Those from the Pea Aphid

Figure 1 shows the phylogenetic relationship of the S-type and N-type cathepsin B genes from the social aphids ofthe genus Tuberaphis to the cathepsin B genes from thenonsocial aphid A. pisum on the basis of deduced aminoacid sequences. The S-type genes from the social aphidsformed a well-supported and distinct clade in the phylog-

eny, so did the N-type genes from the social aphids. The S-type clade clustered with Ap84 sequence from A. pisum,although statistical support for the grouping was weak.The N-type clade clustered with Ap16D1, Ap16a,Ap912, and Ap3098 sequences from A. pisum.

Phylogenetic Relationship of the Cathepsin B Genesfrom the Social Aphids and Their Expression in DifferentCastes

Figure 2A shows the phylogenetic relationship of theS-type and N-type cathepsin B genes from the social aphidsdeduced from amino acid sequences. For each of Tubera-phis species, the major sequence type among the 10 clonesexamined was subjected to the phylogenetic analysis. TheS-type clade clustered with the Ap84 clade consisting ofsequences from nonsocial aphids A. pisum and Tuberaphiscitricida, whereas the N-type clade showed affinity to theAp16D1 clade encompassing sequences from these nonso-cial aphids. The phylogenetic relationships were largelycongruent with the mitochondrial phylogeny of the

FIG. 1.—Molecular phylogenetic analysis of the soldier-specific type (S-type) and nonspecific (N-type) cathepsin B protein sequences from fivesocial aphids of the genus Tuberaphis and 14 cathepsin B protein sequences from the EST and genomic data of the pea aphid Acyrthosiphon pisum. Atotal of 219 aligned amino acid sites were subjected to the analyses. Cathepsin B protease sequences from other insects and animals were used asoutgroup. An NJ tree is shown, whereas an ML tree and a BA tree are substantially congruent with the tree topology. On each of the nodes, bootstrapprobabilities for NJ, support values for ML, and posterior probabilities for BA are indicated. Asterisks indicate statistical values less than 50. Forsequences from Tuberaphis species, clone frequencies in 10 clones examined are indicated in parentheses. In brackets are sequence accession numbers.In rightmost parentheses are organisms from which the sequences were obtained. On the right side, cluster names designated by Kutsukake et al. (2004)and Rispe et al. (2008) are shown.




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Tuberaphis aphids (supplementary fig. S1, SupplementaryMaterial online).

Figure 2B shows the expression levels of the cathepsinB genes in the social aphids evaluated by a semiquantitativeRT-PCR method. In Tuberaphis species, the S-type geneswere expressed strikingly higher (.1,000 times, data notshown) in soldier nymphs than in normal insects, whereasthe N-type genes were expressed irrespective of the castes.In A. spinocephala, both the S-type and N-type genes wereexpressed in both the castes. In A. styracophila, unexpect-edly, the N-type gene exhibited higher expression in soldiernymphs (around 64 times, data not shown) than in normalinsects, whereas the S-type gene was expressed in a consti-tutive manner. In C. jamuritsu, not only the N-type genebut also the S-type gene was, although pseudogenized, ex-pressed in both the castes. Cloning and sequencing of theRT-PCR products confirmed that messenger RNA (mRNA)of the S-type gene containing a stop codon was certainlytranscribed in C. jamuritsu (data not shown).

Accelerated Amino Acid Substitution in the S-TypeCathepsin B Genes in Tuberaphis Species

In Tuberaphis species, KA/KS values of the S-type ca-thepsin B genes (0.63–1.76) were remarkably higher than

those of the N-type cathepsin B genes (0.02–0.21). In par-ticular, in the lineages of T. styraci, T. coreana, andT. taiwana, KA/KS values of the S-type genes were largerthan 1 (1.75–1.76), suggesting that positive selection hasbeen acting on the molecules. In the lineages of T. suma-trana and T. takenouchii, KA/KS values of the S-typegenes were smaller than 1 (0.63–0.83). In Astegopteryxspecies, KA/KS value of the S-type cathepsin B genewas only 0.21, which was almost equivalent to KA/KS

value of the N-type cathepsin B gene, 0.31 (table 2;‘‘All coding’’ lines).

Partitioning of Amino Acid Residues of the S-Type andN-Type Cathepsin B Proteins according to SolventAccessibility

Three-dimensional models of the S-type and N-typecathepsin B mature proteins were inferred by using the crys-tal structure of the bovine cathepsin B protein as template.By computing SAS on the structural models, each of theamino acid residues of the S-type and N-type proteinswas classified into exposed (red), partially exposed (pink),or buried (uncolored) (fig. 3). By comparing amino acid

FIG. 2.—(A) Molecular phylogenetic analysis of the S-type and N-type cathepsin B protein sequences from social aphids of the genera Tuberaphis,Astegopteryx, and Cerataphis and those from nonsocial aphids Acyrthosiphon pisum and Tuberaphis citricida. A total of 323 aligned amino acid siteswere subjected to the analysis. A cathepsin B protease sequence from the rice planthopper Nilaparvata lugens was used as outgroup. An NJ tree isshown, whereas an ML tree and a BA tree are substantially congruent with the tree topology. On each of the nodes, bootstrap probabilities for NJ,support values for ML, and posterior probabilities for BA are indicated. In brackets are sequence accession numbers. (B) Semiquantitative RT-PCR ofthe S-type and N-type cathepsin B genes. S, soldier nymphs; N, normal 2nd instar nymphs; and A, adults.




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sequences of the proteins between T. styraci, T. coreana,T. taiwana, and T. sumatrana, each of the amino acid res-idues was categorized into either ‘‘constant’’ (normal lettersin fig. 3A and B) or ‘‘variable’’ (circled letters in fig. 3A andB and spheres in fig. 3C and D). The S-type protein con-tained more variable residues than the N-type protein (33vs. 11) (fig. 3).

Accelerated Amino Acid Substitution in the S-TypeCathepsin B Genes Preferentially at Exposed Residues

When KA/KS values were separately calculated for ex-posed, partially exposed, and buried residues, striking pat-terns emerged in the S-type cathepsin B genes ofTuberaphis species (table 2). In the lineages of T. styraci,T. coreana, and T. taiwana, KA/KS values of the S-typegenes were extremely high, ranging from 2.12 to 3.77 atexposed and partially exposed residues, whereas low val-ues (0.37–0.56) were observed at buried residues. In thelineage of T. sumatrana, KA/KS values were higher than1 (1.30–1.39) at exposed and partially exposed residues,whereas buried residues exhibited a low KA/KS value of0.30. In the lineage of T. takenouchii, KA/KS values wereless than 1 (0.87–0.93) at exposed and partially exposedresidues but still higher than value at buried residues(0.37). In the S-type cathepsin B genes of Astegopteryx spe-cies, KA/KS values were low irrespective of residue types,although the values at exposed and partially exposedresidues (0.31–0.51) were higher than the value at buriedresidues (0.00) (table 2).

Partitioning of Amino Acid Residues of the S-Type andN-Type Cathepsin B Proteins according to Proximity toCatalytic Cleft

Three-dimensional models of the S-type and N-typecathepsin B mature proteins were inferred from the crystal-lography of the CA074-binding bovine cathepsin B protein,and each of the amino acid residues was categorized intoeither near catalytic within 10 A from the catalytic cleft(blue) or far catalytic over 10 A apart from the catalytic cleft(uncolored) (fig. 4).

No Significant Differences in Amino Acid SubstitutionRates between Near-Catalytic and Far-Catalytic Residues

In all the Tuberaphis and Astegopteryx species and ir-respective of the S-type and N-type cathepsin B proteins,no significant differences in KA/KS values were detectedbetween near-catalytic residues and far-catalytic residues(table 3). Even when exposed, partially exposed, and buriedresidues were separately considered (cf. supplementaryfig. S2, Supplementary Material online), no significant dif-ferences in KA/KS values were detected between near-catalytic residues and far-catalytic residues (supplementarytable S1, Supplementary Material online).

Mapping of Synonymous and NonsynonymousSubstitutions in S-Type and N-Type Cathepsin B Genesin the Evolutionary Course of Tuberaphis Species

On the phylogeny of Tuberaphis species, occurren-ces of synonymous and nonsynonymous substitutions of

Table 2KA and KS Values of Exposed, Partially Exposed, and Buried Regions Obtained from Comparisons of the S-Type and theN-Type Cathepsin B Genes from Tuberaphis and Astegopteryx Species

RegionS-Type N-Type

KS KA KA=K�S KS KA KA=K

�S

Tuberaphis coreana versus Tuberaphis taiwanaAll coding 0.013 0.022 1.76 0.059 0.002 0.03Exposed 0.024 0.048 3.77* 0.060 0.000 0.00Partially exposed 0.028 0.027 2.12 0.083 0.000 0.00Buried 0.000 0.007 0.56 0.049 0.003 0.06

Tuberaphis styraci versus (T. coreana, T. taiwana)All coding 0.019 0.034 1.75 0.049 0.001 0.02Exposed 0.036 0.065 3.38* 0.094 0.000 0.00Partially exposed 0.042 0.055 2.83* 0.041 0.000 0.00Buried 0.000 0.007 0.37 0.036 0.002 0.03

Tuberaphis sumatrana versus (T. styraci, T. coreana, T. taiwana)All coding 0.052 0.043 0.83 0.097 0.021 0.21Exposed 0.082 0.072 1.39 0.040 0.020 0.21Partially exposed 0.074 0.067 1.30 0.087 0.050 0.51Buried 0.026 0.015 0.30 0.128 0.007 0.07

Tuberaphis takenouchii versus (T. styraci, T. coreana, T. taiwana, T. sumatrana)All coding 0.182 0.115 0.63 0.232 0.042 0.18Exposed 0.190 0.158 0.87 0.129 0.079 0.34Partially exposed 0.215 0.169 0.93 0.211 0.070 0.30Buried 0.163 0.067 0.37 0.290 0.011 0.05

Astegopteryx styracophila versus Astegopteryx spinocephalaAll coding 0.066 0.014 0.21 0.102 0.031 0.31Exposed 0.132 0.034 0.51 0.125 0.077 0.76Partially exposed 0.055 0.020 0.31 0.083 0.050 0.50Buried 0.039 0.000 0.00 0.100 0.000 0.00

NOTE.—KS, number of nucleotide substitutions per synonymous site; KA, number of nucleotide substitutions per nonsynonymous site; KS value of the all-coding region

in each comparison was used as K�S in this study. Asterisks indicate significantly higher KA/KS value than 1 (bootstrap test): P , 0.05.




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the S-type and N-type cathepsin B genes were estimatedand mapped on each of the branches (fig. 5). As for theS-type genes, very high KA/KS values were observed atexposed and partially exposed residues in the branchesleading to T. coreana (3.3–4.4), T. taiwana (1.1–3.3),and T. styraci (2.2–2.7). These values were remarkablyhigher than 1.0, but the differences from 1.0 were statis-tically not significant probably because of limited num-bers of nucleotide sites and substitutions subjected to theanalysis. KA/KS values at exposed and partially exposedresidues were consistently around 1.0 in the commonancestors of T. coreana, T. taiwana, and T. styraci(1.1) and also in the lineages leading to T. sumatrana(0.90–0.99) and T. takenouchii (0.91–1.1). At buriedresidues, KA/KS values were consistently low (0.0–0.58) in all the lineages but the lineage of T. taiwana(1.1) (fig. 5A). As for the N-type genes, by contrast,KA/KS values were consistently low (0.0–0.56) in allthe lineages and irrespective of the residue types, exceptfor at partially exposed residues in the lineage of T. su-matrana (1.2) (fig. 5B).

DiscussionAncient Origin of the S-Type and N-Type Cathepsin BGenes in Aphids

The S-type and N-type cathepsin B genes were de-tected not only from social aphids of the genus Tuberaphisbut also from social aphids of other genera Astegopteryxand Cerataphis and, strikingly, also from phylogeneticallydistant nonsocial aphids A. pisum and T. citricida (figs. 1and 2). Considering that, in the aphid evolution, the sub-family Hormaphidinae embracing the social aphids basallydiverged from the subfamily Aphidinae consisting exclu-sively of nonsocial aphids (Heie 1987; Von Dohlen andMoran 1995), the origin of the S-type and N-type cathepsinB genes is likely to be quite ancient, possibly dating back tothe common ancestor of extant aphids 80–150 MYA (VonDohlen and Moran 2000). Here it should be noted that, al-though the N-type cathepsin B genes from the nonsocialaphids formed a good clade with those from the socialaphids, statistical supports for the monophyly of the S-typecathepsin B genes from the nonsocial and social aphids

FIG. 3.—Solvent accessibility of each of the amino acid residues in the S-type and N-type cathepsin B proteins of Tuberaphis styraci, inferred fromthe crystal structure of the bovine cathepsin B protease. (A) The primary structure of the mature S-type cathepsin B protein. (B) The primary structure ofthe mature N-type cathepsin B protein. (C) The inferred tertiary structure of the mature S-type cathepsin B protein. (D) The inferred tertiary structure ofthe mature N-type cathepsin B protein. In (A) and (B), circled letters indicate variable amino acid residues among T. styraci, Tuberaphis coreana,Tuberaphis taiwana, and Tuberaphis sumatrana, whereas normal letters show nonvariable amino acid residues. In (C) and (D), spheres indicatevariable amino acid residues. In (A–D), colors represent the inferred solvent accessibility for each of the amino acid residues: red, exposed; pink,partially exposed; and uncolored, buried.




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were not significant (figs. 1 and 2). Hence, the possibilityseems also likely that the S-type genes of the social aphidsdo not form a clade with but have a distinct evolutionaryorigin from the S-type genes of the nonsocial aphids. Any-way, it appears plausible that the massive amplification ofcathepsin B genes in aphids (Rispe et al. 2008) has predis-posed the evolution of venomous protease in the socialaphids.

The S-Type Cathepsin B Gene Acquired VenomFunction in the Genus Tuberaphis

Among the social aphids examined in this study, sol-dier-specific expression of the S-type cathepsin B gene wasdetected only in the genus Tuberaphis (fig. 2B). Acceler-ated amino acid substitution of the S-type cathepsin Bwas observed only with Tuberaphis species (table 2). Nei-ther such remarkable levels of soldier-specific expressionnor accelerated molecular evolution of the S-type gene wereidentified in Astegopteryx and Cerataphis species (fig. 2;table 2), although these social aphids belong to the sameaphid group Cerataphidini and produce 2nd instar soldiersin their galls as Tuberaphis species do (Stern and Foster1996). These results suggest that, although the S-type

and N-type cathepsin B genes are commonly present inthe social aphids, the soldier-specific expression and thevenom function of the S-type gene evolved after divergenceof the genus Tuberaphis.

Positive Selection Acting on Molecular Surface of S-Type Cathepsin B Protein

When the S-type and N-type cathepsin B protein se-quences were partitioned into exposed, partially exposed,and buried amino acid residues and were subjected to mo-lecular evolutionary analyses, high KA/KS values were de-tected at exposed and partially exposed residues in S-typecathepsin B sequences of Tuberaphis species (table 2). Theevolutionary pattern strongly suggests that positive selec-tion has been acting on the molecular surface of the S-typecathepsin B proteins in the evolutionary course of the socialaphids. It appears likely that the rapid evolution at the sur-face residues of the S-type cathepsin B protease is relevantto its venom function. Surface structure of the venomousprotease must be important for recognition of its target mol-ecules. Structural change in the lethal target molecules ofpredatory insects would result in their resistance to thevenom. Here coevolutionary arms race between soldier’s

FIG. 4.—Proximity to the catalytic cleft of each of the amino acid residues in the S-type and N-type cathepsin B proteins of Tuberaphis styraci,inferred from the crystal structure of the CA074-bound bovine cathepsin B protease. (A) The primary structure of the mature S-type cathepsin B protein.(B) The primary structure of the mature N-type cathepsin B protein. (C) The inferred tertiary structure of the mature S-type cathepsin B protein. (D) Theinferred tertiary structure of the mature N-type cathepsin B protein. In (A) and (B), circled letters indicate variable amino acid residues among T. styraci,Tuberaphis coreana, Tuberaphis taiwana, and Tuberaphis sumatrana, whereas normal letters show nonvariable amino acid residues. In (C) and (D),spheres indicate variable amino acid residues. Blue, near-catalytic residues; uncolored, far-catalytic residues.




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venomous protease and predators’ target molecules mightoccur, which would lead to accelerated evolution preferen-tially on the molecular surface. Alternatively, predatory in-sects might have immune/detoxifying mechanisms against

the venomous protease, which recognize the surface struc-ture of the molecule and inactivate it. If so, accelerated evo-lution of the protease at the molecular surface would befavored for escaping the immune/detoxifying mechanisms.Similar accelerated molecular evolution preferentially atsurface residues has been reported from other venomousenzymes such as phospholipase A2 of snakes (Kini andChan 1999), wherein diversity in surface structure of theenzymes is suggested to be involved in their altered targetspecificity to various cells and tissues, resulting in a widevariety of pharmacological effects including neurotoxicity,myotoxicity, cardiotoxicity, anticoagulant effects, etc (Kini2003).

Soldier-Specific Expression of the S-Type Cathepsin BGene Preceded Evolutionary Acceleration in the GenusTuberaphis

Soldier-specific expression of the S-type cathepsin Bgene was observed in all the Tuberaphis species examined(fig. 2B). Meanwhile, the molecular evolution of the S-typecathepsin B gene was fast in T. styraci, T. coreana, and T.taiwana; moderate in T. sumatrana; and slower in T. taken-ouchii (table 2). In the genus Tuberaphis, T. styraci, T. cor-eana, and T. taiwana formed a compact clade, whereas T.sumatrana and T. takenouchii constituted basal lineages(figs. 2; supplementary fig. S1, Supplementary Material on-line). KA/KS values were certainly higher in the lineages ofT. styraci, T. coreana, and T. taiwana than in the lineages ofT. sumatrana and T. takenouchii (fig. 5). These patternssuggest that 1) soldier-specific expression of the S-type ca-thepsin B gene probably preceded evolutionary accelera-tion of the gene and 2) positive selection acting on thegene might have been stronger in the younger lineages than

FIG. 5.—Estimated numbers of synonymous and nonsynonymoussubstitutions in cathepsin B genes mapped on the phylogeny ofTuberaphis species. (A) S-type, (B) N-type. Four numbers above eachof the branches from left to right indicate: number of nonsynonymoussubstitutions per sequence per branch at exposed residues, that at partiallyexposed residues, that at buried residues; and number of synonymoussubstitutions per sequence per branch at all the residues. Three numbersbelow each of the branches from left to right indicate: KA/KS ratio in thebranch at exposed residues, that at partially exposed residues, and that atburied residues. Branch lengths are arbitrarily shown.

Table 3KA and KS Values of Near-Catalytic and Far-Catalytic Regions Obtained from Comparisons of the S-Type and the N-TypeCathepsin B Genes from Tuberaphis and Astegopteryx Species

Region

S-Type N-Type

KS KA KA=K�S KS KA KA=K

�S

Tuberaphis coreana versus Tuberaphis taiwanaAll coding 0.013 0.022 1.76 0.059 0.002 0.03Near catalytic 0.030 0.028 2.18 0.027 0.000 0.00Far catalytic 0.008 0.021 1.58 0.070 0.002 0.04

Tuberaphis styraci versus (T. coreana, T. taiwana)All coding 0.019 0.034 1.75 0.049 0.001 0.02Near catalytic 0.015 0.036 1.81 0.013 0.000 0.00Far catalytic 0.021 0.033 1.69 0.061 0.001 0.02

Tuberaphis sumatrana versus (T. styraci, T. coreana, T. taiwana)All coding 0.052 0.043 0.83 0.097 0.021 0.21Near catalytic 0.039 0.059 1.11 0.042 0.035 0.34Far catalytic 0.055 0.038 0.72 0.116 0.017 0.16

Tuberaphis takenouchii versus (T. styraci, T. coreana, T. taiwana, T. sumatrana)All coding 0.182 0.115 0.63 0.232 0.042 0.18Near catalytic 0.159 0.143 0.80 0.197 0.048 0.21Far catalytic 0.189 0.107 0.60 0.243 0.040 0.17

Astegopteryx styracophila versus Astegopteryx spinocephalaAll coding 0.066 0.014 0.21 0.102 0.031 0.31Near catalytic 0.029 0.007 0.10 0.087 0.014 0.15Far catalytic 0.077 0.016 0.23 0.104 0.036 0.38

NOTE.—KS, number of nucleotide substitutions per synonymous site; KA, number of nucleotide substitutions per nonsynonymous site; KS value of the all-coding region

in each comparison was used as K�Sin this study.




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in the older lineages. It is currently obscure why such evo-lutionary patterns are observed with the S-type cathepsin Bgene in the genus Tuberaphis. One possibility is that, al-though speculative, evolutionary arms race between sol-diers and predators has become severer in the newlyevolved lineages than in the older lineages. Alternatively,in the older lineages, molecular evolution of the venom pro-tease could have been slowed down after establishment oftheir stable ecological niche. Even the possibility should betaken into account that the apparent evolutionary patternsmight be due to methodological artifact, wherein acceler-ated molecular evolution was not properly estimated forthe genetically distant basal lineages. To address whichof these hypotheses is the most appropriate, further analysesof more Tuberaphis and allied social aphids are needed.

Gut-Specific Expression of the S-Type Cathepsin B Genein Social and Nonsocial Aphids

In T. styraci, the S-type cathepsin B protease is pro-duced in the midgut epithelium of soldier nymphs, secretedinto the midgut cavity, vomited through the stylet, and in-jected into the body cavity of enemies (Kutsukake et al.2004). The intestinal expression and functioning of theS-type cathepsin B protease are probably found in soldiernymphs of Tuberaphis species in common. Here it shouldbe noted that Ap84 cathepsin B gene of the pea aphid,which is phylogenetically related to the S-type cathepsinB genes of the social aphids (figs. 1 and 2), is also highlyexpressed in the midgut (Rispe et al. 2008). Hence, the gut-specific expression of the S-type cathepsin B gene in Tuber-aphis soldiers might reflect the ancestral expression patternof the protease gene.

Why Are Diverse Cathepsin B Genes Expressed inAphid Gut?

The sole food source for aphids, which is plant phloemsap, contains much sugar and some nonessential aminoacids but is devoid of lipids and proteins. Conventionally,it has been believed that aphids substantially have no intes-tinal digestion of proteins (Terra 1990; Douglas 2003).However, recently studies have uncovered the presenceof a variety of proteins in plant phloem fluid (e.g., Kehr2006) and also the presence of intestinal proteases in somephloem-feeding insects (e.g., Foissac et al. 2002; Cristofo-letti et al. 2003; Deraison et al. 2004). In this context, thediscovery of massive amplification of aphid cathepsin Bprotease genes and their preferential expression in the mid-gut is intriguing (Rispe et al. 2008). Why aphids haveevolved so many intestinal protease genes is currently anenigma. One possibility is that, although speculative,aphid–plant coevolution might be involved in the process.Some plants express inhibitors that are effective against in-sect herbivores (Koiwa et al. 1997; Lawrence and Koundal2002). For example, aphid infestation on sorghum wasshown to trigger overexpression of defense genes includingprotease inhibitors (Zhu-Salzman et al. 2004). Some of theplant protease inhibitors are themselves proteins and thus

could be inactivated by aphid proteases. In this context,it is tempting to assume that, although speculative, suchherbivore–plant arms races resulted in the diversificationand rapid evolution of the cathepsin B genes in aphids,which might have predisposed the evolution of the venom-ous protease in the social aphid lineage.

Dynamic Evolution of the S-Type and N-Type CathepsinB Genes in Social Aphids: Relaxed FunctionalConstraints due to Gene Duplication?

In C. jamuritsu, the S-type cathepsin B gene was, al-though containing a nonsense mutation, structurally almostintact and still transcribed into mRNA (fig. 2B), suggestinga recent pseudogenization of the gene copy. In A. styraco-phila, not the S-type gene but the N-type gene was prefer-entially expressed in soldier nymphs (fig. 2B), although it iscurrently unknown whether the upregulated N-type geneproduct is involved in the defensive role. These dynamicevolutionary patterns of the cathepsin B genes in the socialaphids are probably relevant to relaxed functional con-straints in the multigene family generated through the mas-sive gene amplification in aphids (Rispe et al. 2008).

The S-Type Cathepsin B Protease as Novel Componentof Aphid Venom

In T. styraci, the S-type cathepsin B protease is pro-duced in a soldier-specific manner and functions as a majorvenom component for attacking natural enemies (Kutsukakeet al. 2004). This study suggests that the S-type cathepsinB protease plays a defensive role in soldier nymphs ofTuberaphis species but probably not in soldier nymphsof Astegopteryx and Cerataphis species. Meanwhile, itwas observed that soldier nymphs of A. styracophila,A. spinocephala, and C. jamuritsu are able to attack andkill other insects effectively (Aoki et al. 1998; Kurosuet al. 1998, 2006). Attacks by soldiers of these social aphidsto human skin cause unpleasant itch (T. Fukatsu, personalobservations), indicating that the soldiers inject some toxiccompounds other than the cathepsin B protease into ene-mies. In general, animal venom is a mixture of bioactivecompounds such as amines, peptides, phospholipases, hy-aluronidases, proteases, and others, and these moleculessynergistically exert poisonous activities (Habermann1972). On the basis of these lines of evidence, we suggestthat the S-type cathepsin B protease is a novel venom com-ponent acquired in the lineage of Tuberaphis species, andmany other bioactive compounds are to be discovered in theaphid venom. Other social aphids like Astegopteryx spp.and Cerataphis spp. probably use toxic compounds otherthan cathepsin B for attacking enemies.

Gene Duplication, Accelerated Molecular Evolution, andAcquisition of Novel Function as Venomous Protein

Thus far, venomous proteins have been identified andinvestigated mainly in carnivorous animal groups such as




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snakes, scorpions, cone snails, etc., wherein the toxic mol-ecules are used for paralyzing, killing, and/or digesting theirvictims. Most of the venomous proteins are encoded asmultigene families in the animal genomes that have beengenerated through gene duplications and also exhibit accel-erated molecular evolution due to positive selection actingon the molecules. From snake venoms, phospholipases A2

(Nakashima et al. 1993, 1995; Ogawa et al. 1995, Nobuhisaet al. 1996; Kordis and Gubensek 1996, 1997), serine pro-teases (Deshimaru et al. 1996), metalloproteases (Moura-da-Silva et al. 1996), Kunitz/BPTI (bovine pancreatic tryp-sin inhibitor) proteins (Zupunski et al. 2003), three-fingeredtoxins (Lachemanon et al. 1998; Ohno et al. 1998; Gonget al. 2000), and C-type lectins (Tani et al. 2002; Ogawaet al. 2005) have been identified as multiple copied and pos-itively selected venomous proteins. Similar evolutionarypatterns have been found in scorpion sodium channel toxins(Zhu et al. 2004) and Conus conotoxins (Duda and Palumbi1999). In many of these cases, the recruited proteins haveevolved novel toxic activities in addition to the ancestralactivities. In snakes, such recruitment events from nontoxicbody proteins are estimated to have occurred at least 24times (Fry 2006). In the case of phospholipase A2, the en-zyme has evolved a toxic site, irrelevant to the catalytic site,at a C-terminal region of the molecule (Krizaj et al. 1989;Lomonte et al. 1994; Paramo et al. 1998; Nunez et al. 2001;Prijatelj et al. 2002). In this study, we demonstrated thata family of cysteine proteases has acquired a novel venomfunction and exhibited such evolutionary patterns in socialaphids, a herbivorous animal group living exclusively onplant sap. In this case, however, considering that the accel-erated molecular evolution is not related to the catalyticcenter of the protease but associated with the molecular sur-face (tables 2 and 3), the venom activity was probably ac-quired by relatively minor modifications on the molecularsurface structure rather than by generation of a novel activesite. Our finding favors the notion that gene duplication fol-lowed by accelerated molecular evolution comprises a gen-eral and important evolutionary process that enablesacquisition of novel gene functions (Ohno 1970; Hughes1994; Zhang et al. 1998).

Evolutionary Origin of Novel Traits Underpinning InsectSociality

Major social insect groups such as bees, wasps, ants,and termites exhibit a number of novel traits that are essen-tial for maintaining their complex social systems. Recentstudies have unveiled some molecular aspects underlyingnovel traits unique to social insects, such as royal jelly pro-teins for larval nursing in honeybee (Drapeau et al. 2006),an odorant-binding protein Gp-9 that governs the monog-yny/polygyny reproductive modes of fire ant colonies(Krieger and Ross 2002), and antifungal proteins for main-tenance of fungal garden in termites (Bulmer and Crozier2004), wherein gene duplication and accelerated molecularevolution are observed. In a phylogenetically distant insectgroup, social aphids, we also found that the evolution ofa novel venomous protease has been realized by gene du-plication and subsequent accelerated molecular evolution

due to positive selection. For evolution of complex bio-logical systems including insect sociality, gene duplica-tions, accelerated molecular evolution, and acquisitionof novel gene function must have played considerableroles in general.

Supplementary Material

Supplementary figures S1 and S2 and table S1 areavailable at Molecular Biology and Evolution online(http://www.mbe.oxfordjournals.org/).

Acknowledgments

We thank S. Aoki and T. Miura for critically readingthe manuscript and U. Kurosu, S. Aoki, C. C. Wang, H. J.Lee, and Y. Tohsaka for aphid samples. This work was sup-ported by the Japan–France Integrated Action Program SA-KURA of the Japan Society for the Promotion of Science toT.F. and J.C.S.

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Yoko Satta, Associate Editor

Accepted September 9, 2008




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Evolution of Soldier-Specific Venomous Protease in Social Aphids

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