MBE Advance Access originally published online on September 26, 2008
Molecular Biology and Evolution 2008 25(12):2627-2641; doi:10.1093/molbev/msn203
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Research Articles |
Evolution of Soldier-Specific Venomous Protease in Social Aphids
* 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
Institut National de la Recherche Agronomique (INRA), Unité Mixte de Recherche (UMR)1099 BIO3P, Le Rheu, France
E-mail: t-fukatsu{at}aist.go.jp.
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Abstract |
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 TOP Abstract IntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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In social aphids of the genus Tuberaphis a cysteine protease gene of the family cathepsin B exhibits soldier-specific expression and intestinal protease production. The product is orally excreted and injected by soldier nymphs into natural enemies, thereby exerting an insecticidal activity. In an attempt to gain insights into when and how the novel venomous protease 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 genera Tuberaphis, Astegopteryx, and Cerataphis, possessed both the S-type and N-type genes. Phylogenetically distant nonsocial aphids also possessed cathepsin B genes allied to the S-type and the N-type, indicating the evolutionary origin of these genes in the common ancestor of extant aphids. In Tuberaphis species the S-type genes exhibited significant soldier-specific expression and accelerated molecular evolution whereas the N-type genes did not. In Astegopteryx and Cerataphis species, meanwhile, both the S-type and N-type genes exhibited neither remarkable soldier-specific expression nor accelerated molecular evolution. These results suggest that the S-type gene acquired the soldier-specific expression and the venom function after divergence of the genus Tuberaphis. On the structural model of the S-type protease of Tuberaphis styraci the accelerated molecular evolution was associated with the molecular surface rather than the catalytic cleft, suggesting that the venom activity was probably acquired by relatively minor modifications on the molecular surface rather than by generation of a novel active site. In Cerataphis jamuritsu the S-type gene was, although containing a stop codon, structurally almost intact and still transcribed, suggesting recent pseudogenization of the gene copy and possible relevance to relaxed functional constraint in the highly multiplied protease gene family. On the basis of these results we suggest that the massive amplification in aphid cathepsin B genes might have predisposed the evolution of venomous protease in the social aphid lineage and argue that gene duplication, accelerated molecular evolution, and acquisition of novel gene function must have played considerable roles in the evolution of complex biological systems including insect sociality.
Key Words: social aphid • soldier caste • venom protein • cathepsin B protease • accelerated evolution • gene duplication
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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In colonies of social insects, hundreds, thousands, or millions of individuals are integrated into a highly organized and homeostatic system, comprising one of the most impressive biological entities in nature. Some individuals are engaged in reproduction, whereas others produce few or no offspring and constitute specialized castes that are characterized by distinct morphological, physiological, and behavioral traits for their altruistic functions. Morphologically and reproductively differentiated castes (e.g., queens, workers, soldiers, etc.) are well known from social insect groups such as bees, ants, wasps, and termites (Wilson 1971
), but such castes have also been found in less studied groups like aphids (Stern and Foster 1996).
Some 50 species of aphids, representing two subfamilies Hormaphidinae and Eriosomatinae, are known to produce nymphs that altruistically sacrifice their own reproduction for the benefit of their colony mates. Such nymphs are called "soldiers" because their primary social role is defense, whereas this caste may also play a nondefensive altruistic role such as gall cleaning. In highly social aphids, soldier nymphs are morphologically differentiated from normal nymphs and unable to grow, constituting a sterile caste (Aoki 1987; Ito 1989; Stern and Foster 1996).
The establishment and maintenance of the insect sociality must entail a number of evolutionary novelties that are involved in caste differentiation, division of labor, social communication, colony regulation, nest building, and many other social traits. It is of great interest what molecular mechanisms have been acquired and/or recruited in the evolutionary course of the insect sociality. Despite technical difficulties with the nonmodel insects, recent development in molecular genetics and genomics has unveiled some intriguing molecular aspects relevant to social traits in bees, ants, and termites (Abouheif and Wray 2002; Ben-Shahar et al. 2002; Krieger and Ross 2002, 2005; Bulmer and Crozier 2004; Robinson et al. 2005; Drapeau et al. 2006; Forêt and Maleszka 2006; The Honeybee Genome Sequencing Consortium 2006).
Members of the aphid tribe Cerataphidini, representing the subfamily Hormaphidinae and embracing the genera Tuberaphis, Astegopteryx, Cerataphis, and others, are known as social aphids with soldier caste. They form conspicuous galls on trees of the genus Styrax (Styracaceae), wherein adult females parthenogenetically produce monomorphic 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 but are specialized for altruistic social tasks, colony defense and gall cleaning. At ordinary times, soldier nymphs gather around exit holes of the gall, guarding the openings and discarding the wastes (e.g., honeydew globules, shed skins, corpses) from the holes. Encountering intruders, soldier nymphs aggressively exhibit attacking behavior by stinging with their stylet. Aphid predators such as hoverfly maggots and lacewing larvae are tormented, paralyzed, or killed by the attack and usually drop off the gall surface together with attacking soldier nymphs. Accidental bite of soldier nymphs on human skin causes itch, indicating that they possess some toxic substance and inject it into enemies through their stylet. These remarkable social traits associated with the 2nd instar soldier caste are commonly found in the galling generation of all cerataphidine aphids, suggesting that the soldier caste had already evolved in the common ancestor of the aphid group (Aoki 1987; Ito 1989; Stern and Foster 1996).
Tuberaphis styraci is a representative of cerataphidine social aphids, forming large coral-shaped galls on the tree Styrax obassia and producing 2nd instar soldier nymphs (Aoki and Kurosu 1989, 1990). Owing to the development of an artificial diet rearing system for the species (Shibao et al. 2002), T. styraci has recently been investigated intensively as experimental model of social aphid, which has unveiled the density-mediated control mechanisms over induction and suppression of soldier differentiation (Shibao et al. 2003, 2004a, 2004b, 2004c).
In this and other social aphids, normal nymphs and soldier nymphs are clonal offspring of the same mother and thus genetically identical to each other. Notwithstanding this, they are strikingly different in morphology, physiology, behavior, and reproduction, which must be attributed to differential gene expression between the castes. 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 protease gene of the family cathepsin B (Kutsukake et al. 2004). The soldier-specific type (S-type) cathepsin B gene is expressed about 2,000 times higher in soldier nymphs than in normal nymphs, exhibiting specific localization in the midgut epithelium. The S-type cathepsin B protease is secreted into the midgut cavity, vomited out through the stylet, and injected into the victims, thereby exerting an insecticidal activity. Namely, the S-type cathepsin B is an active component of the aphid venom. Certainly, the S-type cathepsin B gene shows accelerated molecular evolution due to positive selection acting on the molecule, as has been reported for other venomous proteins of snakes, scorpions, and cone snails. Meanwhile, another copy of cathepsin B gene, which is expressed irrespective of the castes and shows no accelerated molecular evolution, was also detected from T. styraci. Both the nonspecific type (N-type) gene and the S-type gene were identified from several other social aphids of the genus Tuberaphis, forming distinct clades in the cathepsin B gene phylogeny, respectively. These results suggest that the S-type gene and the N-type gene had already been duplicated in the common ancestor of the Tuberaphis species (Kutsukake et al. 2004). When and how the cathepsin B genes have been duplicated and specialized for the novel venom function are intriguing for understanding of the evolutionary process of the sociality in the aphid group Cerataphidini.
Another recent study also highlighted the relevance of cathepsin B proteases to the aphid biology and evolution. From the expressed sequence tag (EST) collections and the genome analysis of the pea aphid Acyrthosiphon pisum, a total of 28 cathepsin B gene copies, which were classified into 17 clusters, were identified (Rispe et al. 2008). It should be noted that of six representative gene copies subjected to quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) analysis, five genes exhibited gut-specific expression patterns (Rispe et al. 2008), which is reminiscent of the expression pattern of the S-type cathepsin B in T. styraci. Hence, the relationship of the S-type and N-type cathepsin B genes of the social aphid T. styraci to the diverse cathepsin B genes of the nonsocial aphid A. pisum is evolutionarily quite interesting.
To gain insights into the evolutionary origin and process of the soldier-specific venomous cathepsin B protease gene, we identified here the S-type and N-type cathepsin B genes from diverse cerataphidine social aphids and analyzed their molecular phylogenetic and evolutionary aspects together with members of cathepsin B genes family from nonsocial aphids.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Insect Materials
Table 1 shows the social aphids examined in this study. Galls of the aphids were collected from the host plants, and insects were harvested from the galls and immediately preserved in acetone or 99% ethanol (Fukatsu 1999
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Cathepsin B Genes
Soldier nymphs were subjected to total RNA extraction using RNeasy mini kit (Qiagen GmbH, Hilden, Germany). RNA samples were treated with RQ1 RNase-free DNase (Promega, Madison, WI), reverse transcribed using SuperScript 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 of S-type cathepsin B gene, which contained a complete open reading frame (ORF), was amplified by RT-PCR with primers TUB1-F (5'-GGA CTC CTG TAG ATT TAT TTA CGC GA-3') and TUB1-R (5'-GAT AAA AGC CGC GCA AAA ACT A-3') and was cloned and sequenced. Polymerase chain reaction (PCR) was conducted using Advantage 2 cDNA polymerase (Clontech, Shiga, Japan) under a temperature profile of 94 °C for 2 min, followed by 35 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 3 min, and final extension at 72 °C for 5 min. From Tuberaphis takenouchii, around 0.5-kb cDNA region of S-type cathepsin B gene was amplified by RT-PCR with degenerate primers DEG-F (5'-TGY GGN WSN TGY TGG GCN KT-3') and DEG-R (5'-YKN AYN GCR TGN CCN CC-3') and then the full length of cDNA sequence was obtained by 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-type cathepsin B gene from T. takenouchii was amplified by RT-PCR with primers TKNU1-F (5'-CGG CTC CTG 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 region of N-type cathepsin B gene, containing an almost full length of ORF but three amino acid residues at each of 5' and 3' ends, was amplified by RT-PCR from Tuberaphis species with primers TUB2-F (5'-TTA TTA AAA ACG TCG ACA TGA TTC G-3') and TUB2-R (5'-TTT TCT TCG GTG TTT AAT AGG TCA C-3') and was cloned and sequenced. From Astegopteryx styracophila, Astegopteryx spinocephala, and Cerataphis jamuritsu, around 0.2-kb cDNA region of S-type cathepsin B gene was amplified by RT-PCR with primers KT47-F (5'-AAG CCA ATG GAA CAC AAT CAC A-3') and KT47-R (5'-CCC CAT CCG ATG AGC TTY AC-3') and then the full length of cDNA sequence was obtained by 5'- and 3'-RACE procedures. Around 1.0-kb cDNA region of N-type cathepsin B gene was obtained from A. styracophila, A. spinocephala, and C. jamuritsu as described above for N-type cathepsin B gene of Tuberaphis species.
Mitochondrial rRNA Genes
Total DNA was extracted from an individual insect using QIAamp tissue kit (Qiagen). Around 1.6-kb mitochondrial DNA segment, containing small subunit rRNA gene, tRNA-Val gene, and large subunit rRNA gene, was amplified by PCR using primers MtrA1 (5'-AAW AAA CTA GGA TTA GAT ACC CTA-3') and MtrB1 (5'-TCT TAA TYC AAC AYC GAG GTC GCA A-3') under the temperature profile of 94 °C for 2 min followed by 30 cycles of 94 °C for 1 min, 48 °C for 1 min, and 65 °C for 3 min. The PCR product was cloned and sequenced as described previously (Fukatsu et al. 2001).
Molecular Phylogenetic Analysis
Multiple alignment was performed using the program MAFFT 5.8 (Katoh et al. 2005), followed by manual refinement. Aligned sites that included alignment gaps were omitted from the analysis. Molecular phylogenetic analyses were conducted by three methods, Neighbor-Joining (NJ), maximum likelihood (ML), and Bayesian (BA). NJ trees (Saitou and Nei 1987) were constructed using the program ClustalW (Thompson et al. 1994). Bootstrap values were obtained by generating 1,000 bootstrap replications. ML trees were estimated using the program RAxML-VI-HPC Version 2.2.3 (Stamatakis 2006). In the ML analysis, the PROTCATWAG option and the GTRMIX option were used as substitution models for amino acid sequences and nucleotide sequences, respectively. Bootstrap values were 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 + 
+ Inv model and GTR + + Inv model were used as substitution models for amino acid sequences and nucleotide sequences, respectively. In total, 4,100 trees were obtained (ngen 410,000, samplefreq 100), and the first 2,100 of these were considered as "burn-in" and discarded. We used the program ProtTest v1.4 (Abascal et al. 2005) and Modelgenerator v0.84 (Keane et al. 2006) for the selection of the substitution models of amino acid sequences and nucleotide sequences, respectively. We confirmed that the potential scale reduction factor was around 1.00 for all parameters and that the average standard deviation of split frequencies converged toward zero.
Quantitative RT-PCR
Semiquantitative RT-PCR was conducted as described previously (Kutsukake et al. 2004). For each of the social aphid species, soldier nymphs, normal 2nd instar nymphs, and adult insects were subjected to RNA extraction and reverse transcription as described above, respectively. The cDNA samples were adjusted to the same concentration and subjected to PCR amplification by using primers CATB1-F (5'-TAC GAC GAA CAG GGA AAA AAC ACG-3') and CATB1-R (5'-TCC CCA GAA CTT ACT CCA CGA ATT-3') for S-type cathepsin B gene of Tuberaphis species; primers KT47-F and KT47-R for S-type cathepsin B gene of Astegopteryx and Cerataphis species; primers CATB2-F (5'-ATA AAT GCG GGT TCG GAT GTT CTG-3') and CATB2-R (5'-AAC GCC TGA TTT GTA ACT CGG GAA-3') for N-type cathepsin B gene of Tuberaphis species; and primers F4K1-F (5'-TTC TGY TGT CAC HHG TGC GGA T-3') and F4K1-R (5'-TCA WAC GAT GCT TCG ATA GGT CC-3') for N-type cathepsin B gene of Astegopteryx and Cerataphis species. PCR was conducted with Advantage 2 cDNA polymerase (Clontech) under a temperature profile of 94 °C for 2 min, followed by 34 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min, and final extension at 72 °C for 5 min. Aliquots of the PCR solution were sampled every two cycles from 24 cycles to 34 cycles, the samples were electrophoresed on agarose gels, and quantity of the PCR products was evaluated densitometrically after ethidium bromide staining. By identifying PCR cycles that gave almost the same levels of amplified product in soldier nymphs, normal nymphs, and adult insects, relative expression levels of S-type and N-type cathepsin B genes were evaluated across the soldiers and the nonsoldiers.
Estimation of Solvent Accessibility of Amino Acid Residues in Cathepsin B Proteins
Three-dimensional models of S-type and N-type cathepsin B mature proteins of T. styraci were inferred by SWISS-Model (Schwede et al. 2003; http://swissmodel.expasy.org/) using the crystal structure of bovine cathepsin B mature protein as template (Yamamoto et al. 2000; PDB accession 1qdq
[PDB]
). Solvent-accessible surface area (SAS) for each of amino acid residues was computed from the predicted 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 residues was classified into "exposed," "partially exposed," or "buried" class, according to SAS proportion of the side chain, which was formulated as follows: [SAS of the side chain of the residue calculated from the predicted model]/[standard SAS of the side chain of the residue] x 100. Standard SAS of residue X was defined as the average SAS of X in the tripeptide Gly-X-Gly in an ensemble of 30 random conformations. Each of the residues was regarded as exposed when the SAS proportion exceeded 50%, as partially exposed when the proportion was 20–50%, and as buried when the ratio was less than 20%.
Estimation of Proximity of Amino Acid Residues to Catalytic Cleft of Cathepsin B Proteins
For estimating amino acid residues forming the catalytic center, the distances between each of the atoms of the bovine cathepsin B protein and the inhibitor CA074, which was cocrystallized with the protein and occupied the catalytic cleft (Yamamoto et al. 2000), were calculated on the basis of the three-dimensional positions of the atoms. Each of the amino acid residues was categorized into either "near catalytic" when the residue had atoms within 10 Å distance from the inhibitor or "far catalytic" when the residue had no such atoms. In reference to the categorization, corresponding amino acid residues in the S-type and N-type cathepsin B mature proteins of T. styraci were assigned on the basis of the alignment of the amino acid sequences for the phylogenetic inference.
Synonymous and Nonsynonymous Substitution Rates
Synonymous substitutions per site (KS) and nonsynonymous substitutions per site (KA) were calculated as described (Miyata and Yasunaga 1980). Multiple substitutions were corrected by Kimura's two-parameter method (Kimura 1980). For each of the species, the predominant sequence type among the 10 clones sequenced was subjected to the analysis. To define clusters used for the comparison, the phylogenetic relationship of Tuberaphis species inferred from mitochondrial rRNA gene sequences was used. For comparison between the clusters, mean evolutionary distances between the gene clusters were computed according to the phylogenetic information based on mitochondrial rRNA gene sequences. For example, we calculated KS[T. sumatrana vs. (T. styraci, T. coreana, T. taiwana)], which means KS between T. sumatrana and (T. styraci, T. coreana, T. taiwana), by (KS[T. sumatrana vs. T. styraci] + KS[T. sumatrana vs. T. coreana] + KS[T. sumatrana vs. T. taiwana]) x 1/3. We calculated KA in a similar way and estimated the ratio KA/KS, defining the ratio for each of the clusters. In the estimation of KA/KS ratios for each of the partitions defined on the three-dimensional models of cathepsin B mature proteins, we assumed that synonymous rates are constant among codon sites. Thus, KS values calculated from the full-coding regions of the cathepsin B mature proteins were used for calculation of KA/KS ratios for each of the partitions. Statistical significance of the obtained KA/KS value was tested against a bootstrap distribution of KA/KS values, which was generated by 10,000 bootstrap resamplings of codons from the original alignment.
Synonymous and Nonsynonymous Substitutions Estimated on Tree Branches
The phylogenetic relationship inferred from mitochondrial rRNA gene sequences was adopted as the backbone phylogeny of the Tuberaphis species (cf. supplementary fig. S1, Supplementary Material online). To reconstruct the ancestral nucleotide sequences on each of the internal nodes and to compute the numbers of synonymous and nonsynonymous nucleotide differences for each of the branches of the tree, the codeml program of the PAML software package (Yang 1997) was used under the following settings: the average nucleotide frequencies at the three codon positions (CodonFreq = 2) were used as a model of codon frequency; equal amino acid distance (aaDist = 0) was assumed as a codon substitution model; and one nonsynonymous/synonymous substitution rate ratio (
) was applied to all the branches (model = 0). Model selection did not affect our results (data not shown). The statistical analysis was conducted by the method of Zhang et al. (1998), which is based on the Fisher's exact test in a 2 x 2 contingency table with the numbers of nonsynonymous sites and synonymous sites as rows and the numbers of changed sites and the numbers of unchanged sites as columns.
Accession Numbers
The nucleotide sequences determined in this study were deposited in the DDBJ/EMBL/GenBank nucleotide sequence databases (for accession numbers, see table 1).
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Soldier-Specific and Nonspecific Cathepsin B Genes in Social Aphids
From each of T. styraci, T. coreana, T. taiwana, T. sumatrana, and T. takenouchii, S-type cathepsin B gene was amplified by RT-PCR and cloned. For each of the species, 10 clones were sequenced. In T. styraci and T. takenouchii, all 10 sequences were identical. In T. coreana, T. taiwana, and T. sumatrana, two types of sequences were identified, wherein numbers of nucleotide differences were 25, 6, and 1, respectively.
Similarly, N-type cathepsin B gene from each of the Tuberaphis species was analyzed. Of 10 sequenced clones, a single sequence was identified in T. taiwana, two types of sequences 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 detected from T. styraci (differences in 16 sites between N1 and N2, 21 sites between N1 and N3, and 5 sites between N2 and N3).
The slightly different S-type and N-type gene sequences from the same Tuberaphis species are plausibly due to allelic polymorphisms, although the possibilities of recent gene duplications and/or gene conversions cannot be ruled out.
From A. styracophila, A. spinocephala, and C. jamuritsu, S-type cathepsin B gene was obtained by 5'- and 3'-RACE methods. It should be noted that the S-type gene sequence from C. jamuritsu was disrupted by a nonsense mutation located at a 5' region. All six clones determined by 5' RACE method exhibited the same sequence, and three clones of the full-length cDNA we inspected also represented the same sequence. Hence, it is unlikely that the nonsense mutation is due to an experimental artifact like PCR error. Except for the stop codon mutation, the S-type gene was structurally intact. The S-type gene sequences from A. styracophila and A. spinocephala contained a complete ORF.
From the Astegopteryx and Cerataphis species, N-type cathepsin B gene was amplified and cloned. For A. styracophila, A. spinocephala, and C. jamuritsu, 3, 5, and 9 clones were sequenced, respectively. All the sequences were identical within the aphid species.
Phylogenetic Relationship of the Cathepsin B Genes from 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 of the genus Tuberaphis to the cathepsin B genes from the nonsocial aphid A. pisum on the basis of deduced amino acid sequences. The S-type genes from the social aphids formed a well-supported and distinct clade in the phylogeny, 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.
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Phylogenetic Relationship of the Cathepsin B Genes from the Social Aphids and Their Expression in Different Castes
Figure 2A shows the phylogenetic relationship of the S-type and N-type cathepsin B genes from the social aphids deduced from amino acid sequences. For each of Tuberaphis species, the major sequence type among the 10 clones examined was subjected to the phylogenetic analysis. The S-type clade clustered with the Ap84 clade consisting of sequences from nonsocial aphids A. pisum and Tuberaphis citricida, whereas the N-type clade showed affinity to the Ap16D1 clade encompassing sequences from these nonsocial aphids. The phylogenetic relationships were largely congruent with the mitochondrial phylogeny of the Tuberaphis aphids (supplementary fig. S1, Supplementary Material online).
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Figure 2B shows the expression levels of the cathepsin B genes in the social aphids evaluated by a semiquantitative RT-PCR method. In Tuberaphis species, the S-type genes were expressed strikingly higher (>1,000 times, data not shown) in soldier nymphs than in normal insects, whereas the N-type genes were expressed irrespective of the castes. In A. spinocephala, both the S-type and N-type genes were expressed in both the castes. In A. styracophila, unexpectedly, the N-type gene exhibited higher expression in soldier nymphs (around 64 times, data not shown) than in normal insects, whereas the S-type gene was expressed in a constitutive manner. In C. jamuritsu, not only the N-type gene but also the S-type gene was, although pseudogenized, expressed in both the castes. Cloning and sequencing of the RT-PCR products confirmed that messenger RNA (mRNA) of the S-type gene containing a stop codon was certainly transcribed in C. jamuritsu (data not shown).
Accelerated Amino Acid Substitution in the S-Type Cathepsin B Genes in Tuberaphis Species
In Tuberaphis species, KA/KS values of the S-type cathepsin B genes (0.63–1.76) were remarkably higher than those of the N-type cathepsin B genes (0.02–0.21). In particular, in the lineages of T. styraci, T. coreana, and T. taiwana, KA/KS values of the S-type genes were larger than 1 (1.75–1.76), suggesting that positive selection has been acting on the molecules. In the lineages of T. sumatrana and T. takenouchii, KA/KS values of the S-type genes were smaller than 1 (0.63–0.83). In Astegopteryx species, KA/KS value of the S-type cathepsin B gene was 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).
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Partitioning of Amino Acid Residues of the S-Type and N-Type Cathepsin B Proteins according to Solvent Accessibility
Three-dimensional models of the S-type and N-type cathepsin B mature proteins were inferred by using the crystal structure of the bovine cathepsin B protein as template. By computing SAS on the structural models, each of the amino acid residues of the S-type and N-type proteins was classified into exposed (red), partially exposed (pink), or buried (uncolored) (fig. 3). By comparing amino acid sequences of the proteins between T. styraci, T. coreana, T. taiwana, and T. sumatrana, each of the amino acid residues was categorized into either "constant" (normal letters in fig. 3A and B) or "variable" (circled letters in fig. 3A and B and spheres in fig. 3C and D). The S-type protein contained more variable residues than the N-type protein (33 vs. 11) (fig. 3).
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Accelerated Amino Acid Substitution in the S-Type Cathepsin B Genes Preferentially at Exposed Residues
When KA/KS values were separately calculated for exposed, partially exposed, and buried residues, striking patterns emerged in the S-type cathepsin B genes of Tuberaphis species (table 2). In the lineages of T. styraci, T. coreana, and T. taiwana, KA/KS values of the S-type genes were extremely high, ranging from 2.12 to 3.77 at exposed and partially exposed residues, whereas low values (0.37–0.56) were observed at buried residues. In the lineage of T. sumatrana, KA/KS values were higher than 1 (1.30–1.39) at exposed and partially exposed residues, whereas buried residues exhibited a low KA/KS value of 0.30. In the lineage of T. takenouchii, KA/KS values were less than 1 (0.87–0.93) at exposed and partially exposed residues but still higher than value at buried residues (0.37). In the S-type cathepsin B genes of Astegopteryx species, KA/KS values were low irrespective of residue types, although the values at exposed and partially exposed residues (0.31–0.51) were higher than the value at buried residues (0.00) (table 2).
Partitioning of Amino Acid Residues of the S-Type and N-Type Cathepsin B Proteins according to Proximity to Catalytic Cleft
Three-dimensional models of the S-type and N-type cathepsin B mature proteins were inferred from the crystallography of the CA074-binding bovine cathepsin B protein, and each of the amino acid residues was categorized into either near catalytic within 10 Å from the catalytic cleft (blue) or far catalytic over 10 Å apart from the catalytic cleft (uncolored) (fig. 4).
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No Significant Differences in Amino Acid Substitution Rates between Near-Catalytic and Far-Catalytic Residues
In all the Tuberaphis and Astegopteryx species and irrespective of the S-type and N-type cathepsin B proteins, no significant differences in KA/KS values were detected between near-catalytic residues and far-catalytic residues (table 3). Even when exposed, partially exposed, and buried residues were separately considered (cf. supplementary fig. S2, Supplementary Material online), no significant differences in KA/KS values were detected between near-catalytic residues and far-catalytic residues (supplementary table S1, Supplementary Material online).
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Mapping of Synonymous and Nonsynonymous Substitutions in S-Type and N-Type Cathepsin B Genes in the Evolutionary Course of Tuberaphis Species
On the phylogeny of Tuberaphis species, occurrences of synonymous and nonsynonymous substitutions of the S-type and N-type cathepsin B genes were estimated and mapped on each of the branches (fig. 5). As for the S-type genes, very high KA/KS values were observed at exposed and partially exposed residues in the branches leading to T. coreana (3.3–4.4), T. taiwana (1.1–3.3), and T. styraci (2.2–2.7). These values were remarkably higher than 1.0, but the differences from 1.0 were statistically not significant probably because of limited numbers of nucleotide sites and substitutions subjected to the analysis. KA/KS values at exposed and partially exposed residues were consistently around 1.0 in the common ancestors 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 buried residues, 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 all the lineages and irrespective of the residue types, except for at partially exposed residues in the lineage of T. sumatrana (1.2) (fig. 5B).
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Discussion |
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Ancient Origin of the S-Type and N-Type Cathepsin B Genes in Aphids
The S-type and N-type cathepsin B genes were detected not only from social aphids of the genus Tuberaphis but also from social aphids of other genera Astegopteryx and Cerataphis and, strikingly, also from phylogenetically distant nonsocial aphids A. pisum and T. citricida (figs. 1 and 2). Considering that, in the aphid evolution, the subfamily Hormaphidinae embracing the social aphids basally diverged from the subfamily Aphidinae consisting exclusively of nonsocial aphids (Heie 1987
; Von Dohlen and Moran 1995), the origin of the S-type and N-type cathepsin B genes is likely to be quite ancient, possibly dating back to the common ancestor of extant aphids 80–150 MYA (Von Dohlen and Moran 2000). Here it should be noted that, although the N-type cathepsin B genes from the nonsocial aphids formed a good clade with those from the social aphids, statistical supports for the monophyly of the S-type cathepsin B genes from the nonsocial and social aphids were not significant (figs. 1 and 2). Hence, the possibility seems also likely that the S-type genes of the social aphids do not form a clade with but have a distinct evolutionary origin from the S-type genes of the nonsocial aphids. Anyway, it appears plausible that the massive amplification of cathepsin B genes in aphids (Rispe et al. 2008) has predisposed the evolution of venomous protease in the social aphids.
The S-Type Cathepsin B Gene Acquired Venom Function in the Genus Tuberaphis
Among the social aphids examined in this study, soldier-specific expression of the S-type cathepsin B gene was detected only in the genus Tuberaphis (fig. 2B). Accelerated amino acid substitution of the S-type cathepsin B was observed only with Tuberaphis species (table 2). Neither such remarkable levels of soldier-specific expression nor accelerated molecular evolution of the S-type gene were identified in Astegopteryx and Cerataphis species (fig. 2; table 2), although these social aphids belong to the same aphid group Cerataphidini and produce 2nd instar soldiers in their galls as Tuberaphis species do (Stern and Foster 1996). These results suggest that, although the S-type and N-type cathepsin B genes are commonly present in the social aphids, the soldier-specific expression and the venom function of the S-type gene evolved after divergence of 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 sequences were partitioned into exposed, partially exposed, and buried amino acid residues and were subjected to molecular evolutionary analyses, high KA/KS values were detected at exposed and partially exposed residues in S-type cathepsin B sequences of Tuberaphis species (table 2). The evolutionary pattern strongly suggests that positive selection has been acting on the molecular surface of the S-type cathepsin B proteins in the evolutionary course of the social aphids. It appears likely that the rapid evolution at the surface residues of the S-type cathepsin B protease is relevant to its venom function. Surface structure of the venomous protease must be important for recognition of its target molecules. Structural change in the lethal target molecules of predatory insects would result in their resistance to the venom. Here coevolutionary arms race between soldier's venomous protease and predators’ target molecules might occur, which would lead to accelerated evolution preferentially on the molecular surface. Alternatively, predatory insects might have immune/detoxifying mechanisms against the venomous protease, which recognize the surface structure of the molecule and inactivate it. If so, accelerated evolution of the protease at the molecular surface would be favored for escaping the immune/detoxifying mechanisms. Similar accelerated molecular evolution preferentially at surface residues has been reported from other venomous enzymes such as phospholipase A2 of snakes (Kini and Chan 1999), wherein diversity in surface structure of the enzymes is suggested to be involved in their altered target specificity to various cells and tissues, resulting in a wide variety of pharmacological effects including neurotoxicity, myotoxicity, cardiotoxicity, anticoagulant effects, etc (Kini 2003).
Soldier-Specific Expression of the S-Type Cathepsin B Gene Preceded Evolutionary Acceleration in the Genus Tuberaphis
Soldier-specific expression of the S-type cathepsin B gene was observed in all the Tuberaphis species examined (fig. 2B). Meanwhile, the molecular evolution of the S-type cathepsin B gene was fast in T. styraci, T. coreana, and T. taiwana; moderate in T. sumatrana; and slower in T. takenouchii (table 2). In the genus Tuberaphis, T. styraci, T. coreana, and T. taiwana formed a compact clade, whereas T. sumatrana and T. takenouchii constituted basal lineages (figs. 2; supplementary fig. S1, Supplementary Material online). KA/KS values were certainly higher in the lineages of T. styraci, T. coreana, and T. taiwana than in the lineages of T. sumatrana and T. takenouchii (fig. 5). These patterns suggest that 1) soldier-specific expression of the S-type cathepsin B gene probably preceded evolutionary acceleration of the gene and 2) positive selection acting on the gene might have been stronger in the younger lineages than in the older lineages. It is currently obscure why such evolutionary patterns are observed with the S-type cathepsin B gene in the genus Tuberaphis. One possibility is that, although speculative, evolutionary arms race between soldiers and predators has become severer in the newly evolved lineages than in the older lineages. Alternatively, in the older lineages, molecular evolution of the venom protease could have been slowed down after establishment of their stable ecological niche. Even the possibility should be taken into account that the apparent evolutionary patterns might be due to methodological artifact, wherein accelerated molecular evolution was not properly estimated for the genetically distant basal lineages. To address which of these hypotheses is the most appropriate, further analyses of more Tuberaphis and allied social aphids are needed.
Gut-Specific Expression of the S-Type Cathepsin B Gene in Social and Nonsocial Aphids
In T. styraci, the S-type cathepsin B protease is produced in the midgut epithelium of soldier nymphs, secreted into the midgut cavity, vomited through the stylet, and injected into the body cavity of enemies (Kutsukake et al. 2004). The intestinal expression and functioning of the S-type cathepsin B protease are probably found in soldier nymphs of Tuberaphis species in common. Here it should be noted that Ap84 cathepsin B gene of the pea aphid, which is phylogenetically related to the S-type cathepsin B genes of the social aphids (figs. 1 and 2), is also highly expressed in the midgut (Rispe et al. 2008). Hence, the gut-specific expression of the S-type cathepsin B gene in Tuberaphis soldiers might reflect the ancestral expression pattern of the protease gene.
Why Are Diverse Cathepsin B Genes Expressed in Aphid Gut?
The sole food source for aphids, which is plant phloem sap, contains much sugar and some nonessential amino acids but is devoid of lipids and proteins. Conventionally, it has been believed that aphids substantially have no intestinal digestion of proteins (Terra 1990; Douglas 2003). However, recently studies have uncovered the presence of a variety of proteins in plant phloem fluid (e.g., Kehr 2006) and also the presence of intestinal proteases in some phloem-feeding insects (e.g., Foissac et al. 2002; Cristofoletti et al. 2003; Deraison et al. 2004). In this context, the discovery of massive amplification of aphid cathepsin B protease genes and their preferential expression in the midgut is intriguing (Rispe et al. 2008). Why aphids have evolved so many intestinal protease genes is currently an enigma. One possibility is that, although speculative, aphid–plant coevolution might be involved in the process. Some plants express inhibitors that are effective against insect herbivores (Koiwa et al. 1997; Lawrence and Koundal 2002). For example, aphid infestation on sorghum was shown to trigger overexpression of defense genes including protease inhibitors (Zhu-Salzman et al. 2004). Some of the plant protease inhibitors are themselves proteins and thus could be inactivated by aphid proteases. In this context, it is tempting to assume that, although speculative, such herbivore–plant arms races resulted in the diversification and rapid evolution of the cathepsin B genes in aphids, which might have predisposed the evolution of the venomous protease in the social aphid lineage.
Dynamic Evolution of the S-Type and N-Type Cathepsin B Genes in Social Aphids: Relaxed Functional Constraints due to Gene Duplication?
In C. jamuritsu, the S-type cathepsin B gene was, although containing a nonsense mutation, structurally almost intact and still transcribed into mRNA (fig. 2B), suggesting a recent pseudogenization of the gene copy. In A. styracophila, not the S-type gene but the N-type gene was preferentially expressed in soldier nymphs (fig. 2B), although it is currently unknown whether the upregulated N-type gene product is involved in the defensive role. These dynamic evolutionary patterns of the cathepsin B genes in the social aphids are probably relevant to relaxed functional constraints in the multigene family generated through the massive gene amplification in aphids (Rispe et al. 2008).
The S-Type Cathepsin B Protease as Novel Component of Aphid Venom
In T. styraci, the S-type cathepsin B protease is produced in a soldier-specific manner and functions as a major venom component for attacking natural enemies (Kutsukake et al. 2004). This study suggests that the S-type cathepsin B protease plays a defensive role in soldier nymphs of Tuberaphis species but probably not in soldier nymphs of Astegopteryx and Cerataphis species. Meanwhile, it was observed that soldier nymphs of A. styracophila, A. spinocephala, and C. jamuritsu are able to attack and kill other insects effectively (Aoki et al. 1998; Kurosu et al. 1998, 2006). Attacks by soldiers of these social aphids to human skin cause unpleasant itch (T. Fukatsu, personal observations), indicating that the soldiers inject some toxic compounds other than the cathepsin B protease into enemies. In general, animal venom is a mixture of bioactive compounds such as amines, peptides, phospholipases, hyaluronidases, proteases, and others, and these molecules synergistically exert poisonous activities (Habermann 1972). On the basis of these lines of evidence, we suggest that the S-type cathepsin B protease is a novel venom component acquired in the lineage of Tuberaphis species, and many other bioactive compounds are to be discovered in the aphid venom. Other social aphids like Astegopteryx spp. and Cerataphis spp. probably use toxic compounds other than cathepsin B for attacking enemies.
Gene Duplication, Accelerated Molecular Evolution, and Acquisition of Novel Function as Venomous Protein
Thus far, venomous proteins have been identified and investigated mainly in carnivorous animal groups such as snakes, scorpions, cone snails, etc., wherein the toxic molecules are used for paralyzing, killing, and/or digesting their victims. Most of the venomous proteins are encoded as multigene families in the animal genomes that have been generated through gene duplications and also exhibit accelerated molecular evolution due to positive selection acting on the molecules. From snake venoms, phospholipases A2 (Nakashima et al. 1993, 1995; Ogawa et al. 1995, Nobuhisa et al. 1996; Kordi
and Gubenek 1996, 1997), serine proteases (Deshimaru et al. 1996), metalloproteases (Moura-da-Silva et al. 1996), Kunitz/BPTI (bovine pancreatic trypsin inhibitor) proteins (
upunski et al. 2003), three-fingered toxins (Lachemanon et al. 1998; Ohno et al. 1998; Gong et al. 2000), and C-type lectins (Tani et al. 2002; Ogawa et al. 2005) have been identified as multiple copied and positively selected venomous proteins. Similar evolutionary patterns have been found in scorpion sodium channel toxins (Zhu et al. 2004) and Conus conotoxins (Duda and Palumbi 1999). In many of these cases, the recruited proteins have evolved novel toxic activities in addition to the ancestral activities. In snakes, such recruitment events from nontoxic body proteins are estimated to have occurred at least 24 times (Fry 2006). In the case of phospholipase A2, the enzyme has evolved a toxic site, irrelevant to the catalytic site, at a C-terminal region of the molecule (Kri
aj et al. 1989; Lomonte et al. 1994; Páramo et al. 1998; Núñez et al. 2001; Prijatelj et al. 2002). In this study, we demonstrated that a family of cysteine proteases has acquired a novel venom function and exhibited such evolutionary patterns in social aphids, a herbivorous animal group living exclusively on plant sap. In this case, however, considering that the accelerated molecular evolution is not related to the catalytic center of the protease but associated with the molecular surface (tables 2 and 3), the venom activity was probably acquired by relatively minor modifications on the molecular surface structure rather than by generation of a novel active site. Our finding favors the notion that gene duplication followed by accelerated molecular evolution comprises a general and important evolutionary process that enables acquisition of novel gene functions (Ohno 1970; Hughes 1994; Zhang et al. 1998).
Evolutionary Origin of Novel Traits Underpinning Insect Sociality
Major social insect groups such as bees, wasps, ants, and termites exhibit a number of novel traits that are essential for maintaining their complex social systems. Recent studies have unveiled some molecular aspects underlying novel traits unique to social insects, such as royal jelly proteins for larval nursing in honeybee (Drapeau et al. 2006), an odorant-binding protein Gp-9 that governs the monogyny/polygyny reproductive modes of fire ant colonies (Krieger and Ross 2002), and antifungal proteins for maintenance of fungal garden in termites (Bulmer and Crozier 2004), wherein gene duplication and accelerated molecular evolution are observed. In a phylogenetically distant insect group, social aphids, we also found that the evolution of a novel venomous protease has been realized by gene duplication and subsequent accelerated molecular evolution due to positive selection. For evolution of complex biological systems including insect sociality, gene duplications, accelerated molecular evolution, and acquisition of novel gene function must have played considerable roles in general.
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Supplementary Material |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Supplementary figures S1 and S2 and table S1 are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
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Acknowledgements |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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We thank S. Aoki and T. Miura for critically reading the manuscript and U. Kurosu, S. Aoki, C. C. Wang, H. J. Lee, and Y. Tohsaka for aphid samples. This work was supported by the Japan–France Integrated Action Program SAKURA of the Japan Society for the Promotion of Science to T.F. and J.C.S.
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Footnotes |
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Yoko Satta, Associate Editor
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References |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
|---|
Abascal F, Zardoya R, Posada D. ProtTest: selection of best-fit models of protein evolution. Bioinformatics (2005) 21:2104–2105.
Abouheif E, Wray GA. Evolution of the gene network underlying wing polyphenism in ants. Science (2002) 297:249–252.
Aoki S. Evolution of sterile soldiers in aphids. In: Animal societies: theories and facts—Ito Y, Brown JL, Kikkawa J, eds. (1987) Tokyo (Japan): Japan Scientific Societies Press. 53–65.
Aoki S, Kurosu U. Soldiers of Astegopteryx styraci (Homoptera, Aphidoidea) clean their gall. Jpn J Entomol (1989) 57:407–416.
Aoki S, Kurosu U. Biennial galls of the aphid Astegopteryx styraci on a temperate deciduous tree, Styrax obassia. Acta Phytopathol Entomol Hung (1990) 25:57–65.
Aoki S, Kurosu U. The gall, soldiers and taxonomic position of the aphid Tuberaphis taiwana (Homoptera). Jpn J Entomol (1993) 61:361–369.
Aoki S, Kurosu U, Fukatsu T, Ishikawa H. Cerataphis jamuritsu, a subtropical aphid producing soldiers in large, hard galls (Homoptera). Entomol Sci (1998) 1:327–333.
Aoki S, Kurosu U, Nagashima T. Tuberaphis leeuweni (Homoptera), a tropical, monoecious, gall-forming aphid with soldier-like nymphs. Jpn J Entomol (1995) 63:77–86.
Aoki S, Usuba S. Rediscovery of "Astegopteryx" takenouchii (Homoptera, Aphidoidea), with notes on its soldiers and hornless exules. Jpn J Entomol (1989) 57:497–503.
Ben-Shahar Y, Robichon A, Sokolowski MB, Robinson GE. Influence of gene action across different time scales on behavior. Science (2002) 296:741–744.
Bulmer MS, Crozier RH. Duplication and diversifying selection among termite antifungal peptide. Mol Biol Evol (2004) 21:2256–2264.
Cristofoletti PT, Ribeiro AF, Deraison C, Rahbé Y, Terra WR. Midgut adaptation and digestive enzyme distribution in a phloem feeding insect, the pea aphid Acyrthosiphon pisum. J Insect Physiol (2003) 49:11–24.[CrossRef][Web of Science][Medline]
Deraison C, Darboux I, Duportets L, Gorojankina T, Rahbé Y, Jouanin L. Cloning and characterization of a gut-specific cathepsin L from the aphid Aphis gossypii. Insect Mol Biol (2004) 13:165–177.[Medline]
Deshimaru M, Ogawa T, Nakashima K, et al, (11 co-authors). Accelerated evolution of crotalinae snake venom gland serine proteases. FEBS Lett (1996) 397:83–88.[Medline]
Douglas AE. The nutritional physiology of aphids. Adv Insect Physiol (2003) 31:73–140.[CrossRef]
Drapeau MD, Albert S, Kucharski R, Prusko C, Maleszka R. Evolution of the yellow/major jelly protein family and the emergence of social behavior in honey bees. Genome Res (2006) 16:1385–1394.
Duda T, Palumbi S. Molecular genetics of ecological diversification: duplication and rapid evolution of toxin genes of the venomous gastropod Conus. Proc Natl Acad Sci USA (1999) 96:6820–6823.
Foissac X, Edward MG, Du JP, Gatehouse AMR, Gatehouse JA. Putative protein digestion in a sap-sucking homopteran plant pest (rice brown plant hopper; Nilaparvata lugens: Delphacidae)—identification of trypsin-like and cathepsin B-like proteases. Insect Biochem Mol Biol (2002) 32:967–978.[CrossRef][Web of Science][Medline]
Forêt S, Maleszka R. Function and evolution of a gene family encoding odorant binding-like proteins in a social insect, the honey bee (Apis mellifera). Genome Res (2006) 16:1404–1413.
Fraczkiewicz R, Braun W. Exact and efficient analytical calculation of the accessible surface areas and their gradients for macromolecules. J Comp Chem (1998) 19:319–333.[CrossRef]
Fry BG. From genome to "venom": molecular origin and evolution of the snake venom proteome inferred from phylogenetic analysis of toxin sequences and related body proteins. Genome Res (2006) 15:403–420.
Fukatsu T. Acetone preservation: a practical technique for molecular analysis. Mol Ecol (1999) 8:1935–1945.[CrossRef][Medline]
Fukatsu T, Aoki S, Kurosu U, Ishikawa H. Phylogeny of Cerataphidini aphids revealed by their symbiotic microorganisms and basic structure of their galls: implications for host-symbiont coevolution and evolution of sterile soldier castes. Zool Sci (1994) 11:613–623.
Fukatsu T, Shibao H, Nikoh N, Aoki S. Genetically distinct populations in an Asian soldier-producing aphid, Pseudoregma bambucicola (Homoptera: Aphididae), identified by DNA fingerprinting and molecular phylogenetic analysis. Mol Phylogenet Evol (2001) 18:423–433.[CrossRef][Medline]
Gong N, Armugam A, Jeyaseelan K. Molecular cloning, characterization and evolution of the gene encoding a new group of short-chain 
-neurotoxins in an Australian elapid, Pseudonaja textilis. FEBS Lett (2000) 473:303–310.[Medline]
Habermann E. Bee and wasp venoms. Science (1972) 177:314–322.
Heie OE. Paleontology and phylogeny. In: Aphids: their biology, natural enemies and control. World crop pests vol. 2A—Minks AK, Harrewijn P, eds. (1987) Amsterdam (The Netherlands): Elsevier. 367–391.
Hughes AL. The evolution of functionally novel proteins after gene duplication. Proc R Soc Lond B Biol Sci (1994) 256:119–124.[Medline]
Ito Y. The evolutionary biology of sterile soldiers in aphids. Trends Ecol Evol (1989) 4:69–73.[CrossRef]
Katoh K, Kuma K, Toh H, Miyata T. MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Res (2005) 33:511–518.
Keane TK, Creevey CJ, Pentony MM, Naughton TJ, McInerney JO. Assessment of methods for amino acid matrix selection and their use on empirical data shows that ad hoc assumptions for choice of matrix are not justified. BMC Evol Biol (2006) 6:29.[CrossRef][Medline]
Kehr J. Phloem sap proteins: their identities and potential roles in the interaction between plants and phloem-feeding insects. J Exp Bot (2006) 57:767–774.
Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide-sequences. J Mol Evol (1980) 16:111–120.[CrossRef][Web of Science][Medline]
Kini RM. Excitement ahead: structure, function and mechanism of snake venom phospholipase A2 enzymes. Toxicon (2003) 42:827–840.[Medline]
Kini RM, Chan YM. Accelerated evolution and molecular surface of venom phospholipase A2 enzymes. J Mol Evol (1999) 48:125–132.[CrossRef][Web of Science][Medline]
Koiwa H, Bressan RA, Hasegawa PM. Regulation of protease inhibitors and plant defense. Trends Plant Sci (1997) 2:379–384.[CrossRef][Web of Science]
Kordi D, Gubenek F. Ammodytoxin C gene helps to elucidate the irregular structure of Crotalinae group II phospholipase A2 genes. Eur J Biochem (1996) 240:83–89.[Web of Science][Medline]
Kordi D, Gubenek F. Bov-B long interspersed repeated DNA (LINE) sequences are present in Vipera ammodytes phospholipase A2 genes and in genomes of Viperidae snakes. Eur J Biochem (1997) 246:772–779.[Web of Science][Medline]
Krieger MJB, Ross K. Identification of a major gene regulating complex social behavior. Science (2002) 295:328–332.
Krieger MJB, Ross K. Molecular evolutionary analyses of the odorant-binding protein gene Gp-9 in fire ants and other Solenopsis species. Mol Biol Evol (2005) 22:2090–2103.
Kriaj I, Turk D, Ritonja A, Gubenek F. Primary structure of ammodytoxin C further reveals the toxic site of ammodytoxin. Biochim Biophys Acta (1989) 999:198–202.[CrossRef][Medline]
Kurosu U, Buranapanichpan S, Aoki S. Astegopteryx spinocephala (Hemiptera: Aphididae), a new aphid species producing sterile soldiers that guard eggs laid in their gall. Entomol Sci (2006) 9:181–190.
Kurosu U, Matsumono K, Aoki S. Host alternation of two tropical gall-forming aphids, Astegopteryx styracophila and A. pallida (Homoptera). Entomol Sci (1998) 1:21–26.
Kutsukake M, Shibao H, Nikoh N, Morioka M, Tamura T, Hoshino T, Ohgiya S, Fukatsu T. Venomous protease of aphid soldier for colony defense. Proc Natl Acad Sci USA (2004) 101:11338–11343.
Lachemanon R, Armugam A, Tan CH, Jeyaseelan K. Structure and organization of the cardiotoxin genes in Naja naja sputatrix. FEBS Lett (1998) 433:119–124.[CrossRef][Medline]
Lawrence PK, Koundal KR. Plant inhibitors in control of phytophagous insects. Electron J Biotechnol (2002) Available at: http://www.scielo.cl/fbpe/img/ejb/v5n1/03/3.pdf.
Lomonte B, Moreno E, Tarkowsi A, Hanson LÅ, Maccarana M. Neutralizing interaction between heparins and myotoxin II, a lysine 49 phospholipase A2 from Bothrops asper snake venom. Identification of a heparin-binding and cytolytic toxin region by the use of synthetic peptides and molecular modeling. J Biol Chem (1994) 269:29867–29873.
Miyata T, Yasunaga T. Molecular evolution of messenger-RNA—a method for estimating evolutionary rates of synonymous and amino-acid substitutions from homologous nucleotide-sequences and its application. J Mol Evol (1980) 16:23–36.[CrossRef][Web of Science][Medline]
Moura-da-Silva AM, Theakston RD, Crampton JM. Evolution of disintegrin cysteine-rich and mammalian matrix-degrading metalloproteinases: gene duplication and divergence of a common ancestor rather than convergent evolution. J Mol Evol (1996) 43:263–269.[Web of Science][Medline]
Nakashima K, Nobuhisa I, Deshimaru M, et al, (11 co-authors). Accelerated evolution in the protein-coding regions is universal in crotalinae snake-venom gland phospholipase A2 isozyme genes. Proc Natl Acad Sci USA (1995) 92:5605–5609.
Nakashima K, Ogawa T, Oda N, Hattori M, Sakaki Y, Kihara H, Ohno M. Accelerated evolution of Trimeresurus flavoviridis venom gland phospholipase A2 isozymes. Proc Natl Acad Sci USA (1993) 90:5964–5968.
Nobuhisa I, Nakashima K, Deshimaru M, Ogawa T, Shimohigashi Y, Fukumaki Y, Hattori S, Kihara H, Ohno M. Accelerated evolution of Trimeresurus okinavensis venom gland phospholipase A2 isozyme-encoding gene. Gene (1996) 172:267–277.[CrossRef][Web of Science][Medline]
Núñez CE, Angulo Y, Lomonte B. Identification of the myotoxic site of the Lys49 phospholipase A2 from Agkistrodon piscivorus snake venom: synthetic C-terminal peptides from Lys49, but not from Asp49 myotoxins, exert membrane-damaging activities. Toxicon (2001) 39:1587–1594.[Medline]
Ogawa T, Chijiwa T, Oda-Ueda N, Ohno M. Molecular diversity and accelerated evolution of C-type lectin-like proteins form snake venom. Toxicon (2005) 45:1–14.[Medline]
Ogawa T, Kitajima M, Nakashima K, Sasaki Y, Ohno M. Molecular evolution of group II phospholopase A2. J Mol Evol (1995) 41:867–877.[Medline]
Ohno M, Menez R, Ogawa T, et al, (12 co-authors). Molecular evolution of snake toxins: Is the functional diversity of snake toxins associated with a mechanism of accelerated evolution? Prog Nucleic Acid Res Mol Biol (1998) 59:307–364.[Web of Science][Medline]
Ohno S. Evolution by gene duplication (1970) New York: Springer.
Páramo L, Lomonte B, Pizarro-Cerdá J, Bengoechea JA, Gorvel JP, Moreno E. Bactericidal activity of Lys49 and Asp49 myotoxic phospholipase A2 from Bothrops asper snake venom. Synthetic Lys49 myotoxin II-(115-129)-peptide identifies its bactericidal region. Eur J Biochem (1998) 253:452–461.[Medline]
Prijatelj P, Kriaj I, Kralj B, Gubenek F, Punger
ar J. The C-terminal region of ammodytoxins is important but not sufficient for neurotoxicity. Eur J Biochem (2002) 269:5759–5764.[Medline]
Rispe C, Kutsukake M, Doublet V, Hudaverdian S, Legeai F, Simon JC, Tagu D, Fukatsu T. Large gene family expansion and variable selective pressures for cathepsin B in aphids. Mol Biol Evol (2008) 25:5–17.
Robinson GE, Grozinger CM, Whitfield CW. Sociogenomics: social life in molecular terms. Nat Rev Genet (2005) 6:257–270.[CrossRef][Web of Science][Medline]
Ronquist F, Huelsenbeck JP. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics (2003) 19:1572–1574.
Saitou N, Nei M. The neighbor-joining method—a new method for reconstructing phylogenetic trees. Mol Biol Evol (1987) 4:406–425.[Abstract]
Schwede T, Kopp J, Guex N, Peitsch MC. SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res (2003) 31:3381–3385.
Shibao H, Kutsukake M, Fukatsu T. Density triggers soldier production in a social aphid. Proc R Soc Lond B Biol Sci (2004a) 271:S71–S74.
Shibao H, Kutsukake M, Fukatsu T. The proximate cue of density-dependent soldier production in a social aphid. J Insect Physiol (2004b) 50:143–147.[CrossRef][Web of Science][Medline]
Shibao H, Kutsukake M, Fukatsu T. Density-dependent induction and suppression of soldier differentiation in an aphid social system. J Insect Physiol (2004c) 50:995–1000.[Medline]
Shibao H, Kutsukake M, Lee JM, Fukatsu T. Maintenance of soldier-producing aphids on an artificial diet. J Insect Physiol (2002) 48:495–505.[Medline]
Shibao H, Lee JM, Kutsukake M, Fukatsu T. Aphid soldier differentiation: density acts on both embryos and newborn nymphs. Naturwissenschaften (2003) 90:501–504.[CrossRef][Web of Science][Medline]
Stamatakis A. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics (2006) 22:2688–2690.
Stern DL, Foster WA. The evolution of soldiers in aphids. Biol Rev (1996) 71:27–79.[Medline]
Tani A, Ogawa T, Nose T, Nikandrov NN, Deshimaru M, Chijiwa T, Chang CC, Fukumaki Y, Ohno M. Characterization, primary structure and molecular evolution of anticoagulant protein from Agkistrodon actus venom. Toxicon (2002) 40:803–813.[Medline]
Terra WR. Evolution of digestive systems of insects. Annu Rev Entomol (1990) 35:181–200.[CrossRef][Web of Science]
The Honeybee Genome Sequencing Consortium. Insights into social insects from the genome of the honeygee Apis mellifera. Nature (2006) 443:931–949.[CrossRef][Medline]
Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressivemultiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res (1994) 22:4673–4680.
Von Dohlen CD, Moran NA. Molecular phylogeny of the Homoptera—a paraphyletic taxon. J Mol Evol (1995) 41:211–223.[Medline]
Von Dohlen CD, Moran NA. Molecular data support a rapid radiation of aphids in the Cretaceous and multiple origins of host alternation. Biol J Linn Soc (2000) 71:689–717.[Web of Science]
Wilson EO. The insect societies (1971) Cambridge, MA: Harvard University Press.
Yamamoto A, Tomoo K, Hara T, Murata M, Kitamura K, Ishida T. Substrate specificity of bovine cathepsin B and its inhibition by CA074, based on crystal structure refinement of the complex. J Biochem (2000) 127:635–643.
Yang ZH. PAML: a program package for phylogenetic analysis by maximum likelihood. Comput Appl Biosci (1997) 13:555–556.
Zhang JZ, Rosenberg HF, Nei M. Positive selection after gene duplication in primate ribonuclease genes. Proc Natl Acad Sci USA (1998) 95:3708–3713.
Zhu S, Bosmans F, Tytgat J. Adaptive evolution of scorpion sodium channel toxins. J Mol Evol (2004) 58:145–153.[CrossRef][Web of Science][Medline]
Zhu-Salzman K, Salzman RA, Ahn JE, Koiwa H. Transcriptional regulation of sorghum defense determinants against a phloem-feeding aphid. Plant Physiol (2004) 134:420–431.
upunski V, Kordi D, Gubenek F. Adaptive evolution in the snake venom Kunitz/BPTI protein family. FEBS Lett (2003) 547:131–136.[CrossRef][Web of Science][Medline]
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