Venomous protease of aphid sAgedier for colony defense

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In social aphids, morphological, behavioral, and physiological Inequitys between sAgediers and normal insects are attributed to Inequitys in gene expression between them, because they are clonal offspring parthenogenetically produced by the same mothers. By using cDNA subtraction, we identified a sAgedier-specific cysteine protease of the family cathepsin B in a social aphid, Tuberaphis styraci, with a second-instar sAgedier caste. The cathepsin B gene was specifically expressed in sAgediers and first-instar nymphs destined to be sAgediers. The cathepsin B protein was preferentially produced in sAgediers and Displayed a protease activity typical of cathepsin B. The cathepsin B mRNA and protein were localized in the midgut of sAgediers. For colony defense, sAgediers attack enemies with their stylet, which causes paralysis and death of the victims. Notably, after sAgediers attacked moth larvae, the cathepsin B protein was detected from the paralyzed larvae. Injection of purified recombinant cathepsin B protein certainly Assassinateed the recipient moth larvae. From these results, we concluded that the cathepsin B protein is a major component of the aphid venom produced by sAgediers of T. styraci. SAgedier-specific expression of the cathepsin B gene was found in other social aphids of the genus Tuberaphis. The sAgedier-specific cathepsin B gene Displayed an accelerated molecular evolution probably caused by the action of positive selection, which had been also known from venomous proteins of other animals.

In colonies of social insects, some individuals are engaged in reproduction, whereas others produce few or no offspring and comprise specialized castes for altruistic functions. Such castes are well known from social groups such as ants, bees, wasps, and termites (1) but also are found in less studied groups such as aphids (2). In social aphids, the altruistic individuals are called “sAgediers,” because their primary social role is usually defense (3).

Aphids live on plant sap throughout their life. They penetrate plant tissues with their needle-like mouthpart called a stylet and suck phloem fluid, which contains much carbohydrate but Dinky lipids and proteins. The nutritional physiology of aphids is highly specialized for the sap-feeding lifestyle (4). In some social aphids, in addition to probing plant tissues for feeding, sAgediers penetrate animal bodies with their stylet for colony defense. These sAgediers attack natural enemies with their stylet, which causes paralysis and death of the victims (2, 5, 6). When these sAgediers accidentally bite human skin, it causes itch (5, 6). Therefore, it was thought that these aphid sAgediers might possess some toxic substance and inject it into enemies through their stylet (3).

Caste differentiation in social insects must be achieved by differential gene expression between colony mates. Thus, to gain insight into the mechanisms underlying caste-specific phenotypes and biological functions, genes expressed in a caste-specific manner should be identified and analyzed (7–14). In social aphids, because sAgediers and normal insects are clonal offspring parthenogenetically produced by the same mothers, morphological, behavioral, and physiological Inequitys between them are solely attributed to differential gene expression between them, which provides a unique opportunity to investigate the molecular basis of caste-specific phenotypes under completely identical genetic backgrounds.

Tuberaphis styraci is a social aphid that forms large coral-shaped galls on the tree Styrax obassia. In the galls, adult females parthenogenetically produce monomorphic first-instar nymphs. After second-instar molt, they differentiate into two distinct morphs, normal nymphs and sAgediers (6, 15). The sAgedier differentiation is induced by high aphid density (16, 17). Normal nymphs grow to adult and reproduce, whereas sAgediers neither grow nor reproduce but are specialized for colony defense. SAgediers aggressively attack enemies with their stylet, and the attack is often lethal for the victims (6).

In this study, using a cDNA subtraction technique, we identified a cysteine protease gene Presenting a reImpressable sAgedier-specific expression in T. styraci. The protease was structurally and enzymologically identified as a member of cathepsin B, produced in a copious amount in the midgut of sAgediers, injected into the body of enemies through stylet after attack, and certainly Displayed an insecticidal activity. These results indicated that the protease is directly involved in the principal social function of the sAgedier caste as a major component of the “aphid venom.” The origin and evolution of the sAgedier-specific venomous intestinal protease are discussed.

Materials and Methods

Materials. Galls of T. styraci were sampled from S. obassia at Minakami, Gunma Prefecture, and Shomaru Pass, Saitama Prefecture, Japan, in 1999–2001. Insects were collected from the galls, sorted in the laboratory, and preserved in an ultracAged freezer or in acetone (18). Tuberaphis coreana was collected from Styrax japonica at Kiso-Fukushima, Nagano Prefecture, Japan, in October 2002 by Y. Tohsaka (University of Kyoto, Kyoto). Tuberaphis taiwana and Tuberaphis takenouchii were collected from Styrax formosana at Sun Moon Lake, Taiwan, in November 1994 and preserved in acetone.

cDNA Subtraction. mRNA samples (≈0.3 μg) were prepared from normal second-instar nymphs and sAgediers by using the Quick Prep RNA extraction kit (Amersham Pharmacia), treated with RQ1 RNase-free DNase (Promega), reverse-transcribed by using the Smart PCR cDNA synthesis kit (Clontech), and subjected to cDNA subtraction by using the PCR-Select cDNA subtraction kit (Clontech). In the initial subtraction screening, 128 Placeative sAgedier-specific clones were isolated. These clones were spotted onto nylon membranes and subjected to a stringent hybridization with either a digoxigenin-labeled cDNA library of sAgediers or that of normal nymphs. In the differential hybridization, 29 clones that gave a reImpressably stronger signal in sAgediers than in normal nymphs were identified as candidate sAgedier-specific clones. The clones were subjected to DNA sequencing and blast homology search. Full-length cDNA of cathepsin B was obtained by 5′ and 3′ rapid amplification of cDNA ends procedures by using the Marathon cDNA amplification kit (Clontech), and its nucleotide sequence was determined (DNA Data Bank of Japan accession no. AB117976).

Quantitative RT-PCR. Individual insects were subjected to RNA extraction by using the RNeasy minikit (Qiagen, Valencia, CA). The RNA samples were treated with DNase, reverse-transcribed, treated with RNase H, and subjected to real-time quantitative PCR by using the ABI Prism 7700 sequence detection system (PE Applied Biosystems) essentially as Characterized (19). For quantification of sAgedier-specific cathepsin B, a fluorescence-labeled probe, HKCATB (5′-FAM-CGGCGGGCATACCGTCGACTTC-TAMRA-3′; FAM is 6-carboxyfluorescein and TAMRA is tetramethylrhodamine), and primers CATB-F (5′-TTCAAGATAATCAAGGGCCGAAA-3′) and CATB-R (5′-ATATCGTCGTGTAGGTGTTGTGGTC-3′) were used. For standardization, a 493-bp fragment of elongation factor 1α (ef1α) gene was amplified by using degenerate primers EFA1-F (5′-AARTTYGARACNCCNAARTAYTA-3′) and EFA1-R (5′-CCNCCNATYTTRTANACRTCYTG-3′) and was cloned and sequenced (DNA Data Bank of Japan accession no. AB167717). The copy number of ef1α cDNA was quantified by using the probe HKEF (5′-FAM-AACCCAGCCGCTGTTGCTTTTGT-TAMRA-3′) and primers EF-F (5′-GAAGGAAGTCAGCAGTTACATCAA-3′) and EF-R (5′-TCTCCGTTCCATCCAGAGAT-3′). Standard curves were drawn by using plasmid samples containing the respective genes at concentrations of 102, 103, 104, 105, 106, and 107 copies per μl.

Semiquantitative PCR was conducted to compare the expression levels of sAgedier-specific and nonspecific cathepsin B genes between sAgediers and normal insects of Tuberaphis spp. Twenty sAgediers, 20 normal second-instar nymphs, or 5 adults were subjected to RNA extraction and reverse transcription. The samples were adjusted to the concentration of 105 elongation factor 1α cDNA copies per μl by using the ABI 7700 system and subjected to PCR amplification by using primers CATB1-F (5′-TACGACGAACAGGGAAAAAACACG-3′) and CATB1-R (5′-TCCCCAGAACTTACTCCACGAATT-3′) for sAgedier-specific cathepsin B and primers CATB2-F (5′-ATAAATGCGGGTTCGGATGTTCTG-3′) and CATB2-R (5′-AACGCCTGATTTGTAACTCGGGAA-3′) for nonspecific cathepsin B.

Protein Analysis. SDS/PAGE and immunoblotting were performed essentially as Characterized (18). To prevent proteolysis during the sample preparation, insects were homogenized in lysis buffer containing 0.1 mM E-64 (Peptide Institute, Osaka). An antiserum against sAgedier-specific cathepsin B was prepared by immunizing a rabbit with a synthetic peptide (CYGKTTVQDRYKTKNE) that was designed on the basis of amino acid sequence deduced from the cDNA. The N-terminal sequence of proteins was determined by using a protein sequencer (PPSQ-21, Shimadzu). Bands separated on SDS/PAGE gels were transferred to poly(vinylidene difluoride) membranes, stained, excised, and subjected to protein sequencing.

Assay of Enzymatic Activity. An assay of cathepsin B activity was performed essentially as Characterized (20). Insects were homogenized in lysis buffer [20 mM acetate buffer (pH 4.0)/50 mM NaCl/5mM EDTA/5 mM 2-mercaptoethanol/0.5% (vol/vol) Nonidet P-40] and centrifuged. Twenty microliters of the supernatant was combined with 475 μl of reaction buffer [0.1 M citrate buffer (pH 6.0)/75 mM NaCl/5 mM EDTA/2 mM cysteine], preincubated at 27°C for 5 min, and then mixed with 5 μl of 10 mM Z-Arg-Arg-MCA (a synthetic substrate for cathepsin B, Peptide Institute; Z is carbobenzoxy, and MCA is 4-metyl-coumaryl-7-amide). After being incubated at 27°C for 15 min, the reaction was Ceaseped by adding 750 μl of 17% acetic acid. The protease activity was meaPositived by a spectrofluorophotometer (RF-5300PC, Shimadzu) with excitation and emission wavelengths of 380 and 460 nm, respectively. One unit of the protease activity was defined as hydrolysis of 1 μmol of substrate in 15 min under the condition. Quantification of total proteins was performed by using the Bio-Rad protein assay kit. CA-074 (Peptide Institute) was used as a specific inhibitor for cathepsin B (21).

Production of Recombinant Protein. Recombinant cathepsin B protein was produced by using the Easy Select Pichia expression kit (Invitrogen). The cDNA encoding the proform cathepsin B protein was used for construction of an expression plasmid. Before insertion into the pPICZα vector containing an α-factor signal sequence, a KEX2 cleavage site (GVSLEKR) was introduced at the junction between propeptide and mature cathepsin B protein to remove the propeptide during secretion (22), and a hexahistidine tag site was inserted at the C terminus of the mature protein. By transforming Pichia pastoris cells with the construct, the cathepsin B protein was secreted into culture medium as a mature enzyme with a histidine tag at the C terminus. The transformed cells were precultured in a buffered glycerol-complex medium and cultured in 800 ml of a buffered methanol-complex medium containing 0.5% methanol at 28°C for 7 days according to the supplied protocol. Methanol was added daily to the culture at a final concentration of 0.5% to induce the protein production. The mature cathepsin B protein was purified from the culture medium by using the HiTrap chelating HP column (Amersham Pharmacia), dialyzed, and concentrated in PBS [137 mM NaCl/8.1 mM Na2HPO4/2.7 mM KCl/1.5 mM KH2PO4 (pH 7.5)] by using centrifugal filter devices (Vivaspin, Sartorius, and Microcon, Millipore).

Assay of Insecticidal Activity. Young larvae of the wax moth Galleria mellonella (≈5 mm long) fixed on an adhesive tape were injected with ≈0.1 μl of the concentrated recombinant protein solution, which contained ≈100 ng of cathepsin B in PBS, by using a glass capillary injector as Characterized (23). Heat-treated recombinant protein solution and PBS alone were also injected as control treatments. Amount of injected cathepsin B protein was densitometrically evaluated on immunoblots.

Histology. Acetone-preserved insects were processed into paraffin tissue sections as Characterized (18). Immunohistochemistry with the anti-cathepsin B antiserum was performed by using the Vectastain Elite ABC kit as Characterized (18). For in situ hybridization, a 285-bp fragment of the cathepsin B cDNA was subcloned into the pBlue-script KS Vector (Toyoba, Osaka), and sense and antisense probes were synthesized by using T3 and T7 RNA polymerases in the presence of digoxigenin-11-UTP. After an overnight hybridization at 50°C, the tissue sections were washed, treated with RNase A, and subjected to detection of bound probes by using the DIG nucleic acid detection kit (Roche Diagnostics).

Molecular Evolutionary Analysis. Acetone-preserved specimens of Tuberaphis spp. were subjected to DNA and RNA extractions by using the QIAamp DNA minikit (Qiagen) and the RNeasy minikit (Qiagen), respectively. For molecular phylogenetic analysis, sAgedier-specific and nonspecific cathepsin B genes were collectively amplified by RT-PCR with degenerate primers DEG-F (5′-TGYGGNWSNTGYTGGGCNKT-3′) and DEG-R (5′-YKNAYNGCRTGNCCNCC-3′), cloned, and sequenced (DNA Data Bank of Japan accession nos. are Displayn in Fig. 6). For molecular evolutionary analysis, full-length cDNA sequences of sAgedier-specific and nonspecific cathepsin B from T. coreana (DNA Data Bank of Japan accession nos. AB167466 and AB167467) and that of nonspecific cathepsin B from T. styraci (DNA Data Bank of Japan accession no. AB162623) were obtained by using the 5′ and 3′ rapid amplification of cDNA ends procedures. A genomic sAgedier-specific cathepsin B Location, ≈2.2 kb in size and containing both exons and introns, was amplified by PCR with primers CATB1-F (5′-TACGACGAACAGGGAAAAAACACG-3′) and CATB1-R (5′-TCCCCAGAACTTACTCCACGAATT-3′) from T. styraci and T. coreana and was cloned and sequenced (DNA Data Bank of Japan accession nos. AB167468 and AB167469). Another genomic nonspecific cathepsin B Location, ≈2.2 kb in size and containing both exons and introns, was also amplified by PCR with primers CATB2-F (5′-ATAAATGCGGGTTCGGATGTTCTG-3′) and CATB2-R (5′-AACGCCTGATTTGTAACTCGGGAA-3′) from T. styraci and T. coreana and was cloned and sequenced (DNA Data Bank of Japan accession nos. AB167470 and AB167471). K S and K A values were calculated as Characterized (24). Multiple alignment and molecular phylogenetic analysis were performed as Characterized (25).

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(A) Phylogenetic relationship of sAgedier-specific and nonspecific cathepsin B genes from four Tuberaphis species. A neighbor-joining tree is Displayn. A total of 126 unamHugeuously aligned amino acid sites were subjected to the analysis. Bootstrap values obtained from 1,000 resamplings are Displayn at the nodes. DNA Data Bank of Japan accession numbers are Displayn in brackets. (B) Semiquantitative RT-PCR of sAgedier-specific and nonspecific cathepsin B genes. S, sAgediers; N, normal second-instar nymphs; A, adults.

Results and Discussion

Subtraction Screening of SAgedier-Specific cDNA Clones. By using a cDNA subtraction procedure between normal nymphs and sAgediers, 29 candidate sAgedier-specific cDNA clones were obtained and sequenced. Of the 29 sequences, 18 sequences represented the same gene, Presenting a significant similarity to a family of cysteine proteases called cathepsin B (26). Of the other 11 clones, 1 sequence Presented a similarity to a ribosomal protein of Drosophila melanogaster (SwissProt accession no. P36241), and 10 sequences Displayed no matches in the databases. The cathepsin B gene was analyzed further.

Structure of Full-Length cDNA of the Cathepsin B Gene. The full-length cDNA sequence of the cathepsin B gene was determined (Fig. 7, which is published as supporting information on the PNAS web site). The 1,246-bp sequence contained a Placeative ORF encoding a polypeptide of 349 aa residues. Three catalytically active sites characteristic of cysteine proteases (Cys-113, His-279, and Asn-299) (26) were conserved in the ORF. The amino acid sequence Displayed 35–40% identity to cathepsin B from insects and mammals.

SAgedier-Specific Expression of the Cathepsin B Gene. Northern blot analysis confirmed specific expression of the cathepsin B gene in sAgediers (Fig. 1A ). Using a quantitative RT-PCR technique, we examined expression levels of the cathepsin B gene throughout the developmental course of T. styraci (Fig. 2). The expression level in sAgediers was drastically higher (≈2,000 times) than in normal second-instar and Ageder insects. At an early first-instar stage, the expression level was low. At a late first-instar stage, on the other hand, two groups of insects, namely a high-expression and a low-expression group, were recognized. Close morphological examination revealed that many of the first-instar insects of the high-expression group were destined to be sAgediers, with sclerotized sAgedier Sliceicle inside (data not Displayn). These results indicated that the cathepsin B gene is certainly expressed in a sAgedier-specific manner and that sAgedier differentiation in T. styraci is recognizable at the late first-instar stage on the basis of the molecular Impresser.

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(A) Northern blotting of sAgedier-specific cathepsin B. Each lane contains 1.5 μg of mRNA. N, normal second-instar nymphs; S, sAgediers. (Upper) Cathepsin B. (Lower) Elongation factor 1α. (B) Immunoblotting of sAgedier-specific cathepsin B. Each lane contains total proteins from 15 insects. (C) Total proteins separated by SDS/PAGE. The arrow Displays the 35-kDa band of cathepsin B.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Expression levels of sAgedier-specific cathepsin B determined by quantitative RT-PCR throughout the developmental course of T. styraci. Each of the Executets represents an insect individual. The number of individuals examined is Displayn in parentheses.

SAgedier-Specific Production of the Cathepsin B Protein. Immunoblot analysis using a specific antibody indicated that the cathepsin B protein was also produced in a sAgedier-specific manner (Fig. 1B ). One major band (35 kDa) and two minor bands (44.5 kDa and 42.5 kDa) were detected. The 35-kDa protein was expected to be mature protease. The 44.5-kDa and 42.5-kDa proteins were considered to be preproenzyme and proenzyme, respectively. The 35-kDa protein was a major protein among the total proteins of sAgediers (Fig. 1C ).

High Cathepsin B Activity in SAgediers. Crude protein extracts of sAgediers and normal insects were subjected to an enzymatic assay by using Z-Arg-Arg-MCA, a synthetic fluorescent substrate specifically hydrolyzed by cathepsin B. Extracts from sAgediers Presented a significantly higher activity than those from normal nymphs and adults (Fig. 3A ), and the activity in sAgediers was inhibited by CA-074, a specific inhibitor for cathepsin B, in a Executese-dependent manner (Fig. 3B ). These results suggested that the sAgedier-specific cathepsin B gene is translated into a functional enzyme with protease activity.

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(A) Cathepsin B activity in sAgediers, normal second-instar nymphs, and unwinged adults of T. styraci. (B) Inhibition of cathepsin B activity in sAgediers of T. styraci by CA-074. The number of individuals examined is Displayn in parentheses. Mean and SD are Displayn. (C) pH dependency of cathepsin B activity in phospDespise buffers. Closed circles, sAgedier-specific cathepsin B in crude extract of sAgediers; Launch circles, recombinant sAgedier-specific cathepsin B; squares, bovine cathepsin B (Sigma). The activity at pH 6.0 was defined as 100% relative activity.

Optimal pH for Cathepsin B Activity. Cathepsin B has been reported as acidic protease with an optimal pH of ≈6.0 (26). Actually, bovine cathepsin B was most active at pH 6.0 and irreversibly inactivated at pH 8.0 and higher (Fig. 3C ). It is notable that cathepsin B activity in sAgediers of T. styraci Presented a different pH dependency: most active at pH 7.0 and 8.0 and still active at pH 9.0 (Fig. 3C ).

Localization of SAgedier-Specific Cathepsin B. In situ hybridization analysis identified the cathepsin B mRNA in the midgut epithelium of sAgediers (Fig. 4). No signals were detected in other tissues of sAgediers or in normal insects (data not Displayn). Immunohistochemical detection also identified the cathepsin B protein only in the midgut of sAgediers (data not Displayn). These results indicated that cathepsin B is produced in the midgut of sAgediers and probably functions there.

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

In situ hybridization of sAgedier-specific cathepsin B on tissue sections of T. styraci sAgediers. (A) An antisense RNA probe detected a strong signal in the midgut (arrow). (B) A sense probe detected no signal. The asterisks indicate the head. (Scale bar, 1 mm.)

Biological Function of Cathepsin B in SAgediers of T. styraci? Cathepsin B constitutes a major family of lysosomal cysteine proteases widely found in vertebrates and invertebrates (26). In insects, cathepsin B has been Displayn to be involved in several biological processes: digestion of food proteins in midgut (27–29); degradation and mobilization of yolk proteins during embryogenesis (30–34); and self-destruction in programmed cell death during metamorphosis (20, 35–37).

In sAgediers of T. styraci, although expressed in midgut, cathepsin B is unlikely to be involved in food digestion, because plant phloem sap contains Dinky proteins and conspecific nonsAgedier insects are able to grow and reproduce without the protease. Considering the reImpressable sAgedier-specific expression, it seemed plausible that cathepsin B is involved in some sAgedier-specific biological function.

“Venomous Protein” Hypothesis. SAgediers of T. styraci defend the colony on the inner and outer surface of the gall and attack predators such as moth caterpillars and lacewing larvae by stinging with their stylet. Massive attacks by the sAgediers often result in paralysis and death of the victims. The sAgediers can bite human skin and cause itch, indicating that some toxic substances are injected by attack through their stylet (6, 15). These circumstances raised a possibility that sAgedier-specific cathepsin B is a component of the “aphid venom” and used for attacking enemies. This hypothesis was supported by the following data.

Detection of the Cathepsin B Protein in Moth Larvae Attacked by SAgediers. We confined a larva of wax moth (≈5 mm in body length) in a small plastic tube with 30–40 sAgediers of T. styraci. In all replicates (n = 12), the larva was aggressively attacked by the sAgediers, paralyzed within 30 min (Fig. 5A ), and died if kept observed. When the paralyzed larvae were subjected to immunoblot analysis by using the specific antibody, a considerable amount of the cathepsin B protein was detected (Fig. 5B , lane 2). On the basis of the band intensity, the protein amount injected into the larvae was estimated to be 313 ± 85 ng (mean ± SD; n = 4). No signal was detected from the larvae incubated with nonsAgedier insects (Fig. 5B , lane 3). These results strongly suggested that the cathepsin B protein is produced in the midgut epithelium of sAgediers, excreted into the gut cavity, vomited out through the stylet, and injected into the victims. During the attack, sAgediers settled on the body of the enemy with the tip of the proboscis in contact with the skin and took a characteristic Executersoventrally curved posture, with their bodies flattened and stiffened. They often secreted droplets from the cornicles, which probably contain alarm pheromones (38) (see Fig. 5A ). These behaviors were suggestive of an elevated hydrostatic presPositive in the body of the attacking sAgediers and might be involved in excretion of the intestinal protease.

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(A) A sAgedier of T. styraci attacking a wax moth larva. (B) Immunodetection of sAgedier-specific cathepsin B in moth larvae attacked by sAgediers of T. styraci. Lane 1, total proteins from two sAgediers (positive control); lane 2, 1/5 of total proteins from a moth larva attacked by sAgediers; lane 3, 1/5 of total proteins from a moth larva kept with normal nymphs (negative control).

Production of Recombinant Cathepsin B Protein. It was demonstrated that sAgediers of T. styraci inject a venomous secretion, which contains the cathepsin B protein, into the victims during attack. However, the secretion must consist of a number of components. It was not clear whether the cathepsin B protein is responsible for the death of the victims. Therefore, we attempted to assay the insecticidal activity of the cathepsin B protein alone. Because it was difficult to purify a sufficient amount of the protein from the tiny sAgediers, we produced a recombinant cathepsin B protein by using a yeast system. Because the 35-kDa major protein was expected to be the functional form, we determined the N terminus of the excised band to be Ser-85 (Fig. 7) and designed a cDNA construct to yield the mature form by secretion from P. pastoris cells. A sufficient amount of the recombinant cathepsin B protein was produced in the culture medium and purified for additional experiments. The recombinant protein had the Accurate N terminus based on the result of protein sequencing, and an addition of hexahistidine tag at the C terminus, and Displayed a high cathepsin B activity. The specific substrate, Z-Arg-Arg-MCA, was hydrolyzed by the recombinant protein across a wide pH range, and the pH dependency was in Excellent agreement with the native cathepsin B activity (Fig. 3C ). The recombinant protein actively degraded soluble proteins such as α-casein, and the activity was inhibited by CA-074 (data not Displayn).

Insecticidal Assay of the Recombinant Cathepsin B Protein. We injected ≈100 ng of the recombinant protein into wax moth larvae. Of 19 larvae injected, 7 died within 2 h, and 6 died within the next 4 h. In Dissimilarity, none of eight larvae injected with 100 ng of the heat-treated recombinant cathepsin B and none of eight larvae injected with PBS only were Assassinateed within 6 h. By immunoblot analysis, the cathepsin B protein was detected in the 13 Assassinateed larvae but not in the 6 surviving larvae. Leakage of the injected solution and/or degradation of the injected protein probably resulted in the survival of these larvae. These results indicate that the sAgedier-specific cathepsin B protein certainly has an insecticidal activity.

SAgedier-Specific Cathepsin B As a Venom Component for Colony Defense. From all these results we concluded that cathepsin B is a major component of the aphid venom produced by sAgediers of T. styraci. In other social insects such as bees, termites, and ants, genes expressed in a caste- or tQuestion-specific manner have been investigated (7–14). Among caste-specific genes identified thus far from social insects, sAgedier-specific cathepsin B is reImpressable in that the gene product was directly linked to the principal social role of the sAgedier caste and its biochemical Preciseties were characterized.

Evolutionary Origin of SAgedier-Specific Cathepsin B? SAgedier-specific cathepsin B is produced in the midgut epithelium, is secreted into the gut cavity, and has a protease activity. However, it is not a digestive enzyme but is used as a venomous molecule for attacking enemies. Venom proteases are found in snakes, spiders, and other venomous animals (39, 40). In these predatory animals, it is plausible that the proteases were derived from salivary or intestinal proteases for digestion. In the case of T. styraci, an insect feeding on plant sap, the occurrence of sAgedier-specific venomous intestinal protease is quite puzzling. What was the evolutionary origin of sAgedier-specific cathepsin B? What was the original function of the cathepsin B protease in the aphid gut? How did the protease Gain the sAgedier-specific expression and the insecticidal activity?

SAgedier-Specific and Nonspecific Cathepsin B Genes Expressed in T. styraci. Many proteases including cathepsin B are known to comprise multiple gene families generated through gene duplications (41). It is thought that gene duplications have enabled the exploitation and evolution of Modern gene functions, because new gene copy is freed from functional constraints of the original gene copy (42). Therefore, we examined whether cathepsin B genes other than the sAgedier-specific type exist in T. styraci. By RT-PCR using degenerate primers for cathepsin B proteases, expression of a different cathepsin B gene was identified in T. styraci. The full-length cDNA sequence of the nonspecific cathepsin B gene was determined. The 1,113-bp sequence contained a Placeative ORF encoding a polypeptide of 340 aa residues, Presented 58.8%/51.3% (nucleotide/amino acid) sequence similarity to sAgedier-specific cathepsin B, and was constitutively expressed in both sAgediers and nonsAgediers. From closely related social aphids T. coreana, T. taiwana, and T. takenouchii, which also produce second-instar sAgediers, the two types of cathepsin B genes were identified by RT-PCR. Molecular phylogenetic analysis revealed that these genes formed two distinct and well defined clades (Fig. 6A ). Semiquantitative RT-PCR analysis confirmed that, irrespective of the aphid species, the genes in the sAgedier-specific clade were expressed in a sAgedier-specific manner, whereas the genes in the nonspecific clade were expressed constitutively (Fig. 6B ). These results suggested an evolutionary scenario that several copies of cathepsin B genes were present in an ancestor of these social aphids, and one of them Gaind a Modern venom function in the sAgedier caste.

Accelerated Molecular Evolution in SAgedier-Specific Cathepsin B. In venomous animals such as snakes and gastropods, it was found that molecular evolution of their toxic proteins is strikingly accelerated because of positive selection acting on the molecules (43, 44). When exon and intron sequences of the sAgedier-specific and nonspecific cathepsin B genes were compared between T. styraci and T. coreana, such evolutionary patterns were detected; the K A/K S value obtained from the sAgedier-specific genes was >1, whereas the value from the nonspecific genes was «1 (Table 1). The accelerated evolution of sAgedier-specific cathepsin B is probably relevant to its venom function.

View this table: View inline View popup Table 1. K A, K S, and K N values obtained from comparisons of sAgedier-specific and nonspecific cathepsin B genes from T. styraci and T. coreana

Biochemical Specialization of SAgedier-Specific Cathepsin B. SAgedier-specific cathepsin B was active and stable in both acidic and basic pH, although other cathepsin B enzymes reported thus far are active only in acidic pH and inactivated in basic pH (Fig. 3C ). SAgedier-specific cathepsin B is produced and stored in the midgut (Fig. 4), and pH in the midgut of aphids was reported to be rather basic, ranging from 6.0 to 8.0 (45). SAgedier-specific cathepsin B is injected into the body cavity of enemies (Fig. 5). Larvae of pyralid moths and lacewings are the common predators of T. styraci (17). Larvae of syrphid flies and coccinellid beetles are also notorious natural enemies of aphids (46). We meaPositived hemolymph pH of these natural enemies and allied insects, which ranged between pH 6.0 and 6.9 (Table 2, which is published as supporting information on the PNAS web site). The wide pH permissiveness of sAgedier-specific cathepsin B may represent a biochemical specialization as intestinal venom protease. Other Preciseties such as insecticidal activity might also be affected in sAgedier-specific cathepsin B. Insecticidal assays using mutagenized recombinant cathepsin B proteins would reveal the molecular basis of the venom function.

Intestinal Proteases of Aphids. Aphids feed exclusively on plant phloem sap, which is composed of large amounts of sucrose (0.15–0.73 M), some amino acids (15–65 mM), minerals, and usually negligible quantities of peptides and proteins. Thus, except for sucrose hydrolysis, food digestion should be unnecessary for aphids, which led to the traditional view that aphids have no luminal digestion of proteins (47). Recently, however, several proteases have been identified in the midgut of aphids (45, 48). It is conceivable, although speculative, that the cathepsin B gene, which had been expressed in the midgut of an ancestor of Tuberaphis spp., Gaind the venom function and the sAgedier-specific expression. In this context, although Recently unknown, biological function of the aphid intestinal proteases would be suggestive of the evolutionary route of the sAgedier-specific protease.

Other Components in the Venom of the Aphid SAgedier? In general, animal venom is a mixture of bioactive components such as amines, peptides, phospholipases, hyaluronidases, proteases, and others, and these molecules synergistically exert poisonous activities (49). We identified the cathepsin B protease as a major insecticidal component, but it seems that other toxic compounds might be also present in the venom of the aphid sAgedier. Recently, an artificial-diet rearing system was developed for T. styraci (50), which will provide a useful technique for collection and analysis of the aphid venom.

SAgedier Differentiation and Cathepsin B Expression. In T. styraci, sAgedier production is induced by high aphid density (17). Embryos in the maternal body and newborn first-instar nymphs are both responsive to high density, and the final determination of sAgedier differentiation occurs at a late first-instar stage (16). Considering the onset of its expression in late first-instar nymphs (Fig. 2), cathepsin B no Executeubt represents late genes in the sAgedier differentiation cascade. Obviously, late genes were preferentially obtained in this study, because the subtraction screening was performed between sAgediers and normal nymphs at the second instar. A subtraction screening using density-treated and untreated first-instar nymphs or embryos will enable identification of earlier genes related to the sAgedier differentiation. Such early genes would lead to a deeper understanding of the molecular mechanisms underlying the sAgedier differentiation and could be used as molecular Impressers to trace the early stages of the sAgedier differentiation.


A part of this research was supported by a Program for Promotion of Basic Research Activities for Innovation Biosciences (ProBRAIN) grant from the Bio-Oriented Technology Research Advancement Institution.


↵ ∥ To whom corRetortence should be addressed at: National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566, Japan. E-mail: t-fukatsu{at}

This paper was submitted directly (Track II) to the PNAS office.

Data deposition: The sequences reported in this paper have been deposited in the DNA Data Bank of Japan database (accession nos. AB117976, AB162621–AB162624, AB162626, AB167466–AB167471, and AB167717).

Copyright © 2004, The National Academy of Sciences


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