The venom composition of the parasitic wasp Chelonus inanitus resolved by combined expressed sequence tags analysis and proteomic approach

General overview of the cDNA library of C. inanitus venom gland

The 2111 ESTs from the venom glands of C. inanitus were clustered into 488 clusters (95 contigs and 393 singletons, Table 1). The number of ESTs in each contig ranged from 2 to 534 and these clusters were considered as putative unigenes. The deduced sequences from 250 clusters (56 contigs and 194 singletons, 56.85% of all ESTs) shared significant similarities with protein sequences deposited in non-redundant databases (EMBL/Genbank), a proportion comparable to that found by Crawford et al. [33] which have studied the venom gland transcriptome of the parasitoid wasp Microctonus hyperodae. Of these products, 164 (corresponding to 36 contigs and 128 singletons, 21.5% of all ESTs) shared significant similarity with proteins with assigned molecular functions in the gene ontology database. This relatively low percentage is explained in part by the fact that the function of the most represented sequence (534 ESTs) referred below as Ci-23a, is unknown. At level 2 of the gene ontology system, clusters were classified into 9 molecular functional categories (Figure 1), among which "binding" (GO:0005488) and "catalytic activity" (GO:0003824) categories were over-represented (95 and 82 clusters respectively). Interestingly, these categories were also the most common functional categories assigned to the venom glands ESTs from the saw-scaled viper, Echis ocellatus[41] and from the solitary hunting wasp species, Orancistrocerus drewseni[42]. Catalytic activity and binding categories thus constitute a hallmark of the venom gland transcriptomes analysed to date. A "structural molecule activity" function (GO:0005198) has been assigned to 48 clusters (20 contigs, 28 singletons) that corresponded essentially to genes coding for structural constituents of ribosomes (17 contigs, 21 singletons). In addition, products of genes functionally annotated as "translation regulator activity" (GO:0045182) (8 singletons) and "transcription regulator activity" (GO:0030528) (2 contigs, 15 singletons) were also identified, which encompassed transcriptional regulators, DNA or RNA binding proteins and translation elongation factors. All these proteins presumably reflect the metabolic effort invested by the venom gland for the transcription and translation of secreted products. The 2111 ESTs were used to produce in silico a database of venom gland open reading frames (vgORFs) which were matched to the peptide sequences obtained by nano-LC-MS/MS analysis. An overall of 1279 ESTs (26 contigs and 7 singletons, 60.6% of all ESTs) were then identified as coding for venom proteins of C. inanitus.

	No. of clusters	No. of ESTs	Percentage of total ESTs
Total no. of contigs (clusters with > 1 member)	95	1718	81.38
- coding for proteins identified by MS	26	1272	60.26
- coding for other proteins	69	446	21.13
Total no. of singletons (clusters with 1 member)	393	393	18.62
- coding for proteins identified by MS	7	7	0.33
- coding for other proteins	386	386	18.29
TOTAL	488	2111	100

Identification of the venom proteins of C. inanitus

Venoms of parasitic wasps are reputed to have a low content in small proteins and peptides in comparison to venoms of social Hymenoptera [27, 43]. Upon separation of C. inanitus venom proteins by SDS-PAGE, at least 25 proteins with apparent molecular masses ranging from 14 to 300 kDa had been observed while no bands were seen below 14 kDa. This was also consistent with data previously reported [38] from the analysis of SDS-PAGE and two-dimension electrophoresis gels stained with Silver or Coomassie blue staining methods. For the nano-LC-MS/MS analysis presented here, several gradient gels (4-15%) were run at various conditions to allow excision of all gel bands detectable upon Coomassie blue staining. Figure 2 shows that the analysed bands represent the majority of the venom proteins of C. inanitus. However, the presence in this venom of small amounts of additional proteins and peptides cannot be excluded. For 25 proteins, named Ci-14a to Ci-300, peptide sequences exactly matching sequences of the vgORF database were obtained upon nano-LC-MS/MS analysis (see additional file 1: Table of peptide identification). Furthermore, peptides belonging to four additional proteins were detected, namely Vem7, Vem11, Vem17 and Vem37 (Vem being an abbreviation for Venom Mix). These proteins were found in several gel bands, a situation usually found for very abundant proteins. There was no evident correspondence between the relative abundance of venom protein bands on the gel (Figure 2) and the abundance of the corresponding ESTs in the vgORFs database (Table 2). The detailed list of the identified venom proteins will be discussed in the following sections.

Name	Identification	EMBL Acc. No	Significant matches with Pfam entries	No. of corresponding ESTs	Seq. length (amino acids)
Ci-23a	Similar to venom protein from C. sp. near curvimaculatus	FN908672	-	534	114
Ci-45	Chitinase	FN908682	PF00704	64	387
Ci-48b	IDGF-like protein	FN908684	PF00704	118	440
Ci-23c	Mucin-like peritrophin	FN908674	PF01607 (×2)	22	177
Ci-220	Mucin-like peritrophin	FN908692	PF01607 (×2)	3	229
Vem7	Yellow-e3-like protein	FN908694	PF03022	15	432
Ci-50	Esterase/lipase-like	FN908685	PF00151	2	215p
Ci-300	Metalloprotease-like	FN908693	-	8	230p
Ci-95	ACE-like protein	FN908689	PF01401	19	145p
Ci-80a	C1A protease	FN908687	PF00112	1	165p
Ci-40a	Trypsin-like serine protease	FN908678	PF00089	2	144p
Ci-180	Lectin-like protein	FN908691	PF00059	2	218p
Ci-14a	CSP-like protein	FN908669	PF03392	1	104p
Ci-23b	Protein related to PBP/OBP	FN908673	PF01395	29	144
Ci-48a	Similar to lethal (1) G0193 isoforms	FN908683	-	5	383
Vem17	Similar to lethal (1) G0193 isoforms	FN908696	-	3	220p
Ci-80b	Similar to lethal (1) G0193 isoforms	FN908688	-	1	229p
Ci-14b	Unknown protein	FN908670	-	286	141
Ci-15	Unknown protein	FN908671	-	4	123
Ci-27	Unknown protein	FN908675	-	59	210
Ci-28	Unknown protein	FN908676	-	13	182
Ci-35a	Unknown protein	FN908677	-	5	188p
Ci-35b	Unknown protein	FN908678	-	5	266
Ci-40c	Unknown protein	FN908681	-	64	263p
Ci-60	Unknown protein	FN908686	-	2	205p
Vem11	Unknown protein	FN908695	-	3	217
Vem37	Unknown protein	FN908697	-	4	246
Ci-40b	Acidic ribosomal protein P0	FN908680	PF00466 PF00428	3	316p
Ci-120	alpha-N-acetyl glucosaminidase	FN908690	PF05089	2	165p

Protein names for which a signal peptide has been predicted are shown in bold; p, partial sequence.

Ci-23a and Ci-45, two proteins with similarities with venom proteins from C. sp. near curvimaculatus

The Ci-23a venom protein was encoded by a contig corresponding to the highest number of ESTs in the library (534). It displays 50% of sequence identity (BlastP, E-value = 7e-21) with a 32.5 kDa protein referred as "venom protein from C. sp. near curvimaculatus" [GenBank:ACI70208.1], another chelonine wasp. This latter protein was historically the first to be isolated and sequenced from the venom of a parasitoid Hymenoptera [39]. Although it was found to be necessary for the survival of the parasitoid in the lepidopteran host, Trichoplusia ni[44], it is still not related to any other known protein and its function remains unknown to date. A potential cleavage site for both N-arginine dibasic convertase (pattern: .RK|RR[^KR]) and subtilisin-like proprotein convertase (pattern: [KR]R.) was detected at positions 51 to 53 (RRA) of the Ci-23a sequence. However, sequences coding for such convertases were not found in our vgORFs database. Remarkably, Ci-23a was devoid of the 12 tandem repeats of 14 residues that characterized the C-terminal part of the venom protein of C. sp. near curvimaculatus (see additional file 2: Amino acid sequence alignment of Ci-23a and the venom protein from C. sp near curvimaculatus) [39]. These repeat sequences form several α-helices with strong amphipathic structures supposed to run at the surface of the protein [39] and we found that they contain an unusualy high number of potential glycosaminoglycan attachment sites (14 serine motifs involved in the motif pattern: [ED]{0,3}.(S) [GA]). Thus Ci-23a, which is much shorter than its homologue, is potentially processed by non-venomous convertases and is likely to have a substrate or target site specificity different from that of the 32.5 kDa venom protein of C. sp. near curvimaculatus. Interestingly, the latter is a very abundant venom protein [44] while Ci-23a protein is of low abundance (Figure 2).

The Ci-45 venom protein, which gives a strong band upon Coomassie staining (Figure 2), shows high similarity (BlastP, 79% identity, E-value = 2e-141) to a 52 kDa chitinase from venom of C. sp. near curvimaculatus [GenBank:AAA61639.1] [40]. Both proteins possess a predicted signal peptide and a glycosyl hydrolase family 18 domain (Pfam: PF00704) with four highly conserved regions present in all known insect chitinases [45–47] (see additional file 3: Amino acid sequence alignment of representative chitinases from different insect species). Members of glycosyl hydrolases 18 family show an eight-stranded α/β barrel catalytic core structure [48]. The 17 residues featuring this functional domain were all found in the Ci-45 sequence, notably those of the second conserved region implicated in catalysis, as shown by previous site-directed mutagenesis studies (consensus sequence: (F/L)DG(L/I)DLD(W/I)EYP)) [47, 49]. Four cysteine residues involved in two disulfide bonds are conserved in the two Cheloninae enzymes and the secondary structures of both proteins were predicted to be highly similar in the placement of α-helix, β-strand and coil structure (data not shown). Ci-45 differs from its homologue by the absence of a C-terminal chitin-binding Peritrophin-A domain (CBM_14, Pfam: PF01607), but this domain does not appear to be essential for the chitinolytic activity of chitinases in Arthropods [46, 50–52]. The Ci-45 contig is thus most likely coding for an active venom chitinase and might be responsible for the chitinase activity previously detected in the venom of C. inanitus[38]. According to the classification proposed by Zhu et al. [47], it belongs to a group of conserved insect chitinases containing a single catalytic domain (group IV). Our phylogenetic analysis (Figure 3) grouped the chitinases from venom glands of C. inanitus and C. sp. near curvimaculatus in a monophyletic clade with that from teratocytes of T. nigriceps [GenBank:AAX69085.1]. Teratocytes are parasitoid wasp secretory cells that circulate into the parasitized host haemolymph, and represent a different way for the wasp to deliver virulence factors into the host. The teratocyte released chitinase from T. nigriceps is hypothesized to contribute to the avoidance of microbial contamination of the host's haemocoel or to facilitate the emergence of parasitoid larvae through the host's cuticle [53]. Other examples of chitinases produced by venom glands were previously reported from spiders [54, 55] and from the parasitoid wasps M. hyperodae[33] and N. vitripennis[28]. More recently, a chitinase produced by the posterior salivary glands of the cephalopod Octopus kaurna has also been described [56]. Venom and salivary glands chitinases described are thus restricted to invertebrate species. Interestingly, our phylogenetic analysis shows that venom chitinases belong to distinct clades comprising also non venomous enzymes. This suggests chitinases have been selected for production in invertebrate venom glands through multiple independent recruitment events.

Ci-48b, an IDGF-like protein

Like Ci-45, Ci-48b possesses a glycosyl hydrolase family 18 domain (see additional file 4: Amino acid sequence alignment of Imaginal disc Growth Factors (IDGFs)-like proteins from different insect species). However, in the second conserved region, a glutamine residue (Q157) replaces the glutamic acid residue that plays a key role in active insect and bacterial chitinases, in being the putative proton donor during the catalytic mechanism [49, 57, 58]. This feature is shared by members of the imaginal disc growth factors (IDGFs) family which have presumably evolved from chitinases to gain new functions [59]. Interestingly, Ci-48b shows high sequence similarity to IGDFs from several insect species, namely a venom gland protein of honey bee workers [NCBI Reference Sequence: XP_396769.2] and a hemocyte aggregation inhibitor protein from the lepidopteran species Manduca sexta [GenBank:ACW82749.1] (Blast P 38% identity; E-value = 2e-76) [60]. Thus Ci-48b may contribute to the survival and development of C. inanitus eggs once oviposited into their host either by acting as a growth factor or alternatively, by modulating the cellular immune response of young S. littoralis host larvae.

Ci-23c and Ci-220, two putative mucin-like peritrophins

The sequence of the Ci-23c venom protein is 177 amino acid long, giving a theoretical molecular mass of 19.6 kDa (see additional file 5: Amino acid sequence alignment of Ci-23c, Ci-220 and AD-873). It possesses two Chitin-binding peritrophin domains 14 (CBM_14, positions 26-78 and 92-144) and a predicted glycosaminoglycan attachment site (positions 155-158). Another venom protein, Ci-220 (229 amino acids, theoretical molecular mass 26.3 kDa), also contains two CBM_14 domains (positions 29-84 and 82-135) and a predicted glycosaminoglycan attachment site (positions 138-141). The two proteins share an overall 32% identity (BlastP, E-value = 1e-16) and show sequence similarities to a wide variety of CBM_14 domains containing proteins, including a venom component from N. vitripennis[28]. Moreover they share the domain organization of the mucine-like peritrophin AD-873 identified from the salivary gland transcriptome of the mosquito Anopheles darlingi [GenBank:ACI30179.1]. Interestingly this peritrophin is speculated to contribute to the maintenance of the structure of the mouthparts and/or salivary canal of A. darlingi[61]. In C. inanitus, oviposition is accompanied by intense contractions of the abdomen, which are necessary to push the venom from the reservoir into the oviduct, since the venom reservoir is located at the distal end of one of the gland filaments and has a very thin wall without a muscle layer [38]. It is thus possible that the venom proteins Ci-23c and Ci-220, as the mosquito peritrophin, contribute to keeping the reservoir in shape.

Vem7, an ancient yellow-e3-like venom protein

Vem7 shares high sequence similarity to the yellow-e3 protein [GenBank:ABB82366.1] from A. mellifera (BlastP, 42% identity, E-value = 2e-81). The yellow-e3 gene is highly expressed in the head and hypopharyngeal gland of honey bee workers and is considered as the progenitor of all genes of major royal jelly proteins (MRJPs) of A. mellifera. Located in the same genomic region and sharing a similar exon/intron organization, MRJPs would have been generated via recent duplications [62, 63]. Recently, two members of the MRJPs family, MRJP8 [GenBank:ACD84799.1] and MRJP9 [GenBank:ACD84800.1], were identified in the venom gland proteome of A. mellifera[5]. It is noteworthy that phylogenetic analyses put them at the basis of the MRJPs tree, meaning that they are the most ancient members of the MRJPs family and suggesting a venomous "pre-royal jelly" function for the MRJPs progenitor originating from yellow-e3 [5, 63]. Our phylogenetic analysis shows that Vem7 forms a monophyletic group with yellow-e-3 protein (Figure 4) and thus represents the first example of a venomous protein in this clade. This supports the hypothesis that a yellow-e-3 gene progenitor of MRJPs may have encoded a venomous protein like the Vem7 gene.

Putative enzymes

Ci-50 is a venom protein belonging to the lipase family (Pfam: PF00151). One carboxyl-esterase and two other lipases were previously identified in the venom proteome of N. vitripennis[28] and a lipase activity has been found in the venom of P. hypochondriaca[64]. In N. vitripennis, venom lipases might participate in the alteration of host's lipid metabolism to the benefit of the developing parasitoid eggs [65] and a similar function is conceivable for C. inanitus.

The partial sequence of the Ci-300 venom protein shares 39% sequence similarity (BlastP, E-value = 2e-05) with a Zinc-dependent metalloprotease identified in the venom of P. hypochondriaca [GenBank:CAD21587.1]. However, Ci-300 lacks the functional characteristic Zn²⁺-binding motif of HExxHxxGxxH and a distal located methionine [66] found in venom metalloproteinases from the parasitoid species P. hypochondriaca[67], M. aethiopoides[33], Eulophus pennicornis[68] and N. vitripennis[28] (see additional file 6: Partial amino acid sequence alignment of Ci-300 with insect metalloproteases). The sequence of Ci-300 could have thus considerably diverged from an ancestral Zinc-dependent metalloprotease-like protein to acquire an original function in the venom of C. inanitus.

The Ci-95 venom protein shows significant sequence similarity to various angiotensin converting enzymes (ACEs, Pfam: PF01401) and notably to an ACE-like protein from A. mellifera [NCBI Reference Sequence:XP_393561.2] (BlastP, 46% identity, E-value = 2e-29). In addition, upon Western analysis with an antibody made against recombinant Drosophila ACE (kindly provided by Dr. Elwyn Isaac, University of Leeds, UK) a clear band was seen (data not shown). ACE is a dipeptidyl carboxydipeptidase with a broad in vitro substrate specificity that is best known, in mammals, for its role in converting inactive angiotensin I to the vasoconstrictor, angiotensin II, and the inactivation of bradykinin [69]. In insects, ACE-like proteins appear to have a wide tissue distribution, from embryos to adult stages [70, 71] and some have been implicated in the metabolic inactivation of neuropeptides in the central nervous system [72]. Dani et al. [73] have detected an ACE-like enzymatic activity in the venom of P. hypochondriaca and have speculated that venomous ACE could be involved in the processing of peptide precursors in the venom reservoir. More recently, another ACE-like enzyme has also been identified in the venom of N. vitripennis[28].

Ci-80a, a venom protein encoded by a single transcript identified in our VgORFs database, belongs to the peptidase family C1, sub-family C1A (papain family, clan CA, Pfam: PF00112). The partial sequence of Ci-80a contains two out of the four conserved residues of the active site of C1A proteases and all the residues involved in the S2 subsite, which is involved in specificity for the dominant substrate of papain-like cysteine proteases. Interestingly, viral cystatins encoded by the genome of the bracovirus CcBV, associated with the parasitic wasp Cotesia congregata, were shown to target some C1A proteases of the host M. sexta[74]. Cathepsins and their inhibitors may play an important role, yet undetermined, in the context of host-parasitoid physiological relationships.

The Ci-40a venom protein contains a partial trypsin-like serine protease domain (Pfam: PF00089) but displays low sequence similarities with other known proteases (see additional file 7: Amino acid sequence alignment of Ci-40a with serine protease homologs (SPHs)). At least one of the three residues involved in the catalytic triad for serine protease (aspartate residue in position 111) is present in the partial sequence of Ci-40a. Several serine protease homologs were already reported from parasitoid venoms. In the endoparasitoid C. rubecula, the venom protein Vn50 [GenBank:AAP49428.1] has been found to be a mutated serine protease acting as an inhibitor of the defensive reaction of melanization of host hemolymph [21]. In addition, members of the serine protease protein family were recently identified in the venom proteomes of N. vitripennis[28] and P. puparum[29].

The Ci-120 protein shares significant sequence similarity with insect alpha-N-acetyl glucosaminidases (Pfam: PF05089, EC: 3.2.1.50) and might play a role in proteoglycan metabolism.

Lectin-like venom protein

A C-type lectin domain (Pfam: PF00059) was found in the sequence of the Ci-180 venom protein. The domain extends from positions 76 to 199 of the partial sequence. An immunosuppressive function has been proposed for a lectin with a similar C-type lectin domain, encoded by the genome of the bracovirus CpBV, associated with the parasitic wasp Cotesia plutellae[75]. Since parasitoid venoms and PDVs are used by the wasps to manipulate parasitized host physiology, it might not be surprising that common molecules have been selected for delivery into the host via different pathways.

CSP-like and OBP-like proteins

The Ci-14a venom protein belongs to the A10/OS-D insect pheromone-binding protein family (Pfam: PF03392). A high sequence similarity was observed between Ci-14a and Csp3 from A. mellifera (BlastP, 56% identity, E-value = 5e-27), another member of the A10/OS-D protein family. Csp3 [GenBank:ABH88171.1] is a chemosensory protein (CSP) ubiquitously expressed in adults and pre-imaginal stages of the honeybee in which it may play a role in cuticle maturation [76].

Ci-23b is a venom protein containing a pheromone binding protein/general-odorant binding protein (PBP/GOBP) domain (Pfam: PF01395). Homology searches revealed that Ci-23b has highly diverged from known PBPs and GOBPs. In particular, it only shows 4 out of the 6 cystein residues strictly conserved between PBPs and GOBPs [77, 78]. Other OBP-like venom proteins, unrelated to Ci-23b, have previously been identified in the venom proteomes of A. mellifera workers and N. vitripennis females (NCBI Reference Sequences: NP_001035313.1 and NP_001155150.1, respectively) [5, 28]. Beside the involvement of PBPs and OBPs in chemical communication, it is possible that in Hymenoptera, some OBP-like proteins fulfil other roles in relation with the venomous functions.

Venom proteins similar to lethal (1) G0193 isoforms

Ci-48a, Vem17 and Ci-80b venom proteins share sequence similarities with members of a group of proteins similar to protein isoforms A [GenBank:AAF46301.1] and B [GenBank:AAS65275.1] encoded by the lethal (1) G0193 gene from D. melanogaster. This group of cystein-rich proteins notably includes several venom and salivary gland proteins of unknown functions reported from various insect species (see additional file 8: Sequence similarities between Ci-48a, Vem17 and Ci-80b and proteins similar to isoforms of lethal (1) G0193). Ci-80b possesses the least conserved amino acid sequence towards lethal (1) G0193 isoforms suggesting it might have diverged as a virulence factor involved in host-parasite interactions, which are often characterized by a high level of divergence.

New lineage-specific proteins

In addition to Ci-23a and Ci-45 which are conserved in the venom of two Chelonus species, ten C. inanitus venom proteins did not show any significant sequence similarity to known proteins (Ci-14b, Ci-15, Ci-27, Ci-28, Ci-35a, Ci-35b, Ci-40c, Ci-60, Vem11 and Vem 37). This is reminiscent of observations on twenty three venom proteins in N. vitripennis which also have no similarity to known proteins and appear to be lineage- and/or species specific [28]. Given that Chelonines are egg-larval parasitoids it is possible that new C. inanitus proteins have evolved to cope with this particular parasitic context. Although they do not contain conserved domains, they may play an important role during host-parasite interaction and notably Ci-14b, the second most abundant sequence of the transcriptome (286 ESTs).

Additional putative venom proteins identified from the vgORFs database

In addition to proteins identified by mass spectrometry, several ESTs encoding putative venom proteins were identified in our vgORFs database (Table 3). Five ESTs encoded partial sequences of at least three different hyaluronidase-like proteins that show significant sequence similarities to venom hyaluronidases described from several hymenopteran species [79]. One EST encoded a venom allergen 5-like protein (Figure 5). Venom allergen 5 proteins (also called antigens 5) are commonly found in venoms of social Hymenoptera of the superfamilly Vespoidea [80]. They belong to a wider group of proteins expressed by salivary and venom glands of distant animal species, recently gathered under the proposed term of CAP proteins for "Cystein-rich secretory proteins, Antigen 5 and Pathogenesis-related proteins" [56]. CAP domain proteins are the dominant allergy-inducing toxins in hymenopteran venoms [81], and a related CAP protein has previously been identified from the venom of M. hyperodae[33].

Sequence name	EMBL Acc. no.	Best-matched Protein (Acc. no.)	Species	Id. (%)	E-value
A6YN21CM1	FN985040	Hyaluronidase [GenBank:ACD61711.1]	Orancistrocerus drewseni	37	2e-16
A4YE08CM1	FN985041	Hyaluronidase [GenBank:AAA27730.1]	Apis mellifera	46	7e-07
A3YM10CM1	FN985042	Hyaluronidase 2 [GenBank:AAK51798.2]	Apis cerana cerana	45	8e-36
A2YM17CM2	FN985043	Hyaluronidase [GenBank:BAF93867.1]	Anoplius samariensis	37	6e-13
A1YB14CM3	FN985044	Hyaluronidase [GenBank:AAA27730.1]	Apis mellifera	42	7e-43
A1YI24CM3	FN985045	Allergen 5 [GenBank:AAA28303.1]	Dolichovespula arenaria	48	7e-36

Amino acid sequences encoded by the mentioned ESTs were compared to protein sequences deposited in NCBI nr database using BLASTP algorithm. Id.: identity between the amino acid sequence deduced from a given C. inanitus transcript and the corresponding best-matched protein.

Insects

C. inanitus (Hymenoptera: Braconidae), a solitary egg-larval parasitoid, was reared on one of its natural hosts S. littoralis. Adult S. littoralis were kindly provided by Syngenta AG, Stein, Switzerland. They were raised at 27°C at a LD photoperiod of 14 h:10 h and fed with an artificial diet. Diet was prepared from dry powder (Beet Army Worm Diet, Bio Serv, Frenchtown, New Jersey, USA).

Collection of venom glands and RNA isolation

Female wasps were anaesthetized on ice for several minutes and then shortly rinsed in 70% ethanol. The abdominal organs were gently pulled out with forceps and the reproductive apparatus was placed in 50 μl sterile Insect Ringer. Then, the venom gland filaments were dissected, washed in sterile Ringer, put into an Eppendorf tube containing 200 μl of RNAlater™(Qiagen) and stored at - 80°C until RNA isolation. The morphology of the C. inanitus venom gland was previously described by Kaeslin et al. [38]. For RNA isolation, venom gland filaments from 60 female wasps were used; no homogenization was necessary and 0.45 ml Lysis buffer RLT (Qiagen) including 143 mM β-mercaptoethanol was added followed by a proteinase K digestion as described by Johner and Lanzrein [86]. The RNA isolation (RNeasy Mini Kit, Qiagen) including an on column DNase digestion (RNase-free DNase I) was performed according to the manufacturer's protocol. Total RNA was eluted from the column with 100 μl RNase-free water. A second digestion with DNase I was carried out and the RNA was then extracted with acidic phenol; the RNA was precipitated as described in [87]. The yield was 9 μg of total RNA.

cDNA library construction

A cDNA library was constructed with the Creator SMART cDNA Library Construction Kit (BD Biosciences, Ozyme, France) following the supplier's instructions. The first strand cDNA was synthesized from 481 ng of total RNA extracted from C. inanitus venom glands. The cDNA were ligated into pDNR-LIB (BD Biosciences Clontech) and ligation products were transformed into ElectroMax DH10B-T1 Phage Resistant Escherichia coli Competent Cells (Invitrogen, Fisher Scientific, France).

Sequencing and ESTs quality control

To obtain an unbiased overview of the venom gland transcriptome, colonies were amplified with the ϕ29 DNA polymerase by rolling circle amplification. Sequencing was done on a ABI sequencer using the standard M13 forward primer and BigDye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA). Initially 384 clones from the library were analyzed for the presence of venom proteins-related sequences. These were detected and a sequencing of a total of 2500 clones was performed. The EST sequences obtained were analysed for quality control; base calling step was performed with the Phred program f(S9). Low quality bases (phred score < 20) were masked and sequences with more than 30% n-content, or shorter than 60 bp (after vector/adaptator sequences removing) were removed.

ESTs clustering and assembly

After discarding the poor-quality sequences, 2111 high-quality ESTs were subjected to clustering using the TIGR software TGI Clustering tool (TGICL) [88]. The clustering was performed by a modified version of NCBI's megablast. EST sequences were assigned to clusters based on identity: the clustering parameters were 98% minimum percent identity for overlaps, for a minimum overlap length of 40 nt and a maximum length of unmatched overhangs of 20 nt.

Sequences from each cluster were assembled into consensus sequences called "contigs" using the CAP3 assembly program [89] available in TGICL. Sequences from a cluster containing only one sequence were called "singletons".

ESTs annotation

To identify similarities with known proteins, the sequences of contigs and singletons were searched using the BLASTX algorithm against a local non-redundant protein database (NR, NCBI) with a cut-off E-value of 1e-5. To define the function of the contigs and singletons, we used the Gene Ontology (GO) controlled vocabulary [90] and more particularly GOSlim, a subset of GO terms, which provides a higher level of annotations and allows a more global view of the dataset. To this end, an automated GO-annotation of the sequences of contigs and singletons that showed a significant similarity with a Uniprot entry was achieved using the Blast2go software [91], with a stringency cut-off of 1e-6.

Collection of venom, SDS-polyacrylamide gel electrophoresis and protein identification

Wasps were anaesthetized on ice and their venom apparatus (venom gland filaments and reservoir) was dissected. For each protein separation the venom of four wasps was collected. The venom was collected by piercing the reservoirs with a glass capillary. The sucked up venom was collected in 10 μl sterile H₂O and mixed with 10 μl sample buffer (0.125 M Tris-HCl pH 6.5, 4% (w/v) SDS, 10% (v/v) glycerol, 0.01% bromophenol blue, 5% (v/v) β-mercaptoethanol) and heated at 90°C for 6 minutes. For separation of proteins, precast linear gradient READY GEL (BIO RAD) 4-15%Tris-HCl gels with 10 wells were used. Two equivalents of venom (i.e. the amount of venom collected from two reservoirs) were loaded per lane. Electrophoresis was in 0.02 M Tris-HCl pH 8.8, 0.19 M glycine, and 0.1% (w/v) SDS. A constant voltage of 150 V was applied. As marker the High-Range Rainbow™Molecular Weight Marker (GE Healthcare) was used. Staining was done with SERVA blue R (Coomassie) Brilliant Blue R-250 according to the manufacturer's protocol. For protein identification, gel slices were cut out, transferred into Eppendorf tubes and covered with 20 μl ethanol (20%). Treatment of gel slices and nano-LC-MS/MS analysis was as described in [82]. CID spectra were extracted into data files by Bioworks (Rev.3.3.1, Thermo Scientific) without any filters applied. Combined dta files were automatically matched to our personal database of vgORFs by Phenyx software, Version 2.5 (Genebio SA, Geneva, Switzerland). N-terminal sequencing was done by Edman degradation.

Sequence analysis

Each cluster of nucleotide sequences was annotated by being searched against GenBank NCBI database [92] with BLAST algorithms. Since amino acid sequences are more useful to detect homology over long periods, the assembled sequences were translated on-line into the correct open reading frames (ORFs) using ORFINDER tool from NCBI [93] and compared to the sequences in the NCBI nr and Swissprot protein databases. Sequences that did not match were further compared against the Genbank nucleotide databases (Blastn).

The signalP algorithm [94] was accessed online to predict the presence of signal peptides. The deduced amino acid sequences of all the proteins identified by nano-LC-MS/MS analysis were annotated by searching against Pfam protein families database [95]. Modification, cleavage and functional sites were predicted by the ELM server [96].

For amino acid sequence alignments, sequences were retrieved from NCBI nr database, aligned with sequences from C. inanitus with the program MAFFT version 6.0 [97] and edited with the program Jalview [98]. Alignment refinement was done with Gblocks software (version 0.91b) [99]. Phylogenetic relationships were estimated by Bayesian MCMC analyses using the program MrBayes 3.1.2 [100] available online [101, 102]. For each set of aligned sequences, we implemented a mixed model of amino acid substitution, with gamma-correction for heterogeneity rate among residues and correction for invariable residues. Model of protein evolution was selected using ProtTest 2.4 software [103].