Venomics of the ectoparasitoid wasp Bracon nigricans

- Venomics of the ectoparasitoid wasp Bracon nigricans.
- Background: Venom is one of the most important sources of regulation factors used by parasitic Hymenoptera to redirect host physiology in favour of the developing offspring.
- Results: Here we present a combined transcriptomic and proteomic study of the venom of the generalist idiobiont wasp Bracon nigricans, an ectophagous larval parasitoid of different lepidopteran species, for which we recently described the host regulation strategy and the functional role of the venom in the induction of physiological changes in parasitized hosts.
- The experimental approach used led to the identification of the main components of B.
- Parasitic Hymenoptera are part of one of the most spe- ciose insect orders, which includes the largest number of insect natural enemies [1].
- In most cases, these wasps have a fairly broad host range and are idiobionts, since they induce a rapid paralysis of the host which is quickly exploited as a static source of nutrients [4, 5].
- In contrast, koinobionts, which in most cases develop as endophagous parasitoids (i.e., inside the body of the host), show more complex and subtle host regulation strategies, allowing a prolonged interaction of.
- However, in both cases the growth and development of parasitoids is dependent on the physiological regulation of the host, me- diated by a wide range of parasitic factors, some of which are present both in ectoparasitic and endoparasitic wasps (i.e., venom and larval secretions), while others only occur in endoparasitoids (e.g., polydnaviruses and teratocytes .
- 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0.
- Full list of author information is available at the end of the article.
- A significant part of the research efforts are focused on host paralysis, which is the most evident and dramatic symptom in- duced by the venom of ectoparasitic wasps.
- Studies on other host regulation properties of ectopar- asitoid venom are limited, with the exception of the pupal ectoparasitoid Nasonia vitripennis, for which a more comprehensive analysis of host alterations has been carried out, taking into consideration developmen- tal arrest, metabolic changes and immunosuppression [35–37].
- This latter generalist species that parasitizes a number of moth larvae was one of the first studied for its venom composition, which includes neurotoxic proteinaceus components, only partially characterized .
- Main results and features of the assembly are presented in Table 1..
- BLASTx similarity search results revealed that about 52.5% of the total assembled transcripts (22,218 se- quences) have at least one match with the UniProtKB database.
- The functional annotation of the tran- scripts, performed using BLAST2GO, shows that 22,222 sequences (52.48% of the total assembly) share signifi- cant similarity to proteins with assigned Gene Ontology (GO) terms.
- This was performed by filtering the proteome for the presence of the signal peptide identified by SignalP tool (approximately 22% of the 109 total proteins) and, among these, we focused on those in the upper third of RPKM values distribution, exceeding the threshold value of 70.
- Only 10 Table 1 Overview of the de novo transcriptome assembly of.
- 2 N 50 value, which represent the threshold delimiting 50% of the contigs in the entire assembly which are equal to or larger than the reported value.
- To experimentally corroborate the validity of the ap- proach used in the identification of the major functional players in the venom blend of B.
- nigricans, a qRT-PCR experiment was performed on total RNA extracted from venom glands, whole adult males and females deprived of the venom apparatus, focusing on a sample set of 8 genes encoding proteins selected among those showing similarity with sequences deposited in the UniProt/Swis- sProt database and/or highly represented in the venom gland transcriptome.
- 2 Histograms reporting Gene Ontology (GO) level 2 annotation of the transcripts from Bracon nigricans venom glands.
- For each category, the bars represent the transcripts assigned to a GO term as percentages of the total number of transcripts with GO assignments.
- Concurrent analysis of venom gland transcriptome and venom proteome revealed the presence of a large propor- tion of sequences (47.5% of the total transcripts) showing no similarity with those available in the UniProtKB data- base.
- List of proteins identified by SDS-PAGE and LC-MS/MS of crude venom extract, sorted by descending order of RPKM values, filtered for the presence of the signal peptide and highly represented in the venom gland transcriptome (i.e., in the upper third of RPKM values distribution).
- comp22364_c1_seq2) identified by our analysis resulted highly expressed in venom glands, in accordance with the wide occurrence of this enzyme, which is one of the main venom components of Hymenoptera, including honeybees [64] and many para- sitic wasps, such as E.
- BnPLA2 shows 48% of identity, along 99% of the protein length, with two putative PLA2s of B.
- comp22420_c0_seq1) exhibits 32% of identity, along 90% of the protein length, with a.
- These enzymes perform essential roles in the digestion, transport and processing of dietary lipids in most living organisms and might participate in the breakdown of the energy stores contained in the fat body of the host, in order to increase the nutritional suitability of its body fluids ingested by parasitoid larvae [79, 80].
- Moreover, these enzymes may contribute by lipid hydrolysis to the generation of the toxic molecules mentioned above..
- Con- sidering that de novo lipid synthesis is energetically ex- pensive [82], the mobilization and consumption of host lipids, through the action of the abovementioned lipo- lytic venom hydrolases, could provide a selective advan- tage for B.
- BnTRY shows 52% of identity, along 100% of the protein length, with a putative trypsin- like serine protease of B.
- More- over, this protein exhibits a 23% of identity, along 90% of the sequence length, with a N.
- A classical function exerted by serine proteases is digestion, as occurs in the larvae of the ectoparasitoid Euplectrus separatae, which releases a salivary secretion containing a trypsin-like enzyme to digest the host tissues [84].
- When highly expressed in venom glands, serine proteases may also play important roles in interfering with the immune response of the host by altering the proteolytic cascades activated by the detection of non-self intruders [85, 86]..
- However, the disruption of the proteolytic activating cascade involved in the mela- nization of host hemolymph can be also induced by a mu- tated serine protease with inhibitory effects, such as reported for the endoparasitoid Cotesia rubecula [85]..
- EC 3.4.11.1) shows 24% identity, along 98% of the sequence length, with an aminopeptidase N-like pro- tein (GenBank: EFN65598.1) from Camponotus floridanus (Hymenoptera, Formicidae).
- Thus, it is reasonable to assume that BnLCA may participate to the general degradation of host tissues, perhaps increasing their permeability to other venom components and facilitating nutritional exploit- ation of the host, as suggested for other venom zinc- metallopeptidases [92, 93]..
- case of the larvae of Trichogramma australicum and E..
- ervi, which determine the cytolytic degradation of the formed embryos of the aphid Acyrthosiphon pisum [101].
- nigricans can be likely involved also in a more subtle regulation of the host immunity.
- IPR006170) results one of the most abundant components in the venom of B.
- nigricans, as suggested by the strong intensity of the corresponding band (number 27, Fig.
- identity, along the 62% of the sequence length, with the translated transcript GECT01010095 (GenBank) from the endoparasitoid wasp P.
- OBPs participate in solubilization and transport of small hydrophobic odorant molecules and pheromones, and are characterized by six conserved cysteine residues, forming three disulfide bonds that stabilize the folded structure of the protein [103]..
- of the sequence length, with a recently annotated PDI from the genome of the parasitoid wasp Diachasma alloeum (NCBI Reference Sequence: XP .
- Therefore, the PDI presence in the venom of B.
- The integration of transcriptomics and proteomics used in the present work provides the first description of the main components of B.
- nigricans venom and contributes to the expansion of the limited information available for the venom of ectophagous parasitoids, that permanently paralyze and rapidly suppress their victims (i.e., idiobiont parasitoids).
- nigricans, mainly esterases and proteases likely in- volved in paralysis (BnPLA2) and digestion of the host’s tissues, are in line with a typical ectoparasitic idiobiont life-style.
- Enzymes with lipolytic activity are likely in- volved in the mobilization of storage nutrients from fat bodies and/or in the release of neurotoxic fatty acids inducing paralysis, while enzymes related to carbohy- drate catabolism could be responsible for the alteration of glycoconjugants which mediate the recognition of al- tered self domains mediating wound healing, and may induce the observed changes of the carbohydrate titer in the host hemolymph [54].
- hebetor are somewhat limited, it is quite evident that these two closely related species, attacking the same group of in- sect hosts, rely upon a molecular toolkit for host regula- tion and exploitation which is only partly conserved, suggesting the occurrence of an ancestral broad molecu- lar biodiversity of the venom blend, which has been one of the major pre-requisites allowing the evolutionary di- versification of parasitic Hymenoptera..
- Indeed, the func- tional characterization of the venom proteins identified in the present study will offer new tools to develop bioinspired strategies for pest control, based on the use of natural antagonists beyond the organism level, as a source of insecticide molecules..
- nigricans was reared on larvae of the noctuid moth S..
- The de novo transcriptome assembly of the cleaned reads was performed with Trinity, setting the k-mer length at the default value .
- The assembled and annotated venom gland transcriptome was used to gen- erate a custom-made protein database, by translating the six reading frames of the nucleotide sequences in their corresponding amino acid sequences by SEQtools (http://www.seqtools.dk.
- Protein concentration of the venom ex- tracted was assessed by Bradford method [133]..
- 4 Specificity of expression in the venom glands of selected genes.
- Peptide analysis was performed using data-dependent acquisition of one MS scan (m/z range from 400 to 1600 Da/e), followed by MS/MS scans of the three most abundant ions in each MS scan.
- Dynamic exclu- sion was used to acquire a more complete survey of the pep- tides by automatic recognition and temporary exclusion (2 min) of ions from which definitive mass spectral data had previously been acquired.
- Moreover, a permanent exclusion list of the most frequent peptide contaminants (keratins and trypsin peptides) was included in the acquisition method in order to focus the analyses on significant data..
- Differential relative expression of the target genes was measured by one-step qRT-PCR, using the SYBR Green PCR Kit (Ap- plied Biosystems), according to the manufacturer’s in- structions.
- For validation of the ΔΔCt method, the difference between the Ct value of the target and the Ct value of RPS3 transcripts [ΔCt = Ct(target gene)-Ct (RPS3)] was plotted versus the log of ten-fold serial dilutions and 0.01 ng) of the purified RNA samples.
- The plot of log total RNA in- put versus ΔCt displayed a slope less than 0.1, indicating that the efficiencies of the two amplicons were approxi- mately equal.
- The relative expression of the target genes in female body deprived of the venom glands was used as calibrator (relative expression = 1).
- Putative homologous sequences of the most representative venom proteins were identified by sequence similarity searches through a BlastP analysis versus the non- redundant NCBI database (nr NCBI, release October 2019) and Swiss-Prot (release 2019_10 of 13-Nov-19)..
- To reconstruct phylogeny, alignments were manually trimmed to avoid comparisons of non-conserved regions present only in a subset of the taxa.
- List of the most abundant (RPKM>100) annotated transcripts encoding putatively secreted proteins (i.e., positive to SignalP analysis).
- The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript..
- Cloning and expression of the gene for an insect haemocyte anti-aggregation protein (VPr3), from the venom of the endoparasitic wasp, Pimpla hypochondriaca.
- melanogaster in response to long gland components of the parasitoid wasp Leptopilina boulardi: a rho-GAP protein as an important factor.
- Purification of pimplin, a paralytic heterodimeric polypeptide from venom of the parasitoid wasp Pimpla hypochondriaca, and cloning of the cDNA encoding one of the subunits.
- Physiological and biochemical analysis of factors in the female venom gland and larval salivary secretions of the ectoparasitoid wasp Eupelmus orientalis.
- Purification and characterization of insecticidal toxins from venom glands of the parasitic wasp, Bracon hebetor.
- Redirection of metabolism in the flesh fly, Sarcophaga bullata, following envenomation by the ectoparasitoid Nasonia vitripennis and correlation of metabolic effects with the diapause status of the host.
- Venom-induced alterations in fly lipid metabolism and its impact on larval development of the ectoparasitoid Nasonia.
- Venom proteins of the parasitoid wasp Nasonia vitripennis: recent discovery of an untapped pharmacopee.
- Evaluating the evolution and function of the dynamic Venom Y protein in ectoparasitoid wasps.
- Venom gland components of the ectoparasitoid wasp, Anisopteromalus calandrae.
- Deciphering the main venom components of the ectoparasitic ant- like bethylid wasp, Scleroderma guani.
- Susceptibility of different species of insects to an extract of the venom gland of the wasp Microbracon hebetor (say).
- Biology and developmental strategies of the Palaearctic parasitoid Bracon nigricans (Hymenoptera: Braconidae) on the Neotropical moth Tuta absoluta (Lepidoptera: Gelechiidae).
- The salivary protein repertoire of the polyphagous spider mite Tetranychus urticae: a quest for effectors.
- First extensive characterization of the venom gland from an egg parasitoid:.
- Insights into the venom composition of the ectoparasitoid wasp Nasonia vitripennis from bioinformatic and proteomic studies.
- Hydrolase activity in the venom of the pupal endoparasitic wasp, Pimpla hypochondriaca.
- The venom composition of the parasitic wasp Chelonus inanitus resolved by combined expressed sequence tags analysis and proteomic approach.
- The venom gland transcriptome of the parasitoid wasp Nasonia vitripennis highlights the importance of novel genes in venom function.
- A serine proteinase homolog venom protein from an endoparasitoid wasp inhibits melanization of the host hemolymph.
- Identification of the main venom protein components of Aphidius ervi, a parasitoid wasp of the aphid model Acyrthosiphon pisum.
- Proteomic analysis of the venom of the predatory ant Pachycondyla striata (Hymenoptera: Formicidae).
- Anatomy of the mouthparts and digestive tract during feeding in larvae of the parasitoid wasp Trichogramma australicum Girault (Hymenoptera : Trichogrammatidae).
- Disulfide structure of the pheromone binding protein from the silkworm moth, Bombyx mori.
- Venom of the parasitoid wasp Pteromalus puparum contains an odorant binding protein.
- Proteomic analysis of the venom and venom sac of the woodwasp, Sirex noctilio - towards understanding its biological impact.
- Fatty acid solubilizer from the oral disk of the blowfly.
- Protein disulfide isomerase: the multifunctional redox chaperone of the endoplasmic reticulum.
- Rapid expansion of the protein disulfide isomerase gene family facilitates the folding of venom peptides.
- Proteins of the PDI family:.
- The action of the campaniform sensilla on the legs

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