Identification and Functional Characterization of Toxoneuron nigriceps Ovarian Proteins Involved in the Early Suppression of Host Immune Response

Abstract

The endophagous parasitoid Toxoneuron nigriceps (Viereck) (Hymenoptera, Braconidae) of the larval stages of the tobacco budworm Heliothis virescens (Fabricius) (Lepidoptera, Noctuidae) injects the egg, the venom, the calyx fluid, which includes a Polydnavirus (T. nigriceps BracoVirus: TnBV) and the Ovarian Proteins (OPs) into the host body during oviposition. The host metabolism and immune system are disrupted prematurely shortly after parasitization by the combined action of the TnBV, venom, and OPs. OPs are involved in the early suppression of host immune response, before TnBV infects and expresses its genes in the host tissues. In this work, we evaluated the effect of HPLC fractions deriving from in toto OPs. Two fractions caused a reduction in hemocyte viability and were subsequently tested to detect changes in hemocyte morphology and functionality. The two fractions provoked severe oxidative stress and actin cytoskeleton disruption, which might explain the high rate of hemocyte mortality, loss of hemocyte functioning, and hence the host’s reduced hemocyte encapsulation ability. Moreover, through a transcriptome and proteomic approach we identify the proteins of the two fractions: eight proteins were identified that might be involved in the observed host hemocyte changes. Our findings will contribute to a better understanding of the secreted ovarian components and their role in parasitoid wasp strategy for evading host immune responses.

An Overview of Ovarian Calyx Fluid Proteins of Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae): An Integrated Transcriptomic and Proteomic Approach

“…Overall, OPs, in association with PDV and venom, contribute to the evasion of the host immune response by Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae). Moreover, OPs' HPLC fractions have been tested and, for two fractions, the same effects have been observed as shown in the previous work [35]. In addition, we identified the proteins of these two fractions through a combination of transcriptomic and proteomic approaches, resulting in the identification of eight proteins that could be involved in the observed alterations of the host hemocytes.…”

“…The ovarian calyx tissue was placed in a 1.5 mL centrifuge tube (Eppendorf, Hamburg, DE, USA) containing TRI Reagent (Sigma, St. Louis, MO, USA) and stored at −80 • C for RNA extraction and subsequent transcriptomic analysis [23], while the fluid was transferred into a 1.5 mL tube (Eppendorf) and centrifuged at 2000× g for 5 min at 4 • C to remove debris. As previously described [35,36], the supernatant, containing ovarian calyx fluid proteins and the polydnavirus, was further purified with 0.45 µm Millex PVDF filters followed by washing 3 times with 1 mL of 1× PBS. To separate the polydnavirus, a centrifugation was carried out at 30,000× g for 1 h at 4 • C and subsequently the supernatant containing the ovarian calyx fluid proteins was concentrated by ultrafiltration in a column with cut-off 3000 MW, by centrifuging the sample at 3000× g until a volume of around 500 µL was obtained.…”

“…The success of parasitism is also assured by embryonic factors, such as the teratocytes [26]. In the host-parasitoid system, Heliothis virescens-Toxoneuron nigriceps, venom, teratocytes, and some PDV genes have already been studied [27][28][29][30][31][32][33][34] and recently the effects of the OPs on the host immune system have been reported [35,36]. The OPs have been shown to induce several alterations on hemocytes, including cellular oxidative stress, modifications of actin cytoskeleton, vacuolization, and the inhibition of encapsulation capacity of hemocytes, thus inducing both a loss of hemocyte functionality and cell death [36].…”

Immunosuppressive, antimicrobial and insecticidal activities of inhibitor cystine knot peptides produced by teratocytes of the endoparasitoid wasp Cotesia flavipes (Hymenoptera: Braconidae)

“…For instance, defensins are broad-spectrum antibacterial peptides released by teratocytes in order to maintain an infection-free environment for successful parasitoid larval development (Gao et al, 2016). Several other antimicrobial proteins are released by teratocytes such as chitinases (Falabella, 2018), chitolectins (Rossi et al, 2012), apolipoproteins (Salvia et al, 2022), and serine protease inhibitors (Gu et al, 2021). In this study, we associated a new class of molecules, the ICK peptides, as antimicrobial peptides produced by the teratocytes of the parasitoid C. flavipes.…”

In Vitro Evaluation of the Antibacterial Activity of the Peptide Fractions Extracted from the Hemolymph of Hermetia illucens (Diptera: Stratiomyidae)

“…Eluents A and B have the following composition: 2% ACN LC-MS grade and 0.2% HCOOH, and 95% ACN LC-MS grade and 0.2% HCOOH, respectively. The MS/MS method was set up in a data-dependent acquisition mode (DDA), with a full scan ranging from 300 to 1800 m/z range, followed by fragmentation in CID modality of the top 5 ions (MS/MS scan) selected by intensity and charge state (+2, +3, +4 charges), and applying a dynamic exclusion time of 40 s [ 58 ]. The peak list generated was uploaded in Mascot software (version 2.4.0) and research was performed by using the in-house database named the “ Hermetia illucens database”.…”