240 ISJ 15: 240-255, 2018 ISSN 1824-307X REVIEW The mechanism utilized by Toxoneuron nigriceps in inhibiting the host immune system P Falabella Department of Sciences, University of Basilicata, Via dell’Ateneo Lucano 10, 85100 Potenza, Italy Accepted June 26, 2018 Abstract Insect parasitic factors of both maternal and embryonic origin allow the endoparasitoid progeny to elude the humoral and cellular immune system responses of their host. Endoparasitoid wasps of lepidopteran larval stages inject, along with venom and ovarian proteins, Polydnavirus particles acting in synergy with all other factors in host regulation for parasitism success. To date, the molecular mechanisms used by endoparasitoid to circumvent the host immune system are little known. Neverthless, several of these strategies are conserved through the wasp parasitoid species. Heliothis virescens is a noctuid moth, host of the endophagous parasitoid Toxoneuron nigriceps. The first observed effect of parasitism is immune system suppression, as direct consequence of the array of host regulation factors, both of embryonic and maternal origin. This review describes the contribution of all the parasitic components during alterations of the host immune response observed after oviposition by T. nigriceps. Key Words: Heliothis virescens, immunomodulation, endoparasitoid, polydnavirus, venom, teratocytes Introduction The relationship between parasitoids and their hosts can be very complex, involving physiological interactions at different and multiple levels. This association is the result of a long coevolutionary history that in some cases led to a very intimate interaction and to fine regulation of the host physiology by the parasitoid. In the ectoparasitoids, whose larvae feed on the host body from outside, the parasitism is based on the use of venom, able to paralyse the host, allowing the larvae to benefit. In endoparasitoids, whose larvae feed within the host body, parasitism involves several factors, modulating the physiology of the host, in order to obtain a more suitable environment for the development of juvenile stages of their progeny (Schmidt et al., 2001; Federici and Bigot, 2003; Rivers et al., 2005; Vinson, 2012). Endoparasitoid insects, belonging to the Hymenoptera order, have developed strategies to parasitise their hosts, through specialised mechanisms generated by long adaptive processes that occurred within host/parasitoid physiological ___________________________________________________________________________ Corresponding author: Patrizia Falabella Department of Sciences University of Basilicata Via dell’Ateneo Lucano 10, 85100 Potenza, Italy E-mail: patrizia.falabella@unibas.it interactions (Vinson and Scott, 1974; Vinson and Iwantsch, 1980; Godfray, 1994; Quicke, 1997). The first evidence for the physiological changes observed in parasitised hosts is the inactivation of humoral and cellular defences, that prevents parasitoid egg melanization and encapsulation (Beck et al., 2000; Asgari et al., 2003a; Zhang et al., 2006). The insect immune system is generally able to respond to the intruders within few minutes after injuries. Parasitoids, as a consequence, have evolved their ability to alter the host defence with different strategies, some of which acting very fast. For this reason, during oviposition, parasitoid females introduce maternal factors such as ovarian fluid into the host body (Asgari and Schmidt, 1994; Webb and Luckhart, 1994, 1996; Tanaka et al., 2002), venom (Beckage and Gelman, 2004; Zhang et al., 2004; Kohler et al., 2007) and proteins that cover the egg surface (Asgari and Schmidt, 1994). All of these factors, along with embryonic ones, necessarily mediate immunosuppression during the early stages of parasitism, contributing to the success of parasitism (Strand and Burke, 2015). Among maternal factors, polydnaviruses (PDVs) are obligate symbionts of endoparasitoid wasps, attacking exclusively larval stages of their lepidopteran hosts (Webb et al., 2000; Webb and Strand, 2005). PDVs have a double-stranded segmented circular DNA, integrated into the mailto:patrizia.falabella@unibas.it 241 genome of the parasitoid. PDVs infect hosts and express their genes in tissues without any replication, inducing alterations in host physiology. These alterations allow parasitoid larvae survival and growth, to finally pupate in silken cocoons (Strand and Burke, 2014). PDVs replicate exclusively in wasp ovaries, where their circular genome is generated from linear DNA copies of wasp chromosomes (Varricchio et al., 1999, Volkoff et al., 2010; Dupuy et al., 2011) and vertically transmitted to the next wasp generation (Strand, 2010). In several host/parasitoid systems the maternal and embryonic factors of parasitic origin can suppress the host immune system. The innate immune system of insects includes several mechanisms that sometimes are arbitrarily divided into humoral and cellular responses, interacting at different levels to provide defence against invading intruders, both pathogens and parasites. The first physical barrier is the tegument, with the cuticle and the epithelium beneath. When an external organism overcomes this barrier and reaches the haemocoel, humoral and cellular defence reactions are specifically and sequentially activated, such as soluble molecules and haemocytes circulating in the haemolymph (Lemaitre and Hoffmann, 2007). Among the humoral responses, melanization is a very fast reaction due to the activation of serine protease cascade, induced by several types of elicitors (microbial surface molecules). This pathway regulates the coagulation or development of melanine both upon injury and on the surface of the foreign invaders. The production of antimicrobial peptides (Boman et al., 1991; Hoffmann et al., 1993; Hultmark, 1993; Cociancich et al., 1994; Lowenberger, 2001) and reactive intermediates of oxygen or nitrogen (Bogdan et al., 2000; Vass and Nappi, 2001) are also part of the humoral response. Cellular responses are mediated by different types of haemocytes and include encapsulation against parasitic eggs and larvae, phagocytosis of bacterial and yeast cells and nodulations against grouped cells of pathogens, as bacteria or yeasts (Schmidt et al., 2001; Lavine and Strand, 2002). A very common effect of parasitism is host immune response suppression. Endoparasitoid wasps belonging to Braconidae and Ichneumonidae families induce significant immunosuppression of parasitised hosts. Host immunosuppression is usually attributed to the wasp’s ability to avoid or suppress encapsulation which is the major host defence against parasite egg invasion (Summers and Dib-Hajj, 1995). Eggs and larvae of endoparasitoids are able to evade host immune defences either passively and/or by active suppression of the host immune system (Schmidt et al., 2001; Lavine and Strand, 2002). Host defence regulation by the parasitoid is mediated by female secretions injected during oviposition (venom, PDV, ovarian calyx fluid) and embryonic (teratocyte) factors that may act alone and/or in synergy. For example, the Microplitis demolitor bracovirus (MdBV) alone inhibits encapsulation and suppresses other host immune defences, including phagocytosis, melanization of haemolymph and inducible expression of other humoral defence molecules (Strand and Pech, 1995 a, b; Beck and Strand, 2005; Thoetkiattikul et al., 2005; Strand et al., 2006). For the endoparasitoid Campoletis sonorensis, Edson et al (1981) showed that purified viable polydnavirus was responsible for suppressing the host’s (Heliothis virescens) ability to encapsulate the wasp eggs. A cysteine rich gene from Campoletis chlorideae polydnavirus is responsible for the disruption of encapsulation and haemocytes cytoskeleton degradation (Zhang and Wang, 2003). Early genes of Cotesia congregata bracovirus (CcBV) cause haemocyte inactivation and apoptosis (Lavine and Beckage, 1995; Le et al., 2003). The venom of the braconid Apanteles glomeratus (Kitano, 1982) and accessory gland secretions produced by Pimpla turionella and the cynipid Leptopilina heterotoma, specifically inhibit host encapsulation (Osman, 1978; Rizki and Rizki, 1984; Parkinson et al., 2001). The venom protein Vn50 of Cotesia rubecula blocks melanization of its host Pieris rapae (Asgari et al., 2003b) and a layer of calyx fluid glycoproteins protects the developing wasp during embryogenesis (Asgari and Schmidt, 1994). Also teratocytes (Kitano, 1969, 1974; Vinson, 1972) or the egg itself (Kitano and Nakatsuji, 1978) can produce soluble substances to suppress host immune system. This review provides an overview of the interaction between the host/parasitoid model system Heliothis virescens/Toxoneuron nigriceps, focusing on the immune system inhibition of H. virescens by T. nigriceps during parasitism. The host/parasitoid system: Heliothis virescens/Toxoneuron nigriceps Toxoneuron nigriceps (Hymenoptera: Braconidae) is a solitary braconid endoparasitoid wasp of the tobacco budworm larval stages Heliothis virescens (Lepidoptera, Noctuidae). During oviposition, T. nigriceps females inject into the host larva, along with the egg, the venom and the ovarian calyx fluid that include the ovarian proteins and a symbiotic polydnavirus (PDV) named T. nigriceps bracovirus (TnBV) (Fig. 1). Parasitism can occur at all host larval stages and parasitised caterpillars can reach the stage of mature larvae but they fail to pupate (Lewis and Vinson, 1968; Pennacchio et al., 1993). As in other endoparasitoid wasps, the integration of parasitic factors of maternal origin (ovarian calyx fluid, PDV and venom) with embryonic origin factors (teratocytes) causes physiological alterations resulting in host developmental arrest, crucial for T. nigriceps larvae that grow and finally pupate in silken cocoons (Lewis and Vinson, 1968; Vinson and Iwantsh, 1980). TnBV T. nigriceps is associated with a polydnavirus (PDV), T. nigriceps bracovirus (TnBV), injected during oviposition together with the egg. The TnBV genome is composed of DNA circles of different 242 Fig. 1 Heliothis virescens (Lepidoptera, Noctuidae) last (fifth) instar larvae, parasitised by Toxoneuron nigriceps (Hymenoptera: Braconidae). During parasitisation (c), T. nigriceps female (b) injects into H. virescens (a - https://commons.wikimedia.org/w/index.php?curid=1215079) the egg, stored in the ovaries (c-1), the ovarian calyx (c-2) fluid, that includes the ovarian proteins and a symbiotic polydnavirus named T. nigriceps bracovirus (TnBV), and the venom, stored in the reservoir (c-3) and released by venom glands (c-4). The alteration of the neuroendocrine and the immune systems of the host prevents its final pupation and promotes the parasitoid larva development. This strategy leaves the host alive since the mature parasitoid larva egression (d). Figures b and d were kindly provided by Dr. Vinson lab. sizes, ranging from 3.4 to 13.3 kb (Fig. 2). The coding sequences include open reading frames (ORFs) encoding putative proteins that for the majority belongs to 4 gene families: ANK (protein with ankyrin repeat motif) (Falabella et al., 2007) PTP (protein tyrosine phosphatase) (Provost et al., 2004; Falabella et al., 2006), UDP (glucose-6- dehydrogenase and sugar transporter) and BEN domain proteins (Falabella et al., unpublished data). In synergy with other parasitic factors, the symbiotic PDV seems to play the major role in inducing the disruption of host physiology. In particular, the injection and active transcription of TnBV genes are responsible for the alteration of the host neuro-endocrine balance, through the inactivation of host prothoracic glands (PGs) (Tanaka and Vinson, 1991; Pennacchio et al., 1997, 1998 a, b). The consequent block of ecdysteroidogenesis in the last instar larvae inhibits pupation. Although the involvement of both phosphoinositide 3-kinase/protein kinase B/target of rapamycin (PI3K/Akt/TOR) and mitogen-activated protein kinase (MAPK) pathways in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in H. virescens PGs has been demonstrated (Scieuzo et al., 2018), to date only the effect of TnBV infection on PI3K/Akt/TOR cellular signalling is known (Falabella et al., unpublished data). The viral genes potentially involved in this process have been identified (Falabella et al., unpublished data). Moreover, through a transcriptomic approach, viral genes differentially expressed in PGs were identified. These genes could be involved in modulating ecdysteroidogenesis pathways. TnBV gene expression in haemocytes of H. virescens parasitised larvae is primarily responsible for inducing immunosuppression. The exact mechanism of host immune response inactivation by TnBV is not completely elucidated (Varricchio et al., 1999; Pennacchio et al., 2001; Falabella et al., 2003; Malva et al., 2004; Provost et al., 2004; Lapointe et al., 2005). Indeed, the in vitro and in vivo investigation of the specific function of TnBV genes expressed in H. virescens immune cells is difficult. Nevertheless, the functional https://commons.wikimedia.org/w/index.php?curid=1215079 243 Fig. 2 Viral genes expressed in haemocytes of Heliothis virescens parasitised by Toxoneuron nigriceps. Gel profile of Toxoneuron nigriceps bracovirus (TnBV) showing a segmented genome, with circular double-stranded DNA molecules of different size ranging from 3.4 to 13.3 kilobases. TnBV genes expressed in haemocytes of parasitised Heliothis virescens larvae are located on different viral circles (Provost et al., 2004; Falabella et al., unpublished data), conventionally named from 1 to 6, in order from the higher to the lower molecular weight annotation of several isolated TnBV genes (Fig. 2) may indicate their possible role in disruption of haemocytes-mediated encapsulation reactions by the host (Asgari et al., 1997; Provost et al., 2004). Among TnBV genes actively expressed in the haemocytes of parasitised larvae, the gene TnBV1 seems to be involved in the host immunosuppression (Lapointe et al., 2005). In order to characterise the possible involvement of TnBV1 in immunosuppression of H. virescens parasitised larvae, this gene was heterologously expressed in various lepidopteran cell lines using different systems and the effects of gene expression on cell morphology and viability were evaluated. TnBV1 induced apoptosis when expressed in Sf21 cells derived from Spodoptera frugiperda and when expressed in High Five cells derived from the lepidopteran Tricoplusia ni (Lapointe et al., 2005). However, when TnBV1-recombinant baculovirus was used to in vivo infect the host haemocytes, the expression of TnBV1 does not exert any apoptotic effect, leaving the role of TnBV1 expression in parasitised host larvae only partly understood. It is possible that during parasitism TnBV1 protein may interact with other viral or host proteins, inducing the apoptotic pathway in synergy with them. On the other hand, all the effects observed in two different cell lines, both derived from Lepidoptera and expressing TnBV1 alone, overlap with general effects observed in haemocytes of parasitised host. Ferrarese et al (2005) demonstrated that following parasitism the total numbers of haemocytes transiently decreased and among them granulocytes showed actin cytoskeleton disruption and loss of adhesion properties. All these evident morphological changes point towards an induction of apoptosis, at least in granulocytes of parasitised host larvae (Lapointe et al., 2005). The same apoptotic effects were observed in a polyclonal Drosophila S2 cell line stably expressing the TnBV viral protein coding by theTnBVank1 gene, and in haemocytes of H. virescens larvae, both after natural parasitism and after TnBVank1 in vivo transient transfection (Salvia et al., 2017). TnBVank1 belongs to the TnBV gene family including ankyrin motif proteins (ANKs) (Falabella et al., 2007). The ANK gene family, together with PTP encoding genes, is the largest and most conserved bracovirus gene family (Bitra et al., 2012). These proteins show a high levels of amino acid identity to 244 the ankyrin motif of the Drosophila transcription factor Nuclear -Factor- kappa-light-chain-enhancer of activated B cells (NF-kB) inhibitor (IκB)-related protein cactus (Meng et al., 1999; Falabella et al., 2007). IkB, a member of the Toll and IMD pathways, is part of the immune defence system of insects, and in presence of some key molecules belonging to bacteria and fungi, activates the transcription factor NF-kB, that regulates the expression of several genes involved in immunity, for example the antimicrobial peptides (AMPs) (Dushay et al., 1996; Lemaitre et al., 1996; Han and Ip, 1999; De Gregorio et al., 2001; Uvell and Engstrom, 2003). Three ankyrin-like open reading frames (ORFs) were identified in the TnBV genome and were named TnBVank1, TnBVank2 and TnBVank3 (Falabella et al., 2007). All the TnBVank genes contain the ankyrin motif but the phosphorylable serine residues at the N-terminus, essential for the IKB protein function, are absent (Falabella et al., 2007). Moreover, all of putative proteins encoded by the TnBVank genes lack the C-terminal PEST domain, characteristic in all actives IκB like proteins and essential for protein degradation (Ghosh et al., 1998, Ghosh and Karin, 2002; Webb and Strand, 2005) and for the protein turnover control (Rogers et al., 1986). On the basis of the predicted structure of TnBVank genes, it was demonstrated that the TnBVank1 protein binds NF-kB/Rel transcription factors of the tumor necrosis factor (TNF)/Toll immune pathway altering the signal transduction cascade (Falabella et al., 2007). Indeed, in parasitised H. virescens larvae, after bacterial challenge, the nuclear import of NF-κB was inhibited (Falabella et al., 2007). Moreover, the expression of TnBVank1 gene in human HeLa cells reduced the efficiency of the TNF-α-induced expression of a reporter gene under NF-κB transcriptional control, strongly suggesting that TnBVank1 protein is involved in the suppression of the insect immune response (Bitra et al., 2012; Falabella et al., 2007; Thoetkiattikul et al., 2005). The same function was demonstrated for ANK family members of Microplitis demolitor bracovirus (MdBV) which, competing with the host IkB for binding NF-kB/Rel, reduce the expression of AMPs (Bitra et al, 2012). The early expression of the TnBVank1 gene (three hours after parasitism) in haemocytes of H. virescens last instar larvae and the presence of transcripts several hours after parasitism, corroborate TnBVank1 involvement during early immune response suppression of parasitised host, also through the activation of a kind of cell death attributable to a process of apoptosis (Falabella et al., 2007). As reported before (Salvia et al., 2017), TnBVANK1 induces cell death in polyclonal Drosophila S2 cells, that stably express TnBVank1 and in haemocytes derived from both H. virescens parasitised larvae or in vivo transfected using TnBVank1. Coimmunoprecipitation and coimmunolocalisation experiments in TnBVank1 polyclonal Drosophila S2 cells showed that TnBVANK1 interacts with ALG-2 interacting protein X (Alix). Alix is a multifunctional protein and it was first characterised as interactor of apoptosis-linked gene protein 2 (ALG-2), that is a Ca2+-binding protein necessary for cell death (Missotten et al., 1999). It was demonstrated that TnBVANK1-Alix interaction is required to induce apoptosis in all cells expressing this gene, both in vitro (S2 cells) and in vivo (H. virescens larval haemocytes). Indeed, upon Alix silencing by RNA interference (RNAi), TnBVANK1 was no longer able to induce apoptosis (Salvia et al., 2017). Among the gene families present in bracoviruses of different braconid wasp subfamilies, the PTP gene family is probably an ancient component of the ancestral bracovirus of both Microgastrinae and Cardiochilinae subfamilies. PTPs are enzymes that catalyse the phosphorylation of specific protein target at tyrosine residues. Together with protein tyrosine kinase, PTPs regulate the phosphorylation level of cellular proteins (Neel and Tonks, 1997). The functional characterisation of bracovirus PTPs expressed in their hosts may mean that most of them have a role in the success of parasitism. Although in some cases PTP genes do not codify for functionality active enzymes, they could contribute to host/parasitoid interactions performing different functions, for example binding phosphorylated proteins and inhibiting their activities (Provost et al., 2004). Bracovirus PTPs are numerous and characterised by a high degree of diversity, regarding also the target substrate, at least for PTPs characterised until now. This heterogeneousness indicates that they may act altering several cell pathways in parasitised host, especially those regulating the host immunity and endocrine balance (Provost et al., 2004). In particular, regarding parasitised host immune suppression, it is possible to hypothesise that bracovirus PTPs could alter the haemocyte cytoskeleton, strongly reducing their ability to encapsulate the intruders and losing their phagocytic ability. In this last case we could compare the activity of bracovirus PTPs with the ability of some pathogens, bacteria in particular, in using PTPs to alter the control of actin dynamics in infected host cells, resulting in inhibition of phagocytosis (Bleves and Cornelis, 2000; De Vinney et al., 2000; Ernst, 2000; Cornelis, 2002; Gruenheid and Finlay, 2003; Singh et al., 2003; Mustelin et al., 2005). Among bracoviruses, the MdBV suppresses Spodoptera frugiperda immune system inactivating phagocytosis, encapsulation and melanization reaction of haemolimph (Strand and Pech, 1995a,b; Beck and Strand, 2005; Thoetkiattikul et al., 2005; Strand et al., 2006; Suderman et al., 2008). The putative proteins encoded by MdBV include 13 members of PTP gene family, but only 5 PTPs show a potentially active catalytic domain, while the other 8 members of MdBV PTPs seem to encode functionally inactive enzymes, since their sequence analysis showed modification not compatible with tyrosine phosphatase activity (Pruijssers and Strand, 2007). Among the potentially active MdBV PTPs, PTP-H2 and PTP-H3 are preferentially expressed in host haemocytes and localise to focal adhesions, also 245 when they are expressed in haemocyte-like cells, such as Trichoplusia ni High Five and Drosophila S2 cell lines. In these cases, they strongly contribute to disruption of adhesion and inhibition of phagocytosis, complementing the activity of glc1.8, another MdBV gene with antiadhesive and antiphagocytic properties (Suderman et al., 2008). Moreover, MdBV PTP-H2 also induces apoptosis of Sf21 cells. The transient expression of PTP-H2 in this cell line induced the depolarisation of mitochondrial membrane and caspase-dependent apoptosis (Suderman et al., 2008). On the basis of these results Suderman and colleagues (2008) hypothesised that PTP-H2 contributes to immunosuppression of hosts defences also with this activity. The TnBV genome includes 13 genes encoding putative classical PTPs (Falabella et al., 2006). All identified PTP genes have a specific domain including 10 conserved motifs found in vertebrates (Andersen et al., 2001). No relation was found with dual-specificity PTPs present in baculovirus or poxvirus (Provost et al., 2004), challenging the hypothesis of a baculovirus as possible progenitor of BVs (Drezen et al., 2003; Federici and Bigot, 2003). Sequence analysis of the 13 TnBV identified PTPs showed that 8 PTP genes have a complete and potentially active protein tyrosine phosphatase domain. The other 5 PTPs encode incomplete putative proteins or they are interrupted by a stop codon or by a frameshift (Provost et al., 2004). Among the TnBV gene families, PTP is the largest one. Its members lack introns, similarly to the Cotesia congregata bracovirus (CcBV) PTP family (Provost et al., 2004). Using a multiple approach 4 PTPs were identified as expressed in haemocytes of H. virescens parasitised larvae (Falabella et al., 2006; Falabella et al., unpublished data) (Fig. 2). All PTP cDNAs from TnBV encode putative classical PTPs with cytosolic localisation. The expression of these PTPs in haemocytes of H. virescens parasitised larvae suggests their contribution towards host immune system suppression induced by TnBV. Although no experimental data support this conclusion, we propose TnBV PTPs as responsible for the alteration of cytoskeleton dynamics during the cellular immune response. In particular, this event could occur during encapsulation and phagocytosis, similar to the mechanism adopted by some pathogens that, expressing their PTPs as virulence factors, alter the host cell signal transduction pathway, modifying the actin rearrangements thus inducing inhibition of phagocytosis (Bleves and Cornelis, 2000; De Vinney et al., 2000; Ernst, 2000; Pruijssers and Strand, 2007) Although similar data were found in case of PTPs belonging to other bracovirus, where PTPs are actually involved in alteration of host immunity, this remains to be verified experimentally. Venom The venom of endoparasitod wasps is a mixture of compounds, mainly proteins, produced by venom glands, organs located in the female reproductive system in Hymenoptera (Fig. 1) (Billen, 1987; Dorémus et al., 2013). Venom proteins start to be synthesised during the wasp pupal stages in the venom glands, connected to a reservoir where the venom is collected and stored (Jones and Wozniak, 1991) (Fig. 1). The reservoir is directly attached to the terminal part of the oviduct, where the venom is mixed with the ovarian calyx fluid produced in the swollen base of the ovary (Fig. 1). This fluid is composed of a protein mixture (ovarian proteins) and in case of endoparasitoids of lepidopteran larval stages, of polydnavirus (PDV) particles (Webb, 1998). Venom is one of the parasitic factor of maternal origin, injected together with the egg and other factors (i.e. ovarian protein and/or PDV) into the host body during parasitism (Moreau and Asgari, 2015). In synergy with all the parasitic factors, venom acts to assure an adequate environment, allowing the growth of progeny (Beckage and Gelman, 2004; Moreau and Asgari, 2015). In endoparasitoids associated with PDVs, venom regulates host physiology and no paralytic or lethal effects are observed, in contrast to the effects of venom injected into hosts by ectoparasitoids or by endoparasitoid lacking PDVs (Wharton, 1993; Quistad et al., 1994; Parkinson and Weaver, 1999; Moreau et al., 2002; Nakamatsu and Tanaka, 2003; Webb and Strand, 2005; Asgari, 2006). In many host/parasitoid systems, venom complements the activity of PDVs (Kitano, 1986; Tanaka, 1987) and in several instances some venom components provide protection for the eggs against the host immune system during the period between oviposition and expression of PDV genes (Webb and Luckhart, 1994). In endoparasitoids, venom can induce developmental alterations, host castration (Digilio et al., 1998, 2000) and almost always plays an immunosuppression role in the host (Webb and Luckhart, 1994; Luckhart and Webb, 1996; Asgari et al., 1998). The venom of T. nigriceps plays a fundamental role for the success of parasitism, indeed it was demonstrated that, when venom glands were removed from female wasps, no parasitoids emerged from host larvae, H. virescens (Tanaka and Vinson, 1991). On the other hand, in vitro experiments on H. virescens prothoracic glands (PGs) demonstrated that the T. nigriceps bracovirus (TnBV) alone was able to induce the biosynthetic inactivation of PGs (Pennacchio et al., 1998b). Regarding parasitoid wasp venom composition, it has thus far been analysed in at least 20 species belonging to different families, using different approaches (Vincent et al. 2010; Asgari and Rivers, 2011; Doremus et al. 2013; Colinet et al., 2013a,b; 2014; Shaina et al., 2016; Yan et al., 2016; Cusumano et al., 2018). The most recent studies combine transcriptomics of venom glands and proteomics of venom to identify the proteins inject by wasps into the hosts during parasitism (Colinet et al. 2013b). Among the different proteins identified in parasitoid venoms, many share significant homology with proteins which functions are known. 246 Simultaneously, no experimental data exist for the vast majority of parasitoid venom proteins, so their specific role in parasitism generally remains unknown. Indeed, most of the available data focus on proteins that are highly abundant or structurally related to genes in databases with predicted functions, but, when venom proteins are studied in a non-model organism, the interpretation of the results is very difficult (Poirié et al., 2014). Some of these factors, including serine proteases, metalloproteases or esterases (Asgari and Rivers, 2011) are shared by many wasp species, while others are known from only one or a small number of species. Some enzymes are also present in hosts at very low concentrations, indicating that focusing exclusively on abundant venom components can potentially lead to overlooking factors that could have important roles in parasitism. The mechanism of entry into the host cells and the mode of action of parasitoid venom proteins are unknown, except in a few species whose venoms contain some virus-like particles (VLPs). The best known VLPs belong to Leptopilina boulardi. They are produced in the venom gland and injected into host larvae, Drosophila melanogaster, contributing to host immunosuppression. Following injection into host larvae, VLPs target haemocytes, leading to apoptosis and/or morphological changes (Poirié et al. 2014). The main venom protein components of T. nigriceps have been identified through the combination of next-generation transcriptome sequencing and bottom-up proteomics (Laurino et al., 2016). This integrated proteomic and transcriptomic approach allows to identify proteins and biological processes also in non-model organisms (Escoubas et al., 2006; Tang et al., 2010; Safavi-Hemami et al., 2014; Labella et al., 2015). Beside proteins similar to known venom components or annotated proteins (Asgari and Rivers, 2011; Doremus et al., 2013; Moreau and Asgari, 2015), novel proteins, with no similarity in databases, were identified in T. nigriceps venom. Although no functional data are available, we hypothesise on possible involvement of T. nigriceps venom components in alteration of host physiology and in particular in host immune system suppression. A total of 31 different proteins were identified in T. nigriceps venom. Some of these proteins resulted similar to already described proteins in venom from other parasitoids, while others resulted identified for the first time in a venom. Among the known proteins, hydrolases, followed by transferases, oxidoreductases, ligases, lyases and isomerases, was the major family similarly to that reported for venom of other parasitoids (Asgari and Rivers, 2011). It was speculated that some of the identified proteins could be involved in the host immune suppression directly, in synergy to each other and/or to the other parasitic factors (Table 1). Based on the MEROPS database (http://merops.sanger.ac.uk), the T. nigriceps venom metalloproteases belong to the M12 (M12B subfamily) and M13 families (Laurino et al., 2016). These enzymes could be involved in several biological and disease-related processes (Van Goor et al., 2009). Regarding immunosuppression, there seems to be a parallel between the function of different subfamilies of microbial metalloproteases and the venom metalloproteases. Indeed, entomopathogenic microorganisms use metalloproteases in order to suppress the insect cellular defence, inducing the degradation of host protection molecules (Griesch and Vilcinskas, 1998; Liehl et al., 2006). This observation may indicate that T. nigriceps venom metalloproteases might have similar functions in host immune suppression, in addition to their possible activity in host protein and tissue degradation. Besides, Serine protease homologue (SPH) and Calreticulin were also found in T. nigriceps venom (Laurino et al., 2016). These well-studied proteins are distributed in a wide range of organisms including insects, and it has been reported that they inhibit host cell encapsulation and act as immune suppressor factors in insect haemolymph (Choi et al., 2002; Asgari et al., 2003a; Asgari and Schmidt, 2003; Zhang et al., 2004; Kanost and Jiang, 2015). In particular, SPHs have a serine protease catalytic domain but lack one of the amino acid of the catalytic triad, essential for the proteolytic activity (Hedstrom, 2002). SPHs are frequently present as components of parasitoid venoms (Hedstrom, 2002; De Graaf et al., 2010) where they block the phenoloxidase (PO) cascade at different levels (Zhang et al., 2004), inducing the inhibition of melanization. Possibly the serine protease homologue identified in T. nigriceps venom could play a similar crucial role in suppression of host humoral defences. The identification of a calreticulin-like protein in the venom of T. nigriceps is notable. Indeed calreticulins, molecular chaperones acting as lectin and Ca2+-binding proteins, have been previously identified as a component of several venoms of other parasitoids (De Graaf et al., 2010; Zhu et al., 2010; Asgari et al., 2003b). In particular, a calreticulin in the venom of Cotesia rubecula blocked Pieris rapae haemocyte diffusion, inhibiting the encapsulation response (Zhang et al. 2006). The calreticulin present in T. nigriceps venom could bind the intracellular calcium modifying its balance and altering all the cellular pathways in which Ca2+ is involved, such as apoptosis, inflammation and activation of hydrolytic enzymes. In addition, a Ci-48 like protein was identified in T. nigriceps venom (Laurino et al., 2016), similar to the venom protein Ci-48 previously identified in Chelonus inanitus (Vincent et al., 2010) and in Microplitis demolitor venom glands (Burke and Strand, 2014). It was speculated that, in all these cases, the protein could have functions in the early phases upon parasitism that generally involve the inhibition of the host's immune response. Regarding the function of Elongation factor 1- alpha (EF1-alpha) like protein, recovered in T. nigriceps venom (Laurino et al., 2016), although its putative function in host/parasitoid interaction is still unknown, a similar protein was found in the venom http://merops.sanger.ac.uk/ 247 Table 1 Proteins identified in the Toxoneuron nigriceps protein database venom putatively involved in the alteration of immune system of the host Heliothis virescens (from Laurino et al., 2016) T. nigriceps protein involved in immunity suppression Contig Corresponding Acc. N. NCBI protein indicated in annotations obtained by T. nigriceps custom- made database Corresponding Acc. N. protein obtained by BlastP search Membrane metallo-endopeptidaselike1-like 4489 gij340723203jrefjXP_003399984.1j E2AFA5 Venom metalloproteinase 3-like 7042 gij665805846jrefjXP_008551135.1j F4X7T0 Serine protease homolog 90 isoform x1 18408 gij315131321jembjCBM69269.1j A0A034V0K7 Calreticulin 5341 gij665788653jrefjXP_008559929.1j Q8IS63 Venom protein ci-48° 18596 gij665819695jrefjXP_008558721.1j E6ZCK2 Elongation factor 1-alpha 4154 gij665799004jrefjXP_008547400.1j K7IVS1 Spermine oxidase-like 14112 gij665801233jrefjXP_008548621.1j V9IIS9 Ovalbumin-related protein x-like 4452 - Q8IS84 of parasitoid Leptopilina heterotoma (Colinet et al., 2013b). Moreover, it was also found as a secreted product in Leishmania protozoan parasites, where it seems to be involved in the induction of host macrophage deactivation (Nandan et al., 2002). Among other T. nigriceps venom components possibly involved in host immune suppression, the Spermine oxidase-like (SMO) is a FAD-dependent enzyme able to induce the production of reactive oxygen species. It plays several roles in numerous cell functions, and some of them are compatible with an alteration of immune responses (Cervelli et al., 2012). Although its biological role in venom has not been elucidated yet, it is tempting to speculate a similar mechanism regarding the host immune system. The Ovalbumin-related protein x-like is a member of Ovalbumin family and belongs to the ovalbumin serine protease inhibitor family (ov- serpin). The serpin are well known proteins in insects because of their role in inhibiting the activation of the PO cascade and thus the melanization production (Kanost and Gorman, 2008). This function could also be performed in T. nigriceps venom by this protein (Laurino et al., 2016). Among the 31 identified proteins of T. nigriceps venom, the 8 annotated proteins mentioned above (Table 1) likely play a key role in host immune system suppression. This is in agreement both with the function of several other endoparasitoid venoms and with previous researches reported in case of T. nigriceps venom by Tanaka and Vinson (1991). Even this finding supports possible role of T. nigriceps venom in the regulation of host immune system, to confirm what has been speculated so far, functional characterisation of venom components is required. Teratocytes Teratocytes are specialised cells deriving from the dissociation of the extraembryonic membrane, named serosa, during parasitoid egg hatching in the host haemolymph (Dahlman, 1990, 1991; Dahlman and Vinson, 1993; de Buron and Beckage, 1997; Ali et al., 2013). Teratocytes are produced only by endoparasitoid wasps in a restricted number of species belonging to Braconidae, Scelionidae, Mymaridae, Trichogrammatidae, Aphelinidae and Platygastridae families (Strand et al., 1986; Dahlman and Vinson, 1993; Quicke, 1997; Hotta et al., 2001; Basio and Kim, 2005). All known teratocytes, once in the hemolymph, rapidly increase in size without undergoing any cell division and thus becoming highly polyploid. Probably this characteristic optimises their secretory function, in order to provide nutrients to the parasitoid progeny larvae, directly or indirectly (Strand and Wong, 1991). Teratocytes, indeed, release several molecules impacting physiology, development and nutritional suitability of the host. They are involved in several different functions, such as the manipulation of host development, through the inhibition of protein synthesis, or the disruption of the endocrine balance, which often modulates host biochemical changes that are nutritionally relevant for the developing parasitoid larvae. The nutritional role of the teratocytes is more direct and evident in host/parasitoid associations, where these cells perform an extra-oral digestion of selected host tissues, in order to allow the release of nutrients in a suitable form for the developing sister larvae (Falabella et al., 2000; Nakamatsu et al., 2002; Falabella et al., 2005; Grimaldi et al., 2006; Falabella et al., 2009; Caccia et al., 2012; Grossi et al., 2016). 248 In addition, in several host/parasitoid systems, they contribute to host immunosuppression and/or in host immune system modulation for the success of parasitism (Nakamatsu et al., 2002; Rana et al., 2002; Burke and Strand, 2014; Ali et al., 2015). In the Cotesia kariyai and Cotesia glomerata, teratocytes (taken at 4 days post parasitism) seem to play a role in preventing encapsulation during the early stages of parasitism by interfering with the phenoloxidase (PO) cascade. Nevertheless, in C. kariyai it is reported that they act in synergy with ovarian calyx fluid and venom: only when all 3 parasitoid factors were injected into unparasitised hosts along with first-instar parasitoid larvae, the encapsulation was inhibited (Kitano et al., 1990; Tanaka and Wago, 1990). Few works have addressed to the role of T. nigriceps teratocytes (Dahlman and Vinson 1993; Pennacchio et al., 1992, 1994a,b; Consoli et al., 2005, 2007; Rossi et al., 2012). Their functions may be nutritive, immunosuppressive, or secretory and may be involved in regulating host development (Dahlman and Vinson, 1993). It has been reported that they provide an important contribution to prevent H. virescens pupation. Indeed, as previously reported in parasitised H. virescens last instar larvae, the inactivation of the biosynthetic activity of PGs, induced by Toxonigriceps nigriceps Bracovirus (TnBV) infection, results in an evident reduction in ecdysone titre in haemolymph (Tanaka and Vinson, 1991; Pennacchio et al., 1998 a,b). The alteration of host ecdysteroid metabolism is mediated, at least in part, also by T. nigriceps teratocytes (Pennacchio et al., 1994b). Indeed, when teratocytes of T. nigriceps were injected into unparasitised H. virescens 1 day old last instar larvae, they inhibited host pupation (Pennacchio et al., 1992). Results suggested that teratocytes acted by affecting the haemolymph ecdysteroid titre, through inactivation of the 20- hydroxyecdysone, which is converted into polar inactive metabolites (Pennacchio et al., 1994b; Bloch et al., 2002). To date, only 1 protein has been identified in T. nigriceps teratocytes: it is an isolated and functionally characterised putative chitinase (Consoli et al., 2005, 2007; Rossi et al., 2012). Chitinases are well known enzymes that catalyse the hydrolysis of chitin, but they can also be produced as a defence mechanism, as it has been shown by the expression of chitinases in transgenic plants challenged with micro-organisms, nematodes, and insects (Broglie and Broglie, 1993; Lin et al., 1995). It has been hypothesised that T. nigriceps chitinase, released by teratocytes, may either play a putative protective role preventing any possible microbial infection in the host haemocoel, since host-immune defences are suppressed. Moreover, it could promote host cuticle digestion, allowing the parasitoid larva to emerge from the host body, since the parasitoid larva does not possess powerful mandibles at egression (Lewis and Vinson, 1968). Recently, we established a de novo transcriptome of T. nigriceps teratocytes (Falabella et al., unpublished data). The functional annotation and analysis of contigs allowed us to identify several transcripts coding for putative proteins possible involved in the success of parasitism, through the modulation of host immunity. In particular, similarly to the transcriptomic analysis of Microplitis demolitor teratocytes, where the expression of several antimicrobial peptides (AMPs) was found (Burke and Strand, 2014), we identified AMPs, belonging to defensins, attacins and lysozyme families, putatively expressed by T. nigriceps teratocytes and potentially released in the host haemolymph (Falabella et al., unpublished data). Teratocytes might prevent host infection by pathogen intruders supporting the immunosuppressed host by the production of AMPs. Moreover, the same analysis allowed us to identify putative proteins belonging to ovalbumin- related X-like, serpin and Rho GAP families (Falabella et al., unpublished data). Transcriptome analysis of Cotesia plutellae teratocytes reported that these cells express several putative immunosuppressive factors, including several serpins and RhoGAPs proteins, involved in the inhibition of the prophenoloxidase (PPO) cascade system, inducing host immunosuppression (Ali and Kim, 2015; Kanost, 1999). Taken together, this information may indicate that also T. nigriceps teratocytes may have a role in host immunosuppression. However, the specific functions remain to be elucidated requiring further studies. Concluding remarks Although insects have only an innate immune system, they have evolved several complex strategies to defend themselves against pathogens. On the other hand, hymenopteran parasitoids, especially endoparasitoid, have coevolved an arsenal of factors that allow the physiology of the host to be synergistically regulated, in order to ensure the complete development of their progeny. These parasitic factors induce diverse and complex reactions against the host defence mechanisms. To date, these intriguing processes are still poorly understood. This review reports an overall picture describing the strategy used by T. nigriceps in inhibiting the immune system of its host, the lepidopteran H. virescens (Fig. 3). The findings reported in previous works and some preliminary results showed here, highlight the connection and the fine regulation performed by all parasitic factors in host immunity inactivation. Almost all the identified T. nigriceps bracovirus (TnBV) genes are expressed in host haemocytes and, as consequence, can be considered putatively involved in the host immunity alteration (Fig. 2). To date the genome of TnBV is being sequenced and this could lead to the identification of new genes. Once TnBV genome will be functional annotated, we will obtain an overview on the possible roles of putative viral proteins expressed in host tissues will be available. However a functional characterization of all the viral genes will be required. The role of each viral gene will be studied by in vivo transfection in H. virescens haemocytes as previously reported for TnBVANK1 (Salvia et al., 2017). 249 Fig. 3 Overview of Toxoneuron nigriceps parasitic factors and their effect on the host immune system. Overall scheme of T. nigriceps parasitic factors of maternal and embryonic origin and their role in Heliothis virescens immunosuppression. Moreover, 8 out of 31 venom proteins are putatively responsible for host immune suppression (Table 1). Further analysis of these proteins are required to understand their function and their contribution to the success of parasitism and more specifically to immune suppression. Possible strategies could be the injection of each purified venom protein into the host larvae (Digilio et al., 1998, 2000) and/or with a more advanced approach, the silencing of their expression into T. nigriceps venom gland (Lynch and Desplan, 2006; Colinet et al., 2014). At last, also the teratocytes, a parasitic factor of embryonic origin, release molecules clearly involved in the control of the immune response of the parasitised host. The functional annotation of the de novo transcriptome may indicate the involvement of antimicrobial peptides released by teratocytes in the regulation of host immunity. Neverthless only the functional characterization of each of them, produced by chemical synthesis or recombinantly, according to their size, will provide most of the information about their role in the host immune system regulation. What emerges from the information reported in this review is that the regulation of the host immune system by parasitoids is not limited to its inhibition, but also to keep the host alive and in good condition, since it represents the environment and the nutritional resource for the adequate and complete development of the parasitoid offspring. Concluding, T. nigriceps female secretions injected at the oviposition, in synergism with embryonic factors (Fig. 1), induce diverse and partially overlapping mechanisms that inhibit cellular and humoral responses, solving the first problem that parasitoids have to face up: the immune reaction against its egg and the following larval stages (Schmidt et al., 2001; Lavine and Strand, 2002). Acknowledgements This review is dedicated to Professor Enzo Ottaviani, that strongly promoted and encouraged the writing of this work. I would like to thank Dr. Rosanna Salvia, Dr. Carmen Scieuzo, Dr. Andrea Scala and Dr. Antonio Moretta for critically reading the manuscript, Dr. Feng Zhu for the original drawing in Fig. 1c and Dr. Santoro Salvatore for the graphical restitution of this picture after vectorization and Dr. Asha Rao for kindly providing pictures in Fig. 1b and 1d. References Ali MR, Lim J, Kim Y. Transcriptome of a specialized extra-embryonic cell, teratocyte, and its host immunosuppressive role revealed by ex vivo RNA interference. Insect. Mol. Biol. 24: 13-28, 2015. Ali MR, Seo J, Lee D, Kim Y. Teratocyte-secreting proteins of an endoparasitoid wasp, Cotesia plutellae, prevent host metamorphosis by altering endocrine signals. Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 166: 251-62, 2013. Andersen JN, Mortensen OH, Peters GH, Drake PG, Iversen LF, Olsen OH et al. 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