Table 3 Psyttalia venom proteins with a putative function: Biochemical function, occurrence in venom of parasitoids and previously demonstrated or proposed role in parasitism.

Annexin

Annexins are a family of Ca2⁺-dependent lipid binding proteins believed to be engaged in membrane transport processes, although recent work suggests a more complex set of functions. Annexins normally lack signal sequences for secretion, but some members of the family have been identified extracellularly where they can act as receptors⁴⁴. Annexins had never been described so far in the venom of parasitoids. However, some data suggest that different mammalian parasite clades possess annexins with unique properties that can be secreted and are likely involved in host-parasite interactions and host immune-modulation⁴⁵. Moreover, some parasitic nematodes secrete an annexin-like effector into host root cells that may mimic plant annexin function during the parasitic interaction⁴⁶. At last, it has been shown that annexins are also involved in the binding and internalization of toxins in eukaryotic cells⁴⁷.

Arginine kinase

Arginine kinase plays a crucial role in the energy metabolism of insects and other invertebrates through the use of ATP to catalyze the phosphorylation of arginine in phosphoarginine. This enzyme was detected in the venom of Pteromalus puparum⁴⁸ and Leptomastix dactylopii⁴⁹, but its role in the host-parasitoid interaction is unknown.

Calreticulin

Calreticulin is a calcium (Ca2⁺)-binding protein with multifunctional properties including chaperone functions⁵⁰. Calreticulin was shown to inhibit host cell encapsulation in Cotesia rubecula⁵¹ and P. puparum⁵², although the mechanism is still unclear. Calreticulin was found in the venom of several phylogenetically distant species⁴ and seems thus largely shared among parasitoids.

Endoplasmin

Endoplasmin (alternative names: HSP90B1, GP96, GRP-94), which belongs to the heat shock protein 90 family, is a molecular chaperone located in the ER and involved in the final processing and export of secreted proteins⁵³. Among parasitoids, endoplasmin has only been detected so far in the venom gland of Aphidius ervi¹³. This venom protein was suggested to play a role in the secretion, stabilization, transport and host cell targeting of the different A. ervi venom proteins.

Enolase

Enolase is a key enzyme in cell metabolism which is also associated with virulence of several pathogens⁵⁴. An extracellular enolase was recently described in the oviposition injecta from A. ervi⁵⁵ and the venom of Toxoneuron nigriceps⁵⁶. Enolase is also released by teratocytes surrounding the A. ervi embryo⁵⁷. This enzyme was proposed to play an important role in the regulation of the host physiology and the host nutritional exploitation^57,58.

Esterase/lipase-like

Esterases and lipases belong to a superfamily of hydrolytic enzymes that act on carboxylic esters⁵⁹. Proteins belonging to this functional class were previously found in the venom of several phylogenetically distant species^3,4 and appear to be common in parasitoids. The functions of these hydrolase enzymes in host-parasitoid interactions have not been investigated yet.

GH1 β-glucosidases

GH1 β-glucosidases are a family of enzymes found from bacteria to mammals that hydrolyze glycosidic bonds from glycosides and oligosaccharides, and remove non reducing terminal glucosyl residues⁶⁰. Among parasitoids, a β-glucosidase enzymatic activity was detected in the venom of Pimpla hypochondriaca⁶¹. A member of this enzyme family was also recently identified, but not abundant, in the venom of Microplitis demolitor¹² and, in a low quantity, in a transcriptomic study of the A. ervi venom apparatus¹³. GH1 β-glucosidases include myrosinases that play a central role in the glucosinolate-myrosinase system, one of the best-studied activated plant defense system³³. Some insects have co-opted this system to defend themselves against enemies, by sequestering plant-derived glucosinolates and producing their own myrosinase-like enzyme^36,37,38.

Heat shock protein 70

Heat shock proteins 70 (Hsp70; alternative name: GRP-78) are a family of chaperones with distinct sub-cellular localization and function⁶². An Hsp70 protein was identified in the venom of the parasitoid P. puparum⁴⁸ whose function remains to be elucidated.

Leucine-rich repeat protein

Leucine-rich repeats (LRRs) are motifs involved in protein-protein interactions⁶³. LRRs are generally composed of 20-29 amino acid stretches rich in leucine. To our knowledge, LRR domain-containing proteins were only identified in the venom of A. ervi until now¹³. They were suggested to act as scavengers for the pea aphid Toll-like receptors, thus impairing the host immune response via the Toll pathway.

Neprilysin-like metalloprotease

Neprilysin-like (NEP) proteins are zinc-dependent metalloproteases belonging to the M13 peptidase family. They are involved in the degradation of a number of regulatory peptides in the nervous or immune system of mammals⁶⁴ and insects⁶⁵. Although they are typically membrane-bound, ectopeptidases such as NEP may also be shed from the membrane through a proteolytic process and found in the surrounding fluid⁶⁶. NEP-like proteins were detected in the venom of the Braconidae A. ervi¹³, Microctonus hyperodae²⁰, M. demolitor¹² and T. nigriceps⁵⁶, as well as of the Figitidae Leptopilina boulardi⁷. They were also found associated with the VLPs produced in the ovary of Venturia canescens⁶⁷. NEP-like proteins have been hypothesized to modulate the host immune system by degrading immune-specific peptides⁶⁷.

Phospholipase A2

Secreted phospholipases A2 (PLA2s) are a family of relatively stable enzymes found in venoms. PLA2 has in vitro and in vivo immunomodulatory effects in bee venom⁶⁸, and neurotoxic and myotoxic effects in snake venoms⁶⁹. This enzyme was recently detected in the venom of M. demolitor¹² and T. nigriceps⁵⁶, but its function in the host-parasitoid interaction is unknown.

Protein disulfide isomerase

Protein disulfide isomerases (PDIs) are enzymes involved in the folding and stabilizing of nascent polypeptides in the endoplasmic reticulum (ER) through catalysis of disulfide bond formation and isomerization⁷⁰. Although this protein is normally recycled back to the ER from the Golgi via its C-terminal KDEL motif, secreted PDIs can escape this turnover mechanism⁷¹. Among parasitoids, PDIs have only been detected so far in the venom gland of A. ervi¹³. They have a broad substrate specificity and are involved in the folding of toxin peptides in different venomous organisms^72,73.

Reprolysin-like metalloprotease

Reprolysin-like (REP) proteins are zinc-dependent metalloproteases belonging to the M12 peptidase family, commonly found as constituents of snake venom. They were previously detected in the venom of the parasitoids Pimpla hypochondriaca²², Eulophus pennicornis²³, Chelonus inanitus¹¹, M. demolitor¹² and T. nigriceps⁵⁶. A recombinant E. pennicornis venom REP-like protein was demonstrated to display toxicity toward the host and to manipulate host development²³.

Serine carboxypeptidase

Classical serine carboxypeptidases are enzymes that hydrolyze a peptide bond at the C-terminal end of peptides and proteins. A related enzyme (Scpep1) that do not show proteolytic activity but is involved in other functions was described in mice⁷⁴. To our knowledge, serine carboxypeptidase has only been identified so far in the venom of M. demolitor¹² and T. nigriceps⁵⁶. Interestingly, serine carboxypeptidases have also been described as candidate virulence factors in several pathogens⁷⁵.

Serpin

Serpins (serine protease inhibitors) are a large family of functionally diverse protease inhibitors. They share a conserved structural architecture with an exposed reactive center loop (RCL) of about 20 amino acids, which acts as bait for target serine proteases⁷⁶. The L. boulardi venom serpin LbSPNy was previously shown to be involved in host immune suppression⁷⁷: it interferes with melanization in the Drosophila host through inhibition of the phenol oxidase activation. More recently, serpins were described in the venom of A. ervi¹³ and M. demolitor¹² but their role in parasitism success is unknown.