The immunological interaction between Drosophila melanogaster and its larval parasitoids has been thoroughly investigated, however, little is known about the interaction between the host and its pupal parasitoids. Pachycrepoideus vindemmiae, a pupal ectoparasitoid of D. melanogaster, injects venom into its host while laying eggs on the puparium, which regulates host immunity and interrupts host development. To resist the invasion of parasitic wasps, various immune defense strategies have been developed in their hosts as a consequence of co-evolution. In this study, we mainly focused on the host immunomodulation by P. vindemmiae and thoroughly investigated cellular and humoral immune response, including cell adherence, cell viability, hemolymph melanization and the Toll, Imd, and JAK/STAT immune pathways. Our results indicated that venom had a significant inhibitory effect on lamellocyte adherence and induced plasmatocyte cell death. Venom injection and in vitro incubation strongly inhibited hemolymph melanization. More in-depth investigation revealed that the Toll and Imd immune pathways were immediately activated upon parasitization, followed by the JAK/STAT pathway, which was activated within the first 24 h post-parasitism. These regulatory effects were further validated by qPCR. Our present study manifested that P. vindemmiae regulated the cellular and humoral immune system of host D. melanogaster in many aspects. These findings lay the groundwork for studying the immunological interaction between D. melanogaster and its pupal parasitoid.

Background: Pteromalus puparum (Hymenoptera: Pteromalidae) is an endoparasitoid wasp that parasitizes many butterfly species, including a Brassicaceae pest, Pieris rapae (Lepidoptera: Pieridae), the small white cabbage butterfly. P. puparum females inject venom along with their eggs into hosts to ensure successful parasitism. The venom regulates host development and behavior, suppresses host immunity, and influences host metabolism. It has been shown that the venom contains ⊍-amylases, a group of hydrolytic enzymes that act in insect sugar metabolism. So far, three ⊍-amylases have been identified in P. puparum (Pteromalus puparum ⊍-amylases, PpAmys) and the function of PpAmy1 has been reported. However, the functions of PpAmy2 and PpAmy3 remain unknown.Results: We studied the functions of an ⊍-amylase highly expressed in muscle-rich tissues (PpAmy2) and an ⊍-amylase highly expressed in venom apparatus (PpAmy3) using RNAi and GC-TOF-MS techniques. Knockdown of PpAmy3 by RNAi reduced the body length and weight of 1-day old larval offspring while there was no significant effect when PpAmy2 was knocked down. Compared to the control injected with siGFP, many metabolites in P. puparum changed when PpAmy2 was knocked down, while the injection of PpAmy3 recombinant protein into host induced metabolite changes in the P. rapae hemolymph.Conclusion: Our study demonstrated that PpAmy2 acts in metabolism in the muscles of the parasitoid while PpAmy3 could influence the host metabolism and may support the development of parasitic wasp offspring.

BACKGROUND Midgut and salivary gland α‐amylases are digestive enzymes required for the development of insects and have been investigated in some insect species. However, α‐amylases in the endoparasitioid wasps have not been reported. Pteromalus puparum (Hymenoptera: Pteromalidae) is a dominant endoparasitioid wasp that parasitizes many butterfly species, including the Brassicaceae pest Pieris rapae (Lepidoptera: Pieridae). Here, we studied the characteristics and functions of three α‐amylases in P. puparum. RESULTS We cloned three genes encoding α‐amylases in P. puparum, PpAmy1, PpAmy2 and PpAmy3. The full length of the PpAmy1 cDNA is 1872 bp, encoding 496 amino acids, the PpAmy2 cDNA is 1863 bp long, encoding 518 amino acids, and PpAmy3 cDNA consists of 1802 bp encoding 521 amino acids. PpAmys are highly similar in amino acid sequences, but they have separate tissue distributions. Phylogenetic results show that gene duplications may occur between PpAmy2 and PpAmy3. PpAmy1 and PpAmy3 are most highly expressed in the digestive tract and the venom apparatus, respectively, while PpAmy2 is broadly expressed in all tissues. We report that PpAmy1 acts in the digestive tract, where it influences lifespan as demonstrated using RNAi and α‐amylase rescue analyses, and there is no significant difference in longevity when PpAmy2 and PpAmy3 are knocked down. CONCLUSION PpAmys probably have roles in carbohydrate metabolism of P. puparum and its host/parasitoid relationships. The characterization and functional study of PpAmys lays the foundation for the protection and utilization of parasitoid resources, and the biological control of agricultural pests. © 2019 Society of Chemical Industry