| Japanese beetle, Popillia japonica, is a natural host and major biological control target of the entomopathogenic nematodes Steinernema glaseri and Heterorhabditis bacteriophora. However, field trials and laboratory assays have tended to yield variable results. To enhance the biological control potential of these entomopathogenic nematodes, information about the interaction between host defense mechanisms and the nematode infection strategies is needed. Using petri dish and glass plate assays, we observed that larvae of P. japonica respond to attack by infective juveniles of H. bacteriophora with aggressive and evasive behavioral defenses. Brushing with the legs and rubbing with the abrasive raster (sometimes killing nematodes) were key aggressive behaviors associated with nematode attack in Petri dish assays. Larvae in soil assays were observed to respond to the presence of nematodes with evasive behavior. Remarkably, contact with even a single live nematode triggered behavioral countermeasures directed at eliminating the attacker. The aggressive behavior is effective in defense against nematode infection. Using an in vitro assay, we found that defensive chemicals exist in P. japonica larval gut fluid. The gut fluid is effective in immobilizing infective juveniles of H. bacteriophora, but not S. glaseri. With injection and dissection techniques, we investigated the host immune encapsulation response to different nematode species including S. carpocapsae and S. scapterisci. The larvae of P. japonica had a strong encapsulation and melanization response to all species except S. glaseri. In studies of nematode infection strategies against P. japonica larvae, we observed that both nematode species S. glaseri and H. bacteriophora possess similar behavioral responses to host and host environmental cues. However, H. bacteriophora, which is susceptible to host gut fluid and induces a strong immune encapsulation, tends to penetrate into the host hemocoel via membranous areas of cuticle and release its symbiotic bacteria during or soon after penetration. By contrast, S. glaseri, which is resistant to gut fluid and can avoid immune encapsulation, tend to penetrate via the midgut and release its symbiotic bacteria later than H. bacteriophora. To investigate the mechanism of immune avoidance of S. glaseri in P. japonica larvae, we extracted the nematode surface coat proteins and tested for activities in suppression of the host immune system. At least one protein of the nematode surface coat (SCP3a) is responsible for lysing hemocytes and suppressing encapsulation and phagocytosis. |
