## 6 NP Immunomodulators
### Nematodes and Nematcomplexes  
Entomopathogenic nematodes (EPN) are parasitic nematodes (e.g., nematode family Steinnermatidae) which form mutualistic nematocomplexes with certain bacteria (_Xenorhabditis_ genus) [73]. This synergistic relationship elicits a damaging immunocompromising effect in the insect host and consequently has been exploited as potent bio-insecticides in pest control. Juvenile nematodes carry the symbiont bacterial host and together they infect and overcome their insect host immune system which, in turn, kills the insect, allowing the nematode to reproduce in the cadaver and serve as a vector for continued spread of the bacteria. _G. mellonella_ are a commonly used model to test this insecticidal activity but by inference also endorses the potential of this model to study and elucidate host-pathogen immune responses.  
Infection of _G. mellonella_ (last (7 th) instar; \(n=30\)) with the _Steinerema feltiae_ (_S. feltiae_) EPN alone reduced the in vitro haemocyte phagocytic activity of bacteria (_E. coli_) by approximately 50% which was attributed to sequestering soluble haemolymph immune molecules (e.g., opsonins and PRR such as LPS-binding protein) by binding to the lipid-rich cuticle surface [44]. The role of the _Steinerema_ cell surface cuticle in dampening immune responses was further demonstrated when [PERSON] et al. (2012) [74] showed an 80% reduction in vivo phagocytosis of fluorescent beads coated with crude extract of nematode surface coat proteins; however, this property was lost when purified cuticle-derived recombinant Sg-ENOL was used. The authors demonstrated that rSg-ENOL was a virulence factor accelerating bacterial symbiont infection (_X. poinarii_) by accelerating plasmin-mediated tissue infiltration in the insect host.  
In another study, a putative mechanism of immunosuppression was elucidated in response to injection of purified nematode cuticles (from _S. carpocapsae_ or _H. bacterioplora_) into _G. mellonella_ (early 5 th instar; \(n=20\)). Administration with either cuticle type caused a significant reduction in haemocyte density (approximately 50-70% versus negative control; \(p<0.05\)) and a similar reduction in haemocyte in vitro phagocytosis of fluorescent bacteria [50]. Haemolymph plasma taken from the cuticle-treated insects failed to prevent infection in another bacterial model, suggesting that haemolymph soluble anti-microbial molecules (e.g., opsonins and AMPs) were depleted by EPN pre-treatment corroborating the observation by [PERSON] et al. (2010). This inhibitory effect was seen following treatment with the PLA\({}_{2}\)-inhibitor DEX (see previous section) and was blocked by the eicosanoid substrate arachidonic acid, which strongly implicates the eicosanoid biosynthesis pathway in this broad-spectrum immunosuppression. The immunosuppressive effect of extracts from the nematode _S. carpocapsae_ outer layer proteins (SoP) and its symbiont bacteria _X. nematophila_ (XoP) cell wall were evaluated in _G. mellonella_ (late-stage caterpillars; \(n=\) unpublished). The study determined that prior treatment of larvae with bacterial extracts (XoP) alone was responsible for the marked inhibition of in vivo haemocyte phagocytosis of fluorescent bacteria (_S. aureus_) (\(p<0.0001\)), resulting in a halving of the phagocytosis ability [75].  
Use of the _G. mellonella_ in vivo model demonstrated that the EPN were rapidly acting, broad spectrum immunosuppressive agents in insects whose activity is mediated by the eicosanoid pathway common to vertebrates and invertebrates alike. The nematodal cuticle plays a role in immune evasion by sequestering soluble immune molecules (e.g., opsonins and AMPs) from the haemolymph, and both the nematode and symbiont bacteria directly compromise haemocyte phagocytic activity. Indeed, in a recent study, EPN and other bacterial feeding nematodes were found to ingest and destroy haemocytes [76].