Behavioural infection tolerance of malaria is negatively correlated with pro-inflammatory cytokine expression in the brain of an invasive songbird

Invasive species are often freed from the co-evolved parasites of their native range (“enemy release hypothesis”), leaving little benefit for the host to induce self-harming inflammatory responses against novel pathogens in the invading range. Instead, selection may favour the allocation of energetic resources to traits that facilitate growth and reproduction to increase competitive ability (“evolution of increased competitive ability hypothesis”). In extension of these hypotheses, we tested whether the globally invasive house sparrow (Passer domesticus; n=21) exhibits sickness behaviours during acute experimental inoculation with Plasmodium relictum. Because sickness behaviours are a consequence of inflammation, we quantified cytokine expression in the liver and brain. Overall, we did not detect any changes in feeding duration, preening duration, or activity in malaria-infected sparrows (n=10) during acute malaria infection and infected sparrow behaviour did not differ from malaria-resistant sparrows (n=5) or sham controls (n=6). However, pro-inflammatory IFN-γ expression in the brain, but not the liver, was negatively correlated with infected sparrow activity, suggesting individuals that mount strong inflammatory responses are more likely to show sickness behaviours. Disease transmission is often linked with host behaviour; if invasive species behaviourally tolerate infection and maintain normal activity and sociality, they may disproportionately facilitate the spread of disease.

Methods
We used an experimental inoculation with Plasmodium relictum (avian malaria parasites) and quantified overall activity, feeding, and preening to assess sickness behaviour. Behaviour was quantified from two-hour long video recordings but the first 30 minutes of each video was not scored to avoid behaviour disruptions caused by researchers setting up the recording system. Three observers, blind to treatement, scored the videos for activity (the number of hops and flights), time spent preening (seconds), and time spent feeding (seconds). 

We assessed cytokine expression of two pro-inflammatory cytokines (IFN-γ & TNF-α) and two anti-inflammatory cytokines (IL-10 and TGF-β) that are implicated in the pathology of malaria in mammals. We extracted RNA from liver and cerebelum tissue obtained 13 days post-inoculation when sparrows experience peak parasite loads. We converted RNA to cDNA before conducting Taqman qPCR. Probes and primers for cytokines and references were validated in both tissues, including the selesction of the most stable reference gene (TBP in the liver, ACTB in the cerebellum). Data are reported as relative fold expression following Livak & Schmittgen (2001, "Analysis of relative gene expression data using real-time quantitative PCR and the 2^-delta delta Ct" in Methods, 25, 402-408).