Experiment 2 –Timeline for Diet Selection Experiment
To investigate whether birds alter feeding behavior to optimize responses to infection we conducted a second experiment, in which we simulated an infection in established zebra finch pairs using the bacterial endotoxin lipopolysaccharide (LPS), and quantified feeding behavior in immune challenged and control individuals, as well as birds housed near either a control pair (no immune threat), or birds housed near a pair given an immune challenge with LPS (social cue of heightened infection risk). To investigate how an immune challenge and perceived immune threat shape macronutrient preference, birds were provided with two isocaloric diets with varied lipid and protein ratios and their consumption was recorded. Birds were kept on a 14 L: 10 D light cycle and housed in 24”x16”x16” cages that were divided down the center into two separate 12”x16”x16” cage sections that each housed one pair of birds. Birds were housed in previously established pairs with one female and one male per cage section. Each cage section had two perches, a water dish, and two food dishes in which birds were fed ad libitum . Birds were given a choice of diet where each cage contained one high lipid food block and one high protein food block, whose composition was the same as in Experiment 1. Diet type was randomly assigned to either the left or right food dish to avoid confounding effects of cage side preference. To assess whether an immune threat altered the amount and type of diet that birds consumed, birds were provided with a choice in diet for seven days prior to experimental treatment and for five days following experimental treatment. Diets were weighed daily and replaced every other day. Three desiccation controls for each diet type were also weighed daily and the average desiccation values for each diet type (high lipid or high protein) were subtracted from feeding values to account for daily changes in food mass due to desiccation.
Pairs housed on one side of the cage were injected with either LPS or Saline (Figure 1; LPS-injected: N=24 birds or Saline-injected: N=24 birds), whereas pairs housed on the other side of the cage were unmanipulated (focal pairs; LPS-focal: N=24 birds or Saline-focal: N=24 birds). Injected pairs provided social cues to the focal pair. Solid opaque dividers were placed on both sides of each 24 x 16 x 16 cage to ensure that birds housed in each double cage could only see one another. To assess how an immune challenge and social cue of heightened infection risk shape feeding behavior, we injected previously established pairs with either lipopolysaccharide (LPS), a non-replicating antigen that activates the immune system and induces sickness behaviors, or a saline solution (control). Specifically, we injected stimulus birds intra-abdominally with either 50 μL of 2 mg/kg LPS (Sigma-Aldrich #L7261, Salmonella enterica serotype typhimurium) or 50 μL of phosphate-buffered saline (sham control, Sigma-Aldrich #P3813). Body mass and fat score data were collected on all stimulus and focal birds prior to treatment and on days 1, 2, and 5 post-treatment.
To assess whether heightened infection risk altered complement activity or baseline corticosterone concentrations, we collected blood samples from all focal birds 3 days prior to stimulus bird injections and 1, 2, and 5 days following stimulus bird injections. All blood samples for baseline corticosterone were collected within 3 minutes of entering the room. We collected additional blood samples from all focal males to assess changes in plasma testosterone concentrations in response to a heightened cue of infection 4 days prior to and 3 days following injection of the stimulus pairs. Immediately after collection, blood samples were centrifuged and blood plasma was separated and frozen at -20°C. To determine how treatment affected zebra finch gut microbial diversity and composition, cloacal swab samples were collected from all birds prior to treatment (day 0) and on day 5 following stimulus bird injections. Cloacal swabs were placed into 300 µl of RNAlater (Invitrogen, Thermo Fisher Scientific) and frozen at -80℃. All research protocols were approved by the Oklahoma State University Institutional Animal Care and Use Committee.