DOI: 10.5061/dryad.bg79cnp77
title: A fruit diet rather than invertebrate diet maintains a robust innate immunity in an omnivorous tropical songbird

description: 1. Diet alteration may lead to nutrient limitations even in the absence of
 food limitation, and this may affect physiological functions, including
 immunity. Nutrient limitations may also affect the maintenance of body
 mass and key life history events that may affect immune function. Yet,
 variation in immune function is largely attributed to energetic trade-offs
 rather than specific nutrient constraints. 2. To test the effect of diet
 on life history traits, we tested how diet composition affects innate
 immune function, body mass and moult separately and in combination with
 each other, and then used path analyses to generate hypotheses about the
 mechanistic connections between immunity and body mass under different
 diet composition. 3. We performed a balanced parallel and crossover design
 experiment with omnivorous Common Bulbuls Pycnonotus barbatus in out-door
 aviaries in Nigeria. We fed 40 wild-caught bulbuls ad libitum on fruits or
 invertebrates for 24 weeks, switching half of each group between
 treatments after 12 weeks. We assessed innate immune indices (haptoglobin,
 nitric oxide and ovotransferrin concentrations, and haemagglutination and
 haemolysis titres), body mass and primary moult, fortnightly. We
 simplified immune indices into three principal components (PCs), but we
 explored mechanistic connections between diet, body mass and each immune
 index separately. 4. Fruit fed bulbuls had higher body mass, earlier moult
 and showed higher values for two of the three immune PCs compared to
 invertebrate fed bulbuls. These effects were reversed when we switched
 bulbuls between treatments after 12 weeks. Exploring the correlations
 between immune function, body mass and moult, showed that an increase in
 immune function was associated with a decrease in body mass and delayed
 moult in invertebrate fed bulbuls, while fruit fed bulbuls maintained body
 mass despite variation in immune function. Path analyses indicated that
 diet composition was most likely to affect body mass and immune indices
 directly and independently from each other. Only haptoglobin concentration
 was indirectly linked to diet composition via body mass. 5. We
 demonstrated a causal effect of diet composition on innate immune
 function, body mass and moult: bulbuls were in better condition when fed
 on fruits than invertebrates, confirming that innate immunity is nutrient
 specific. Our results are unique because they show a reversible effect of
 diet composition on wild adult birds whose immune systems are presumably
 fully developed and adapted to wild conditions – demonstrating a
 short-term consequence of diet alteration on life history
 traits. 
description: This dataset includes information from a diet manipulation
 experiment on Common Bulbuls in out-door aviaries in Nigeria. We caught 40
 adult Common Bulbuls using mist nets around the A. P. Leventis
 Ornithological Research Institute (APLORI) in Nigeria (09°52’N, 08°58’E)
 between 28 October to 7<sup> </sup>November 2016 and housed
 them in groups of 10 birds in four adjacent out-door aviaries at
 APLORI. Birds were fed fruits and invertebrates in captivity until the
 experiment started on the 2 December 2016. Birds were supplied water and
 food ad libitum before and throughout the experiment. All birds were
 sampled for blood, assessed for moult and weighed to determine baseline
 body mass and innate immune function on 1 or 2 December, before diet
 restriction commenced on 2 December. During the experiment, birds in two
 aviaries were fed fruits , and the other two were fed invertebrates and
 sampled fortnightly. After 12 weeks of diet treatment, five birds from
 each aviary were switched between treatments, and the other five birds of
 each aviary remained on the same treatment. Switched birds replaced each other in
 aviaries with the alternative diet treatment, so we maintained four
 aviaries with the same diet treatment throughout the experiment.
 In one of the fruit treatment aviaries, we moved only four birds to the
 invertebrate treatment because we had nine birds left in this aviary. The
 experiment continued for another 12 weeks. Thus, we grouped individuals
 as: invertebrate throughout, invertebrate to fruit, fruit to invertebrate
 and fruit throughout. There were six females and 14 males on
 fruit diet and nine females and 11 males on invertebrate diet at the start
 of the experiment, but we were blind to the sex of individuals
 during the experiment, because sexes were only determined molecularly
 after the experiment. All birds were sexed using gel
 electrophoresis. Birds were sampled between 6:00 and
 10:00 hours daily in two consecutive days per sampling session. Two
 aviaries of alternate diet treatments were sampled per day, with sampling
 order rotating between sampling sessions.Plasma and blood cells were stored at
 -20<sup>° </sup>C for one week and then moved to
 -80<sup>° </sup>C until transported for immune assays in
 Groningen, the Netherlands. Haptoglobin, nitric oxide and
 ovotransferrin concentration were carried by colorimetric
 assays, absorbance were measured using a Versamax plate reader (Molecular
 Devices Sunnyvale, California, US).  Natural antibody-mediated
 haemagglutination and complement-mediated haemolysis titres of plasma
 samples against 1% rabbit red blood cells (Envigo RMS (UK) Ltd.) in
 phosphate buffered saline were measured as described by Matson et al.
 (2005). Additonal morphometric measurements,
 including wing length, tarsus length and body mass are included in the
 dataset. Moult status and scores of feathers for each individual are also
 included in the dataset.  Haematocrit
 measurements (PCV) and occurrence of ectoparasite, and microfilaria are
 also recorded in the data where available, although these were not
 analysed for the current manuscript.
description: Notes are provided as comments where necessary for each column of
 data in the separate excel worksheets. This provides information about
 measurement units and scoring. Each worksheet contains separate data for
 either sampling information or immune assays and can be linked by the Ring
 number of each bird.