Gut microbiota of homing pigeons shows summer-winter variation under constant diet indicating a substantial effect of temperature.

BACKGROUND: Gut microbiotas play a pivotal role in host physiology and behaviour, and may affect host life-history traits such as seasonal variation in host phenotypic state. Generally, seasonal gut microbiota variation is attributed to seasonal diet variation. However, seasonal temperature and day length variation may also drive gut microbiota variation. We investigated summer-winter differences in the gut bacterial community (GBC) in 14 homing pigeons living outdoors under a constant diet by collecting cloacal swabs in both seasons during two years. Because temperature effects may be mediated by host metabolism, we determined basal metabolic rate (BMR) and body mass. Immune competence is influenced by day length and has a close relationship with the GBC, and it may thus be a link between day length and gut microbiota. Therefore, we measured seven innate immune indices. We expected the GBC to show summer-winter differences and to correlate with metabolism and immune indices. RESULTS: BMR, body mass, and two immune indices varied seasonally, other host factors did not. The GBC showed differences between seasons and sexes, and correlated with metabolism and immune indices. The most abundant genus (Lachnoclostridium 12, 12%) and associated higher taxa, were more abundant in winter, though not significantly at the phylum level, Firmicutes. Bacteroidetes were more abundant in summer. The Firmicutes:Bacteroidetes ratio tended to be higher in winter. The KEGG ortholog functions for fatty acid biosynthesis and linoleic acid metabolism (PICRUSt2) had increased abundances in winter. CONCLUSIONS: The GBC of homing pigeons varied seasonally, even under a constant diet. The correlations between immune indices and the GBC did not involve consistently specific immune indices and included only one of the two immune indices that showed seasonal differences, suggesting that immune competence may be an unlikely link between day length and the GBC. The correlations between the GBC and metabolism indices, the higher Firmicutes:Bacteroidetes ratio in winter, and the resemblance of the summer-winter differences in the GBC with the general temperature effects on the GBC in the literature, suggest that temperature partly drove the summer-winter differences in the GBC in homing pigeons.

DNA metabarcoding quantifies the relative biomass of arthropod taxa in songbird diets: Validation with camera-recorded diets.

Ecological research is often hampered by the inability to quantify animal diets. Diet composition can be tracked through DNA metabarcoding of fecal samples, but whether (complex) diets can be quantitatively determined with metabarcoding is still debated and needs validation using free-living animals. This study validates that DNA metabarcoding of feces can retrieve actual ingested taxa, and most importantly, that read numbers retrieved from sequencing can also be used to quantify the relative biomass of dietary taxa. Validation was done with the hole-nesting insectivorous Pied Flycatcher whose diet was quantified using camera footage. Size-adjusted counts of food items delivered to nestlings were used as a proxy for provided biomass of prey orders and families, and subsequently, nestling feces were assessed through DNA metabarcoding. To explore potential effects of digestion, gizzard and lower intestine samples of freshly collected birds were subjected to DNA metabarcoding. For metabarcoding with Cytochrome Oxidase subunit I (COI), we modified published invertebrate COI primers LCO1490 and HCO1777, which reduced host reads to 0.03%, and amplified Arachnida DNA without significant changing the recovery of other arthropod taxa. DNA metabarcoding retrieved all commonly camera-recorded taxa. Overall, and in each replicate year (N = 3), the relative scaled biomass of prey taxa and COI read numbers correlated at R = .85 (95CI:0.68-0.94) at order level and at R = .75 (CI:0.67-0.82) at family level. Similarity in arthropod community composition between gizzard and intestines suggested limited digestive bias. This DNA metabarcoding validation demonstrates that quantitative analyses of arthropod diet is possible. We discuss the ecological applications for insectivorous birds.

A fruit diet rather than invertebrate diet maintains a robust innate immunity in an omnivorous tropical songbird.

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. 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 compositions. 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. 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. We demonstrated a causal effect of diet composition on innate immune function, body mass and moult: bulbuls were in a 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.

Prenatal Transfer of Gut Bacteria in Rock Pigeon.

Vertebrates evolved in concert with bacteria and have developed essential mutualistic relationships. Gut bacteria are vital for the postnatal development of most organs and the immune and metabolic systems and may likewise play a role during prenatal development. Prenatal transfer of gut bacteria is shown in four mammalian species, including humans. For the 92% of the vertebrates that are oviparous, prenatal transfer is debated, but it has been demonstrated in domestic chicken. We hypothesize that also non-domestic birds can prenatally transmit gut bacteria. We investigated this in medium-sized Rock pigeon (Columba livia), ensuring neonates producing fair-sized first faeces. The first faeces of 21 neonate rock pigeons hatched in an incubator, contained a microbiome (bacterial community) the composition of which resembled the cloacal microbiome of females sampled from the same population (N = 5) as indicated by multiple shared phyla, orders, families, and genera. Neonates and females shared 16.1% of the total number of OTUs present (2881), and neonates shared 45.5% of their core microbiome with females. In contrast, the five females shared only 0.3% of the 1030 female OTUs present. These findings suggest that prenatal gut bacterial transfer may occur in birds. Our results support the hypothesis that gut bacteria may be important for prenatal development and present a heritability pathway of gut bacteria in vertebrates.

Unusual patterns in ¹5N blood values after a diet switch in red knot shorebirds.

When a diet switch results in a change in dietary isotopic values, isotope ratios of the consumer's tissues will change until a new equilibrium is reached. This change is generally best described by an exponential decay curve. Indeed, after a diet switch in captive red knot shorebirds (Calidris canutus islandica), the depletion of (13)C in both blood cells and plasma followed an exponential decay curve. Surprisingly, the diet switch with a dietary (15)N/(14)N ratio (δ(15)N) change from 11.4 to 8.8 ‰ had little effect on δ(15)N in the same tissues. The diet-plasma and diet-cellular discrimination factors of (15)N with the initial diet were very low (0.5 and 0.2 ‰, respectively). δ(15)N in blood cells and plasma decreased linearly with increasing body mass, explaining about 40 % of the variation in δ(15)N. δ(15)N in plasma also decreased with increasing body-mass change (r (2)=.07). This suggests that the unusual variation in δ(15)N with time after the diet switch was due to interferences with simultaneous changes in body-protein turnover.

When the seasons don't fit: speedy molt as a routine carry-over cost of reproduction.

The failure of animals to fit all life-cycle stages into an annual cycle could reduce the chances of successful breeding. In some cases, non-optimal strategies will be adopted in order to maintain the life-cycle within the scope of one year. We studied trade-offs made by a High Arctic migrant shorebird, the red knot Calidris canutus islandica, between reproduction and wing feather molt carried out in the non-breeding period in the Dutch Wadden Sea. We compared primary molt duration between birds undertaking the full migratory and breeding schedule with birds that forego breeding because they are young or are maintained in captivity. Molt duration was ca. 71 days in breeding adults, which was achieved by an accelerated feather replacement strategy. Second-year birds and captive adults took ca. 22% and 27% longer, respectively. Second-year birds start molt in late June, more than four weeks before captive adults, and almost seven weeks before adults that return from breeding in late July-August. Adults finish molt in October when steeply increasing thermostatic costs and reductions in food availability occur. Primary molt duration was longer in female than in male knots (all ages), which was accordance with the somewhat larger body size of females. Since fast growth leads to lower quality feathers, the speedy wing molt shown by Arctic-breeding birds may represent a time constraint that is an unavoidable and routine cost of reproduction. So far it was hypothesized that only birds over 1 kg would have difficulty fitting molt within a year. Here we show that in birds an order of magnitude smaller, temporal imperatives may impose the adoption of non-optimal life-cycle routines in the entire actively breeding population.

Cost-benefit analysis of mollusc-eating in a shorebird. II. Optimizing gizzard size in the face of seasonal demands.

Aiming to interpret functionally the large variation in gizzard masses of red knots Calidris canutus, we experimentally studied how the digestive processing rate is influenced by the size of the gizzard. During their non-breeding season, red knots feed on hard-shelled molluscs, which they ingest whole and crush in their gizzard. In three experiments with captive birds we tested predictions of the hypothesis that gizzard size, via the rate of shell crushing and processing, constrains intake rate in red knots (against the alternative idea that external handling times constrain intake rate). Gizzard size within individual birds was manipulated by varying the hardness of the diet on offer, and was confirmed by ultrasonography. The results upheld the "shell-crushing hypothesis" and rejected the "handling time hypothesis". Intake rates on with-shell prey increased with gizzard size, and decreased with shell mass per prey. Intake rates on soft (without shell) prey were higher than on with-shell prey and were unaffected by gizzard size. Offering prey that were heavily shelled relative to their flesh mass led to energy intake rates that were marginally sufficient to balance the daily energy budget within the time that is naturally available in a tidal system. We predicted the optimal gizzard sizes that are required to either (1) balance energy income with energy expenditure, or (2) to maximise net daily energy intake. The gizzard mass of free-living red knots in the Wadden Sea is such that it maximises daily net energy intake in spring when fuelling for migration, while it balances energy budget throughout the remainder of the year.