Using live videography observation and Bayesian isotope mixing model to identify food composition and dietary contribution to inorganic mercury and methylmercury intake by songbird nestlings.

Mercury (Hg) exposure is increasing in terrestrial birds; however, studies on its sources are scarce. In the present study, we elucidated the food composition of green-backed tit nestlings from three urban forest parks (CPL, AHL, and LCG) using live videography observation (LVO). Furthermore, the daily dietary intakes of inorganic Hg (IHg) (MDIIHg) and methylmercury (MeHg) (MDIMeHg) were determined using the Bayesian isotope mixing model (BIMM) to uncover the nestlings' specific dietary Hg contribution. Both LVO and BIMM indicated that Lepidoptera (primarily caterpillar) constituted the primary food source for the nestlings in the three forests, accounting for approximately 60% of their diet in all three forest parks. The estimated MDI of Hg revealed that lepidopterans and spiders primarily contributed to IHg exposure, with a co-contribution ratio of 71.8%-97.7%. Unexpectedly, dietary MeHg was mostly derived from spiders; the highest contribution ratio of 93.6% was recorded at CPL, followed by another peak ratio of 92.9% at LCG. However, the dietary exposure was primarily IHg, accounting for 69.8% (AHL), 62.0% (LCG), and 61.3% (CPL) of the nestlings. Our study findings highlight the importance of dietary IHg transfer in evaluating the effects of Hg in nestlings. LVO, coupled with BIMM, is an effective tool for determining the food compositions of songbird nestlings and estimating the contribution of specific diets.

Effects of ambient temperature during the nestling stage on a stress indicator in nestling pied flycatchers Ficedula hypoleuca.

Long-term and short-term changes in ambient temperature can cause stress in birds, leading to changes in the level of hematological parameters. The H:L ratio (heterophil-to-lymphocyte ratio) is a hematological index that allows for the assessment of the stress induced by environmental changes, including weather conditions. In this paper, we examined the influence of temperatures and the sum of precipitation on the health of nestling pied flycatchers (Ficedula hypoleuca) by using the H:L ratio reflecting the body's response to stress. All examined temperature indicators influenced the H:L ratio, yet the average value of daily minimum temperature during the first 12 days of nestling life had the strongest influence, maximum temperature had the weakest effect, while precipitation had no significant influence. Our research indicates that even a small increase in temperature caused a stress reaction in nestling pied flycatchers, which was reflected by an increase in the H:L ratio. The increase in the stress index (H:L ratio) was probably a result of poor weather conditions (precipitation, low temperature), which prevented the adult birds from actively foraging and properly feeding the nestlings.

Sequence subtyping of Trichomonas gallinae from Bonelli's eagle (Aquila fasciata) during four years (2014-2017) reveals that MLS type is associated with lesions.

Avian trichomonosis is a parasitic disease that affects wild birds. The objective of this work was to determine the importance of avian trichomonosis in Bonelli's eagles to improve conservation measures in this population. One hundred and eighty-eight birds were studied: 181 chicks, two juveniles, one subadult and four adults. The birds were externally examined and gross lesions at the oropharynx registered. Samples from the oropharyngeal cavity were obtained for Trichomonas spp. detection by culture and PCR, and positive samples were subjected to a multilocus sequence typing approach, including the ITS1/5.8S/ITS2 region (ITS), ribosomal RNA small subunit (18S) and Fe-hydrogenase gene (FeHyd). Global prevalence of T. gallinae infection was 37.8% in total, 45.5% in nestlings. Thirty-three percent of the birds developed lesions that ranged from mild (n = 41) to moderate (n = 14) or severe (n = 7). Multilocus sequence typing analysis showed five different MLS types, ITS-A/18S-VI/FeHyd-A1 and ITS-D/18S-II/Fe-C4 being the most frequent. An association between ITS-A/18S-VI/FeHyd-A1 and moderate or severe lesions was observed, but birds with type ITS-A/18S-VI/FeHyd-A2 also developed lesions. On the contrary, birds with MLS type ITS-D/18S-II/FeHyd-C4 displayed only a low proportion of mild lesions. Chicks raised in nests were at higher risk for T. gallinae infection and development of lesions than chicks raised in captivity. Discordances between samples cultured in TYM and samples subjected to PCR from oropharyngeal swabs were observed, swab-ITS-PCR being more sensitive.RESEARCH HIGHLIGHTS 45.5% of Bonelli's eagles in the nest carried T. gallinae and 39.4% showed lesions.PCR from oral swabs showed higher sensitivity than culture in TYM for detection of T. gallinae.MLS types ITS-A/18S-VI/Fe-A1 (and A2) are a risk factor for the development of lesions.

Oxidative status of blue tit nestlings varies with habitat and nestling size.

Oxidative status has been proposed as an important ecological and evolutionary force given that pro-oxidant metabolites damage molecules, cells and tissues, with fitness consequences for organisms. Consequently, organisms usually face a trade-off between regulating their oxidative status and other physiological traits. However, environmental stressors and the availability of dietary-derived antioxidants vary according to local conditions and, thus, organisms inhabiting different habitats face different oxidative pressures. Still, there is little information on how different environmental conditions influence the oxidative status of animals inhabiting terrestrial environments. In this work, we examined the variation in oxidative status in the blue tit (Cyanistes caeruleus), a bird species with hatching asynchrony. Specifically, we examined the oxidative status of the largest and the smallest nestlings in the brood, inhabiting four forests differing in food availability and ectoparasite prevalence. We measured lipid peroxidation (malondialdehyde; MDA) as a marker of oxidative damage, total antioxidant capacity (Trolox-equivalent antioxidant capacity; TEAC) and antioxidant enzymatic activity (catalase, glutathione S-transferase, glutathione peroxidase) in blood samples. The glutathione peroxidase (GPX) activity differed among the forests, being the highest in the pine forest and the lowest in a mixed oak (Quercus) forest in the most humid area. Lipid peroxidation was higher in larger nestlings, suggesting higher oxidative damage with an increasing growth rate. Neither brood size, laying date, nor ectoparasites were related to the oxidative status of nestlings. These results suggest that nest rearing conditions might shape the oxidative status of birds, having consequences for habitat-dependent variation in regulation of oxidative status.

Influence of Natural Asymmetric Embryonic Visual Afferentation on the Neuronal Activations in the Caudomedial Mesopallium during the Freezing Response in Altricial Nestlings.

The study was designed to investigate the role of asymmetric prenatal visual stimulation on the activation of caudomedial mesopallium (CMM) neurons in nine-day-old pied flycatcher nestlings during auditory-guided freezing. Four groups of nestlings were studied: groups 1 and 2 included nestlings with normal vision and visually deprived, respectively, that were incubated and hatched in normal light environment; groups 3 and 4, nestlings with normal vision and visually deprived, respectively, that were incubated and hatched in the dark. The eyes of visually deprived nestlings were covered with non-transparent cups 2 h before the experiment. C-Fos expression was studied. It was shown that densities of neurons activated during freezing response differed in right vs. left CMM only in the group of visually deprived nestlings incubated under light. This suggests that the presence or absence of the asymmetric embryonic visual afferentation may result in the development of different strategies of the visual system integration into defense behavior.

Nest predation and adult mortality relationships with post-natal metabolic rates and growth among songbird species.

Metabolism is thought to mediate the connection between environmental selection pressures and a broad array of life history tradeoffs, but tests are needed. High juvenile predation correlates with fast growth, which may be achieved via fast juvenile metabolism. Fast offspring metabolism and growth can create physiological costs later in life that should be minimized in species with low adult mortality. Yet, relationships between juvenile metabolism and mortality at offspring versus adult stages are unexplored. We found that post-natal metabolism was positively correlated with adult mortality but not nest predation rates among 43 songbird species on three continents. Nest predation, but not adult mortality, explained additional variation in growth rates beyond metabolism. Our results suggest that metabolism may not be the mechanism underlying the relationships between growth and mortality at different life stages.

Perfluoroalkyl acids and sulfonamides and dietary, biological and ecological associations in peregrine falcons from the Laurentian Great Lakes Basin, Canada.

Perfluoroalkyl substances (PFAS) are a large, diverse group of chemicals and several perfluoroalkyl acids (PFAAs) are known environmental contaminants. Wildlife exposure to PFAAs and precursors has been shown, but less is known regarding replacements such as shorter-chain PFAS. In the present study, exposure to a suite of PFAAs and associations with dietary, biological and ecological factors were investigated in populations of a sentinel apex species - the peregrine falcon (Falco peregrinus). Nestling blood (n = 57) and sibling eggs (n = 9) were sampled in 2016 and 2018 from nests in rural and urban regions across the Laurentian Great Lakes Basin, Canada. PFSAs (perfluorinated sulfonic acids) including PFHxS, PFOS, and PFDS were detected in most egg and plasma samples, whereas 11 PFCAs (perfluorinated carboxylic acids; C5-C14, C16) compared to eight PFCAs (C8-C14, C16) were detected in most eggs and plasma, respectively. Shorter-chain C8-C10 PFCAs were more dominant in plasma and longer-chain C12-C14 PFCAs in eggs, but profiles were similar for PFOS, PFDS, PFUdA and PFHxDA. The exposure to PFAAs in peregrine falcons is likely mediated by dietary factors such as foraging location (δ13C and δ34S) and trophic position (δ15N) given the associations observed in eggs and nestling plasma, respectively. Moreover, significant relationships were observed for circulating ΣPFCAs and region (rural/urban), and nestling body condition after adjusting for sampling year and dietary tracers, suggesting that compared to rural nestlings, urban nestlings may be more exposed to ΣPFCAs and prone to their potential physiological impacts. Our findings highlight the importance of integrating dietary, biological and ecological factors when studying PFAS exposure in birds.

Patterns and trends in lead (Pb) concentrations in bald eagle (Haliaeetus leucocephalus) nestlings from the western Great Lakes region.

Most studies examining bald eagle (Haliaeetus leucocephalus) exposure to lead (Pb) have focused on adults that ingested spent Pb ammunition during the fall hunting season, often at clinical or lethal levels. We sampled live bald eagle nestlings along waterbodies to quantify Pb concentrations in 3 national park units and 2 nearby study areas in the western Great Lakes region. We collected 367 bald eagle nestling feather samples over 8 years during spring 2006-2015 and 188 whole blood samples over 4 years during spring 2010-2015. We used Tobit regression models to quantify relationships between Pb concentrations in nestling feathers and blood using study area, year, and nestling attributes as covariates. Pb in nestling feather samples decreased from 2006 to 2015, but there was no trend for Pb in blood samples. Pb concentrations in nestling feather and blood samples were significantly higher in study areas located closer to and within urban areas. Pb in feather and blood samples from the same nestling was positively correlated. Pb in feathers increased with nestling age, but this relationship was not observed for blood. Our results reflect how Pb accumulates in tissues as nestlings grow, with Pb in feathers and blood indexing exposure during feather development and before sampling, respectively. Some nestlings had Pb concentrations in blood that suggested a greater risk to sublethal effects from Pb exposure. Our data provides baselines for Pb concentrations in feathers and blood of nestling bald eagles from a variety of waterbody types spanning remote, lightly populated, and human-dominated landscapes.

Female begging calls reflect nutritional need of nestlings in the hen harrier Circus cyaneus.

BACKGROUND: Most birds exhibit bi-parental care with both sexes providing food for their young. Nestling signal food needs through begging. However, for some species, males rarely visit the nest, so have limited opportunity for gaining information directly from the chicks. Instead, females beg when males deliver food. We tested whether this calling signalled nutritional need and specifically the needs of the female (Breeder Need hypothesis) or that of their chicks (Offspring Need hypothesis). RESULTS: We observed begging and provisioning rates at 42 nests of hen harrier (Circus cyaneus) in Scotland, explored the factors associated with variation in begging rate and the relationship between begging and provisioning. We also tested the impact of food on begging and provisioning through a feeding experiment. Female begging rate increased up to a chick age of 3 weeks and then tailed off. In addition, begging increased when broods were large. CONCLUSIONS: Our data provided support for the Offspring Need hypothesis. At nests where adlib food was provided females reduced their begging rate. These patterns suggested that female begging was an honest signal of need. However, begging continued even with adlib food and was only weakly associated with greater provisioning by males, suggesting that these calls may also play an additional role, possibly reflecting sexual or parent-offspring conflict.

Does oxidative stress shorten telomeres?

Oxidative stress shortens telomeres in cell culture, but whether oxidative stress explains variation in telomere shortening in vivo at physiological oxidative stress levels is not well known. We therefore tested for correlations between six oxidative stress markers and telomere attrition in nestling birds (jackdaws Corvus monedula) that show a high rate of telomere attrition in early life. Telomere attrition was measured between ages 5 and 30 days, and was highly variable (average telomere loss: 323 bp, CV = 45%). Oxidative stress markers were measured in blood at age 20 days and included markers of oxidative damage (TBARS, dROMs and GSSG) and markers of antioxidant protection (GSH, redox state, uric acid). Variation in telomere attrition was not significantly related to these oxidative stress markers (|r| ≤ 0.08, n = 87). This finding raises the question whether oxidative stress accelerates telomere attrition in vivo The accumulation of telomere attrition over time depends both on the number of cell divisions and on the number of base pairs lost per DNA replication and, based on our findings, we suggest that in a growing animal cell proliferation, dynamics may be more important for explaining variation in telomere attrition than oxidative stress.

Neither artificial light at night, anthropogenic noise nor distance from roads are associated with oxidative status of nestlings in an urban population of songbirds.

Increasing urbanization is responsible for road-related pollutants and causes an unprecedented increase in light and noise pollution, with potential detrimental effects for individual animals, communities and ecosystems. These stressors rarely act in isolation but studies dissecting the effects of these multiple stressors are lacking. Moreover, studies on urban stressors have mainly focused on adults, while exposure in early-life may be detrimental but is largely ignored. To fill this important knowledge gap, we studied if artificial light at night, anthropogenic noise and road-related pollution (using distance from roads as a proxy) explain variation in oxidative status in great tit nestlings (Parus major) in an urban population. Artificial light at night, anthropogenic noise and distance from roads were not associated with variation of the nine studied metrics of oxidative status (superoxide dismutase-SOD-, glutathione peroxidase-GPX, catalase-CAT-, non-enzymatic total antioxidant capacity-TAC-, reduced glutathione-GSH-, oxidized glutathione-GSSG-, ratio GSH/GSSG, protein carbonyls and thiobarbituric acid reactive substances-TBARS). Interestingly, for all oxidative status metrics, we found that there was more variation in oxidative status among individuals of the same nest compared to between different nests. We also showed an increase in protein carbonyls and a decrease of the ratio GSH/GSSG as the day advanced, and an increase of GPX when weather conditions deteriorated. Our study suggests that anthropogenic noise, artificial light at night and road-related pollution are not the most important sources of variation in oxidative status in great tit nestlings. It also highlights the importance of considering bleeding time and weather conditions in studies with free-living animals.

Differential transcriptional responses underlie dietary induction of intestinal carbohydrase activities in house sparrow nestlings.

Many species show diet-induced flexibility of activity of intestinal enzymes; however, molecular and genetic mechanisms responsible for such modulation are less known, particularly in altricial birds. The goal of our study was to test whether a diet-induced increase in activity of intestinal maltase and sucrase in house sparrow nestlings is matched with an increase in maltase-glucoamylase (MG) and sucrase-isomaltase (SI) complex mRNAs respectively. Both enzyme activities were significantly higher in mid-intestine of nestlings fed a medium-starch (MS) diet compared to those fed a starch-free (SF) diet. In contrast to the similar pattern of dietary induction for both enzyme activities, diet MS elevated significantly only the level of MG mRNA, but not SI mRNA. The coordinated increase in activity of maltase and in MG mRNA is consistent with the hypothesis that dietary induction of this enzyme is under transcriptional control. In contrast, the lack of such coordination for changes in activity of sucrase and SI mRNA suggests that upregulation of this enzyme may be achieved by post-translational factor(s). We conclude that genetic mechanisms responsible for diet-induced flexibility of digestive enzymes in birds may differ from that observed in mammals.

Parental behavior correlates to baseline corticosterone of mates and offspring in nesting eastern bluebirds (Sialia sialis).

Raising nestlings in a biparental species involves a complex and dynamic interaction of physiology and behavior among a group of organisms. Parents may be predicted to vary their behaviors based not only upon their own state, but also in relation to the states of both offspring and the other parent. In this study we explore the relationships between parental feeding behaviors and family member condition in eastern bluebirds, with a special emphasis on baseline corticosterone, a hormone associated with energy mediation and stress. We found that the overall number of feeding trips made by both male and female parents were positively correlated to the corticosterone levels of nestlings. Maternal, but not paternal, baseline corticosterone levels were positively correlated to nestling baseline corticosterone levels. Additionally, adult males' feeding behavior was positively correlated to adult females' baseline corticosterone levels. These findings suggest a complex interplay between parental behavior and physiological state not only within a given organism, but also across organisms operating within a family unit. In addition, these results provide evidence that paternal and maternal efforts are influenced by related but distinct pressures, and that male and female parenting may be governed differentially even in species with relatively equitable biparental care.

Nestling telomere length does not predict longevity, but covaries with adult body size in wild barn swallows.

Telomere length and dynamics are increasingly scrutinized as ultimate determinants of performance, including age-dependent mortality and fecundity. Few studies have investigated longevity in relation to telomere length (TL) in the wild and none has analysed longevity in relation to TL soon after hatching, despite the fact that telomere shortening may mostly occur early in life. We show that TL in nestling barn swallows (Hirundo rustica) in the wild does not predict longevity. However, TL positively covaries with body size, suggesting that individuals with large TL can afford to grow larger without paying the cost of reduced TL, and/or that benign rearing conditions ensure both large body size and low rates of telomere shortening. Overall, our study hints at a role of TL in developmental processes, but also indicates a need for further analyses to assess the expectation that TL in young individuals predicts longevity in the wild.

Detection and genetic characterization of blaESBL-carrying plasmids of cloacal Escherichia coli isolates from white stork nestlings (Ciconia ciconia) in Spain.

OBJECTIVES: This study aimed to characterize Escherichia coli isolates from cloacal samples of white stork nestlings, with a special focus on extended-spectrum ß-lactamases (ESBLs)-producing E. coli isolates and their plasmid content. METHODS: Cloacal samples of 88 animals were seeded on MacConkey-agar and chromogenic-ESBL plates to recover E. coli and ESBL-producing E. coli. Antimicrobial susceptibility was screened using the disc diffusion method, and the genotypic characterization was performed by polymerase chain reaction (PCR) and subsequent sequencing. S1 nuclease Pulsed-Field-Gel-Electrophoresis (PFGE), Southern blotting, and conjugation essays were performed on ESBL-producing E. coli, as well as whole-genome sequencing by short- and long-reads. The four blaESBL-carrying plasmids were completely sequenced. RESULTS: A total of 113 non-ESBL-producing E. coli isolates were collected on antibiotic-free MacConkey-agar, of which 27 (23.9%) showed a multidrug-resistance (MDR) phenotype, mainly associated with ß-lactam-phenicol-sulfonamide resistance (blaTEM/cmlA/floR/sul1/sul2/sul3). Moreover, four white stork nestlings carried ESBL-producing E. coli (4.5%) with the following characteristics: blaSHV-12/ST38-D, blaSHV-12/ST58-B1, blaCTX-M-1/ST162-B1, and blaCTX-M-32/ST155-B1. Whole-genome sequencing followed by Southern blot hybridizations on S1-PFGE gels in ESBL-positive isolates proved that the blaCTX-M-1 gene and one of the blaSHV-12 genes were carried by IncI1/pST3 plasmids, while the second blaSHV-12 gene and the blaCTX-M-32 gene were located on IncF plasmids. The two blaSHV-12 genes and the two blaCTX-M genes had similar but non-identical close genetic environments, as all four genes were flanked by a variety of insertion sequences. CONCLUSION: The role played by several genetic platforms in the mobility of ESBL genes allows for interchangeability on a remarkably small scale (gene-plasmid-clones), which may support the spread of ESBL genes.

Microsatellite Instability Assay as a Potential Approach to Evaluate Genotoxicity: Lead Exposure in a Nestling Passerine Bird at the Stage of Intensive Erythropoiesis.

Although many avian studies have investigated the toxic effects of lead on important biochemical and physiological processes, organ and system function, and behavior, studies evaluating the specific genotoxic effects of exposure to lead are scarce. Nowadays, rapid technological advances can provide new molecular techniques in this regard. In this study, as a novel approach in bird studies, we used a panel of ten microsatellite loci to investigate the microsatellite instability (MSI) in response to experimental lead intoxication in a common hole-nesting species, the great tit Parus major. For this purpose, an experiment based on an intentional single supplementation of a lead (II) acetate trihydrate compound was conducted, with the use of two different doses, applied to randomly chosen great tit nestlings from randomly selected broods, being at the stage of intensive erythropoiesis. Although this preliminary study did not find any MSI in the seven microsatellite markers retained for the final comparison, it contributes to the examination of this molecular technique in field conditions as being potentially applicable in ecotoxicological bird studies. We believe that certain issues should be considered in finding an explanation for our result. First, the single doses of lead used in this study may have been too weak to induce genetic instability. Second, the panel of microsatellite markers studied may have been unsusceptible to lead genotoxicity in general. Third, the relatively short time interval (5 days) between the experimental procedure (lead exposure) and the sampling of post-exposure material (blood) for genetic analyses could have limited the effect of lead genotoxicity. Further analyzes are needed to verify these findings and to evaluate the scope of application of the MSI analysis in wild bird population studies.

Adaptive Response to Gillnets Bycatch in a North Sardinia Mediterranean Shag (Gulosus aristotelis desmarestii) Population.

Mediterranean Shag (Gulosus aristotelis desmarestii) is a seabird endemic to the Mediterranean and Black Seas, recently included in the IUCN list of threatened Species. Most of the reproductive colonies are hosted in Sardinia and surrounding islets. Bycatch in fishing nets is one of the most significant threats for this population. Our work aimed to assess alterations in the sex ratio caused by bycatch and to study the adaptive response of the population to a skewed adult sex ratio. The sex ratio of Mediterranean Shags found drowned in the gillnets near the colonies and that of the nestlings of the Corcelli (northeast Sardinia) colony was determined using the sex-linked polymorphism of the gene Chromobox-Helicase-DNA-binding 1. The data of the shags found drowned in gillnets evidenced a high mortality rate (83.3%; p < 0.001) and a larger size of males (35% heavier than females, p < 0.05) compared to females, supporting the theory that heavier individuals are able to forage at great depths. With 64.8% of the nestlings being male, the sex ratio of nestlings was statistically different from parity (p < 0.05). Furthermore, it was related to the brood size. In one- and two-chick broods, 73% and 70% of nestlings, respectively, were males, while in three-chick broods, only 33% were males. Our data identify the higher rate of male shags drowned in gillnets as a factor causing an alteration of the sex ratio in the Mediterranean Shag population. According to the Sex Allocation Theory, an adaptive adjustment of sex made by adult females restores the Mendelian sex ratio in the population.

The mercury flow through a terrestrial songbird food chain in subtropical pine forest: Elucidated by Bayesian isotope mixing model and stable mercury isotopes.

In order to comprehend the transfer of inorganic mercury (IHg) and methylmercury (MeHg) within food chains in terrestrial pine forests, we collected samples of Great Tit nestlings, common invertebrates, plants, and soil in a subtropical pine forest and used Bayesian isotope mixing model analysis, Hg daily intake, and stable Hg isotopes to elucidate the flow of MeHg and IHg in these food chains. Results indicate that caterpillars and cockroaches are the predominant prey items for nestlings, accounting for a combined contribution of 81.5%. Furthermore, caterpillars, cockroaches, and spiders were found to contribute the most (∼80%) of both IHg and MeHg that dietary accumulated in nestlings. The provisoned invertebrates tend to supply more IHg and diluting the proportion of MeHg as total Hg (MeHg%). Notably, nestling feathers displayed the highest Δ199Hg values but a relatively lower MeHg%, suggesting an imbalanced incorporation of Hg from maternal transfer and dietary accumulation during the nestling stage. This study highlights the efficacy of nestlings as indicators for identifying Hg sources and transfers in avian species and food chains. However, caution must be exercised when using Hg isotope compositions in growing feathers, and the contribution of maternally transferred Hg should not be ignored.

Are microbes and metabolites influencing the parental consumption of nestlings' feces in gray-backed shrikes?

The behavioral video recordings of the gray-backed shrike Lanius tephronotus revealed that parent birds eat the feces produced by their nestlings. "Parental nutrition hypothesis" attributes the origin of this behavior to nutrition-recovery and cost-saving, respectively. However, the presence of usable nutrients in the nestlings' feces is unknown because of traditional technology. In this study, we analyzed all the metabolites and the variations in the diversity and content of microbes in the feces of gray-backed shrike nestlings. We aimed to report the changes in microbes and metabolites with the age of nestlings and point out that the parent birds that eat the feces may gain potential nutrition benefits. The results showed that the relative abundances of Proteobacteria, Firmicutes, and Bacteroidota, changed significantly when the nestlings were 6 days old. The relative abundances of 6 probiotics, which are involved in digestion, metabolism, and immunity-related physiological functions, decreased in the nestlings' feces gradually with age; therefore, these probiotics may be obtained by parent birds upon ingestion of the feces of young nestlings. Among the metabolites that were detected, 20 were lipids and some had a role in anti-parasitic functions and wound healing; however, their relative contents decreased with age. These beneficial substances in the nestlings' feces may stimulate the parents to swallow the feces. Moreover, there were many aromatic metabolites in the newly hatched nestlings' feces, but the content of bitter metabolites increased as they grew up. Therefore, our results are in accordance with the nutritional hypothesis.

Comparing western (Megascops kennicottii) and whiskered (M. trichopsis) screech-owl microbiomes in southern Arizona using a novel 16S rRNA sequencing method.

Microbiomes are essential to a host's physiology and health. Despite the overall importance of microbiomes to animal health, they remain understudied in wildlife. Microbiomes function as physical barriers to invading pathogens, and changes in the diversity or composition of microbes within a host may disrupt this barrier. In order to use microbiomes in wildlife ecology, knowledge of the natural variation within and among species is essential. We compare the diversity and composition of two avian species that share the same habitat and niche in our study area, the western screech-owl (Megascops kennicottii) and the whiskered screech-owl (M. trichopsis). We used a targeted 16S sequencing method to improve the taxonomic resolution of microbiomes. We found similar measures of alpha diversity between species and sample types (cloacal samples vs. fecal samples). However, there were significant differences in bacterial species richness among nestlings from different nest boxes, and the composition differed between the two bird species and among nestlings from different nest boxes. Western screech-owls had more variation in alpha diversity and composition and had fewer bacterial species in their core microbiome than whiskered screech-owls. Siblings are likely to yield similar findings for microbiomes; thus, sampling nestlings from different nests may be most informative for monitoring population-level changes.