Volatiles of symbiotic bacterial origin explain ectoparasitism and fledging success of hoopoes.

BACKGROUND: Some parasites use olfactory cues to detect their hosts and, since bacterial symbionts are partially responsible for animal odours, they could influence host parasitism. By autoclaving nest materials of hoopoe (Upupa epops) nests before reproduction started, we explored the hypothetical links between host-associated bacteria, volatiles and parasitism. During the nestling stage, we (i) estimated the level of ectoparasitism by chewing lice (Suborder Mallophaga) in adult hoopoe females and by Carnus haemapterus flies in nestlings, and (ii) characterized microbial communities and volatile profiles of nest environments (nest material and nest cavity, respectively) and uropygial secretions. RESULTS: Experimental nests had less diverse bacterial communities and more diverse volatile profiles than control nests, while occupants experienced lower intensity of parasitism in experimental than in control nests. The experiment also affected beta diversity of the microbial communities of nest material and of the volatiles of the nestling uropygial secretions. Moreover, microbial communities of uropygial secretions and of nest materials covaried with their volatile profiles, while the volatile profile of the bird secretions explained nest volatile profile. Finally, a subset of the volatiles and bacteria detected in the nest material and uropygial secretions were associated with the ectoparasitism intensity of both adult females and nestlings, and with fledging success. CONCLUSIONS: These results show that a component of animal odours is linked with the microbial communities of the host and its reproductive environment, and emphasize that the associations between bacteria, ectoparasitism and reproductive success are partially mediated by volatiles of bacterial origin. Future work should focus on mechanisms underlying the detected patterns.

Winged resistance: Storks and gulls increase carriage of antibiotic resistance by shifting from paddy fields to landfills.

Waterbirds are vectors for the dissemination of antimicrobial resistance across environments, with some species increasingly reliant on highly anthropized habitats for feeding. However, data on the impact of their feeding habits on the carriage of antibiotic resistance genes (ARGs) are still scarce. To fill this gap, we examined the microbiota (16S rRNA amplicon gene sequencing) and the prevalence of ARG (high-throughput qPCR of 47 genes) in faeces from white storks (Ciconia ciconia) and lesser black-backed gulls (Larus fuscus) feeding in highly (landfill) and less (paddy fields) polluted habitats. Faecal bacterial richness and diversity were higher in gulls feeding upon landfills and showed a greater abundance of potential pathogens, such as Staphylococcus. In contrast, faecal bacterial communities from storks were similar regardless of habitat preferences, maybe due to a less intense habitat use compared to gulls. In addition, birds feeding in the landfill carried a higher burden of ARGs compared to the surrounding soil and surface waters. Network analysis revealed strong correlations between ARGs and potential pathogens, particularly between tetM (resistance to tetracyclines), blaCMY (beta-lactam resistance), sul1 (sulfonamide resistance) and members of the genera Streptococcus, Peptostreptococcus, and Peptoclostridium. Our work demonstrates how transitioning from paddy fields to landfills fosters the carriage of ARGs and potential pathogens in the bird gut, shedding light on the ecological role of these avian vectors in antimicrobial resistance dissemination.

Circular and L50-like leaderless enterocins share a common ABC-transporter immunity gene.

Microbes live within complex communities of interacting populations, either free-living in waters and soils or symbionts of animals and plants. Their interactions include the production of antimicrobial peptides (bacteriocins) to antagonize competitors, and these producers must carry their own immunity gene for self-protection. Whether other coexisting populations are sensitive or resistant to the bacteriocin producer will be key for the population dynamics within the microbial community. The immunity gene frequently consists of an ABC transporter to repel its own bacteriocin but rarely protects against a nonrelated bacteriocin. A case where this cross-resistance occurs mediated by a shared ABC transporter has been shown between enterocins MR10A/B and AS-48. The first is an L50-like leaderless enterocin, while AS-48 is a circular enterocin. In addition, L50-like enterocins such as MR10A/B have been found in E. faecalis and E. faecium, but AS-48 appears only in E. faecalis. Thus, using the ABC transporter of the enterocin MR10A/B gene cluster of Enterococcus faecalis MRR10-3 as a cross-resistance model, we aimed to unravel to what extent a particular ABC transporter can be shared across multiple bacteriocinogenic bacterial populations. To this end, we screened the MR10A/B-ABC transporters in available microbial genomes and analyzed their sequence homologies and distribution. Overall, our main findings are as follows: (i) the MR10A/B-ABC transporter is associated with multiple enterocin gene clusters; (ii) the different enterocins associated with this transporter have a saposin-like fold in common; (iii) the Mr10E component of the transporter is more conserved within its associated enterocin, while the Mr10FGH components are more conserved within the carrying species. This is the least known component of the transporter, but it has shown the greatest specificity to its corresponding enterocin. Bacteriocins are now being investigated as an alternative to antibiotics; hence, the wider or narrower distribution of the particular immunity gene should be taken into account for clinical applications to avoid the selection of resistant strains. Further research will be needed to investigate the mechanistic interactions between the Mr10E transporter component and the bacteriocin as well as the specific ecological and evolutionary mechanisms involved in the spread of the immunity transporter across multiple bacteriocins.

Lactiplantibacillus paraplantarum BPF2 and Pediococcus acidilactici ST6, Two Bacteriocinogenic Isolated Strains from Andalusian Spontaneous Fermented Sausages.

Traditional spontaneously fermented foods are well known for their sensory and safety properties, which is mainly due to their indigenous microflora. Within this group of food, Mediterranean dry-cured sausages stand out as a significant source of lactic-acid bacterial strains (LAB) with biotechnological properties, such as their antimicrobial activity. The aim of this study was to investigate the biodiversity of antagonistic LAB strains from different Andalusian traditional sausages, such as salchichón and chorizo. First, a screening was carried out focusing on the antimicrobial activity against foodborne pathogens, such as Listeria monocytogenes, Escherichia coli, Clostridium perfringens, and Staphylococcus aureus, selecting two strains due to their higher antibiosis properties, both in agar and liquid media. These bacteria were identified as Lactiplantibacillus paraplantarum BPF2 and Pediococcus acidilactici ST6. In addition, genomic studies confirmed the presence of certain structural genes related to the production of bacteriocins. Finally, the culture supernatants of both strains were purified and analyzed by LC-MS/MS, obtaining the relative molecular mass and the amino acid sequence and identifying the peptides as the bacteriocins Pediocin-PA and Leucocin K. In conclusion, genomes and antimicrobial substances of P. acidilactici ST6, a Pediocin-PA producer, and Lpb. paraplantarum BPF2, a Leucocin K producer, isolated from Andalusian salchichón and chorizo, respectively, are presented in this work. Although further studies are required, these strains could be used alone or in combination as starters or protective cultures for the food industry.

Seed Size, Not Dispersal Syndrome, Determines Potential for Spread of Ricefield Weeds by Gulls.

Recent field data suggest that migratory gulls disperse many rice field weeds by gut passage (endozoochory), most of which are dry fruited and widely assumed to have no long-distance dispersal mechanisms, except via human activity. We investigated this mechanism with a feeding experiment, in which seeds of five common rice field weeds (in order of increasing seed size: Juncus bufonius, Cyperus difformis, Polypogon monspeliensis, Amaranthus retroflexus, and the fleshy-fruited Solanum nigrum) were fed to seven individuals of lesser black-backed gulls Larus fuscus held in captivity. We quantified seed survival after collecting faeces at intervals for 33 h after ingestion, then extracting intact seeds and running germination tests, which were also conducted for control seeds. All five species showed high seed survival after gut passage, of >70%. Gut retention times averaged 2-4 h, but maxima exceeded 23 h for all species. Germinability after gut passage was 16-54%, and gut passage accelerated germination in J. bufonius and S. nigrum, but slowed it down in the other species. All species had lower germinability after gut passage compared to control seeds (likely due to stratification prior to the experiment), but the loss of germinability was higher in smaller seeds. There was no evidence that the different dispersal syndromes assigned to the five species (endozoochory, epizoochory or barochory) had any influence on our results. In contrast, mean gut retention time was strongly and positively related to seed size, likely because small seeds pass more quickly from the gizzard into the intestines. Non-classical endozoochory of dry-fruited seeds by waterbirds is a major but overlooked mechanism for potential long-distance dispersal, and more research into this process is likely essential for effective weed management.

Allium-Derived Compound Propyl Propane Thiosulfonate (PTSO) Reduces Vibrio Populations and Increases Body Weight of European Seabass (Dicentrarchus labrax) Juveniles.

The global demand for fish products is continuously increasing as the population grows, and aquaculture plays an important role in supplying this demand. However, industrial antibiotic misuse has contributed to the spread of antimicrobial resistance among pathogenic bacteria, therefore, several antibiotic alternatives have been proposed. In this study, we have analyzed the effects of Allium-derived propyl propane thiosulfonate (PTSO) in European seabass juveniles' growth and performance. These effects were tested by measuring the body weight and analyzing the gut microbiome of fish after 89 days of feeding trial. The relative abundance of potentially pathogenic Vibrio in the foregut and hindgut of supplemented fish decreased, while Pseudomonas and Kocuria increased significantly. Alpha diversity indices significantly decreased in both gut regions of fish fed with Allium-derived PTSO supplemented diet, as well as between bacterial community composition. These results may indicate a positive effect of the supplementation in the diet with Allium-derived PTSO, reducing potentially pathogenic Vibrio and increasing body weight at the end of the experiment (89 days). However, this supplementation with Allium-derived PTSO produces changes in the diversity and composition of microbial communities, so further experiments would be necessary to explore bacterial community composition and health relationship.

Beneficial Shifts in the Gut Bacterial Community of Gilthead Seabream (Sparus aurata) Juveniles Supplemented with Allium-Derived Compound Propyl Propane Thiosulfonate (PTSO).

This study analyzes the potential use of an Allium-derived compound, propyl propane thiosulfonate (PTSO), as a functional feed additive in aquaculture. Gilthead seabream (Sparus aurata) juveniles had their diet supplemented with this Allium-derived compound (150 mg/kg of PTSO) and were compared with control fish. The effects of this organosulfur compound were tested by measuring the body weight and analyzing the gut microbiota after 12 weeks. The relative abundance of potentially pathogenic Vibrio and Pseudomonas in the foregut and hindgut of supplemented fish significantly decreased, while potentially beneficial Lactobacillus increased compared to in the control fish. Shannon's alpha diversity index significantly increased in both gut regions of fish fed with a PTSO-supplemented diet. Regarding beta diversity, significant differences between treatments only appeared in the hindgut when minority ASVs were taken into account. No differences occurred in body weight during the experiment. These results indicate that supplementing the diet with Allium-derived PTSO produced beneficial changes in the intestinal microbiota while maintaining the productive parameters of gilthead seabream juveniles.

Enterocin Cross-Resistance Mediated by ABC Transport Systems.

In their struggle for life, bacteria frequently produce antagonistic substances against competitors. Antimicrobial peptides produced by bacteria (known as bacteriocins) are active against other bacteria, but harmless to their producer due to an associated immunity gene that prevents self-inhibition. However, knowledge of cross-resistance between different types of bacteriocin producer remains very limited. The immune function of certain bacteriocins produced by the Enterococcus genus (known as enterocins) is mediated by an ABC transporter. This is the case for enterocin AS-48, a gene cluster that includes two ABC transporter-like systems (Transporter-1 and 2) and an immunity protein. Transporter-2 in this cluster shows a high similarity to the ABC transporter-like system in MR10A and MR10B enterocin gene clusters. The aim of our study was to determine the possible role of this ABC transporter in cross-resistance between these two different types of enterocin. To this end, we designed different mutants (Tn5 derivative and deletion mutants) of the as-48 gene cluster in Enterococcus faecalis and cloned them into the pAM401 shuttle vector. Antimicrobial activity assays showed that enterocin AS-48 Transporter-2 is responsible for cross-resistance between AS-48 and MR10A/B enterocin producers and allowed identification of the MR10A/B immunity gene system. These findings open the way to the investigation of resistance beyond homologous bacteriocins.

Cosmetic coloration of cross-fostered eggs affects paternal investment in the hoopoe (Upupa epops).

The signalling hypothesis suggests that avian eggshell coloration is a sexually selected female signal advertising her quality to its male partner, thereby stimulating his provisioning rate. This hypothesis has been tested for structural eggshell pigments, but not for cosmetic colorations, such as that produced by the uropygial secretion on eggshells. During the breeding season, female hoopoes (Upupa epops) host in their uropygial glands symbiotic bacteria. Females actively smear the eggshells with their secretion, protecting embryos from pathogenic trans-shell infections and changing eggshell coloration. Because the colour of the secretions is related to their antimicrobial potential, cosmetic eggshell coloration may act as a cue or even as a post-mating sexually selected signal if it affects male provisioning rates. To experimentally test this hypothesis, we cross-fostered already-smeared clutches between hoopoe nests, and quantified male feeding behaviour to females before and after the experiment. This approach allows disentanglement of the effects of female quality and of egg coloration on male investment. In accordance with the hypothesis, males adjusted their provisioning rate to the eggshell cosmetic coloration. This is, to our knowledge, the first experimental demonstration that egg colour stained with uropygial secretion could act as a post-mating sexual signal of female quality to males.

Faecal microbiota and antibiotic resistance genes in migratory waterbirds with contrasting habitat use.

Migratory birds may have a vital role in the spread of antimicrobial resistance across habitats and regions, but empirical data remain scarce. We investigated differences in the gut microbiome composition and the abundance of antibiotic resistance genes (ARGs) in faeces from four migratory waterbirds wintering in South-West Spain that differ in their habitat use. The white stork Ciconia ciconia and lesser black-backed gull Larus fuscus are omnivorous and opportunistic birds that use highly anthropogenic habitats such as landfills and urban areas. The greylag goose Anser anser and common crane Grus grus are herbivores and use more natural habitats. Fresh faeces from 15 individuals of each species were analysed to assess the composition of bacterial communities using 16S rRNA amplicon-targeted sequencing, and to quantify the abundance of the Class I integron integrase gene (intI1) as well as genes encoding resistance to sulfonamides (sul1), beta-lactams (blaTEM, blaKPC and blaNDM), tetracyclines (tetW), fluoroquinolones (qnrS), and colistin (mcr-1) using qPCR. Bacterial communities in gull faeces were the richest and most diverse. Beta diversity analysis showed segregation in faecal communities between bird species, but those from storks and gulls were the most similar, these being the species that regularly feed in landfills. Potential bacterial pathogens identified in faeces differed significantly between bird species, with higher relative abundance in gulls. Faeces from birds that feed in landfills (stork and gull) contained a significantly higher abundance of ARGs (sul1, blaTEM, and tetW). Genes conferring resistance to last resort antibiotics such as carbapenems (blaKPC) and colistin (mcr-1) were only observed in faeces from gulls. These results show that these bird species are reservoirs of antimicrobial resistant bacteria and suggest that waterbirds may disseminate antibiotic resistance across environments (e.g., from landfills to ricefields or water supplies), and thus constitute a risk for their further spread to wildlife and humans.

Allium Extract Implements Weaned Piglet's Productive Parameters by Modulating Distal Gut Microbiota.

Antimicrobial resistance (AMR) has risen as a global threat for human health. One of the leading factors for this emergence has been the massive use of antibiotics growth-promoter (AGPs) in livestock, enhancing the spread of AMR among human pathogenic bacteria. Thus, several alternatives such as probiotics, prebiotics, or phytobiotics have been proposed for using in animal feeding to maintain or improve productive levels while diminishing the negative effects of AGPs. Reducing the use of antibiotics is a key aspect in the pig rearing for production reasons, as well as for the production of high-quality pork, acceptable to consumers. Here we analyze the potential use of Allium extract as an alternative. In this study, weaned piglets were fed with Allium extract supplementation and compared with control and antibiotic (colistin and zinc oxide) treated piglets. The effects of Allium extract were tested by analyzing the gut microbiome and measuring different productive parameters. Alpha diversity indices decreased significantly in Allium extract group in caecum and colon. Regarding beta diversity, significant differences between treatments appeared only in caecum and colon. Allium extract and antibiotic piglets showed better values of body weight (BW), average daily weight gain (ADG), and feed conversion ratio (FCR) than control group. These results indicate that productive parameters can be implemented by modifying the gut microbiota through phytobiotics such as Allium extract, which will drive to drop the use of antibiotics in piglet diet.

Allium-Based Phytobiotic Enhances Egg Production in Laying Hens through Microbial Composition Changes in Ileum and Cecum.

Phytobiotics (bioactive compounds extracted from plants) are one of the explored alternatives to antibiotics in poultry and livestock due to their antimicrobial activity and its positive effects on gut microbiota and productive properties. In this study, we supplemented a product based on garlic and onion compounds in the diet to laying hens at the beginning of their productive life (from 16 to 20 weeks post-hatching). The experimental group showed a significant increase in the number of eggs laid and in their size, produced in one month compared to the control. This increase in production was accompanied by microbiota changes in the ileum and cecum by means of high throughput sequencing analyses. These bacterial shifts in the ileum were mainly the result of compositional changes in the rare biosphere (unweighted UniFrac), while in the cecum, treatment affected both majority and minority bacterial groups (weighted and unweighted UniFrac). These changes in the microbiota suggest an improvement in food digestibility. The relative abundance of Lactococcus in the ileum and Lactobacillus in the cecum increased significantly in the experimental group. The relative abundance of these bacterial genera are known to have positive effects on the hosts. These results are very promising for the use of these compounds in poultry for short periods.

Autoclaving Nest-Material Remains Influences the Probability of Ectoparasitism of Nestling Hoopoes (Upupa epops).

Nest bacterial environment influences avian reproduction directly because it might include pathogenic- or antibiotic-producing bacteria or indirectly because predators or ectoparasites can use volatile compounds from nest bacterial metabolism to detect nests of their avian hosts. Hoopoes (Upupa epops) do not build nests. They rather reuse holes or nest-boxes that contain remains of nest-materials from previous breeding seasons. Interestingly, it has been recently described that the nest's bacterial environment partly affects the uropygial gland microbiota of hoopoe females and eggshells. Blood-sucking ectoparasites use chemical cues to find host nests, so we experimentally tested the hypothetical effects of microorganisms inhabiting nest-material remains before reproduction regarding the intensity of ectoparasitism suffered by 8-day-old nestling hoopoes. In accordance with the hypothesis, nestlings hatched in nest-boxes with autoclaved nest-material remains from the previous reproductive seasons suffered less from ectoparasites than those hatched in the control nest-boxes with nonautoclaved nest-material. Moreover, we found a positive association between the bacterial density of nest-material during the nestling phase and ectoparasitism intensity that was only apparent in nest-boxes with autoclaved nest-material. However, contrary to our expectations, nest bacterial load was positively associated with fledgling success. These results suggest a link between the community of microorganisms of nest-material remains and the intensity of ectoparasitism, and, on the other hand, that the nest bacterial environment during reproduction is related to fledging success. Here, we discuss possible mechanisms explaining the experimental and correlative results, including the possibility that the experimental autoclaving of nest material affected the microbiota of females and nestlings' secretion and/or nest volatiles that attracted ectoparasites, therefore indirectly affecting both the nest bacterial environment at the nestling stage and fledging success.

The gut microbiota of brood parasite and host nestlings reared within the same environment: disentangling genetic and environmental effects.

Gut microbiota are essential for host health and survival, but we are still far from understanding the processes involved in shaping their composition and evolution. Controlled experimental work under lab conditions as well as human studies pointed at environmental factors (i.e., diet) as the main determinant of the microbiota with little evidence of genetic effects, while comparative interspecific studies detected significant phylogenetic effects. Different species, however, also differ in diet, feeding behavior, and environmental characteristics of habitats, all of which also vary interspecifically, and, therefore, can potentially explain most of the detected phylogenetic patterns. Here, we take advantage of the reproductive strategy of avian brood parasites and investigate gut microbiotas (esophageal (food and saliva) and intestinal) of great spotted cuckoo (Clamator glandarius) and magpie (Pica pica) nestlings that grow in the same nests. We also estimated diet received by each nestling and explored its association with gut microbiota characteristics. Although esophageal microbiota of magpies and great spotted cuckoos raised within the same environment (nest) did not vary, the microbiota of cloacal samples showed clear interspecific differences. Moreover, diet of great spotted cuckoo and magpie nestlings explained the microbiota composition of esophageal samples, but not of cloaca samples. These results strongly suggest a genetic component determining the intestinal microbiota of host and parasitic bird species, indicating that interspecific differences in gut morphology and physiology are responsible for such interspecific differences.

Replication of the mirror mark test experiment in the magpie (Pica pica) does not provide evidence of self-recognition.

Self-recognition in animals is demonstrated when individuals pass the mark test. Formerly, it was thought that self-recognition was restricted to humans, great apes, and certain mammals with large brains and highly evolved social cognition. However, 1 study showed that 2 out of 5 magpies (Pica pica) passed the mark test, suggesting that magpies have a similar level of cognitive abilities to great apes. The scientific advancement depends on confidence in published science, and this confidence can be reached only after rigorous replication of published studies. Here, we present a close replication of the magpie study but using a larger sample size while following a very similar experimental protocol. Like the previous study, in our experiment, magpies showed both social and self-directed behavior more frequently in front of the mirror versus a control cardboard stimulus. However, during the mark test, self-directed behavior proved more frequent in front of the cardboard than in the mirror. Thus, our replication failed to confirm the previous results. Close replications, while not disproving an earlier study, identify results that should be considered with caution. Therefore, more replication studies and additional experimental work is needed to unambiguously demonstrate that magpies are consistently able to pass the mark test. The existence of compelling evidence of self-recognition in other corvid species is discussed in depth. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

Egg Production in Poultry Farming Is Improved by Probiotic Bacteria.

Antimicrobial resistance (AMR) is one of the most serious threats for human health in the near future. Livestock has played an important role in the appearance of antibiotic-resistant bacteria, intestinal dysbiosis in farming animals, or the spread of AMR among pathogenic bacteria of human concern. The development of alternatives like probiotics is focused on maintaining or improving production levels while diminishing these negative effects of antibiotics. To this end, we supplied the potential probiotic Enterococcus faecalis UGRA10 in the diet of laying hens at a final concentration of 108 Colony Forming Units per gram (CFU/g) of fodder. Its effects have been analyzed by: (i) investigating the response of the ileum and caecum microbiome; and (ii) analyzing the outcome on eggs production. During the second half of the experimental period (40 to 76 days), hens fed E. faecalis UGRA10 maintained egg production, while control animals dropped egg production. Supplementation diet with E. faecalis UGRA10 significantly increased ileum and caecum bacterial diversity (higher bacterial operational taxonomic unit richness and Faith's diversity index) of laying hens, with animals fed the same diet showing a higher similarity in microbial composition. These results point out to the beneficial effects of E. faecalis UGRA10 in egg production. Future experiments are necessary to unveil the underlying mechanisms that mediate the positive response of animals to this treatment.

Bacterial density rather than diversity correlates with hatching success across different avian species.

Bacterial communities within avian nests are considered an important determinant of egg viability, potentially selecting for traits that confer embryos with protection against trans-shell infection. A high bacterial density on the eggshell increases hatching failure, whether this effect could be due to changes in bacterial community or just a general increase in bacterial density. We explored this idea using intra- and interspecific comparisons of the relationship between hatching success and eggshell bacteria characterized by culture and molecular techniques (fingerprinting and high-throughput sequencing). We collected information for 152 nests belonging to 17 bird species. Hatching failures occurred more frequently in nests with higher density of aerobic mesophilic bacteria on their eggshells. Bacterial community was also related to hatching success, but only when minority bacterial operational taxonomic units were considered. These findings support the hypothesis that bacterial density is a selective agent of embryo viability, and hence a proxy of hatching failure only within species. Although different avian species hold different bacterial densities or assemblages on their eggs, the association between bacteria and hatching success was similar for different species. This result suggests that interspecific differences in antibacterial defenses are responsible for keeping the hatching success at similar levels in different species.

Nest Bacterial Environment Affects Microbiome of Hoopoe Eggshells, but Not That of the Uropygial Secretion.

The study of associations between symbiotic bacterial communities of hosts and those of surrounding environments would help to understand how bacterial assemblages are acquired, and how they are transmitted from one to another location (i.e. symbiotic bacteria acquisition by hosts). Hoopoes (Upupa epops) smear their eggshells with uropygial secretion (oily secretion produced in their uropygial gland) that harbors antibiotic producing bacteria. Trying to elucidate a possible role of nest material and cloaca microbiota in determining the bacterial community of the uropygial gland and the eggshells of hoopoes, we characterized bacterial communities of nest material, cloaca, uropygial gland and eggshells by the ARISA fingerprinting. Further, by adding material with scarce bacteria and antimicrobial properties, we manipulated the bacterial community of nest material and thus tested experimentally its effects on the microbiomes of the uropygial secretion and of the eggshells. The experiment did not influence the microbiome of the uropygial secretion of females, but affected the community established on eggshells. This is the first experimental evidence indicating that nest material influences the bacterial community of the eggshells and, therefore, probability of embryo infection. Some of the bacterial strains detected in the secretion were also in the bacterial communities of the nest material and of cloaca, but their occurrence within nests was not associated, which suggests that bacterial environments of nest material and cloaca are not sources of symbiotic bacteria for the gland. These results do not support a role of nest environments of hoopoes as reservoirs of symbiotic bacteria. We discuss possible scenarios explaining bacterial acquisition by hoopoes that should be further explored.

The Microbiome of the Uropygial Secretion in Hoopoes Is Shaped Along the Nesting Phase.

Microbial symbiont acquisition by hosts may determine the effectiveness of the mutualistic relationships. A mix of vertical and horizontal transmission may be advantageous for hosts by allowing plastic changes of microbial communities depending on environmental conditions. Plasticity is well known for gut microbiota but is poorly understood for other symbionts of wild animals. We here explore the importance of environmental conditions experienced by nestling hoopoes (Upupa epops) during the late nesting phase determining microbiota in their uropygial gland. In cross-fostering experiments of 8 days old nestlings, "sibling-sibling" and "mother-offspring" comparisons were used to explore whether the bacterial community naturally established in the uropygial gland of nestlings could change depending on experimental environmental conditions (i.e., new nest environment). We found that the final microbiome of nestlings was mainly explained by nest of origin. Moreover, cross-fostered nestlings were more similar to their siblings and mothers than to their stepsiblings and stepmothers. We also detected a significant effect of nest of rearing, suggesting that nestling hoopoes acquire most bacterial symbionts during the first days of life but that the microbiome is dynamic and can be modified along the nestling period depending on environmental conditions. Estimated effects of nest of rearing, but also most of those of nest of origin are associated to environmental characteristics of nests, which are extended phenotypes of parents. Thus, natural selection may favor the acquisition of appropriated microbial symbionts for particular environmental conditions found in nests.

Laying date, incubation and egg breakage as determinants of bacterial load on bird eggshells: experimental evidence.

Exploring factors guiding interactions of bacterial communities with animals has become of primary importance for ecologists and evolutionary biologists during the last years because of their likely central role in the evolution of animal life history traits. We explored the association between laying date and eggshell bacterial load (mesophilic bacteria, Enterobacteriaceae, Staphylococci, and Enterococci) in natural and artificial magpie (Pica pica) nests containing fresh commercial quail (Coturnix coturnix) eggs. We manipulated hygiene conditions by spilling egg contents on magpie and artificial nests and explored experimental effects during the breeding season. Egg breakage is a common outcome of brood parasitism by great spotted cuckoos (Clamator glandarius) on the nests of magpie, one of its main hosts. We found that the treatment increased eggshell bacterial load in artificial nests, but not in magpie nests with incubating females, which suggests that parental activity prevents the proliferation of bacteria on the eggshells in relation to egg breakage. Moreover, laying date was positively related to eggshell bacterial load in active magpie nests, but negatively in artificial nests. The results suggest that variation in parental characteristics of magpies rather than climatic variation during the breeding season explained the detected positive association. Because the eggshell bacterial load is a proxy of hatching success, the detected positive association between eggshell bacterial loads and laying date in natural, but not in artificial nests, suggests that the generalized negative association between laying date and avian breeding success can be, at least partially, explained by differential bacterial effects.