Tree Species Diversity and Forest Edge Density Jointly Shape the Gut Microbiota Composition in Juvenile Great Tits (Parus major).

Despite the microbiome's key role in health and fitness, little is known about the environmental factors shaping the gut microbiome of wild birds. With habitat fragmentation being recognised as a major threat to biological diversity, we here determined how forest structure influences the bacterial species richness and diversity of wild great tit nestlings (Parus major). Using an Illumina metabarcoding approach which amplifies the 16S bacterial ribosomal RNA gene, we measured gut microbiota diversity and composition from 49 great tit nestlings, originating from 23 different nests that were located in 22 different study plots across a gradient of forest fragmentation and tree species diversity. Per nest, an average microbiome was determined on which the influence of tree species (composition and richness) and forest fragmentation (fragment area and edge density) was examined and whether this was linked to host characteristics (body condition and fledging success). We found an interaction effect of edge density with tree species richness or composition on both the microbial richness (alpha diversity: Chao1 and Shannon) and community structure (beta diversity: weighted and unweighted UniFrac). No significant short-term impact was observed of the overall faecal microbiome on host characteristics, but rather an adverse effect of specific bacterial genera on fledging success. These results highlight the influence of environmental factors on the microbial richness as well as the phylogenetic diversity during a life stage where the birds' microbiota is shaped, which could lead to long-term consequences for host fitness.

Exploring the faecal microbiome of the Eurasian nuthatch (Sitta europaea).

Gastrointestinal microbiota fulfill pivotal roles in providing a host with nutrition and protection from pathogenic microorganisms. Up to date, most microbiota research has focused on humans and other mammals, whereas birds and especially wild birds lag behind. Within the field of the avian gut microbiome, research is heavily biased towards poultry. In this study, we analyzed the gut microbiome of the Eurasian nuthatch (Sitta europaea), using faecal samples of eight nestlings originating from three nuthatch nests in the south of Ghent (Belgium), using Illumina sequencing of the 16S rRNA gene. Relative frequency analysis showed a dominance of Firmicutes and Actinobacteria and to a lesser extent Proteobacteria. Bacteroidetes and other phyla were relatively rare. At higher taxonomic levels, a high degree of inter-individual variation in terms of overall microbiota community structure as well as dominance of certain bacteria was observed, but with a higher similarity for the nestlings sharing the same nest. When comparing the nuthatch faecal microbiome to that of great tit nestlings that were sampled during the same breeding season and in the same forest fragment, differences in the microbial community structure were observed, revealing distinct dissimilarities in the relative abundancy of taxa between the two bird species. This study is the first report on the nuthatch microbiome and serves as a reference study for nuthatch bacterial diversity and can be used for targeted screening of the composition and general functions of the avian gut microbiome.

Forest fragmentation modulates effects of tree species richness and composition on ecosystem multifunctionality.

Forest fragments in highly disturbed landscapes provide important ecosystem services ranging from acting as biodiversity reservoir to providing timber or regulating hydrology. Managing the tree species richness and composition of these fragments to optimize their functioning and the deliverance of multiple ecosystem services is of great practical relevance. However, both the strength and direction of tree species richness and tree species composition effects on forest ecosystem multifunctionality may depend on the landscape context in which these forest remnants are embedded. Taking advantage of an observatory network of 53 temperate forest plots varying in tree species richness, tree species composition, and fragmentation intensity we measured 24 ecosystem functions spanning multiple trophic levels and analyzed how tree species diversity-multifunctionality relationships changed with fragmentation intensity. Our results show that fragmentation generally increases multifunctionality and strengthens its positive relationship with diversity, possibly due to edge effects. In addition, different tree species combinations optimize functioning under different fragmentation levels. We conclude that management and restoration of forest fragments aimed at maximizing ecosystem multifunctionality should be tailored to the specific landscape context. As forest fragmentation will continue, tree diversity will become increasingly important to maintain forest functioning.

Mitigating the impact of microbial pressure on great (Parus major) and blue (Cyanistes caeruleus) tit hatching success through maternal immune investment.

The hatching success of a bird's egg is one of the key determinants of avian reproductive success, which may be compromised by microbial infections causing embryonic death. During incubation, outer eggshell bacterial communities pose a constant threat of pathogen translocation and embryo infection. One of the parental strategies to mitigate this threat is the incorporation of maternal immune factors into the egg albumen and yolk. It has been suggested that habitat changes like forest fragmentation can affect environmental factors and life-history traits that are linked to egg contamination. This study aims at investigating relationships between microbial pressure, immune investment and hatching success in two abundant forest bird species and analyzing to what extent these are driven by extrinsic (environmental) factors. We here compared (1) the bacterial load and composition on eggshells, (2) the level of immune defenses in eggs, and (3) the reproductive success between great (Parus major) and blue (Cyanistes caeruleus) tits in Belgium and examined if forest fragmentation affects these parameters. Analysis of 70 great tit and 34 blue tit eggshells revealed a similar microbiota composition (Enterobacteriaceae, Lactobacillus spp., Firmicutes and Bacteroidetes), but higher bacterial loads in great tits. Forest fragmentation was not identified as an important explanatory variable. Although a significant negative correlation between hatching success and bacterial load on the eggshells in great tits corroborates microbial pressure to be a driver of embryonic mortality, the overall hatching success was only marginally lower than in blue tits. This may be explained by the significantly higher levels of lysozyme and IgY in the eggs of great tits, protecting the embryo from increased infection pressure. Our results show that immune investment in eggs is suggested to be a species-specific adaptive trait that serves to protect hatchlings from pathogen pressure, which is not directly linked to habitat fragmentation.

Usutu Virus Epizootic and Plasmodium Coinfection in Eurasian Blackbirds ( Turdus merula) in Flanders, Belgium.

At the end of the summer of 2016, unusually high levels of mortality were detected in Passeriformes and Strigiformes in Flanders, Belgium, mainly in Eurasian Blackbirds ( Turdus merula). A passive surveillance program demonstrated a widespread Usutu virus outbreak and revealed a coinfection with Plasmodium in 99% of the dead passerine birds that were necropsied.

Investigation of Amphibian Mortality Events in Wildlife Reveals an On-Going Ranavirus Epidemic in the North of the Netherlands.

In the four years following the first detection of ranavirus (genus Ranavirus, family Iridoviridae) infection in Dutch wildlife in 2010, amphibian mortality events were investigated nationwide to detect, characterize and map ranaviruses in amphibians over time, and to establish the affected host species and the clinico-pathological presentation of the disease in these hosts. The ultimate goal was to obtain more insight into ranavirus disease emergence and ecological risk. In total 155 dead amphibians from 52 sites were submitted between 2011 and 2014, and examined using histopathology, immunohistochemistry, virus isolation and molecular genetic characterization. Ranavirus-associated amphibian mortality events occurred at 18 sites (35%), initially only in proximity of the 2010 index site. Specimens belonging to approximately half of the native amphibian species were infected, including the threatened Pelobates fuscus (spadefoot toad). Clustered massive outbreaks involving dead adult specimens and ranavirus genomic identity indicated that one common midwife toad virus (CMTV)-like ranavirus strain is emerging in provinces in the north of the Netherlands. Modelling based on the spatiotemporal pattern of spread showed a high probability that this emerging virus will continue to be detected at new sites (the discrete reproductive power of this outbreak is 0.35). Phylogenetically distinct CMTV-like ranaviruses were found in the south of the Netherlands more recently. In addition to showing that CMTV-like ranaviruses threaten wild amphibian populations not only in Spain but also in the Netherlands, the current spread and risk of establishment reiterate that understanding the underlying causes of CMTV-like ranavirus emergence requires international attention.

Concurrent ranavirus and Batrachochytrium dendrobatidis infection in captive frogs (Phyllobates and Dendrobates species), The Netherlands, 2012: a first report.

A ranavirus infection with concurrent Batrachochytrium dendrobatidis infection and mortality in captive Phyllobates and Dendrobates species is reported. Greyish skin with hepato- and reno-megaly were evident. Microscopically, Batrachochytrium dendrobatidis was present in the stratum corneum of the hyperkeratotic skin. Intracytoplasmic inclusion bodies were present in erythrocytes and multiple organs. All samples examined tested positive using PCR for the major capsid protein (MCP) gene of ranavirus and the ITS-1-5.8S region of B. dendrobatidis. The sequence obtained showed a 99% identity with the deposited sequence of the MCP gene of the common midwife toad virus (CMTV). This is the first report of mortality in captivity in poison dart frogs caused by a ranavirus, CMTV or like virus, and Batrachochytrium dendrobatidis infection.

Prevalence, whole genome characterization and phylogenetic analysis of hepatitis B virus in captive orangutan and gibbon.

BACKGROUND: Hepatitis B virus (HBV) is a public health problem worldwide and apart from infecting humans, HBV has been found in non-human primates. METHODS: We subjected 93 non-human primates comprising 12 species to ELISA screening for the serological markers HBsAg, antiHBs and antiHBc. Subsequently, we detected HBV DNA, sequenced the whole HBV genome and performed phylogenetic analysis. RESULTS: HBV infection was detected in gibbon (4/15) and orangutan (7/53). HBV DNA isolates from two gibbons and seven orangutans were chosen for complete genome amplification. We aligned the Pre-S/S, Pre-C/C and entire genomes with HBV sequences and performed phylogenetic analysis. The gibbon and orangutan viruses clustered within their respective groups. CONCLUSIONS: Both geographic location and host species influence which HBV variants are found in gibbons and orangutans. Hence, HBV transmission between humans and non-human primates might be a distinct possibility and additional studies will be required to further investigate this potential risk.