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Analysis of ultrastructure and microstructure of blackbird (Turdus merula) and song thrush (Turdus philomelos) eggshell by scanning electron microscopy and X-ray computed microtomography.

ABSTRACT: The unique structure of the egg allows for efficient reproduction on land. Although the functions of the egg are ensured by the concomitant cooperation of all its structures, the eggshell also plays a significant role. Apart from maintaining an aqueous environment within the egg along with controlled gas exchange, the color and pigmentation pattern of eggshell contributes to identification and protection. As a result of all these functions, the structure, shape, and pigmentation of eggshell greatly vary across the class of birds, and understanding these three variability-determining factors may aid in better interpretation of evolutionary mechanisms. In this study, we analyzed for the first time the structure, mineral composition, and characteristics of the pigmentation of blackbird (Turdus merula) and song thrush (Turdus philomelos) eggshells. The shell of blackbird eggs is much thicker compared to the shell of song thrush eggs which is due to a much thicker crystalline and palisade layers. In both species, strongly elongated mammillary knobs are observed, which create a large space between the mineralized shell and the egg membranes. The blackbird egg shell has a higher water vapor conductivity which is due to the larger diameter of the circle and the surface area of individual pores. The primary compound entering the mineral composition of the shell in both species is CaCO₃ however, the thrush egg shells contained more Mg in all layers except the crystalline layer, and S in the crystalline and palisade layers. The two species clearly differ in the size and distribution of pigment spots on the eggshell. We suppose that the differences in shell structure and pigmentation presented in this study may in the future provide a basis for explaining the reasons for the much lower reproductive efficiency of song thrush compared to blackbird.

SUBMITTER: Damaziak K

PROVIDER: S-EPMC9279409 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background:In recent years, next generation high throughput sequencing technologies have proven to be useful tools for investigations concerning the genomics or transcriptomics also of non-model species. Consequently, ornithologists have adopted these technologies and the respective bioinformatics tools to survey the genomes and transcriptomes of a few avian non-model species. The Common Blackbird is one of the most common bird species living in European cities, which has successfully colonized urban areas and for which no reference genome or transcriptome is publicly available. However, to target questions like genome wide gene expression analysis, a reference genome or transcriptome is needed. Methods:Therefore, in this study two Common Blackbirds were sacrificed, their mRNA was isolated and analyzed by RNA-Seq to de novo assemble a transcriptome and characterize it. Illumina reads (125 bp paired-end) and a Velvet/Oases pipeline led to 162,158 transcripts. For the annotation (using Blast+), an unfiltered protein database was used. SNPs were identified using SAMtools and BCFtools. Furthermore, mRNA from three single tissues (brain, heart and liver) of the same two Common Blackbirds were sequenced by Illumina (75 bp single-end reads). The draft transcriptome and the three single tissues were compared by their BLAST hits with the package VennDiagram in R. Results:Following the annotation against protein databases, we found evidence for 15,580 genes in the transcriptome (all well characterized hits after annotation). On 18% of the assembled transcripts, 144,742 SNPs were identified which are, consequently, 0.09% of all nucleotides in the assembled transcriptome. In the transcriptome and in the single tissues (brain, heart and liver), 10,182 shared genes were found. Discussion:Using a next-generation technology and bioinformatics tools, we made a first step towards the genomic investigation of the Common Blackbird. The de novo assembled transcriptome is usable for downstream analyses such as differential gene expression analysis and SNP identification. This study shows the importance of the approach to sequence single tissues to understand functions of tissues, proteins and the phenotype.

Project description:BACKGROUND:Passerine birds are frequently infected with diverse haemosporidian parasites. While infections are traditionally considered benign in wild birds, recent studies demonstrated mortalities of passerine species due to exo-erythrocytic development of the parasites, which can damage organs in affected hosts. However, exo-erythrocytic development remains insufficiently investigated for most haemosporidian species and thus little is known about the virulence of tissue stages in wild passerine birds. The aim of the present study was to investigate natural haemosporidian infections in deceased Eurasian blackbirds (Turdus merula) and song thrushes (Turdus philomelos) and to determine parasite burden and associated histological effects. METHODS:For molecular analysis, blood and tissue samples from 306 thrushes were screened for Plasmodium, Haemoproteus and Leucocytozoon parasites by nested PCR. For the detection of parasite stages in organ samples, tissue sections were subjected to chromogenic in situ hybridization (CISH) using genus- and species-specific probes targeting the rRNAs of parasites. Exo-erythrocytic parasite burden was semi-quantitatively assessed and histological lesions were evaluated in haematoxylin-eosin-stained sections. RESULTS:By PCR, 179 of 277 Eurasian blackbirds and 15 of 29 song thrushes were positive for haemosporidians. Parasites of all three genera were detected, with Plasmodium matutinum LINN1 and Plasmodium vaughani SYAT05 showing the highest prevalence. CISH revealed significant differences in exo-erythrocytic parasite burden between lineages in Eurasian blackbirds, with P. matutinum LINN1 frequently causing high exo-erythrocytic parasite burdens in various organs that were associated with histological alterations. Song thrushes infected with P. matutinum LINN1 and birds infected with other haemosporidian lineages showed mostly low exo-erythrocytic parasite burdens. Two Eurasian blackbirds infected with Leucocytozoon sp. TUMER01 showed megalomeronts in various organs that were associated with inflammatory reactions and necroses. CONCLUSION:This study suggests that P. matutinum LINN1, a common lineage among native thrushes, regularly causes high exo-erythrocytic parasite burdens in Eurasian blackbirds, which may result in disease and mortalities, indicating its high pathogenic potential. The findings further illustrate that the same parasite lineage may show different levels of virulence in related bird species which should be considered when assessing the pathogenicity of haemosporidian parasite species. Finally, the study provides evidence of virulent Leucocytozoon sp. TUMER01 infections in two Eurasian blackbirds caused by megalomeront formation.

Project description:Necropsy of two free-ranging common blackbirds (Turdus merula) found dead in central Italy revealed the presence of a high number of cyclocoelid flukes in the coelomatic cavity. Cyclocoelid flukes primarily infect avian respiratory system. Histologically, air sac walls were covered with a fibrinous exudate containing degenerate heterophils, many trematodes and some colonies of Gram-positive cocci. The superficial bronchi and parabronchi were markedly distended, and the adjacent pulmonary parenchyma was congested and collapsed. Trematodes, surrounded by a mild suppurative to pyogranulomatous inflammatory reaction, were also observed on the pericardial, intestinal, kidney and hepatic serosal surfaces. The death of the two examined birds was likely due to the high parasite load and associated severe lesions. At parasitological examination, flukes showed a tongue-shaped elongate body, tapered anteriorly and rounded posteriorly, of 2,088-2,314??m in width and 8,268-11,830??m in length. The mouth was slightly oval and sub-terminal, with a small oral sucker. The oval pharynx measured 250-309??m, and the two caeca joined posteriorly. Two large (550-702??m?×?450-520??m) globular testes were situated obliquely to each other, whereas an oval (250?×?300??m in mean) or round (about 334??m in diameter) intertesticular ovary was placed in a longitudinal straight line with the testes. The ootype was about 110??m in diameter, while the brown-yellow eggs measured 131.5?×?73.9??m in mean. The genital pore was post-pharyngeal, while the symmetrically arranged vitelline glands were not confluent posteriorly. Morphoflogical diagnosis led to the identification of Morishitium polonicum, a cyclocoelid fluke species that typically inhabits the air sacs of blackbirds. The morphological diagnosis was corroborated by molecular phylogenetic analysis of the mitochondrial (CO1, ND1) DNA loci. The present study provides the first report of pathological lesions and death caused by M. polonicum in birds.

Project description:Leucocytozoon parasites (Haemosporida, Leucocytozoidae) are haemosporidians whose diversity, exo-erythrocytic development and potential vectors are the least studied. The knowledge about their exo-erythrocytic development and pathogenicity is fragmentary, resulting in an incomplete comprehension of the impact of these parasites on avian hosts. For a long time, Leucocytozoon infections were considered benign to wild birds, even though they were virulent in poultry and responsible for some wild bird population declines. This study aimed to investigate the presence of Leucocytozoon species exo-erythrocytic stages in song thrushes Turdus philomelos using conventional histological techniques (sections stained by H&E) and chromogenic in situ hybridization (CISH). Tissues from ten birds (seven naturally infected and three opportunistic samplings) were examined using both methods. Parasite lineages were identified from blood samples using PCR-based techniques. Leucocytozoon species meronts were found in five individuals (in four birds using H&E staining protocol, and in three in CISH-treated histological sections). Meronts were found mainly in the kidneys, but some meronts were also present in the lungs. It was possible to observe different maturation stages of meronts in the same bird individual, indicating an asynchronous development. Cytomeres were readily visible in developing meronts. One megalomeront-like structure was present close to a blood vessel in the heart. It was covered with a prominent capsular-like wall. No inflammatory reaction or necrosis was seen in the tissues surrounding the meronts or the megalomeront-like structure. We could confirm the transmission of three Leucocytozoon lineages (lTUPHI14, lSTUR1 and lTUPHI13) in Europe, and add evidence of the transmission of two Plasmodium lineages, including Plasmodium circumflexum (pTURDUS1), and Haemoproteus asymmetricus (hTUPHI01). We call for further research to better understand Leucocytozoon parasite exo-erythrocytic development.

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Analysis of ultrastructure and microstructure of blackbird (Turdus merula) and song thrush (Turdus philomelos) eggshell by scanning electron microscopy and X-ray computed microtomography.

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