RNAscope in situ hybridization reveals microvascular sequestration of Plasmodium relictum pSGS1 blood stages but absence of exo-erythrocytic dormant stages during latent infection of Serinus canaria.

BACKGROUND: Birds chronically infected with avian malaria parasites often show relapses of parasitaemia after latent stages marked by absence of parasites in the peripheral circulation. These relapses are assumed to result from the activation of dormant exo-erythrocytic stages produced during secondary (post-erythrocytic) merogony of avian Plasmodium spp. Yet, there is no morphological proof of persistent or dormant tissue stages in the avian host during latent infections. This study investigated persistence of Plasmodium relictum pSGS1 in birds with latent infections during winter, with the goal to detect presumed persisting tissue stages using a highly sensitive RNAscope® in situ hybridization technology. METHODS: Fourteen domestic canaries were infected with P. relictum pSGS1 by blood-inoculation in spring, and blood films examined during the first 4 months post infection, and during winter and spring of the following year. After parasitaemia was no longer detectable, half of the birds were dissected, and tissue samples investigated for persisting tissue stages using RNAscope ISH and histology. The remaining birds were blood-checked and dissected after re-appearance of parasitaemia, and their tissues equally examined. RESULTS: Systematic examination of tissues showed no exo-erythrocytic stages in birds exhibiting latent infections by blood-film microscopy, indicating absence of dormant tissue stages in P. relictum pSGS1-infected canaries. Instead, RNAscope ISH revealed rare P. relictum blood stages in capillaries of various tissues and organs, demonstrating persistence of the parasites in the microvasculature. Birds examined after re-appearance of parasitemia showed higher numbers of P. relictum blood stages in both capillaries and larger blood vessels, indicating replication during early spring and re-appearance in the peripheral circulation. CONCLUSIONS: The findings suggest that persistence of P. relictum pSGS1 during latent infection is mediated by continuous low-level erythrocytic merogony and possibly tissue sequestration of infected blood cells. Re-appearance of parasitaemia in spring seems to result from increased erythrocytic merogony, therefore representing recrudescence and not relapse in blood-inoculated canaries. Further, the study highlights strengths and limitations of the RNAscope ISH technology for the detection of rare parasite stages in tissues, providing directions for future research on persistence and tissue sequestration of avian malaria and related haemosporidian parasites.

A survey of parasitic infections in Psittaciformes and Passeriformes in Mashhad, Iran.

The health, growth and fertility of avian species can be negatively affected by parasite infection. This survey assesses the presence, variety and distribution of internal and external parasites among parrots and perching birds in Mashhad, Iran. This study examined 751 caged pet birds from different species and regions in Mashhad for faecal samples and 132 oral swabs for digestive tract parasites. Furthermore, skin scrapings were conducted on 14 canaries displaying dishevelled feathers. During the study, mortalities and moribund birds that had been referred underwent necropsies to examine internal parasites. Following the formol ether faecal examination, only one Malango parrot tested positive for Heterakoidea eggs among 751 faecal samples (0.13%). Further, one cockatiel showed evidence of parasitic infection with Eimeria spp. (0.13%). However, neither Cryptosporidium nor Giardia protozoa were detected in the samples (0%). Oral swabs revealed no evidence of Trichomonas (0%). On the other hand, skin scraping revealed the presence of the mite Dermanyssus in 7 out of 14 canaries with dishevelled feathers (50%). Of 25 moribund and weak budgerigars, 2 were infected with Acuaria in their proventriculus (8%). In addition, 3 out of 14 deceased myna birds carried the nematode Diplotriana in their coelomic cavities (21.42%). In conclusion, the rate of internal parasites has been relatively low in ornamental birds of Mashhad, whereas the prevalence of external parasites has been higher.

Evaluation of Non-Vector Transmission of Usutu Virus in Domestic Canaries (Serinus canaria).

Usutu virus (USUV) is a flavivirus transmitted to avian species through mosquito bites that causes mass mortalities in wild and captive bird populations. However, several cases of positive dead birds have been recorded during the winter, a vector-free period. To explain how USUV "overwinters", the main hypothesis is bird-to-bird transmission, as shown for the closely related West Nile virus. To address this question, we experimentally challenged canaries with intranasal inoculation of USUV, which led to systemic dissemination of the virus, provided the inoculated dose was sufficient (>102 TCID50). We also highlighted the oronasal excretion of infectious viral particles in infected birds. Next, we co-housed infected birds with naive sentinels, to determine whether onward transmission could be reproduced experimentally. We failed to detect such transmission but demonstrated horizontal transmission by transferring sputum from an infected to a naive canary. In addition, we evaluated the cellular tropism of respiratory mucosa to USUV in vitro using a canary tracheal explant and observed only limited evidence of viral replication. Further research is then needed to assess if and how comparable bird-to-bird transmission occurs in the wild.

Integrated metabolome, full-length sequencing, and transcriptome analyses unveil the molecular mechanisms of color formation of the canary yellow and red bracts of Bougainvillea × buttiana 'Chitra'.

Bougainvillea is a typical tropical flower of great ornamental value due to its colorful bracts. The molecular mechanism behind color formation is not well-understood. Therefore, this research conducted metabolome analysis, transcriptome analysis, and multi-flux full-length sequencing in two color bracts of Bougainvillea × buttiana 'Chitra' to investigate the significantly different metabolites (SDMs) and differentially expressed genes (DEGs). Overall, 261 SDMs, including 62 flavonoids and 26 alkaloids, were detected, and flavonols and betalains were significantly differentially accumulated among the two bracts. Furthermore, the complete-length transcriptome of Bougainvillea × buttiana was also developed, which contained 512 493 non-redundant isoforms. Among them, 341 210 (66.58%) displayed multiple annotations in the KOG, GO, NR, KEGG, Pfam, Swissprot, and NT databases. RNA-seq findings revealed that 3610 DEGs were identified between two bracts. Co-expression analysis demonstrated that the DEGs and SDMs involved in flavonol metabolism (such as CHS, CHI, F3H, FLS, CYP75B1, kaempferol, and quercetin) and betacyanin metabolism (DODA, betanidin, and betacyanins) were the main contributors for the canary yellow and red bract formation, respectively. Further investigation revealed that several putative transcription factors (TFs) might interact with the promoters of the genes mentioned above. The expression profiles of the putative TFs displayed that they may positively and negatively regulate the structural genes' expression profiles. The data revealed a potential regulatory network between important genes, putative TFs, and metabolites in the flavonol and betacyanin biosynthesis of Bougainvillea × buttiana 'Chitra' bracts. These findings will serve as a rich genetic resource for future studies that could create new color bracts.

Sex differences in song syntax and syllable diversity in testosterone-induced songs of adult male and female canaries.

BACKGROUND: Behavioral sex differences are widespread in the animal world. These differences can be qualitative (i.e., behavior present in one sex but not the other, a true sex dimorphism) or quantitative (behavior is present at a higher rate or quality in one sex compared to the other). Singing in oscine songbirds is associated with both types of differences. In canaries, female rarely sing spontaneously but they can be induced to do so by treatments with steroids. Song in these females is, however, not fully masculinized and exhibits relatively subtle differences in quality as compared with male song. We analyzed here sex differences in syllable content and syllable use between singing male and female canaries. METHODS: Songs were recorded from three groups of castrated male and three groups of photoregressed female canaries that had received Silastic™ implants filled with testosterone (T), with T plus estradiol (E2), or left empty (control). After 6 weeks of hormone treatment, 30 songs were recorded from each of the 47 subjects. Songs were segmented and each syllable was annotated. Various metrics of syllable diversity were extracted and network analysis was employed to characterize syllable sequences. RESULTS: Male and female songs were characterized by marked sex differences related to syllable use. Compared to females, males had a larger syllable-type repertoire and their songs contained more syllable types. Network analysis of syllable sequences showed that males follow more fixed patterns of syllable transitions than females. Both sexes, however, produced song of the same duration containing the same number of syllables produced at similar rates (numbers per second). CONCLUSIONS: Under the influence of T, canaries of both sexes are able to produce generally similar vocalizations that nevertheless differ in specific ways. The development of song during ontogeny appears to be a very sophisticated process that is presumably based on genetic and endocrine mechanisms but also on specific learning processes. These data highlight the importance of detailed behavioral analyses to identify the many dimensions of a behavior that can differ between males and females.

Male canaries normally sing complex songs at high rate while females only rarely sing very simple songs. Testosterone induces active singing in both male and female canaries, but female song is still not fully masculinized by these treatments even if song duration does not differ between the sexes. We analyzed the syllable repertoire and the sequence of use for different syllables in canaries of both sexes treated with testosterone or testosterone supplemented with estradiol. Compared to females, males had a larger syllable-type repertoire and their songs contained more syllable types. Syllable transitions were also more fixed in males. Sex differences in adult singing of canaries are thus a complex mixture of differences that result from the different endocrine condition of males and females (and are thus partially reversed by administration of exogenous testosterone) and of more stable differences that presumably develop during the ontogenetic process under the influence of endocrine and genetic differences and of differential learning processes. Canary song thus represents an outstanding model system to analyze the interaction between nature and nurture in the acquisition of a sophisticated learned behavior as well as the mechanisms controlling sex differences in vocal learning and production.

Attitudes, capacities and perceived barriers in research of nurses of the Canary Health Service.

OBJECTIVE: To evaluate the attitudes, capacities and perceived barriers associated with research among nurses and midwives of the Canary Health Service (SCS). METHODS: Descriptive observational cross-sectional study with an analytical component carried out in the different SCS departments by means of an online survey in which sociodemographic and specific variables, the Spanish version of the Attitudes towards Research and Development within Nursing Questionnaire (ATRDNQ-e) instrument and the BARRIERS scale were collected. Authorisation was obtained from the two provincial ethics committees. A descriptive and inferential analysis (Mann-Whitney U test and Kruskal-Wallis test, post hoc contrast by Dwass-Steel-Critchlow-Fligne test) was performed with JAMOVI® v.2.3.24 software. RESULTS: A total of 512 nurses and midwives with a mean age of 41.82 years participated in the study. Regarding the scores with the ATRDNQ-e instrument, the dimension with the lowest score was «Language of research¼ (mean = 3.55/SD = 0.84) and the highest «Assessment of nursing research and development of the nursing discipline¼ (mean = 4.54/SD = 0.52). The total mean score with the BARRIERS scale was 54.33 (SD = 16.52), with «Organizational characteristics¼ being the highest scoring subscale (mean = 17.25/SD = 5.90). The two highest perceived barriers were «Not enough time at work to implement new ideas¼ (mean = 2.55/SD = 1.11) and «Nursing does not have time to read research¼ (mean = 2.46/ SD = 1.11). CONCLUSIONS: SCS nurses have a positive attitude towards research, although there are some barriers where improvement actions for nursing research should be implemented.

Effects of the depletion of neural progenitors by focal X-ray irradiation on song production and perception in canaries.

The song control nucleus HVC of songbirds has emerged as a widespread model system to study adult neurogenesis and the factors that modulate the incorporation of new neurons, including seasonal state, sex differences or sex steroid hormone concentrations. However, the specific function of these new neurons born in adulthood remains poorly understood. We implemented a new procedure based on focal X-ray irradiation to deplete neural progenitors in the ventricular zone adjacent to HVC and study the functional consequences. A 23 Gy dose depleted by more than 50 percent the incorporation of BrdU in neural progenitors, a depletion that was confirmed by a significant decrease in doublecortin positive neurons. This depletion of neurogenesis significantly increased the variability of testosterone-induced songs in females and decreased their bandwidth. Expression of the immediate early gene ZENK in secondary auditory areas of the telencephalon that respond to song was also inhibited. These data provide evidence that new neurons in HVC play a role in both song production and perception and that X-ray focal irradiation represents an excellent tool to advance our understanding of adult neurogenesis.

Does the syrinx, a peripheral structure, constrain effects of sex steroids on behavioral sex reversal in adult canaries?

We previously confirmed that effects of testosterone (T) on singing activity and on the volume of brain song control nuclei are sexually differentiated in adult canaries: females are limited in their ability to respond to T as males do. Here we expand on these results by focusing on sex differences in the production and performance of trills, i.e., rapid repetitions of song elements. We analyzed >42,000 trills recorded over a period of 6 weeks from 3 groups of castrated males and 3 groups of photoregressed females that received Silastic™ implants filled with T, T plus estradiol or left empty as control. Effects of T on the number of trills, trill duration and percent of time spent trilling were all stronger in males than females. Irrespective of endocrine treatment, trill performance assessed by vocal deviations from the trill rate versus trill bandwidth trade-off was also higher in males than in females. Finally, inter-individual differences in syrinx mass were positively correlated with specific features of trills in males but not in females. Given that T increases syrinx mass and syrinx fiber diameter in males but not in females, these data indicate that sex differences in trilling behavior are related to sex differences in syrinx mass and syrinx muscle fiber diameter that cannot be fully suppressed by sex steroids in adulthood. Sexual differentiation of behavior thus reflects organization not only of the brain but also of peripheral structures.

Chemical 'canaries': Munitions workers in the First World War.

In the early twentieth century, scientific innovations permanently changed international warfare. As chemicals traveled out of laboratories into factories and military locations, war became waged at home as well as overseas. Large numbers of women were employed in munitions factories during the First World War, but their public memories have been overshadowed by men who died on battlefields abroad; they have also been ignored in traditional histories of chemistry that focus on laboratory-based research. Mostly young and poorly educated, but crucial for Britain's military success, these female workers were subjected to procedures of social regulation and consigned to carrying out dangerous chemical procedures causing chronic illness or death; in particular, when TNT died their skin yellow, they were colloquially known as 'canaries.'

Epidemiology of canine cutaneous round cell tumours on the canary archipelago in Spain.

In this study we undertook a comprehensive analysis of a Pet Tumour Registry of the Canary Archipelago (PTR-CA) in Spain to investigate the epidemiology of canine cutaneous round cell tumours. From a database of 2526 tumours collected from 2003 to 2020, we conducted a longitudinal analysis of the main trends in diagnosis, age, multiplicity and anatomical distribution as well as a case-control study comparing these cases with the contemporaneous canine population of the Canary Archipelago to analyse breed distribution. In line with former studies, we found histiocytomas mostly affect young dogs (2, IQR 1-5) and mast cell tumours affect middle-to-old dogs (8, IQR 6-10) with grade 1 affecting at younger ages (6.5, IQR 6-8) than both grade 2 (8, IQR 6-10 years) and grade 3 (9, IQR 7-11). Histiocytomas and plasmacytomas showed a similar anatomical distribution appearing mainly on the face, head and neck regions while mast cell tumours occur mainly on limbs and trunk. Higher risk for mast cell tumours and histiocytomas were found for Bulldog-related breeds such as Boxer (ORMCT = 23.61, CI95%: 19.12-29.15, ORHCT = 10.17, CI95%: 6.60-15.67), Boston Terrier (ORMCT 19.47, CI95%: 7.73-49.05, ORHCT 32.61, CI95%: 11.81-90.07) and Pug (ORMCT 8.10, CI95%: 5.92-11.07, ORHCT 7.87, CI95%: 4.66-13.28) while Chihuahua dogs showed significantly less risk (ORMCT 0.18, CI95%: 0.09-0.33, ORHCT 0.41, CI95%: 0.21-0.78). Notably, the Canarian Mastiff, a local breed, had a low risk of suffering from a mast cell tumour which raises the question of whether this relates to a genetic peculiarity of this breed or some husbandry and environmental factor.

Whole genome sequencing identifies candidate genes and mutations that can explain diluted and other colour varieties of domestic canaries (Serinus canaria).

The domestic canary (Serinus canaria) is one of the most common pet birds and has been extensively selected and bred over the last few centuries to constitute many different varieties. Plumage pigmentation is one of the main phenotypic traits that distinguish canary breeds and lines. Feather colours in these birds, similarly to other avian species, are mainly depended on the presence of two major types of pigments: carotenoids and melanins. In this study, we exploited whole genome sequencing (WGS) datasets produced from five canary lines or populations (Black Frosted Yellow, Opal, Onyx, Opal × Onyx and Mogno, some of which carrying different putative dilute alleles), complemented with other WGS datasets retrieved from previous studies, to identify candidate genes that might explain pigmentation variability across canary breeds and varieties. Sequencing data were obtained using a DNA pool-seq approach and genomic data were compared using window-based FST analyses. We identified signatures of selection in genomic regions harbouring genes involved in carotenoid-derived pigmentation variants (CYP2J19, EDC, BCO2 and SCARB1), confirming the results reported by previous works, and identified several other signatures of selection in the correspondence of melanogenesis-related genes (AGRP, ASIP, DCT, EDNRB, KITLG, MITF, MLPH, SLC45A2, TYRP1 and ZEB2). Two putative causative mutations were identified in the MLPH gene that may explain the Opal and Onyx dilute mutant alleles. Other signatures of selection were also identified that might explain additional phenotypic differences between the investigated canary populations.

Bornaviral infections in Atlantic canaries (Serinus canaria) in Poland.

The presence of canary bornavirus (Orthobornavirus serini) genetic material was tested in organ samples from 157 Atlantic canaries (Serinus canaria) and four hybrids of Atlantic canary and European goldfinch (Carduelis carduelis). The subjects of the research were samples collected in the years 2006-2022. A positive result was obtained in 16 canaries and one hybrid (10.5%). Eleven positive canaries had neurological signs prior to death. Four of them also had atrophic changes in the forebrain, which have not previously been described in canaries and other species of birds infected with avian bornavirus. In one canary, computed tomography without contrast was performed. This study showed no changes, despite advanced forebrain atrophy found on post-mortem examination of the bird. The organs of the studied birds were also tested with PCR tests for the presence of polyomaviruses and circoviruses. There was no correlation between the bornavirus infection and the presence of the other two viruses in the tested canaries.RESEARCH HIGHLIGHTS The incidence of bornaviral infections in canaries in Poland is relatively low.Non-contrast CT is not a useful method for brain atrophy diagnostic in canaries.Neurological signs were found in the majority of birds infected with bornaviruses.Visceral ganglioneuritis was found in a minority of birds infected with bornaviruses.

Identification of chromosome region and candidate genes for canary-yellow flesh (Cyf) locus in watermelon (Citrullus lanatus).

Genetic control of fruit flesh color in watermelon is complex, and significant knowledge gaps still exist. In the present study, we investigated the genetic basis of canary-yellow flesh color in watermelon inbred line PI 635597 using a segregating population derived from a cross between PI 635597 and another inbred line, Cream of Saskatchewan (pale yellow flesh color). We showed that a single dominant gene controls the canary-yellow flesh color for the Cyf (canary-yellow flesh) trait. Bulk segregant analysis (BSA) and fine genetic mapping narrowed down the Cyf locus to a 79.62-kb region on chromosome 6, which harbors 10 predicted genes. Sequence variation analysis in the promoter and coding regions and gene expression analysis in both parental lines and selected watermelon accessions with diverse fruit flesh colors support Cla97C06G122050 (unknown protein) and Cla97C06G122120 (pentatricopeptide repeat) as predicted candidate genes for the Cyf locus. Marker-assisted selection and sequence alignment showed that the Cyf locus could differentiate canary-yellow flesh and pale-yellow flesh. Our results indicate that the Cyf locus might be responsible for canary-yellow flesh color and carotenoid accumulation levels.

Integrative taxonomy for the traditional coccidians (Chromista: Miozoa: Eimeriidae) from island canaries (Aves: Passeriformes: Fringillidae): Worldwide distribution, morphological and molecular characterization, revaluations and establishment of junior synonyms.

Island canaries Serinus canaria (Linnaeus) are finches native to the North Atlantic Islands, however, they have a worldwide distribution in captivity due to their relevance as a pet bird. Coccidians are the most reported parasites of passerines worldwide, both in the wild and in captivity, being frequently associated with disease in passerines kept in rehabilitation centers and commercial breeders. This study aimed to identify coccidians from island canaries kept in captivity in Brazil. Three hundred and fifteen genomic DNA extracted from fecal samples of island canaries from different breeders from Southern and Southeastern Brazil were used to perform a nested PCR assay to amplify a partial fragment of the 28S small subunit ribosomal RNA gene (28S) of Isospora spp. Microscopic screening and morphological identification of Isospora oocysts was performed in fecal samples corresponding to PCR positive DNA samples. Fecal samples have been formalin-stored for approximately four years. Positivity rate for both microscopy and PCR was 10.5% (33/315). Posteriorly, Isospora serini (Aragão, 1933) Box, 1975 and Isospora canaria Box, 1975 were morphologically identified from fresh fecal samples of island canaries maintained by a breeder in the State of São Paulo, Southeastern Brazil, providing a genotypic characterization via sequencing of the mitochondrial cytochrome c oxidase subunit 1 (COI) and 28S genes. The 28S and COI sequences referring to the morphological identification of I. canaria was, respectively, 100% and 99% similar to sequences deposited as Isospora serinuse Yang, Brice, Elliot & Ryan, 2015 from island canaries kept in a rehabilitation center in Australia. The COI sequence referring to the morphological identification of I. serini was 100% similar to a sequence of an extraintestinal Isospora, corroborating this identification/sequencing since I. serini is the first isosporan with an extra-intestinal cycle demonstrated. The comparison of morphological and molecular data from I. canaria and I. serini from this study with published data of Isospora spp. from canaries worldwide, allowed the specific identification from preliminary generic identifications, correction of misidentifications, as well as the establishment of junior synonyms. Finally, this study provides morphological and molecular data that ensure the correct identification of the two Isospora spp. from island canaries in future studies worldwide.

Prosociality and reciprocity according to parental status, communication, and personality in domestic canaries (Serinus canaria).

Prosociality (behaviours that benefit to a recipient without necessarily involving a cost to the actor) has recently been shown to exist in various taxa, including birds. Studies on prosociality in primates found that prosocial tendencies of the subject could be related to sex and parental care, communication from the recipient, cognitive abilities and personality. To investigate the existence of such associations on birds, we conducted a Prosocial Choice Task with domestic canaries (Serinus canaria). In our experiment, the subject could choose between three options: a prosocial, a selfish, and a null option (with no cost for the subject). We also conducted a food sharing experiment and measured several personality traits. Our results highlighted high levels of prosociality and a tendency to reciprocity among reproductive mates. We found a higher propensity to be prosocial in nulliparous individuals than in individuals that have previously been parents, but better sharing abilities in parents than in nulliparous individuals. When they were recipient, parents also used communication more efficiently than nulliparous subjects. Data suggest that parental expertise could enhance subjects' skills in eliciting prosociality. We also highlighted some interaction between prosociality, learning abilities, and some personality traits, proactive individuals being fast learners and more prosocial, while reactive individuals being slow learners and more reciprocal. Our results suggest that prosociality and reciprocity could be linked to personality and cognitive abilities, and that it might be interesting to consider them as parts of individual's cognitive style.

Canary Bornavirus (Orthobornavirus serini) Infections Are Associated with Clinical Symptoms in Common Canaries (Serinus canaria dom.).

While parrot bornaviruses are accepted as the cause of proventricular dilatation disease (PDD) in psittacine birds, the pathogenic role of bornaviruses in common canaries is still unclear. To answer the question of whether canary bornaviruses (species Orthobornavirus serini) are associated with a PDD-like disease in common canaries (Serinus canaria f. dom.), the clinical data of 201 canary bird patients tested for bornaviruses using RT-PCR assays, were analyzed for the presence of PDD-like gastrointestinal or central nervous system signs and for other viruses (mainly circovirus and polyomavirus), yeasts and trichomonads. Canary bornavirus RNA was detected in the clinical samples of 40 out of 201 canaries (19.9%) coming from 28 of 140 flocks (20%). All nucleotide sequences obtained could unequivocally be determined as canary bornavirus 1, 2, or 3 supporting the current taxonomy of the species Orthobornavirus serini. PDD-like signs were found associated with canary bornavirus detection, and to a lesser extent, with circoviruses detection, but not with the detection of polyomaviruses, yeasts or trichomonads. The data indicate that canary bornaviruses contribute to a PDD-like disease in naturally infected canaries, and suggest a promoting effect of circoviruses for the development of PDD-like signs.