Trichobilharzia Species Research Articles

Molecular identification of Trichobilharzia species in recreational waters in North-Eastern Poland.

In Europe, avian schistosomes of the genus Trichobilharzia are the most common etiological agents involved in human cercarial dermatitis (swimmer's itch). Manifested by a skin rash, the condition is caused by an allergic reaction to cercariae of nonhuman schistosomes. Humans are an accidental host in this parasite's life cycle, while water snails are the intermediate, and waterfowl are the final hosts. The study aimed to conduct a molecular and phylogenetic analysis of Trichobilharzia species occurring in recreational waters in North-Eastern Poland. The study area covered three water bodies (Lake Skanda, Lake Ukiel, and Lake Tyrsko) over the summer of 2021. In total, 747 pulmonate freshwater snails (Radix spp., Lymnaea stagnalis) were collected. Each snail was subjected to 1-2 h of light stimulation to induce cercarial expulsion. The phylogenetic analyses of furcocercariae were based on the partial sequence of the ITS region (ITS1, 5.8S rDNA, ITS2 and 28SrDNA). For Radix spp. phylogenetic analyses were based on the ITS-2 region. The prevalence of the Trichobilharzia species infection in snails was 0.5%. Two out of 478 (0.4%) L. stagnaliswere found to be infected with Trichobilharzia szidati. Moreover, two out of 269 (0.7%) snails of the genus Radix were positive for schistosome cercariae. Both snails were identified as Radix auricularia. One of them was infected with Trichobilharzia franki and the other with Trichobilharzia sp. Molecular identification of avian schistosome species, both at the intermediate and definitive hosts level, constitutes an important source of information on a potential threat and prognosis of local swimmer's itch occurrence, and helps to determine species diversity in a particular area.

DNA Barcoding of Trichobilharzia (Trematoda: Schistosomatidae) Species and Their Detection in eDNA Water Samples

We designed and tested species-specific PCR primers to detect Trichobilharzia species via environmental DNA (eDNA) barcoding in selected Austrian water bodies. Tests were performed with eDNA samples from the field as well as with artificial samples from the lab, where snails releasing cercariae were kept in aquariums. From two localities, Trichobilharzia was documented based on the release of cercariae from snails, enabling morphological species identification. In both cases, the corresponding species were detected via eDNA: Trichobilharzia szidati and Trichobilharzia physellae. Nonetheless, the stochasticity was high in the replicates. PCR tests with aquarium water into which the cercariae had been released allowed eDNA detection even after 44 days. As in the PCRs with eDNA samples from the field, positive results of these experiments were not obtained for all samples and replicates. PCR sensitivity tests with dilution series of T. szidati genomic DNA as well as of PCR amplification products yielded successful amplification down to concentrations of 0.83 pg/µL and 0.008 pg/µL, respectively. Our results indicate that the presumed species specificity of PCR primers may not be guaranteed, even if primers were designed for specific species. This entails misidentification risks, particularly in areas with incomplete species inventories.

Diversity of Trichobilharzia in New Zealand with a new species and a redescription, and their likely contribution to cercarial dermatitis.

In response to annual outbreaks of human cercarial dermatitis (HCD) in Lake Wanaka, New Zealand, ducks and snails were collected and screened for avian schistosomes. During the survey from 2009 to 2017, four species of Trichobilharzia were recovered. Specimens were examined both morphologically and genetically. Trichobilharzia querquedulae, a species known from four continents, was found in the visceral veins of the duck Spatula rhynchotis but the snail host remains unknown. Cercaria longicauda [i.e. Trichobilharzia longicauda (Macfarlane, 1944) Davis, 2006], considered the major aetiological agent of HCD in Lake Wanaka, was discovered, and redescribed from adults in the visceral veins of the duck Aythya novaeseelandiae and cercariae from the snail Austropeplea tomentosa. Recovered from the nasal mucosa of Ay. novaeseelandiae is a new species of Trichobilharzia that was also found to cycle naturally through Au. tomentosa. Cercariae of a fourth species of Trichobilharzia were found in Au. tomentosa but the species remains unidentified.

Molecular evidence for distinct modes of nutrient acquisition between visceral and neurotropic schistosomes of birds

Trichobilharzia species are parasitic flatworms (called schistosomes or flukes) that cause important diseases in birds and humans, but very little is known about their molecular biology. Here, using a transcriptomics-bioinformatics-based approach, we explored molecular aspects pertaining to the nutritional requirements of Trichobilharzia szidati (‘visceral fluke’) and T. regenti (‘neurotropic fluke’) in their avian host. We studied the larvae of each species before they enter (cercariae) and as they migrate (schistosomules) through distinct tissues in their avian (duck) host. Cercariae of both species were enriched for pathways or molecules associated predominantly with carbohydrate metabolism, oxidative phosphorylation and translation of proteins linked to ribosome biogenesis, exosome production and/or lipid biogenesis. Schistosomules of both species were enriched for pathways or molecules associated with processes including signal transduction, cell turnover and motility, DNA replication and repair, molecular transport and/or catabolism. Comparative informatic analyses identified molecular repertoires (within, e.g., peptidases and secretory proteins) in schistosomules that can broadly degrade macromolecules in both T. szidati and T. regenti, and others that are tailored to each species to selectively acquire nutrients from particular tissues through which it migrates. Thus, this study provides molecular evidence for distinct modes of nutrient acquisition between the visceral and neurotropic flukes of birds.

Genetic diversity of an avian nasal schistosome causing cercarial dermatitis in the Black Sea-Mediterranean migratory route.

This study is part of an effort to document the diversity of avian schistosomes in ducks and snails in Northern Iran, a major flyway (Black Sea/Mediterranean) for migratory birds and where cercarial dermatitis (CD) is prevalent in rice growing areas. CD is an allergic skin reaction from schistosome trematodes that emerge from aquatic snails. Most CD cases are reported from recreational swimmers or aquaculture farmers. Much of the work on the epidemiology of CD has focused in recreational waters in the Americas and Europe, with fewer studies in aquaculture, particularly in Iran. The artificial environment at aquaculture sites support dense populations of snails that are hosts to schistosomes, as well as domestic ducks. Thus, are domestic ducks reservoir hosts of species of Trichobilharzia, one of the main etiological agents of CD in Northern Iran? This study focused on a survey of domestic ducks for the presence of the nasal schistosome, T. regenti, that has been reported widely in Europe. Trichobilharzia regenti were found in domestic ducks in the Guilan Province of Iran based on morphological and molecular analyses. The presence of this species in Northern Iran indicates that the domestic duck can serve as a reservoir host for this species and that one of the local snail species is likely the intermediate host. The continued study and surveillance of this species is important because it is a neuropathic schistosome that can use a diversity of bird definitive hosts and Radix snails that are widespread across Eurasia.

Phylogenetic analysis of nasal avian schistosomes (Trichobilharzia) from aquatic birds in Mazandaran Province, northern Iran

Nasal schistosomes are trematodes in the family Schistosomatidae, many members of which are causative agents of human cercarial dermatitis (HCD). Little is known about the species diversity and distribution of nasal dwelling schistosomes of water birds, particularly in countries outside of Europe; even less is known in countries like Iran. Nasal schistosomes are of particular interest since these species migrate via the central nervous system to the nasal cavity once they penetrate their host. Thus, there must be efforts to determine the incidence of HCD due to nasal schistosomes. HCD outbreaks are reported seasonally in Mazandaran Province, northern Iran, an area well known for rice cultivation leading to increased person contact with water and infected snails. Such places include favorable habitat for both domestic ducks year round, and wild migratory ducks in the winter through spring. Recent reports have detected the presence of both nasal and visceral schistosomes in ducks in this area but with little species characterization. In this study, we examine a diversity of aquatic birds to determine the distribution, prevalence and bird host use of nasal schistosomes. We apply for the first time a molecular identification and phylogenetic analysis of these schistosomes. From 2012 to 2014, the nasal cavity of 508 aquatic birds from Mazandaran Province were examined that included species in Anseriformes, Gruiformes, Charadriiformes and Phoenicopteriformes. Nasal schistosomes were found in 45 (8.9%) birds belonging to Anseriformes (Anas platyrhynchos and Anas clypeata). Phylogenetic analysis of the nuclear internal transcribed spacer 1 rDNA and the mitochondrial cytochrome oxidase1 gene of isolated eggs revealed that all samples grouped in a sister clade to the European Trichobilharzia regenti. However, Trichobilharzia from this study were more similar to a unique haplotype of Trichobilharzia, isolated from the nasals of an A. clypeata in France. The genetic and phenotypic differences between the species found herein and T. regenti from Europe, may prove with additional data to be a distinct species of Trichobilharzia.

Physa marmorata (Mollusca: Physidae) as a natural intermediate host of Trichobilharzia (Trematoda: Schistosomatidae), a potential causative agent of avian cercarial dermatitis in Brazil

Species of Trichobilharzia are the main etiological agents of cercarial dermatitis in humans, which is considered a re-emerging disease. Despite the diversity and global distribution of species of this genus, studies of Trichobilharzia are scarce in South America. The goal of our investigations is better understand the diversity, distribution and life cycle of avian schistosomes and their likely role in causing dermatitis in Brazil. As part of this effort, cercariae found in naturally infected Physa marmorata were identified by morphological and molecular (mitochondrial cox1, nuclear ITS1 and 28S gene regions) methods as Trichobilharzia sp. These cercariae are similar morphologically to T. jequitibaensis described previously from Brazil and similar genetically to the North American physid transmitted species T. querquedulae and T. physellae. This is the first report of a potential agent of cercarial dermatitis from naturally infected snails from Brazil and first molecular characterization of a South American species of Trichobilharzia. A discussion follows concerning the potential role of this species has in outbreaks of dermatitis in Brazil.

Identification of a major causative agent of human cercarial dermatitis, Trichobilharzia franki (Müller and Kimmig 1994), in southern England and its evolutionary relationships with other European populations

BackgroundTrichobilharzia is the most species rich and widely distributed genus of schistosomes and is known throughout Europe and North America as an agent of human cercarial dermatitis. The disease is caused by an acute allergic reaction in the skin that develops as a consequence of repeated contact with water containing schistosomatid cercariae. However, despite historical outbreaks of the disease, there are no published records of accurately identified Trichobilharzia species from the UK.MethodsTwo hundred Radix auricularia (L.) were sampled from a recreational fishing lake in Hampshire and emerging schistosomatid cercariae were collected for microscopy and DNA extraction. General morphological description of the cercariae was performed, alongside sequencing and phylogenetic analysis of the 28S ribosomal DNA for accurate species identification as well as comparisons of ITS1 in order to identify evolutionary affinities with other European populations. All molecular comparisons were performed using published sequences.ResultsThe phylogenetic analysis of 28S sequences identified the cercariae as Trichobilharzia franki. Two unique British ITS1 haplotypes were identified which were most closely related to haplotypes of T. franki populations from France. Haplotype network analysis indicated the mixing of T. franki populations throughout Europe. It is suggested that parasite distribution is the probable result of the movement of migratory waterfowl.ConclusionsThis is the first accurate record of T. franki in the UK. The movement of T. franki with waterfowl could pose a considerable human health risk, as in mainland Europe, and signifies T. franki-associated human cercarial dermatitis as a re-emerging disease in the UK.

Trichobilharzia mergi sp. nov. (Trematoda: Digenea: Schistosomatidae), a visceral schistosome of Mergus serrator (L.) (Aves: Anatidae)

Parasitological investigations on red-breasted mergansers (Mergus serrator L.) in Iceland revealed digenean flukes of the family Schistosomatidae. Adult worms were detected in blood vessels of the large intestine and eggs were deposited in the mucosa and surrounded by granulomatous reactions. Traditional morphological methods showed that the flukes have very slender filiform bodies, males are equipped with a short gynaecophoric canal and both suckers and spatulate ends are present on each sex. Among characteristics of the flukes which render them morphologically distinct from other Trichobilharzia species are: i) males—well developed vesicula seminalis (v.s.) consisting of a short v.s. externa and a significantly longer (approx. 3 times) v.s. interna, unusually well developed genital papilla and localization of the first testis a relatively long distance posterior to the gynaecophoric canal; ii) eggs—small and elongated with slightly rounded poles and a short terminal spine. DNA taxonomic techniques confirmed that a new species had been identified, Trichobilharzia mergi sp. n. The sequence data were deposited in GenBank under the accession numbers JX456151 to JX456172. Comparison of the results with our previously published data on characterization of DNA of cercariae isolated from freshwater lymnaeid snails showed that larval development of T. mergi is associated with Radix balthica L. (=Radix peregra Müller, 1774;=Radix ovata Draparnaud, 1805).

Molecular taxonomic identification of Schistosomatidae from Naroch Lake and Polonevichi Lake in Belarus

In Belarus, Naroch Lake is the only area with a high incidence of the human cercarial dermatitis (HCD). However, very little is known about the taxonomy of avian schistosomes, the causative agents of the disease, which are found in Naroch Lake and other lakes in Belarus. In this study, we used a molecular approach to investigate the systematic position and biodiversity of avian schistosomes from Naroch Lake and Polonevichi Lake. Based on the sequence analysis of the ITS genomic region, we were able to detect four different species of bird schistosomes in Naroch Lake ( Trichobilharzia szidati, Trichobilharzia franki, Bilharziella polonica and a novel Trichobilharzia species) and two species in Polonevichi Lake ( T. szidati and B. polonica). The data were used to reveal the phylogenetic position of HCD causative cercariae found in Belarusian water reservoirs and to establish their relationships within the group of avian schistosomes. We discuss the possibility of identifying species of Trichobilharzia using the fragment length polymorphism analysis of the ITS region. Possible epidemiological causes of HCD incidence in Belarus are also discussed.

Detection of European Trichobilharzia Schistosomes (T. franki, T. szidati, and T. regenti) Based on Novel Genome Sequences

The most frequent causative agents of cercarial dermatitis in Europe are the avian schistosomes of the genus Trichobilharzia . They preferably parasitize birds of the Anatidae. Trichobilharzia spp. schistosomes are also able to penetrate mammalian skin, posing a health risk to mammals, including humans. Currently several loci from nuclear and mitochondrial genomes are determined for European species of Trichobilharzia . Among them there is 1 genome sequence, ToSau3A, which is suitable for detection of Trichobilharzia spp. infection in aquatic systems. In the present paper, we used a PCR assay to obtain novel genome sequences from cercariae isolates of 3 European bird schistosome species ( Trichobilharzia franki , Trichobilharzia szidati , and Trichobilharzia regenti ) collected from freshwater ponds in Belorussia and Russia. We applied RAPD-fingerprinting using 1 random primer to differentiate 3 trichobilharzian species and subsequently cloned and sequenced putative species-specific RAPD fragments. One of them (410 bp in length), which was obtained for T. franki , revealed 64% homology with the repeat region of Schistosoma mansoni (GenBank FN357352) and turned out to be suitable for designing a specific primer pair (TR98F and TR98R) to detect 7 novel DNA sequences in the genome of 3 European Trichobilharzia species. The newly designed primer pair was found to be potentially suitable for PCR-based detection of trichobilharzian infection in snails. PCR primers TR98F and TR98R amplified only the DNA isolated from cercariae and sporocysts of 3 trichobilharzian species, but neither the DNA of 3 other digenean species ( Bilharziella polonica , Apatemon sp., and Diplostomum sp.) nor the DNA of uninfected host snails ( Lymnaea stagnalis , Radix auricularia , and Radix ovata ).

Final hosts and variability of Trichobilharzia regenti under natural conditions

In Europe, despite of intensive work on avian schistosomes in the past, only one representative of nasal schistosomes has been detected so far, Trichobilharzia regenti, a species that remained undescribed to the scientific community until 1998. Since then, however, T. regenti has gained a considerable attention, not only due to the induction of serious lesions concomitant with the migration of the schistosomulae through the central nervous system of their natural hosts (birds) but also due to their implication in cercarial dermatitis and their neurotrophic behaviour in mammals (mice, experimental) where the parasite migrate to the CNS. From 2001 to 2010, by a parasitic control of various potential final hosts (aquatic birds) living in natural condition in France and in Iceland, we detected T. regenti in six species: Anas platyrhynchos, Cygnus olor, Aythya fuligula, Aythya ferina, Mergus merganser and Anser anser. Although morphological features, mainly of eggs, showed some variations with the original description of the parasite, molecular analysis confirmed their membership to the T. regenti species. Nevertheless, specific molecular and morphological variations were especially observed for nasal parasite isolated from Anas clypeata. For this species, we discuss the possibility for this nasal schistosome to belong to a nasal Trichobilharzia species already described in Africa or even an undescribed nasal species.

Molecular Systematics of the Avian Schistosome Genus Trichobilharzia (Trematoda: Schistosomatidae) in North America

Trichobilharzia is a genus of threadlike schistosomes with a cosmopolitan distribution in birds. Species of Trichobilharzia achieve notoriety as major etiological agents of cercarial dermatitis, or swimmer's itch. There are 40 species described in the literature, for which the majority lacks molecular sequence information. To better understand the phylogenetic relationships, diversity, species boundaries, host use, and geographic distribution of this genus better, we surveyed 378 birds and over 10,000 snails from North America. The phylogenetic analysis was based on nuclear 18S, 28S rDNA, internal transcribed spacer region and mitochondrial cytochrome oxidase I sequence data. Specimens were recovered that could be related to 6 of the 14 described species of Trichobilharzia from North America (Trichobilharzia physellae, Trichobilharzia querquedulae, Trichobilharzia szidati, Trichobilharzia stagnicolae, Trichobilharzia franki, and Trichobilharzia brantae). An additional 5 lineages were found that could not be related directly to previously described species. Trichobilharzia brantae, transmitted by Gyraulus parvus, grouped outside the clade containing the recognized species of Trichobilharzia. A subgroup of the Trichobilharzia clade designated Clade Q was comprised of closely related species whose adults and eggs are similar, yet the European species use lymnaeids whereas the North American species use physids as snail hosts. This molecular phylogeny provides a useful framework (1) to facilitate identification of worms, including those involved in dermatitis outbreaks; (2) to test hypotheses about the evolution, diversification, host-parasite interactions and character evolution of Trichobilharzia; and (3) to guide future taxonomic revision of Trichobilharzia.

Cercarial dimensions and surface structures as a tool for species determination of Trichobilharzia spp.

AbstractCercariae of bird schistosomes are traditionally considered to be very similar in their morphological characteristics. In order to solve the problem, we tested some methods which might be suitable for cercarial differentiation. Fourteen isolates of three Trichobilharzia species (T. szidati, T. franki, T. regenti) occurring sympatrically in Central Europe were used. Dimensions of individual cercariae do not represent a useful criterion for identification, because the intraspecific variability exceeds the interspecific one. On the other hand, chaetotaxy appears a promising way for discrimination, although some sensory papillae do not stain sufficiently with silver nitrate. The papillary pattern (i.e. number and relative position of papillae) is specific for all Trichobilharzia species studied by us. Therefore, we compiled an identification key for the three Trichobilharzia species. In addition, we tried to find species-specific surface saccharide epitopes; none of the labeled lectin probes can be used as a speciesspecific marker.

Unusual finding of Trichobilharzia sp. in Motacilla alba in the Czech Republic

Adult male worms of Trichobilharzia sp. recovered from a pied wagtail (Motacilla alba) in the Czech Republic were found to belong to a new species of the genus. The finding of Trichobilharzia sp. in a passeriform bird in Europe represents an important discovery, as only anseriform birds have thus far been reported as final hosts of the European Trichobilharzia species.

Ultrastructure of pigmented and unpigmented photoreceptors in cercariae of Trichobilharzia ocellata (Plathelminthes, Trematoda, Schistosomatidae): evidence for the evolution of parasitism in Neodermata.

The lens-covered pigment cup ocelli in free-swimming cercariae of Trichobilharzia ocellata were re-examined and the differentiation of the lenticular elements was studied in cercariae still enclosed in sporocysts. Each eye consists of a single rhabdomeric sensory cell, a single cup cell harboring pigment granules and the lens. This lens is developed step by step by the fusion of numerous dark platelets not enclosed by a bordering membrane. The material is presumably proteinaceous. There is no evidence that the lenses are of mitochondrial origin. Besides the pigment cup ocelli, a special type of unpigmented rhabdomeric photoreceptor was discovered. Three unicellular photoreceptors arranged in a three-dimensional configuration exist. These light-sensing organs show a principle of construction similar to that of phaosomous photoreceptors. We hypothesize that both types of photoreceptors are relevant for distinct reactions in the host-finding behaviour of the cercariae. The special type of lensing in the pigmented eyes seems to be restricted to Trichobilharzia species, whereas the phaosomous-like receptors may be more widespread in members of the Schistosomatidae. The obvious absence of any mitochondrial lensing in T. ocellata, and probably all other species of the Digenea, may be correlated with the evolution of endoparasitism in the Trematoda.

The phylogeny of the Schistosomatidae based on three genes with emphasis on the interrelationships of Schistosoma Weinland, 1858.

Schistosomes are digenean flukes, parasitic of birds, mammals and crocodiles. The family Schistosomatidae contains species of considerable medical and veterinary importance, which cause the disease schistosomiasis. Previous studies, both morphological and molecular, which have provided a good deal of information on the phylogenetics of this group, have been limited in the number of species investigated or the type or extent of molecular data used. This paper presents the most comprehensive phylogeny to date, based on the sequences of 3 genes, complete ribosomal small subunit rRNA and large ribosomal subunit rRNA, and mitochondrial cytochrome oxidase 1, sequenced from 30 taxa including at least 1 representative from 10 of the 13 known genera of the Schistosomatidae and 17 of the 20 recognized Schistosoma species. The phylogeny is examined using morphological characters, intermediate and definitive host associations and biogeography. Theories as to the origins and spread of Schistosoma are also explored. The principal findings are that Ornithobilharzia and Austrobilharzia form a sister group to the Schistosoma; mammalian schistosomes appear paraphyletic and 2 Trichobilharzia species, T. ocellata and T. szidati, seem to be synonymous. The position of Orientobilharzia within the Schistosoma is confirmed, as is an Asian origin for the Schistosoma, followed by subsequent dispersal through India and Africa.

Detection of bird schistosomes in lakes by PCR and filter-hybridization

Many lakes around the world are contaminated with bird schistosome cercariae, which penetrate into human skin, causing an itching dermatitis called “swimmers' itch.” Bathers could be forewarned from exposure to the larvae and ecological examinations could be performed, when a sensitive method to detect the parasites in aquatic systems, where lots of organisms hinder microscopic examinations, would be available. For this purpose we cloned, sequenced, and analyzed a 396 bp tandem repeated DNA sequence from Trichobilharzia ocellata (ToSau3A), and employed it for developing molecular detection assays. It hybridized with less than 100 pg DNA from different Trichobilharzia species ( T. ocellata, Trichobilharzia franki, and Trichobilharzia regenti), but not with 10 ng DNA from other related or sympatric trematodes. A PCR assay, amplifying this sequence with the same specificity, detected 100 fg T. ocellata DNA, 1 cercaria in 0.5 g plankton, and 2 cercariae in 0.5 g host snail tissues.

Prevalence of cercariae from Lymnaea stagnalis snails in a pond system in Southern Germany.

Lymnaea stagnalis snails were collected from 174 individual ponds of an extensive pond system in South Germany; 43,441 snails collected during 1980 2000 were examined for shedding cercariae. The species richness (at least 18 species of cercariae) and the high cercarial prevalence (at least 44.9%) may result from the high abundance and diversity of vertebrate and invertebrate hosts in the area. The mean prevalence (% +/- SEM) of most cercarial species increased from May-June to August-October, e.g. in Diplostomum spathaceum from 4.1% to 18.6%, in all echinostomatids from 5.6% to 18.0%, in Pseudechinoparyphium echinatum from 3.0% to 11.2%, in Xiphidiocercariae from 2.2% to 13.4%, and the overall prevalence shifted from 13.7% to 53.5%. There was no change in the prevalence of the different cercarial species over the last 20 years. The agents of cercarial dermatitis showed a constant low prevalence, 0.17% in Trichobilharzia ocellata (43,441 L. stagnalis examined) and 0.24% in other Trichobilharzia species (4,245 Radix examined). Such low prevalences seem to be normal in areas where cercarial dermatitis occurs in humans.

Trichobilharzia regenti n. sp. (Schistosomatidae, Bilharziellinae), a new nasal schistosome from Europe.

Members of the genus Trichobilharzia are parasitic in visceral or nasal body parts of their avian hosts. The examination of water snails in South Bohemia revealed a schistosome infection in Radix peregra snails. The experimental infection of ducklings (Anas platyrhynchos, Cairina moschata) confirmed that a new Trichobilharzia species--T. regenti n. sp.--was found. The adults, eggs and larvae (miracidia and cercariae) of the species were morphologically characterized. The adult occupy the nasal blood vessels where they lay the eggs; the miracidia hatch from the eggs directly in the tissue and leave the host during drinking/feeding of the infected birds.