Parasites are important components of biodiversity and contributors to ecosystem functioning, but are often neglected in ecological studies. Most studies examine model parasite systems or single taxa, thus our understanding of community composition is lacking. Here, the seasonal and annual dynamics of parasites was quantified using a 5-year metabarcoding time-series of freshwater plankton, collected weekly. We first identified parasites in the dataset using literature searches of the taxonomic match and using sequence metadata from the National Center for Biotechnology Information (NCBI) nucleotide database. In total, 441 amplicon sequence variants (belonging to 18 phyla/clades) were classified as parasites. The four phyla/clades with the highest relative read abundance and richness were Chytridiomycota, Dinoflagellata, Oomycota and Perkinsozoa. Relative read abundance of total parasite taxa, Dinoflagellata and Perkinsozoa significantly varied with season and was highest in summer. Parasite richness varied significantly with season and year, and was generally lowest in spring. Each season had distinct parasite communities, and the difference between summer and winter communities was most pronounced. Combining DNA metabarcoding with searches of the literature and NCBI metadata allowed us to characterize parasite diversity and community dynamics and revealed the extent to which parasites contribute to the diversity of freshwater plankton communities.

Currently, the systematics, biology and epidemiology of piscine Cryptosporidium species are poorly understood. Here, we compared Sanger ‒ and next-generation ‒ sequencing (NGS), of piscine Cryptosporidium, at the 18S rRNA and actin genes. The hosts comprised 11 ornamental fish species, spanning four orders and eight families. The objectives were: to (i) confirm the rich genetic diversity of the parasite and the high frequency of mixed infections; and (ii) explore the potential of NGS in the presence of complex genetic mixtures. By Sanger sequencing, four main genotypes were obtained at the actin locus, while for the 18S locus, seven genotypes were identified. At both loci, NGS revealed frequent mixed infections, consisting of one highly dominant variant plus substantially rarer genotypes. Both sequencing methods detected novel Cryptosporidium genotypes at both loci, including a novel and highly abundant actin genotype that was identified by both Sanger sequencing and NGS. Importantly, this genotype accounted for 68·9% of all NGS reads from all samples (249 585/362 372). The present study confirms that aquarium fish can harbour a large and unexplored Cryptosporidium genetic diversity. Although commonly used in molecular parasitology studies, nested PCR prevents quantitative comparisons and thwarts the advantages of NGS, when this latter approach is used to investigate multiple infections.

Tick-borne apicomplexan haemoparasites infect wild and domestic animals, but studies on their distribution among free-living animals are comparatively fewer. Tissues from 241 wild carnivores of eight Mustelidae, two Canidae, one Viverridae and one Felidae species were collected in Northern Spain, and analysed by real-time PCR targeting the 18S rRNA gene and sequencing. Babesia vulpes (formerly known as Theileria annae) was the only piroplasm detected in red foxes (Vulpes vulpes). Badgers (Meles meles) were shown to harbour two novel Babesia sp. sequence types (A and B) that only shared 96.7% homology between them and were closely related (ca. 97–98%) to, but distinct from B. vulpes and other babesia from carnivores. Analysis of PCR-derived sequences also revealed the presence of Cystoisospora sp. and Hepatozoon sp. in badgers and showed that wild cats (Felis silvestris catus) were infected with Cytauxzoon sp. Forty-two per cent of the animals subjected to a detailed external examination were parasitized by ixodid ticks, being Ixodes hexagonus and Ixodes ricinus the most abundant species. This study provided novel data on the different haemoparasites that can infect European wild carnivores and showed that they can be hosts for a range of haemoparasites and pose a potential risk for transmission to domestic animals.

Sarcocystis spp. represent apicomplexan parasites. They usually have a heteroxenous life cycle. Around 200 species have been described, affecting a wide range of animals worldwide, including reptiles. In recent years, large numbers of reptiles have been imported into Europe as pets and, as a consequence, animal welfare and species protection issues emerged. A sample of pooled feces from four confiscated green pythons (Morelia viridis) containing Sarcocystis spp. sporocysts was investigated. These snakes were imported for the pet trade and declared as being captive-bred. Full length 18S rRNA genes were amplified, cloned into plasmids and sequenced. Two different Sarcocystis spp. sequences were identified and registered as Sarcocystis sp. from M. viridis in GenBank. Both showed a 95–97% sequence identity with the 18S rRNA gene of Sarcocystis singaporensis. Phylogenetic analysis positioned these sequences together with other Sarcocystis spp. from snakes and rodents as definitive and intermediate hosts (IH), respectively. Sequence data and also the results of clinical and parasitological examinations suggest that the snakes were definitive hosts for Sarcocystis spp. that circulate in wild IH. Thus, it seems unlikely that the infected snakes had been legally bred. Our research shows that information on the infection of snakes with Sarcocystis spp. may be used to assess compliance with regulations on the trade with wildlife species.

Descriptions of Babesia occultans have previously been restricted to sub-Saharan Africa. Here, we report the finding, for the first time, of this low or non-pathogenic bovine Babesia species in Tunisia, northern Africa. B. occultans DNA was detected by molecular methods in Hyalomma marginatum unfed ticks collected in 3 bioclimatic regions of Tunisia. The near-full-length 18S rRNA gene was sequenced and compared with related sequences retrieved from GenBank. Phylogenetic analysis indicated that other sequences deposited as Babesia sp. could also correspond to B. occultans, suggesting that this species may have a wide distribution in Mediterranean and Asiatic regions, and not only in sub-Saharan Africa as previously described. A B. occultans-specific Reverse Line Blot (RLB) oligonucleotide probe was designed for future epidemiological studies that would help to clarify this possibility.

We have recently proposed revival of the name Entamoeba nuttalliCastellani, 1908 for a virulent amoeba (P19-061405 strain) isolated from a rhesus monkey (Macaca mulatta) and located phylogenetically between E. histolytica and E. dispar. In this study, E. nuttalli was isolated from feces of captive Japanese macaques (M. fuscata) in an open-air corral in Japan. The sequence of the 18S rRNA gene in the isolates differed from the P19-061405 strain in 2 nucleotide positions, but was identical to the EHMfas1 strain isolated previously from a cynomolgus monkey (M. fascicularis). One of the E. nuttalli isolates from Japanese macaques, named the NASA6 strain, was axenized and cloned. In isoenzyme analysis, the mobilities of hexokinase and phosphate glucose isomerase in the NASA6 strain were identical to those in the P19-061405 and EHMfas1 strains, but the mobility of phosphoglucomutase was different. These results were supported by gene analyses of these enzymes. Inoculation of NASA6 strain trophozoites into the liver of hamsters led to formation of an amoebic liver abscess. The liver lesions were characterized by extensive necrosis associated with inflammatory reactions. These results demonstrate that the NASA6 strain is potentially virulent and that E. nuttalli should be recognized as a common parasite in macaques.

Twenty-one marine and freshwater specimens of Hildenbrandia from Europe were compared by analyses of morphometric data and sequences of the rbcL chloroplast gene (which codes for the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase enzyme) and the nuclear 18S ribosomal RNA (rRNA) gene. Eleven freshwater specimens, all corresponding to H. rivularis, were collected from the British Isles, Germany, France, Austria, Spain and Italy. Six marine specimens, representing H. rubra, were obtained from Northern Ireland, Wales, France, Norway, Sweden and the Netherlands, while four marine specimens, corresponding to H. crouanii, were collected from Scotland and Germany. Morphometric analyses of vegetative and reproductive characters distinguished three groups of specimens, which correspond to the three species. Parsimony and distance analyses of the rbcL and 18S rRNA genes were performed on these samples and sequences of North American representatives were included to determine their inter-relationships. The rbcL gene sequence analyses separated the marine and freshwater specimens, with the H. rivularis and H. angolensis representatives forming a well- supported monophyletic clade. The rbcL sequence analyses did not resolve the groupings of marine specimens determined in the morphometric analysis. Analyses of the 18S rRNA gene sequences also supported separation of the marine samples from freshwater samples, although the bootstrap and decay support was not as strong as for the rbcL gene. H. rubra formed a monophyletic clade that was weakly supported. In terms of biogeographic trends, the fairly low intraspecific divergence of H. rivularis (0–1·9% for the rbcL gene and 0–3·6% for the 18S rRNA gene) and the positioning of these samples in the gene trees supports the concept that they are derived from marine populations.

Morphometric analysis and phylogenetic analysis of sequences of the rbcL chloroplast gene (which codes for the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase enzyme) and the nuclear 18S ribosomal RNA (rRNA) gene were carried out on 26 specimens of marine and freshwater Hildenbrandia from North America. Nineteen marine specimens were collected from Alaska to Costa Rica on the Pacific coast and from Newfoundland to Connecticut on the Atlantic coast. Seven freshwater samples were collected from Texas, Costa Rica, St Lucia and Puerto Rico. Three groups of samples were distinguished by morphometric analysis: one containing all freshwater samples (H. angolensis Welwitsch ex West et West), one consisting of a marine sample with parallel tetrasporangial divisions (H. occidentalis Setchell ex Gardner) and one group with non-parallel tetrasporangial divisions (H. rubra (Sommerfelt) Meneghini. These groupings were partially incongruent with those obtained by analyses of the molecular data. Parsimony and distance analyses of the rbcL gene resulted in trees in which Atlantic and Pacific clades were largely resolved. However, an Alaskan sample was included in the Atlantic group, which may indicate a trans-Arctic invasion event. The freshwater samples were paraphyletic for the rbcL gene, among the marine collections, which supports the concept of multiple invasions establishing the freshwater populations in North America. The 18S rRNA gene sequence data indicate that the freshwater samples are monophyletic with the exception of the unresolved position of the H. occidentalis sample. The freshwater samples form a monophyletic clade when multiple outgroups are used. The rbcL data appear to be mutationally saturated above approximately 17% divergence, which makes interpretation of phylogenetic signal among distant groups difficult. This may be a result of the asexual reproduction of the alga.