The techniques employed to collect and store trematodes vary between research groups, and although these differences are sometimes necessitated by distinctions in the hosts examined, they are more commonly an artefact of instruction. As a general rule, we tend to follow what we were taught rather than explore new techniques. A major reason for this is that there are few technique papers in the published literature. Inspired by a collaborative workshop at the Trematodes 2024 symposium, we outline our techniques and processes for collecting adult trematodes from fishes and discuss the improvements we have made over 40 years of dissections of 20,000+ individual marine fishes. We present these techniques for two reasons: first, to encourage unified methods across the globe, with an aim to produce optimally comparable specimens across temporal periods, across geographic localities, and between research groups; and second, as a resource for inexperienced researchers. We stress the importance of understanding differences in host biology and the expected trematode fauna, which ultimately enables organised and productive dissections. We outline our dissection method for each key organ separately, discuss handling, fixation, and storage methods to generate the most uniform and comparable samples, and explore ethical considerations, issues of accurate host identification, and the importance and potential of clear record keeping.

A BioBlitz is a rapid and intensive survey of a specific geographic area that brings together experts and often lay participants to assess biodiversity, typically of macrobiota that are easily observed and identifiable on-site. This concept has become popular across taxonomic fields, attracting interest globally to increase knowledge of local biodiversity. Inspired by the success of the approach, we undertook a ‘ParasiteBlitz’ at an unexplored locality (Stono Preserve, Charleston, South Carolina, USA) to determine its feasibility for parasites, whose assessment of diversity is largely neglected worldwide. We assembled a team of parasitologists with complementary expertise. Over 12 days (3 days in each habitat) in April 2023, we intensively screened fishes and aquatic invertebrates for parasites, and sampled sediment and water for environmental DNA (eDNA) metabarcoding from four aquatic habitats: wetland, freshwater pond, brackish impoundment, and tidal creek. We incorporated assistance from non-parasitologists and students. Details on methodologies and results are provided in individual papers in this Special Collection. Traditional methods revealed the presence of ca. 100 species of seven major metazoan parasite taxa, and the eDNA survey yielded over 1,000 amplicon sequence variants identified as parasites, most with sequences unmatched in GenBank, and resulting in only a few species identified as named species in the one-year post-Blitz timeframe we imposed upon ourselves for identification. Limitations and challenges of the ParasiteBlitz are discussed, and our results support that this approach can be effective for rapid discovery of the dimensions of parasite assemblages in an understudied environment and contribute to parasitology knowledge.

Stephanofilaria is a genus of nematodes that cause ulcerative dermal lesions in large mammals. However, there is a dearth of knowledge on the molecular genetics of Stephanofilaria species infecting critically endangered rhinoceros. This study employed genetic barcoding genes to identify Stephanofilaria species and to determine its genetic diversity and evolution. Phylogenetic analyses on partial genes of the second internal transcribed spacer Ribosomal DNA (ITS-2) and cytochrome c oxidase subunit 1 (Cox-1), revealed a 77% and 93% bootstrap support at the Cox-1 and ITS-2 loci respectively to a clade containing previously identified Stephanofilaria species. Morphological examination also confirmed features diagnostic of Stephanofilaria dinniki previously known to infect rhinoceros. Gene diversity of Cox-1 was 0.931 ± 0.030 and 0.579 ± 0.104 for the ITS-2, whereas nucleotide diversity was 0.008 ± 0.002 and 0.00197 ± 0.0016 for the Cox-1 and ITS-2 genes respectively. Neutrality tests (Fu and Li’s D* and Fu and Li’s F*) were significantly negative (p<0.05) at all loci, whereas Tajima D and Fu’s FS were each statistically significant (p<0.05) at the Cox-1 and ITS-2 loci respectively. The high gene diversity, low nucleotide diversity and negative neutrality tests are consistent with positive selection at the Cox-1 gene. Stephanofilaria infection among rhinoceros is currently restricted to highland sanctuaries compared to a widespread distribution in both lowlands and highlands in the 1960s suggesting an adaptation to vectors thriving in cooler highland temperatures. This is the first genetic identification of S. dinniki, in rhinoceros and will aid in diagnosis, treatment, studies, and rhinoceros conservation.

Equids are infected by a diversity of gastrointestinal nematode parasites, including 64 species of equine strongyle nematodes from19 genera. Despite numerous surveys of horse strongyles worldwide, certain geographic regions and rare species remain understudied. In 1964, a new species of equine strongyle, Cylicocyclus pekingensis, was described from a donkey in China. Subsequently, this species was recorded in horses from Kazakhstan and reclassified as Hsiungia pekingensis (K’ung and Yang, 1964), the only species in this genus. Since then, H. pekingensis has not been reported elsewhere, with limited knowledge on its distribution and phylogeny.

This study documents the first record of H. pekingensis in North America. Adult specimens were recovered from fecal samples of a domestic horse in Alberta, Canada, following treatment with ivermectin. Species identification involved detailed morphological examination, complemented with sequencing of the internal transcribed spacer 1 (ITS1), 5.8S rRNA gene, and the internal transcribed spacer 2 (ITS2) regions of the nuclear genome. Phylogenetic analysis indicated a close evolutionary relationship with species from Poteriostomum and Parapoteriostomum genera. Nemabiome ITS2 sequencing of a paired pre-treatment sample also detected the presence of H. pekingensis in the studied horse. Re-analysis of public equine nemabiome datasets further detected H. pekingensis in feral horses in Alberta, but not in other regions considered. This study expands the known distribution of this rare species and enhances our knowledge of its placement in the phylogeny of equine strongyles. Furthermore, our re-analysis of public nemabiome datasets highlights the value of this approach for studying the global distribution of parasite species.

The parasites of Nile tilapia, Oreochromis niloticus (Linnaeus, 1758) are poorly documented in the United States despite the economic importance and global introduction of this African fish. Only one metazoan parasite (Gyrodactylus cichlidarum Paperna, 1968; Gyrodactylidae) reportedly infects Nile tilapia in the United States. Examining Nile tilapia from a flow-through aquaculture system hydrologically linked to Sougahatchee Creek (Tallapoosa River, Auburn, Alabama), we observed a gill infection by Cichlidogyrus sclerosus Paperna & Thurston, 1969 (Dactylogyridae). This monogenoid was originally described from the gill of Mozambique tilapia, Oreochromis mossambicus (Peters, 1852) from Lake Victoria, Uganda. Specimens of C. sclerosus were studied for morphology and phylogenetic analyses using the 28S and ITS1. We identified our specimens as C. sclerosus because they had the following combination of morphological features: marginal hooks shorter than dorsal anchor length; anchor roots reduced; dorsal anchor point bent; dorsal bar pyriform projections approximately half as long as dorsal bar width; penis short (<100 μm), not coiled, tubular, lacking swelling, having irregularly surfaced heel; and accessory piece straight and bifid. Our 28S and ITS1 phylogenies recovered our C. sclerosus sequences in a clade with conspecific sequences and showed no obvious biogeographic pattern. Cichlidogyrus sclerosus reportedly infects 21 fishes of 11 genera and 3 families from 36 countries in Africa, Asia, North America, South America, and Europe. The study of Nile tilapia parasites, especially those exhibiting direct life cycles and low host specificity, is important because they comprise potential invasive species.

New, well-known and predicted life cycles for trematodes of the Haploporoidea (Haploporidae and Emprostiotrematidae) and three families of the Lepocreadioidea (Enenteridae, Gorgocephalidae, Gyliauchenidae) involve encystment of the metacercaria in the open (usually on vegetation) followed by ingestion by a range of herbivorous or detritivorous fishes. These life cycles appear among relatively highly derived plagiorchiidan trematodes in which three-host life cycles incorporating an animal second intermediate host are dominant. We hypothesise that the two-host life cycles in the Haploporoidea and Lepocreadioidea arose by secondary truncation of a three-host cycle; the second intermediate host was lost in favour of encystment in the open. Modification of a three-host life cycle effective for the infection of carnivores is consistent with the understanding that fishes arose as carnivores and that multiple lineages have secondarily become detritivores and herbivores. Four of the five trematode families involved infect fishes relating to multiple orders, suggesting a complex history of host-switching. In contrast, the Gorgocephalidae, the smallest of the families, has been found only in a single family, Kyphosidae. The timing of the evolutionary events leading to this putative life cycle truncation is yet to be deduced, but the rich developing understanding of the history of the fishes creates a strong template for future analysis.

The synonymies of the echinochasmid genera Mesorchis Dietz, 1909 and Monilifer Dietz, 1909 with Stephanoprora Odhner, 1902 remain contentious and unresolved with morphology. To explore the matter, we herein provide a supplemental description of the type species of Stephanoprora, Stephanoprora ornata Odhner, 1902, based on specimens we collected from the intestine of a Nile crocodile Crocodylus niloticus Laurenti, 1768 (Crocodylia: Crocodylidae) captured in the Kavango River, Namibia. No nucleotide information was available previously for S. ornata. Morphology plus 28S and ITS2 phylogenetic analyses suggested Stephanoprora is a monotypic genus that can be differentiated from other genera by having 26 collar spines. Stephanoprora differs from Mesorchis by the number and distribution of collar spines (26 [2 dorsal spines, 12 lateral spines, 12 corner spines] vs. 22 [2 dorsal spines, 12 lateral spines, 8 corner spines] in Mesorchis), length of the pre-pharyngeal oesophagus, pharynx position posterior to collar (vs. pre-pharyngeal oesophagus short, pharynx anterior to or at level of corner spines), and testes shape (elongate-ovoid, irregular in outline vs. ovoid, atypically elongated). Our 28S and ITS2 phylogenies recovered the new sequence of S. ornata (having 26 collar spines) sister to all remaining echinochasmid sequences, representing species that have 20–24 collar spines. Echinochasmus Dietz, 1909 and Mesorchis were recovered as paraphyletic. We retain Monilifer as a junior subjective synonym of Echinochasmus based a suite of morphological features related to body shape and genitalia and because the designated type for Monilifer was reassigned to Echinochasmus. We accept 25 species of Mesorchis.

Members of the genus Oncicola Travassos, 1916 are generalist parasites able to infect a broad spectrum of carnivorous hosts, such as marsupials, procyonids, felids, and canids, and are distributed globally. Adult specimens were collected from the intestines of three white-nosed coatis (N. narica), whereas cystacanths (larval form) were found in the body cavities of two amphibian species (paratenic hosts) in localities from northern and southeastern Mexico. Morphologically, both stages were identified as O. luehei (Travassos, 1917) on the basis of the following features: trunk cylindrical, narrow anteriorly, enlarging midbody, tapering gradually to narrow posteriorly; proboscis globular with six circular rows of hooks with six hooks each, decreasing in size posteriorly; neck short with sensory papilla; tubular lemnisci long, extending to the posterior region; protonephridia dendritic type; and eight cement glands, compact with a single giant nuclei. Sequences from cytochrome c oxidase subunit 1 from mtDNA were aligned and compared with sequences available in GenBank. Phylogenetic analyses revealed that adults and cystacanths formed a clade with two other isolates identified as Oncicola sp. and O. luehei from Mexico. The intraspecific genetic divergence among the isolates was low, ranging from 0.0% to 3.0%, indicating that the two stages of the life cycle belong to the same species. The haplotype network was inferred with 11 sequences and revealed a lack of shared haplotypes between populations, suggesting a reduced recombination rate and a high pattern of genetic variation among individuals. Finally, these new records of O. luehei increase the distribution range of O. luehei on both coasts of Mexico.

The growth of knowledge and research practices in any discipline is characterised by a trade-off between depth and breadth: we can either invest efforts to learn a little about many things, or learn a lot about few things. In parasitology, breadth of knowledge corresponds to research on biodiversity and taxonomy: the discovery and description of an increasing number of new species. In contrast, depth of knowledge comes from focused research on a few model species, about which we accumulate much detailed information. Breadth and depth of knowledge are equally important for progress in parasitology. In this essay, focusing on trematodes, I demonstrate that current research is rapidly broadening our knowledge (high rate of new trematode species being discovered) but not deepening that knowledge at a comparable rate. The use of model species, with caveats, appears to offer a promising avenue for deeper knowledge. I present a case study illustrating how it is possible to develop new model trematode species at low cost to increase the depth of our understanding in areas including host-parasite ecological dynamics, co-evolution, and responses to environmental and climatic changes. The take-home message serves as a call to action to parasitologists, emphasising the need to focus as much effort on depth of knowledge as we currently invest in breadth of knowledge.

Cystic Echinococcosis (CE) is a zoonotic disease caused by Echinococcus granulosus sensu lato. Diagnosing CE primarily relies on imaging techniques, and there is a crucial need for an objective laboratory test to enhance the diagnostic process. Today, cell-free DNAs (cfDNAs) have gained importance regarding their biomarker potential. This study aims to investigate the diagnostic capabilities of different cfDNA targets (Echinococcus-specific repeat sequences (mgs-4 and mgs-12) and partial fragment of repetitive sequence (EG1 Hae III)) and evaluate their diagnostic effectiveness when compared to a frequently used commercial E.granulosus-specific IgG ELISA. Seventy-six confirmed hepatic CE patients and healthy controls were included in the study. The EG1 Hae III region was assessed using nested PCR, whereas real-time PCR was employed to investigate other cfDNA targets. Analysis of the cfDNA-targeted tests indicated that mgs-4 demonstrated the highest diagnostic efficacy in distinguishing CE patients from healthy controls, achieving a sensitivity of 60.5% (p = 0.002). Combining ELISA with the mgs-4 target led to an increased sensitivity of 72.4% for distinguishing between CE patients and the control group. The sensitivity rates for ELISA and the three cfDNA targets varied among the groups. Active CE patients showed sensitivity rates of 52.9%, 52.9%, 23.5%, and 52.9% for ELISA, mgs-4, mgs-12, and EG1 Hae III assays, respectively. In contrast, inactive cyst patients displayed sensitivity rates of 21.4%, 66.7%, 19%, and 42.9% for the corresponding assays. The mgs-4, either alone or in combination with ELISA, demonstrated notably higher sensitivity values for CE diagnosis in all group comparisons compared to serology.

Surface flow of freshwater on Adriatic islands is rare due to the extreme permeability of the karst terrain. Hence, most helminthological studies of freshwater fishes in the Adriatic drainage have focused on mainland freshwater systems, while data from islands are scarce. We collected minnow, Phoxinus lumaireul (Schinz, 1840), specimens in the Suha Ričina stream on Krk Island and screened them for helminth ectoparasites. Identification of metacercariae cysts (black spots) was carried out by sequencing part of the 28S rDNA gene, and a single monogenean worm was identified by sequencing part of the internal transcribed spacer 2 (ITS2). To estimate the level of infection, the number of black spots was counted to calculate prevalence, mean abundance, and mean intensity. Phylogenetic inference based on maximum likelihood and Bayesian inference revealed a rare black spot disease taxon, genus Uvulifer, which represents one of the first records in Europe on fish (second intermediate host), a first record from a Phoxinus host, a first record for the Adriatic drainage and Croatia, and a first record for Southern Europe in general. Furthermore, the monogenean was identified as Gyrodactylus sp., potentially representing a new species.

The genus Enenterum Linton, 1910 comprises species which parasitize herbivorous kyphosid fish. In the present study, a new species is described based on fresh specimens collected from Kyphosus incisor from Rio de Janeiro. The new species is characterized by having the oral sucker infundibuliform with 10 lobes, prepharynx two times longer than pharynx, presence of oesophagus, testes slightly lobed, round ovary and rectum with muscular sphincter connected to the anus. New genetic sequences include partial 18S and 28S rDNA and ITS1-5.8S-ITS2. The phylogenetic analyses place Enenterum gomesae n. sp. as sister of Enenterum aureum, corroborating the morphological analyses. Enenterum aureum (=E. pimelopteri) previously described from Kyphosus spp. from Rio de Janeiro is now considered E. gomesae n. sp. The new species represents the only South American species so far described for this genus.

Species of the family Echinostomatidae use diverse gastropod taxa as first intermediate hosts. However, identification of echinostomatid larvae often proves difficult because of incomplete information on their life cycles and lack of molecular data that can link larvae to the corresponding known adults. Here, echinostomatids that were isolated from freshwater limpets in South Africa were described using light and scanning electron microscopy, and ribosomal (28S, ITS, and 18S) and mitochondrial (cox1) DNA sequences. The analyses revealed three species: Petasiger radiatus, Petasiger sp., and Echinostomatidae gen. sp. Considering the close morphological resemblance between cercariae of Petasiger spp., the current species were compared with data from literature. The results showed that cercarial size is generally unsuitable for species discrimination. The numbers of flame cells and refractile granules in the excretory system, and penetration gland cell patterns, may indicate, but do not prove species identity. Although papillary patterns were distinct between species, papillae were clearly discernible only using scanning electron microscopy and are known for only a few species. Phylogenetic reconstruction indicated that 28S rDNA sequences of Petasiger on GenBank are for P. exaeretus, P. phalacrocoracis, P. radiatus, and six unnamed species. Furthermore, the results revealed that multiple ITS rDNA and cox1 sequences labelled as Stephanoprora amurensis and P. phalacrocoracis on GenBank, are from isolates whose identities are questionable. Echinostomatidae gen. sp. could not be assigned to any currently known genus. Expansion of the genetic database of the family Echinostomatidae is necessary for the delineation of putative species and elucidation of intergeneric relationships.

This study aimed to determine the prevalence of gastrointestinal parasites in alpacas on selected farms in Poland. In July and August 2019 and August 2021, 223 samples from six commercial farms were examined using coproscopic techniques. The total percentage of alpacas infected with intestinal parasites was 57.7%. Eggs of Nematodirus sp. were found in 28.9%, Trichostrongylus sp. in 15.5%, Strongyloides sp. in 13.4%, Camelostrongylus sp. in 11.3%, other strongyle-type in 12.4%, Trichuris sp. in 3.1%, Capillaria spp. in 2.1%, Oesophagostomum sp. in 1.0% and eggs of Moniezia sp. in 1.0% of individuals. Oocysts of Eimeria macusaniensis were found in 8.2%, Eimeria sp. in 4.1%, and Cryptosporidium sp. in 3.1% of animals. Redundancy analysis showed that parasites and their number in faeces were related to the individual’s country of origin, sex and age. Females had significantly more eggs of parasites than males. More significant parasite infection was recorded in younger individuals. Moreover, the most infected were individuals from Germany. Some of the described parasites in tested alpacas have zoonotic potential. Due to the possibility of introducing parasites native to alpacas and acquiring species parasitising wild and farmed animals in Europe, permanent veterinary monitoring of animals imported from other regions is necessary.

Systematic review and meta-analysis were conducted to provide comprehensive information on the prevalence of amphistome infections in domestic ruminants in sub-Saharan Africa. A systematic search of peer-reviewed articles published between 2002 and 2023 was conducted. Prevalence estimates and meta-analysis were based on 76 peer-reviewed articles which met the inclusion criteria. Of the 55,122 domestic ruminants screened, 12,858 were infected, and the overall pooled prevalence was 22% (95% confidence interval [CI], 10-37). The highest prevalence was recorded in southern Africa 25% (95% CI, 0-62), and central Africa 16% (95% CI, 0-61) the lowest. Cattle were the most frequently sampled hosts (76.56%, n = 42,202) and sheep (8.78%, n = 4838) the lowest, and cattle recorded the highest pooled prevalence of 28% (95% CI, 12-47), and goats the lowest at 5% (95% CI, 0-14). Prevalence rate was the high in males 32% (95% CI, 21-44), adult ruminants 37% (95% CI, 15-62) and animals with poor body condition 47% (95% CI, 34-60), and during the wet season 36% (95% CI, 0-94). The highest pooled prevalence was recorded at postmortem 23% (95% CI, 8-43) compared to coprology 20% (95% CI, 6-39) studies. The meta-regression model demonstrated that the body condition score, host, and period, and the interactions of different factors significantly influenced the prevalence. The lowest prevalence rate was noted for the period between 2013 and 2023. This is the first systematic review and meta-analysis in sub-Saharan Africa that provides a comprehensive review of the prevalence of amphistome infections in domestic ruminants in the past 20 years.

In the winter of 2022–2023, hundreds of the Atlantic puffins (Fratercula arctica) appeared dead in the coast of the Canary Islands, a rare event considering their cold-living habits, normally occupying the North Atlantic Ocean. In this work, investigation about the parasites present in the Atlantic puffins found in the biggest islands of the Archipelago was carried out from a population portion. Necropsies of 39 birds were made and, during the examination of the urinary tracts, helminths were found. Morphoanatomical analysis under microscope allowed to identify them into Renicola genus with high similarity to Renicola sloanei. After that, DNA was extracted and NADH dehydrogenase subunit 1 gene were amplificated by a polymerase chain reaction method followed by sequencing and phylogenetic analysis. The molecular results demonstrated that in fact R. sloanei was the helminth parasite present in the urinary tracts of the Atlantic puffins found in the Canary Islands.

The two stork species that nest in Central Europe, Ciconia ciconia and Ciconia nigra, have been repeatedly shown to host the digenetic trematode Cathaemasia hians (Rudolphi, 1809) in their esophagus and muscular stomach. These host species differ in their habitat and food preferences, and the morphologic characters of C. hians isolates ex Ci. nigra and Ci. ciconia are not identical. These differences led to a previous proposal of two subspecies, Cathaemasia hians longivitellata Macko, 1960, and Cathaemasia hians hians Macko, 1960. We hypothesize that the Cathaemasia hians isolates ex Ci. nigra and Ci. ciconia represent two independent species. Therefore, in the present study, we performed the first molecular analyses of C. hians individuals that were consistent with the diagnosis of C. hians hians (ex Ci. nigra) and C. hians longivitellata (ex Ci. ciconia). The combined molecular and comparative morphological analyses of the central European Cathaemasia individuals ex Ci. nigra and Ci. ciconia led to the proposal of a split of C. hians into C. hians sensu stricto (formerly C. hians hians) and C. longivitellata sp. n. (formerly C. hians longivitellata). Morphological analyses confirmed that the length of the vitellaria is the key identification feature of the two previously mentioned species. Both Cathaemasia spp. substantially differ at the molecular level and have strict host specificity, which might be related to differences in the habitat and food preferences of the two stork species.

The entomopathogenic nematode (EPN), Steinernema yirgalemense, is an effective biological control agent against a variety of important insect pests in South Africa. To develop a South African EPN product feasibly in South Africa, EPNs need to be mass-produced. This study aimed to record the population dynamics of S. yirgalemense with in vitro liquid production in shake flasks, with a protein source of powdered egg yolk. The Erlenmeyer flask results indicated variation between flasks, albeit still achieving high yields. The reasons for attaining such variability in the recovery, yield, and growth stages are unclear, hence requiring further studies seeking to increase consistency. The results obtained indicate that, when IJ recovery is low, yields are also low due to relatively few reproductive adults being present in solution, which, in turn, produces more offspring, which later converts to the desired infective juvenile used for product formulation development. For commercial viability, a consistent production system is required that produces predictable yields. This study showed comparable high yields achieved with the flasks and in an early-stage bioreactor setup, being a positive development for S. yirgalemense mass production. Prior to the bioreactor scale-up process, protocol of mass production, the population and growth dynamics of the nematodes in the flask environment requires understanding. This is a positive step, leading to the future commercialisation of a local EPN product.

Despite a great effort made for almost 90 years, the diversity of freshwater fish trematodes in Mexico is still far from being fully known. The addition of molecular data to the description of trematode diversity in the last two decades added the potential to establish more robust species limits and a more accurate biodiversity estimation, but also led in some instances to the recognition of cryptic species complexes. Here, we used sequences of the large subunit of the nuclear ribosomal gene (28S rRNA) as barcodes, and morphological data, to assess the diversity of freshwater fish trematodes from a lake within a tropical rainforest. Eighty freshwater fish specimens of eight species were studied, and 120 trematode specimens were collected. Morphologically, specimens were allocated into nine genera; molecular phylogenetic analyses along with sequence divergence data provided evidence for recognising 11 trematode taxa, six adults and five metacercariae; six of them were identified to species level. Geographical distribution and host association patterns are briefly discussed for each trematode taxa.