Transmission of apicomplexan parasites-including Eimeria, Toxoplasma, and Cryptosporidium-relies on the environmentally resilient oocyst wall, a bilayered structure whose integrity is maintained by cysteine-rich oocyst wall proteins (OWPs). Yet the functional roles of these conserved proteins remain poorly defined. Here, we characterize EtOWP6, a conserved OWP in Eimeria tenella, the causative agent of avian coccidiosis. Immunofluorescence reveals that EtOWP6 localizes specifically to wall-forming bodies and oocyst/sporocyst walls during sexual development. CRISPR/Cas9-mediated knockout demonstrates that EtOWP6 is essential for sporocyst formation, with deficient parasites exhibiting malformed sporocysts, developmental arrest in vitro, and an in vivo reduction in oocyst output exceeding 85%. The knockout strain is highly attenuated yet retains partial immunogenicity. Transcriptomic analysis reveals that EtOWP6 deletion induces widespread transcriptional reprogramming, disrupting genes critical for developmental regulation and oocyst wall biogenesis. Our findings establish EtOWP6 as a critical regulator coupling structural integrity with developmental progression in Eimeria, and more broadly, highlight OWPs as conserved targets for transmission-blocking interventions across apicomplexan parasites.

Biodiversity loss is a threat to ecosystems for potentially triggering cascades of extinctions that compromise the stability of ecological communities. Host-parasite interaction networks are particularly sensitive to host extinction and represent a significant portion of biodiversity. This study investigates the structural robustness of two distinct small mammal-parasite networks in the Neotropical region: (i) generalist protozoan parasites and (ii) specific nematode parasites. By using sequential host removal simulations based on centrality metrics (degree and betweenness) we compare the decay of parasite richness and network fragmentation with random extinction scenarios. Our results demonstrate that targeted removal of key hosts significantly accelerates network collapse compared to random loss (P < 0.001 in all scenarios), showing that network stability depends on a small number of highly connected species. The protozoan network proved more vulnerable to fragmentation, while the nematode network was more sensitive to the loss of parasite diversity when the most connected hosts were removed. These results highlight the importance of considering network structure and the topological roles of species in conservation strategies, as the loss of key hosts can have disproportionate consequences for maintaining parasite diversity and ecosystem integrity.

Canine leishmaniosis (CanL), by Leishmania infantum, is a global zoonotic disease with complex immune interactions. The non-pathogenic Leishmania tarentolae emerged as a candidate for immunoprophylaxis, yet in vivo evidence demonstrating natural cross-protection in dogs remains scarce. This experimental study evaluated the immunological and clinical outcomes of co-exposure to both parasites. Fourteen purposedly breed dogs were allocated into three groups: Group 1 (G1) and Group 2 (G2) were previously exposed to L. tarentolae, then challenged with L. infantum intravenously (G1) or intradermally (G2). Group 3 (G3) served as unimmunized L. infantum-infected control group, with three dogs challenged intravenously and two intradermally. Animals were longitudinally monitored for 13.5 months through clinical, molecular and serological assessments. While prior exposure to L. tarentolae did not confer sterilizing immunity against L. infantum, differences in clinicopathological and immunological parameters were observed among experimental groups. Unimmunized controls (G3) developed higher UPC values during the later stages of the study, whereas pre-exposed dogs (G1 and G2) generally showed lower UPC values, although considerable inter-individual variability was observed. The intravenous challenge group (G1) developed high parasite burdens, hyperglobulinemia, and inflammatory alterations, whereas intradermally challenged group (G2) showed comparatively lower parasite burdens and fewer clinicopathological alterations. Additionally, in vitro stimulation with L. infantum, on peripheral blood monocytic cells from pre-immunized dogs, revealed a cytokine profile characterized by intermittent pro-inflammatory activation and variable regulatory interleukin-10 expression (IL-10). Overall, this is the first study detailing the longitudinal clinicopathological and immunological profile of L. infantum infection in the canine model, which suggests that prior exposure to L. tarentolae may influence the subsequent course of L. infantum. Hence, further studies are advocated to investigate the potential disease-modifying effect herein observed.

Parasitic flatworms, including cestodes and trematodes, are covered by a specialized syncytial tegument that mediates nutrient uptake and host-parasite interactions. While the tegument of trematodes has been extensively characterized, its molecular composition in cestodes remains largely unknown. In this work, we performed a comparative proteomic analysis of the tegument of three cestode species, including larval and adult stages: Hymenolepis microstoma, Mesocestoides corti (syn. M. vogae) and the human pathogen Echinococcus multilocularis. Using stringent enrichment criteria relative to whole-worm extracts, we identified hundreds of tegument-enriched proteins in each species. Comparative analyses revealed a conserved core of tegumental proteins shared among all three species, including members of the Tegument Allergen-Like (TAL) family, vesicular trafficking components and calcium-sensing proteins, and identified candidates for nutrient uptake activities such as glucose and nucleoside transporters. Further comparative analyses revealed a set of shared tegumental proteins with the trematode Schistosoma mansoni, including conserved proteins that are specific to parasitic flatworms, supporting the existence of a conserved ancestral tegumental proteome. Finally, we confirmed tegumental expression of several genes in H. microstoma and E. multilocularis, and demonstrated regionally restricted gene expression among tegumental cytons, suggesting functional specialization within the syncytial tegument. Altogether, these results reveal an evolutionarily conserved composition of the tegument of parasitic flatworms, providing a foundation for future work targeting this critical host-parasite interface.

The protozoan parasite Perkinsus olseni actively proliferates within marine molluscan hosts and causes serious negative effects on mollusk production worldwide. Recent research revealed that one Japanese isolate requires exogenous glycine betaine (GB) for vegetative proliferation, whereas isolates from some congeneric species do not, suggesting that this requirement is a derived characteristic of this genus. In the present study, we established various isolates from Japan, Europe, and Oceania, and showed that P. olseni isolates from Japan and Europe commonly require exogenous GB, whereas Oceanian isolates do not. This suggests that Oceanian isolates may retain ancestral-like characteristics compared to Japanese and European isolates. Analyses of the non-transcribed spacer region of ribosomal DNA further support the above interpretation that the Oceanian isolates represent a more basal split, and additionally suggest that the Japanese and European isolates belong to lineages that have undergone relatively recent geographic expansion. Morphological comparisons among isolates showed that the Oceanian isolates possessed larger schizonts and trophozoites, producing fewer daughter cells, but formed larger hypnospores capable of producing a greater number of zoospores, although the association between GB requirements and morphological differences remains unknown. The present study demonstrates the existence of distinct lineages of this parasite and provides novel insights into the evolution, geographical dispersion, and shifts in life-history strategies of P. olseni.

Infectious diseases are a powerful ecological force, yet the predictors shaping variation in parasite prevalence across wildlife remain incompletely understood. Birds provide an exceptional system to address this challenge, as their global diversity, ecological breadth, and role as pathogen reservoirs make them excellent models for studying infection dynamics. Among avian parasites, haemosporidians (Plasmodium, Haemoproteus, and Haemoproteus subgenus Parahaemoproteus) are nearly ubiquitous, affecting host physiology and survival while offering a model for testing how ecological and evolutionary factors modulate infection risk. Using a georeferenced global dataset of haemosporidian infection, we evaluated how latitude, bird host abundance, bird habitat specialization, bird species age, and parasite co-occurrence influence infection prevalence across avian communities. Bayesian phylogenetic logistic models revealed that these predictors capture key variation in infection patterns. Latitude emerged as the strongest predictor among ecological and evolutionary variables considered, showing contrasting associations across parasite groups: prevalence decreased with latitude for Plasmodium and Haemoproteus, but increased for Parahaemoproteus. Associations among parasite groups also emerged as important predictors of prevalence. Plasmodium prevalence was negatively associated with the prevalence of both Haemoproteus and Parahaemoproteus, while Parahaemoproteus prevalence was negatively associated with Plasmodium prevalence. In contrast, Haemoproteus prevalence was not significantly associated with the occurrence of either Plasmodium or Parahaemoproteus. Abundance and habitat breadth of avian hosts were not meaningfully associated with prevalence in any of the three parasite groups. Similarly, bird species age showed no supported association with parasite prevalence, suggesting that contemporary ecological and geographic factors and interactions among parasite groups are more important than host evolutionary age in explaining global variation in avian haemosporidian prevalence. Collectively, these results demonstrate that incorporating cross-parasite associations within a phylogenetic framework provides new insights into the mechanisms structuring global spatial patterns of haemosporidian prevalence.

Monogenean parasites are among the most common ectoparasites of marine fishes and can influence host health, fitness, and population dynamics. Despite their ecological importance, historical data on monogenean prevalence and diversity in marine fish are scarce. Here, we report data from a historical survey of 1,518 fish, representing 242 species across 87 families, collected from the Central California coast to the Gulf of California between 1967 and 1980. Monogeneans were identified from gills, skin, and body openings, and prevalence and mean abundance were analyzed across five depth zones: sub-littoral, epipelagic, mesopelagic, bathypelagic, and bathyal. Infections were concentrated in sub-littoral and epipelagic fishes, with prevalence and diversity declining in deeper habitats, likely reflecting host density, environmental conditions, and parasite life history. A total of 42 monogenean species from 14 families were recorded, including several undescribed taxa. This historical dataset provides a rare baseline for Pacific marine fishes and offers a valuable reference for assessing long-term changes in parasite populations and host-parasite dynamics under environmental change.

Alveolate protozoan parasites in the genus Hepatozoon infect a wide range of wild animals and pose a significant health threat to companion animals, mainly dogs and cats. Aiming to create a baseline description of Hepatozoon sp. in wild animals in the northeastern USA, we used 18S rDNA metabarcoding to screen blood from 243 small- and medium-sized wild mammals from 11 species sampled in New Brunswick, NJ, USA. To the best of our knowledge, this is the first use of metabarcoding to examine community-level Hepatozoon prevalence and diversity in North American wildlife. We analyzed amplicon sequence variants (ASVs) using phylogenetic and haplotype network approaches. We detected Hepatozoon DNA in 17.3% of individual mammals but only rodents were positive, with no positive carnivores, lagomorphs, marsupials, or shrews. Thin blood smears from three gray squirrels showed leukocyte parasitemia ranging from 1.5 to 6.9%. We identified 130 distinct ASVs, most of which had a top pairwise identity to Hepatozoon griseisciuri A or B or Hepatozoon sp. DB2220. In the phylogenetic analysis, all ASVs grouped with sequences from rodents and reptiles, separate from the clade containing sequences of parasites known to infect dogs and cats. Contrary to other studies, we found no evidence of Hepatozoon sp. in carnivores such as raccoons and did not detect Hepatozoon species known to infect domestic animals, suggesting the synanthropic mammals examined are not current reservoir hosts for these parasites in the study region.

The polymerase chain reaction (PCR) is widely used to amplify malaria parasite DNA to diagnose Plasmodium infections, but can generate artefactual chimeric sequences through template switching. Here, we examine the occurrence and consequences of PCR-generated chimeras in studies of malaria parasites from humans, macaques and mosquitoes, and highlight two major sources of errors. First, in samples containing mixed-species infections, PCR can produce interspecific chimeras. This problem is particularly acute among closely related species of Plasmodium that infect macaques in southeast Asia, several of which have been transmitted to humans. Reanalysis of published mitochondrial DNA sequences from Thailand and Malaysia revealed numerous chimeric sequences involving P. knowlesi, P. inui, and P. cynomolgi, as well as P. vivax and other related species. Second, amplification of multicopy genes can also generate intra-genomic chimeras. The most widely used PCR template for malaria parasite identification has been the small subunit (18S) ribosomal RNA gene, but Plasmodium genomes contain two divergent forms of the rRNA unit, the A-type and the S-type. We show that several studies have generated intertype rRNA chimeras, which can occur even in the absence of multi-species infection. This has led to difficulties in identifying the relevant species, given an inflated view of sequence diversity, and has confused conclusions regarding zoonotic transmissions. These findings highlight the need for more rigorous approaches to PCR amplification to avoid the generation of chimeras, and for the validation of potentially chimeric sequences before phylogenetic or epidemiological interpretation.

The cumulative number of parasite species known to science keeps increasing steadily year after year, suggesting we are far from the end of our inventory of extant parasite biodiversity. However, what about higher taxa such as genera or families: are their known numbers also still rising? Finding new genera or families represents the discovery of clades with greater evolutionary uniqueness than the discovery of new species belonging to already known lineages. We surveyed the taxonomic literature between 2010 and 2025 to quantify patterns in the discovery of new helminth genera and families. Few new families were proposed during the study period, however 271 new genera were established, with 80% of them being 'true' new genera, i.e. previously unknown lineages based entirely on the discovery of new specimens. Whereas we might expect that the more genera one has already found, the harder it could be to find new ones, we instead found a positive relationship between the number of new genera found per higher taxon and the currently known species richness of that taxon. This supports a sampling hypothesis whereby species-rich taxa like Trematoda are also the ones likely to yield more new genera. Indeed, the cumulative number of new trematode genera found in the 2010-2025 period has increased linearly, suggesting many more genera await discovery in this taxon as well as in Nematoda, Monopisthocotylea and Polyopisthocotylea. In contrast, an asymptote has been reached in Acanthocephala and Cestoda, indicating the final list of genera is at hand in those taxa. Our findings suggest that the quest to document parasite biodiversity is far from complete as we still lack knowledge of many phylogenetically distinct helminths characterised by morphologies that do not fit within currently known genera.

Biomphalaria snails are intermediate hosts of Schistosoma mansoni, and their distribution predicts where intestinal schistosomiasis may occur. Here, we investigated the genetic diversity and distribution patterns of Biomphalaria spp. in the Lake Albert region and assessed their role in the transmission of schistosomes. Snails (n = 6869) were screened for schistosome infections using cercariae shedding and molecular techniques. In total, 29,670 Biomphalaria snails were collected, 90% of which were in the upland sites. For a subset of snails (n = 184) we sequenced a partial cytochrome c oxidase subunit one (COX1) fragment for phylogeographic analyses. Biomphalaria sudanica, Biomphalaria pfeifferi and Biomphalaria stanleyi were identified, with the former two species having haplotypes shared at the lake and upland sites. We also report the presence of a cryptic lineage, tentatively named B. cf. pfeifferi, awaiting further description. Molecular assays revealed higher overall prevalence of S. mansoni (1.9%) and S. rodhaini (1.12%) among Biomphalaria spp. (n = 1619) than inferred from cercarial shedding (0.66%). However, S. mansoni occurred exclusively at lake and nearby stream sites and was hosted predominantly by B. sudanica (24.7%, 22/89) and B. pfeifferi (5.4%; 5/147), while S. rodhaini, hosted mainly by B. cf. pfeifferi, was widely distribution in upland sites allopatric to S. mansoni. The wet season (October and November) had the highest S. mansoni prevalence. We argue that the lack of S. mansoni in upland sites reflects a more nuanced situation than simply the absence of suitable host snails, and suggest taxonomic revision of the B. pfeifferi complex.

Hyperparasitism, in which a parasite exploits another parasite, represents a logical extension of parasite diversity and complexity within ecological networks. While common and ecologically significant in microbial systems, hyperparasitism among metazoan parasites appears comparatively rare. This discrepancy is intriguing given the high diversity and frequent co-occurrence of parasites within hosts. Here, we compile and review published cases of hyperparasitism involving metazoan parasites to assess their taxonomic distribution and ecological characteristics. We identify a wide range of hyperparasitic strategies, including opportunistic associations and highly specialized interactions. However, many published cases appear to be accidental or rare, suggesting that true hyperparasitism is even less frequent than reported. We argue that the relative rarity of hyperparasitism among metazoan parasites results from a combination of ecological and evolutionary constraints, including low encounter probabilities, dependence on multiple hosts, parasite aggregation, limited trophic accessibility, structural simplicity of parasite bodies, the presence of dual immune barriers imposed by both host and parasite, and, especially, energetic scaling constraints. Together, these factors restrict the conditions under which hyperparasitism can evolve and persist. Therefore, despite the vast diversity of parasites, their suitability as hosts is limited, and thus they rarely serve as hosts for other metazoan parasites. In this context, "good parasites" may indeed make "bad hosts."

Anthropogenic climate change is expected to increase not only mean temperatures but also the magnitude and pattern of thermal variability, including the frequency, duration, and predictability of extreme events. While the effects of elevated mean temperatures on disease dynamics are well studied, far less is known about how different patterns of temperature variability shape host-parasite interactions, despite theoretical predictions from the climate variability hypothesis. Here, we used a controlled experimental system (Daphnia magna and two microsporidian parasites, Ordospora colligata and Hamiltosporidium tvaerminnensis) to disentangle the effects of thermal variability. Across two experiments conducted under different thermal regimes, we exposed hosts to cyclic (predictable) and random (unpredictable) heatwaves. Contrary to predictions from the climate variability hypothesis, temperature variability did not uniformly increase infection risk. Instead, infection outcomes depended on the mean temperature, duration of the heatwaves, the pattern of thermal variation, and parasite identity. At lower temperatures (Experiment 1), thermal variability had minimal effects on infection. At higher mean temperatures (Experiment 2), parasites responded in distinct ways: H. tvaerminnensis spore production was sensitive to the pattern of heat events, whereas O. colligata responded primarily to heatwave duration. These results demonstrate that climatic variability can differentially alter host-parasite interactions rather than exerting consistent directional effects on disease, with the effects of temperature variability depending on both species identity and thermal regime. Taken together, these findings suggest that increasing climatic variability may influence parasite communities and disease outcomes under the ongoing global change.

Neospora caninum is a major apicomplexan pathogen causing bovine abortion, resulting in major economic losses to the global livestock industry. Since its initial identification in 1988, N. caninum has emerged as one of the leading infectious causes of bovine abortion worldwide. Protein kinase G (PKG) acts as a central regulator of parasite motility, invasion, and egress; however, the downstream effectors that mediate these processes remain poorly defined. To systematically define the PKG-associated signaling network, we employed TurboID-mediated proximity labeling combined with subcellular localization profiling and phosphoproteomic analysis, identifying 37 high-confidence candidate proteins associated with PKG. These proteins are involved in vesicular trafficking, ion transport and metabolism, transcriptional regulation, and phosphoinositide/cyclic nucleotide signaling. We further identified a previously uncharacterized microneme-localized protein containing ion-transport domains, ITP1, that shows proximity to PKG and exhibits PKG-dependent phosphorylation. Genetic ablation of ITP1 significantly impaired parasite invasion and egress and reduced microneme protein secretion. These findings support a functional link between PKG signaling and ion transport-associated processes in the regulation of microneme exocytosis and host cell invasion in apicomplexan parasites.

Calicophoron daubneyi is an emerging rumen fluke of ruminants whose prevalence and geographic range have increased markedly across Western Europe over recent decades. Despite its growing veterinary importance, population-level genetic studies of this trematode have been hampered by the absence of suitable multilocus markers, with previous work relying on mitochondrial or ribosomal sequences that provide limited resolution for epidemiological inference. In this study, we developed and validated the first panel of polymorphic microsatellite markers for C. daubneyi and applied them to assess genetic diversity, clonality, and population structure in natural populations from the eastern French Pyrenees. Using Illumina-based genomic sequencing, we isolated 16 candidate loci, of which 10 robust and polymorphic microsatellites were retained after stringent quality filtering. We used these markers to genotype 187 adult parasites collected from cattle originating from seven localities spanning a maximum pairwise distance of approximately 50 km. Analyses explicitly accounted for the strong clonal amplification characteristic of trematode life cycles. We observed high levels of genetic diversity and a pronounced but variable clonal signal among sites. After attempting to correct for clonality and null alleles, genetic differentiation among populations was low and no significant isolation by distance was detected, consistent with substantial genetic exchange occurring at the regional spatial scale examined. These findings suggest that definitive host movement contributes importantly in shaping parasite gene flow within this interconnected livestock system. By providing the first validated microsatellite toolkit and baseline population-genetic data for C. daubneyi, this study establishes a foundation for future molecular epidemiological investigations of transmission dynamics, host-parasite interactions, and parasite dispersal in European livestock systems.