Parasitology

Epigenetic regulation plays a central role in the developmental control and antigenic variation of Plasmodium falciparum, yet the functions of many chromatin-modifying enzymes remain poorly understood. Here, we investigated the role of the putative Jumonji C (JmjC) domain-containing enzyme PfJmjC2 (PF3D7_0602800) in maintaining chromatin organization and histone modification balance during the asexual blood stages of the parasite. Using a conditional glmS ribozyme-mediated knockdown system, we achieved a [~]70% reduction in PfJmjC2 transcript and protein levels, which resulted in a marked delay in DNA replication and intraerythrocytic development. Immunoprecipitation assays revealed that PfJmjC2 physically interacts with histone H2A, suggesting an association with the nucleosome core. Upon PfJmjC2 depletion, the balance of epigenetic marks was disrupted, indicating a shift toward a more compact and transcriptionally repressed chromatin state. MNase-ChIP-PCR analysis further showed that PfJmjC2 associates with nucleosome-dense regions, including var gene promoters, although its reduction did not alter var gene transcription or switching patterns. Together, these findings indicate that PfJmjC2 contributes to chromatin organization and nucleosome stability rather than direct transcriptional control, highlighting its potential role in maintaining chromatin compaction and epigenetic homeostasis in P. falciparum.

Seasonality is a ubiquitous feature of wildlife disease ecology, but is determined by a complex interplay of environmental, parasitological and host factors. Gastrointestinal parasites often exhibit strong seasonal dynamics in wild vertebrate populations due to, for example, environmental influences on free-living or vectored life stages, and variation in the physiological and immune status of hosts across their annual cycle. At the same time, wild populations are typically infected with multiple parasites. The seasonal dynamics of co-infecting parasites may differ depending on age and reproductive status, and associations among parasites may be driven by short-term within-individual changes or longer-term interactions that are consistent among hosts. Here, we used faecal samples and egg counts collected repeatedly from individually marked and monitored wild Soay sheep that were part of a long-term study to investigate seasonal dynamics of six gastrointestinal parasite groups (strongyle nematodes, coccidian protozoa, Capillaria, Strongyloides, Nematodirus, and Moniezia). Prevalence and abundance generally tended to be higher spring and summer, and burdens were higher in lambs than adults. Within the highly prevalent strongyle nematode group, we found differences in seasonality of egg counts depending on adult reproductive status. Reproductive ewes had increased counts in spring around the time of birth followed by a drop in abundance in summer, while barren ewes showed little evidence of seasonality. Males showed a sustained rise in egg counts through spring and summer, and sex differences were only strongly apparent in summer. In contrast, in similarly prevalent coccidia we found a peak in faecal oocyst counts in spring but no differences in seasonality among males, barren and pregnant ewes. Using multivariate mixed-effects models, we went on to show that both strongyle and coccidia counts are moderately repeatable across seasons among individuals. We further show that apparent positive correlation between strongyle and coccidia counts was driven by short-term within-individual changes in both parasite burdens rather than long-term among-individual covariation. Overall, our results demonstrate that seasonality varies across demographic and parasite groups and highlight the value of investigating fluctuating susceptibility and exposure over time for understanding epidemiology of a population.

Trypanosoma cruzi, the agent of Chagas disease, must adapt to a diversity of environmental conditions that it faces during its life cycle. The adaptation to these changes is mediated by signaling pathways that coordinate the cellular responses to the new environmental settings. Cyclic AMP (cAMP) and Calcium (Ca2+) signaling pathways regulate critical cellular processes in this parasite, such as differentiation, osmoregulation, host cell invasion and cell bioenergetics. Although the use of CRISPR/Cas9 technology prompted reverse genetics approaches for functional analysis in T. cruzi, it is still necessary to expand the toolbox for genome editing in this parasite, as for example to perform multigene analysis. Here we used an efficient T7RNAP/Cas9 strategy to tag and delete three genes predicted to be involved in cAMP and Ca2+ signaling pathways: a putative Ca2+/calmodulin-dependent protein kinase (CAMK), Flagellar Member 6 (FLAM6) and Cyclic nucleotide-binding domain/C2 domain-containing protein (CC2CP). We endogenously tagged these three genes and determined the subcellular localization of the tagged proteins. Furthermore, the strategy used to knockout these genes allow us to presume that TcCC2CP is an essential gene in T. cruzi epimastigotes. Our results will open new venues for future research on the role of these proteins in T. cruzi.

Advances in experimental and theoretical work increasingly suggest that parasite interactions within a single host can affect the spread and severity of wildlife diseases. Yet empirical data to support predicted co-infection patterns are limited due to the practical challenges of gathering convincing data from animal populations and the stochastic nature of parasite transmission. Here, we investigated co-infection patterns between micro- (bacteria and protozoa) and macroparasites (gastrointestinal helminths) in natural populations of the multimammate mouse (Mastomys natalensis). Fieldwork was performed in Morogoro (Tanzania), where we trapped 211 individual M. natalensis and tested their behavior using a modified open-field arena. All animals were checked on the presence of helminths in their gastrointestinal tract, three bacteria (Anaplasma, Bartonella, and Borrelia) and two protozoan genera (Piroplasma and Hepatozoon). Besides the presence of eight different helminth genera (reported earlier), we found that 21% of M. natalensis were positive for Anaplasma, 13% for Bartonella, and 2% for Hepatozoon species. Hierarchical modelling of species communities was used to investigate the effect of the different host-related factors on these parasites infection probability and community structure. Our results show that the infection probability of Anaplasma and Bartonella was higher in adults than juveniles. We also observed that females and less explorative individuals had a higher infection probability with Bartonella. We found limited support for within-host interactions between micro-and macroparasites, as only animals infected with Bartonella were significantly more likely to be infected with Protospirura, Trichuris, and Trichostrongylidae helminths.

Toxoplasma gondii is a unicellular parasite that can infect most warm-blooded animals. In humans, it is linked to schizophrenia and other neurological disorders. It is also responsible for congenital toxoplasmosis, which can be lethal or cause severe neurological damage in the developing foetus. More subtle behavioural effects have been observed in various species, including humans, in particular increased risk-taking, which is thought to increase the parasites transmission to feline predators, the definitive hosts. We investigated the prevalence of Toxoplasma gondii in wild eastern chimpanzees (Pan troglodytes schweinfurthii) in the Budongo Forest, Uganda. We tested 45 individuals of two communities, one of which (Sonso) has a home range overlapping with human settlements. In this community, the prevalence was 22.7% (N=5 of 22), whereas in the neighbouring, but more isolated, Waibira community none of the 23 individuals tested were infected. The majority of infected Sonso individuals were adult males and/or high-ranking group members, attributes associated with increased risk-taking behaviour in chimpanzees and other animals. While further work is necessary to reveal the causal relation between infection and behaviour, our findings open new avenues of research for the neural impact of parasites on primate social behaviour.

Schistosomiasis is a neglected tropical disease caused by human-infective schistosomes (Trematoda: Schistosoma). Intestinal schistosomiasis in sub-Saharan Africa and the Neotropics is caused primarily by Schistosoma mansoni and is transmitted by several Biomphalaria planorbid snail species. Adult male and female parasites in the definitive mammalian host pair and reside in the mesenteric vasculature; females lay eggs that traverse the intestinal wall to be excreted, but a significant proportion become trapped in host tissues, especially the liver, eliciting granulomatous immune responses that underlie most disease pathology. S. mansoni is the primary lab model for research and, due to the abundance and ease of harvesting, liver-derived eggs are almost exclusively used to maintain the life cycle and to study miracidia and subsequent larval stages. However, recent evidence shows that eggs from the liver or intestine have key morphometric, transcriptomic, and antigenic differences, which can profoundly affect experimental outcomes. To determine whether these differences extend to the miracidia stage, we compared miracidia hatched from mouse liver and intestine-derived eggs, sequencing their transcriptomes and assessing their unstimulated behaviors over time in an arena allowing for high-resolution tracking of miracidia behavior at a large spatiotemporal scale. We found that while transcriptomic profiles of miracidia are distinguishable based on egg tissue origin, only a small subset of genes is differentially expressed. Further, basic, unstimulated behavior of miracidia that developed in different niches of the definitive host was significantly different. These different behavioral programs may reflect intrinsic developmental programming or differential viability and hardiness related to tissue origin. These findings underscore the importance of egg source in experimental design and interpretation, with significant implications for the maintenance of laboratory life cycles and the use of miracidia in schistosomiasis research.

Trypanosoma cruzi is a protozoan parasite transmitted by triatomine insect vectors, which expel their infectious dejections when they feed, causing Chagas disease in humans. The transmission and incidence of this vector-borne disease depend on the vital traits of its vectors, including Triatoma infestans, the main vector in Southern South America. Being an ectothermic species, its metabolism and its vital traits respond to temperature fluctuations. Here, we evaluated if changes in the average and variability of temperature expected with climate change modify: (i) the extrinsic incubation period (EIP) of T. cruzi within the vector T. infestans, (ii) its parasitic load, and (iii) the probability that its dejections were T. cruzi-positive. We acclimated triatomines infected with Dm28c T. cruzi strain to two constant and two variable temperature treatments and measured T. cruzi in their dejections by qPCR over a 42-day period. We observed that individuals in warm-temperature treatments showed lower EIP and higher parasitic load than cold-temperature treatments. Also, temperature variability can increase the parasitic load peak in cold-temperature treatments. Consequently, in a climate change scenario, there might be an increase in the vector capacity of T. infestans and probably a change in the risk of vectorial transmission of T. cruzi.

The protozoan parasite Leishmania spp. is a causative agent of leishmaniasis, a disease that affects millions of people in more than 80 countries worldwide. Apart from its medical relevance, this organism has a genetic organization that is unique among eukaryotes. Studies of the mechanisms regulating gene expression in Leishmania led us to investigate noncoding RNAs (ncRNAs) as regulatory elements. We previously identified differentially expressed (DE) ncRNAs in Leishmania braziliensis with potential roles in the parasite biology and development. Herein, we present a functional analysis of one such DE ncRNA, the 147-nucleotide-long transcript ncRNA97, which is preferentially expressed in amastigotes, the replicative form within mammalian phagocytes. By RT-qPCR the ncRNA97 was detected in greater quantities in the nucleus under physiological conditions and in the cytoplasm under nutritional stress. Interestingly, the transcript is protected at the 5 end but is not processed by the canonical trypanosomatid trans-splicing mechanism, according to the RNA circularization assay. ncRNA97 knockout (KO) and addback (AB) transfectants were generated and subjected to phenotypic analysis, which revealed that ncRNA97 impairs the starvation response and differentiation to the infective form. Comparative transcriptomics of ncRNA97KO and parental cells revealed that transcripts encoding amastigote-specific proteins were affected. This pioneering work demonstrates that ncRNAs contribute to the developmental regulatory mechanisms of Leishmania.

The production of energy in the form of ATP by the mitochondrial ATP synthase must be tightly controlled. One well-conserved form of regulation is mediated via ATPase inhibitory factor 1 (IF1), which governs ATP synthase activity and gene expression patterns through a cytoprotective process known as mitohormesis. In apicomplexans, the processes regulating ATP synthase activity are not fully elucidated. Using the model apicomplexan Toxoplasma gondii, we found that knockout and overexpression of TgIF1, the structural homolog of IF1, significantly affected gene expression. Additionally, TgIF1 overexpression resulted in the formation of a stable TgIF1 oligomer that increased the presence of higher order ATP synthase oligomers. We also show that parasites lacking TgIF1 exhibit reduced mitochondrial cristae density, and that while TgIF1 levels do not affect growth in conventional culture conditions, they are crucial for parasite survival under hypoxia. Interestingly, TgIF1 overexpression enhances recovery from oxidative stress, suggesting a mitohormetic function. In summary, while TgIF1 does not appear to play a role in metabolic regulation under conventional growth conditions, our work highlights its importance for adapting to stressors faced by T. gondii and other apicomplexans throughout their intricate life cycles. SIGNIFICANCE STATEMENTO_LIToxoplasma gondii is a member of the Apicomplexa, a phylum consisting of parasites responsible for significant global morbidity and mortality. An intact mitochondrial ATP synthase is critical T. gondii survival, but how this enzyme is regulated is not completely understood. C_LIO_LIOur work demonstrates that the T. gondii homolog of ATPase inhibitory factor 1 (TgIF1) does not impact metabolism under standard culture conditions, but plays a role in mitochondrial cristae density and stress responses. C_LIO_LIThis study reveals the role of TgIF1 in regulating ATP synthase activity under stressful conditions and increases our understanding of this divergent enzyme in T. gondii. C_LI

Avian malaria, caused by Plasmodium parasites, poses a significant threat to bird populations worldwide, particularly in vulnerable island ecosystems. Yet, progress in understanding avian malaria transmission dynamics has been hampered by the lack of molecular tools to quantify and sex the transmissible stages of the parasite: the male and female gametocytes. This challenge is compounded by the nucleated erythrocytes of avian hosts and the absence of an in vitro culture system, which have historically hindered the advancement of molecular approaches. Here, we develop and validate the first molecular markers to discriminate between asexual, male, and female gametocytes in the widespread avian malaria parasite Plasmodium relictum. Using single-cell RNA sequencing and orthology-guided stage mapping, we identified conserved, stage-specific transcripts and leveraged this information to develop molecular markers capable of quantifying and distinguishing male and female gametocytes in two parasite cyt-b lineages, pSGS1 and pDELURB. These markers outperformed microscopy in sensitivity, detected gametocytes earlier and for longer, and revealed consistently higher male-to-female gametocyte ratios than estimated by blood smears. The high degree of conservation of these markers across Plasmodium species suggests broad applicability of these markers to other avian lineages.

Helminths are common parasites of wild ungulates that can have substantial costs for growth, mortality, and reproduction. While these costs are relatively well documented for mature animals, knowledge of helminths impacts on juveniles is more limited. Identifying these effects is important because young individuals are often heavily infected, and juvenile mortality is an important process regulating wild populations. Here, we investigated associations between helminth infection and overwinter survival in juvenile wild red deer (Cervus elaphus) on the Isle of Rum, Scotland. We collected faecal samples non-invasively from known individuals and used them to count propagules of three helminth taxa (strongyle nematodes, Fasciola hepatica, and Elaphostrongylus cervi). Using generalised linear models, we investigated associations between parasite counts and overwinter survival for calves and yearlings. Strongyles were associated with reduced survival in both age class, and F. hepatica was associated with reduced survival in yearlings, while E. cervi infection showed no association with survival in either age class. This study provides observational evidence for fitness costs of helminth infection in juveniles of a wild mammal, and suggests that these parasites could play a role in regulating population dynamics.

In wildlife research, wild-caught vertebrates are often given anti-parasitic drugs when brought into captivity to protect animal health and reduce confounding effects of parasitic infection on research outcomes. However, the impacts of antiparasitic drugs on non-target parasite taxa are understudied, especially regarding host sex. To help address this gap, we investigated the impact of an anthelmintic medication on a protozoan gut parasite by quantifying coccidia oocyst burden in the feces of wild-caught dark-eyed juncos (Junco hyemalis) before and after two oral doses of ivermectin. Prior to treatment, males showed higher oocyst burdens than females. Following treatment, ivermectin-treated males showed larger increases in oocyst burden compared to both control males and ivermectin-treated females, while there was no difference in baseline and post-treatment oocyst burden in treated females. Our results suggest that wild-caught songbirds should be housed separately by sex during ivermectin treatment and that male enclosures should be cleaned at a relatively higher frequency due to excessive oocyst shedding. It is unclear whether increases in oocyst burden among males were attributable to helminth removal or a direct impact of ivermectin on host immune function. Further work investigating sex differences in the impact of antiparasitic drugs on non-target parasite taxa is warranted.

Piroplasms, which include the agents of cattle fever and human and dog babesiosis, are a diverse group of blood parasites of significant veterinary and medical importance. The invasive Asian longhorned tick, Haemaphysalis longicornis, is a known vector of piroplasms in its native range in east Asia and invasive range in Australasia. In the US state of Virginia, H. longicornis has been associated with Theileria orientalis Ikeda outbreaks that caused cattle mortality. We examined 667 H. longicornis collected in 2021 from three sites in New Brunswick, New Jersey, the US state where established populations of this species were first detected in 2017. We used primers targeting the 18S small subunit rRNA and the cytochrome b oxidase loci and unveiled the presence of DNA from an unidentified Theileria species (in 1 nymph) and Theileria cervi type F (1 adult, 5 nymphs). In addition, we sequenced a 130 bp fragment of the cytochrome oxidase b locus from Odocoileus virginianus, the white-tailed deer, in a partially engorged questing H. longicornis, supporting the association of this tick species with deer. We also detected DNA from an undescribed Babesia sensu stricto ( true Babesia, 2 adults, 2 nymphs) as well as Babesia sp. Coco (1 adult, 1 nymph). Finally, we detected DNA from Babesia microti S837 (1 adult, 4 nymphs). Babesia microti S837 has been sequenced from striped skunks, Mephitis mephitis, and is closely related to the human pathogen B. microti US-type. The five parasites we are associating with H. longicornis represent a diverse assemblage spanning three clades in the piroplasm phylogeny, two undescribed, raising concerns of transmission amplification of veterinary pathogens as well as spillover of pathogens from wildlife to humans.

Nucleocytoplasmic transport is essential in eukaryotes and mediated by importin (Imp) /{beta} receptors that recognize nuclear localization signals (NLSs) on cargo proteins. The SV40 large T-antigen NLS (SV40-NLS), studied extensively, binds importin with high affinity in the nanomolar range, while modified versions, the Bimax peptides, bind in the picomolar range. Bimax peptides impede nuclear import and reduce viability in yeast and human cells, highlighting the potential of NLS peptides as inhibitors of nuclear transport. In this study, we investigated the potential of the SV40-NLS to target Toxoplasma gondii importin (TgImp). Expression of the SV40-NLS fused to a GFP reporter led to cytotoxicity in T. gondii tachyzoites; this depended on the SV40-NLS sequence and its position within the protein. Over-expression of TgImp rescued parasites from the SV40-NLS-induced cytotoxicity, confirming that the mechanism of action involves disruption of nuclear import. Importantly, the same construct did not affect cell viability in mammalian cells, suggesting a selective vulnerability in importin -mediated nuclear transport in the parasite. The SV40-NLS peptide offers an advantage over small-molecule inhibitors by targeting large interaction surfaces of importin with high specificity, minimizing off-target effects. This study lays the groundwork for a novel peptide-based therapeutic strategy employing NLS motifs to selectively inhibit nuclear import in T. gondii.

Anthropogenic changes to the environment, including altered food resource availability, influence host physiology, behaviour, and population dynamics, which can have strong downstream consequences on wildlife disease dynamics. Additionally, some individuals within a population contribute disproportionately to infection as super-shedders of infection, but the extent to which food availability alters the likelihood of super-shedding and overall parasite infection patterns is poorly understood. We conducted a three-year field experiment in southern Finland to investigate how food supplementation and parasite removal affect nematode infection measures and the relationship between nematode infection and fitness of wild bank voles (Clethrionomys glareolus). Using a factorial design across 12 populations, we manipulated food availability and administered anthelmintic treatments to assess effects on nematode infection status, intensity, and two measures of super-shedding (abundance super-shedding, intensity super-shedding). We also examined parasite impacts on host fitness, including apparent survival probability and reproductive status. Food supplementation did not affect likelihood of infection, intensity or intensity super-shedding, but did reduce the likelihood of abundance super-shedding, suggesting an effect of food availability on infection heterogeneity. We also identified an interaction between nematode infection status and host age on fitness. Notably, infected younger individuals had reduced survival and reproduction, but infected older individuals had greater survival and reproduction compared to their uninfected counterparts. Our study provides novel empirical evidence on how anthropogenic changes in food availability can influence parasite transmission dynamics and the fitness consequences of these sub-lethal parasites in a wildlife system.

Strongyloides nematodes are parasites of livestock, and S. papillosus infects ruminant livestock that can cause disease. Recent genomic analysis of several Strongyloides species is now facilitating the population genomic analyses of natural Strongyloides infections, for example finding that S. ratti in wild UK rats exists as an assemblage of long-lived, asexual lineages. Here we have initiated an investigation into the population genomics of S. papillosus in goats in Pakistan. We find that S. papillosus is common, with a prevalence of 28%; that the population is genetically diverse and that individual goats commonly have mixed-genotype infections; and that there is evidence of only limited admixture. These results now provoke further questions about the host range of different S. papillosus genotypes that can in the future be investigated by further population genomic analyses.

Group-living primates experience the benefits and costs associated with sociality, including an elevated risk of parasite transmission. However, the relative influence of group type (i.e., social structure), group size, and habitat disturbance on parasitic infection remains unclear, particularly in Southeast Asian primates. In this study, the abundance of intestinal parasites in proboscis monkeys (Nasalis larvatus) inhabiting a riverine forest along the Menanggul River, Sabah, Malaysian Borneo, was investigated. Fecal samples (n = 160) were collected from one-male-multifemale and all-male groups in areas with varying levels of anthropogenic disturbance, with efforts made to ensure that each sample originated from a different individual. In addition, the effects of group type, group size, and sampling location on parasite abundance were evaluated using fecal egg counts and Bayesian models. Three dominant parasite species groups (Trichuris sp., Strongyloides fuelleborni, and Oesophagostomum aculeatum) with an overall infection prevalence of 81.25% were identified. Results showed that group type did not significantly affect parasite abundance. However, group size showed a positive correlation with the abundance of Trichuris sp. and a negative correlation with S. fuelleborni and O. aculeatum. In addition, our models revealed that the infection load of Trichuris sp. decreased with increasing distance from the river mouth, which was used as a proxy for a disturbance gradient, whereas O. aculeatum exhibited higher infection load at greater distances, indicating lower prevalence in more disturbed downstream areas. Thus, parasite abundance in proboscis monkeys may be shaped by social and environmental factors, with taxa-specific responses likely reflecting differences in environmental persistence and transmission ecology.

In this study, we employed a total of eight distinct modifications of histone proteins (H3K23ac, H3K27me3, H3K36me3, H3K4me3, H3K9ac, H3K9me3, H4K12ac, and H4K20me1) to discern the various chromatin colors encompassing lncRNA genes in both mature and immature gonads of the human parasite Schistosoma mansoni. Our investigation revealed that these chromatin colors exhibit a tendency to aggregate based on the similarities in their metagene shapes, leading to the formation of less than six distinct clusters. Moreover, these clusters can be further grouped according to their resemblances by shape, which are co-linear with specific regions of the genes and potentially associated with transcriptional stages.

Haemoglobin degradation is crucial for the growth and survival of Plasmodium falciparum in human erythrocytes. Although the process of Hb degradation has been studied in great detail, the mechanisms of Hb uptake remain ambiguous to date. Here, we characterized Heme Detoxification Protein (PfHDP), a protein localized in the parasitophorous vacuole, parasite food vacuole and infected erythrocyte cytosol for its role in Hb uptake. Immunoprecipitation of PfHDP-GFP fusion protein from a transgenic line using anti-GFP antibody and of Plasmodium parasite extract using anti-human Hb antibodies respectively, showed the association of PfHDP/Hb with each other as well as with the members of PTEX translocon complex. Some of these associations such as PfHDP/Hb and PfHDP/Pfexp-2 interactions were confirmed by in vitro protein-protein interaction tools. To know the roles of PfHDP and translocon complex in Hb import into the parasites, we next studied the Hb uptake by the parasite in PfHDP knock-down line using the GlmS ribozyme strategy. PfHDP knock-down significantly reduced the Hb uptake in these parasites in comparison to the wild type parasites. Further, the transient knock-down of one of the members of the translocon complex; PfHSP101 showed considerable reduction in Hb uptake. Morphological analysis of PfHDP-HA-GlmS transgenic parasites in the presence of GlcN showed food vacuole abnormalities and parasite stress, thereby causing a growth defect in the development of these parasites. Together, we implicate the translocon complex in the trafficking of PfHDP/Hb complex in the parasite and suggest a role for PfHDP in the uptake of Hb and parasite development. The study thus reveals new insights into the function of PfHDP, making it an extremely important target for developing new antimalarials.

Exposure to environmental parasites should increase with host population density due to the accumulation of infective parasites in space. However, competition for resources also increases with density, lowering condition and increasing susceptibility, which offers an alternative pathway for density-dependent infection to act. To test how these two processes act independently or together to drive greater parasite counts, we used a long-term study of red deer to examine associations between host density, resource availability, and counts of three common helminth parasites. We found that greater density correlated with reduced resource availability, and while density was positively associated with both strongyle and tissue worm burdens, resource availability was independently and negatively associated with the same burdens, supporting separate roles of density-dependent exposure and susceptibility in driving infection. This study provides evidence that competition for resources is an important driver of infection in higher-density areas, exacerbating the effects of density-dependent increases in exposure.