International Journal for Parasitology: Drugs and Drug Resistance

Short-chain dehydrogenases/reductases (SDRs) constitute a large enzyme superfamily involved in endogenous metabolism and xenobiotic biotransformation. In the parasitic nematode Haemonchus contortus, SDRs may catalyze the carbonyl reduction of a benzimidazole anthelmintic flubendazole (FLU), whose increased reduction is associated with FLU resistance. This study thus investigated the constitutive expression of SDRs and their inducibility by FLU in drug-susceptible and benzimidazole-resistant strains of H. contortus. The expression of 23 sdr genes was analyzed by quantitative PCR, while targeted proteomic assays enabled the quantification of 15 SDR proteins. In adult nematodes, pronounced sex-dependent differences were detected at both transcript and protein levels. Resistance-associated alterations were less pronounced and were observed predominantly in males, with SDR9, SDR12, SDR15, and SDR20 displaying increased protein abundances in the resistant strain. Exposure to FLU induced only minimal transcriptional responses in juvenile stages, whereas adult nematodes exhibited marked sex- and strain-specific responses in the expression of SDRs. The strongest transcriptional effects were detected in resistant males, while significant protein-level changes following FLU treatment were observed exclusively in adults of the drug-susceptible strain. Notably, SDR9 and SDR20 combined resistance-associated expression patterns with responsiveness to FLU exposure. Taking together, the first targeted proteomic characterization of SDRs in H. contortus revealed several SDR isozymes with constitutive overexpression in resistant nematodes and/or inducibility by FLU, suggesting a potential role in adaptation to anthelmintic exposure.

Pyrethroid insecticides on bednets have been the mainstay of malaria control since the millennium by killing mosquitoes. The target of these pyrethroids is the voltage-gated sodium channel (VGSC). Resistance mutations at the pyrethroid binding site (L995F/S) have spread widely, but functional tools to dissect vgsc itself have lagged behind the population-genetic surveillance data. Manipulation of vgsc via knockout or functional mutation would be a vital step towards understanding its contribution to insecticide resistance, as well as exploring its utility as a genetic control target. Here we generate and characterise two CRISPR-edited An. gambiae lines: an exonic knockout (vgscKO) and an intronic CRISPR-mediated cassette exchange intermediate (vgscInt) sited near SNPs linked to L995F. Both insertions are viable and fertile in heterozygosity but homozygous lethal, demonstrating that vgsc is haplosufficient and therefore the locus represents a viable target for population suppression genetic control strategies. These insertions can also be used as balancers for studying vgsc mutations without the confounding effects of wild type alleles. We investigate the insecticide resistance phenotype of vgscKO and demonstrate that knocking out one copy of vgsc produces reduced susceptibility to deltamethrin and DDT at sub-discriminating doses, with implications for vgsc-mediated mechanisms of insecticide resistance.

Beef cattle farming plays a crucial role in the livestock-related economies of both Italy and Argentina. Although both countries have grazing systems of meat production, the heterogeneity of those systems leads to different parasitological scenarios regarding epidemiology and the presence of resistance to commonly used anthelmintic drugs. Considering ivermectin (IVM) is the most widely used anthelmintic for treating gastrointestinal nematode (GIN) infections in cattle, the current studies evaluated the efficacy of ivermectin (IVM) given subcutaneously (SC) to both young calves and adult cows in ten (10) commercial cattle farms from Italy (A to E) and Argentina (F to J). The work was complemented with the assessment of the IVM plasma exposure and disposition kinetics both in young and adult animals. While adult cows and young calves were included in the study performed in Italy (Campania region), only calves aged 8-14 months old were selected to perform the efficacy trials in Argentina (central area of the Buenos Aires province). Fifteen (15) calves/cows naturally infected with GINs were treated with IVM (0.2 mg/kg) in each of the Farms. The therapeutic response (efficacy) was determined at 14 days after treatment by the faecal egg count reduction test. Six (6) adult cows and eight (8) young calves treated with IVM were randomly selected to perform the pharmacokinetic (PK) study with blood samples being taken 2 h and 21 days post-treatment. Drug concentrations were measured by HPLC. Similar IVM PK trends were obtained for both adult and young cattle. The IVM systemic exposure (expressed as AUC) obtained for adult cattle (380{+/-}158 ng.d/mL) was similar to that observed in calves group (313{+/-}85.5 ng.d/mL). No statistical differences between animals from both ages were observed for the most representative PK parameters (P>0.05). Surprisingly, while in Argentina IVM resistance was present in all the farms evaluated (90% CI = 0%-95%), in Italy a fully susceptible nematode population was presented in three farms (90% CI = 96%-100%) and only a very low level of IVM resistance was detected in the rest of the farms (90% CI = 92%-98%). Whereas Cooperia spp. and Haemonchus spp. were identified as the principal genera exhibiting resistance to IVM in Argentina, Cooperia spp., Ostertagia spp., and Oesophagostomum spp. were reported to display low levels of resistance under the Italian field scenario. In conclusion, IVM can be safely and effectively used in both young and adult cattle with similar disposition kinetic patterns. While its therapeutic response is failing under the conditions investigated in the Argentinian farms, its efficacy remains notably high in the Italian region under study. However, the use of IVM, as well any other active antiparasitic principle, should always be preceded by a diagnostic assessment of the nematode populations resistance status.

Extensive use of insecticides is increasing resistance risks that could severely reduce the control of mosquito vectors that transmit infectious agents of Neglected Tropical Diseases. Addressing resistance threats by discovering new insecticides can be challenging because of the limited throughput and translatability of existing in vitro screening pipelines. Acquiring multiple phenotypic endpoints of larvae could alleviate these restrictions by more thoroughly profiling drug effects on whole organisms, providing leads to compounds with novel mechanisms of action, and increasing screening throughput compared to adult-stage assays. Here, we establish a pair of assays for profiling motility and development traits in larval-stage mosquitoes at a higher scale than standard larvicidal screening techniques. We optimized assay parameters and developed novel image processing approaches that enable relatively high throughput screening of chemical compounds on single larvae within a screen with condition replicates. We tested the assay with an insect growth regulator (S-methoprene), a slow-acting pyrrole (chlorfenapyr), and two microbial larvicides (Spinosad and Lysinibacillus sphaericus). These measurements aligned well with known insecticide mechanisms, and dose response curves established assay baselines for comparison in future screens. Finally, we present ways in which the assay design can be modified across different imaging technologies, showing the flexibility of the screening approach.

BackgroundPlasmodium falciparum field isolates exhibit genomic variation, including copy number variation and sequence divergence. In contrast, the P. falciparum 3D7 reference genome (Pf3D7) was derived from a long-term laboratory-adapted strain and does not fully reflect the genomic variation among field isolates. The extent to which the reference genome influences RNA-seq mapping and expression inference in natural infections remains unclear. ResultsWe generated both a reference genome and single cell RNA sequencing (scRNAseq) data from a P. falciparum-infected carrier in Mali. This new ML52 assembly was annotated using Companion with Pf3D7 as the reference. scRNAseq reads from the natural infection isolate were aligned to both the Pf3D7 genome and the isolate-specific ML52 genome, followed by locus-level alignment inspection. For most conserved genes, expression inference was concordant for both references, while genes showing reference-genome-dependent differences were investigated further. Some discrepancies were attributed to mapping artefacts or to reads aligning to unplaced genomic contigs that reflected divergent haplotypes from the co-infecting strains in the naturally infected carrier. While most multigene family loci showed concordant gene expression across both references, var genes exhibited considerable mis-mapping against 3D7 var loci as well as 2/3 of var reads not mapping at all to 3D7. ConclusionscRNAseq expression inference in P. falciparum is robust for conserved genes and most multigene families when comparing to a matched vs unmatched reference genome, but the extremely polymorphic var genes require a matched assembly in order to evaluate expression. These findings highlight the importance of the reference genome for var gene studies and should be considered when interpreting transcriptomic analyses in other organisms possessing highly variable antigenic loci.

Drug repositioning has emerged as an attractive strategy to accelerate the development of new antimalarial therapies, particularly by evaluating compounds already used against pathologies co-endemic with malaria. This approach offers the advantage of leveraging existing pharmacokinetic, toxicological, and safety data, thereby potentially shortening the drug development pipeline. However, transposing a compound from its original therapeutic indication to an antimalarial use is far from straightforward: differences in target biology, parasite stage specificity, pharmacodynamic requirements, and host-parasite interactions can result in a loss of efficacy despite promising in vitro or structural rationale. Rigorous in vivo validation therefore remains indispensable before any repositioning hypothesis can be considered translationally relevant. In this context, we evaluated the blood-stage antimalarial activity of triclabendazole, an antihelminthic drug used against co-endemic fascioliasis, together with its metabolite triclabendazole sulfoxide, and sutezolide, an oxazolidinone antibiotic, in a murine model of Plasmodium berghei ANKA infection following oral administration. None of the three compounds demonstrated significant antimalarial activity under these experimental conditions, contradicting a previously published repositioning hypothesis. Beyond these specific findings, our study is deliberately framed within the 3Rs principles (Replacement, Reduction, Refinement) governing animal experimentation. We argue that publishing negative in vivo results is not only scientifically legitimate but ethically necessary: sharing such data allows research teams working on similar preclinical models to build on existing knowledge, avoid unnecessary experimental duplication, and ultimately reduce the number of animal procedures performed across the field. We advocate for wider dissemination of negative outcomes in antimalarial drug repositioning research as a concrete contribution to more responsible and efficient use of animal models in preclinical pharmacology. Graphical Abstract

Mass drug administration (MDA) using Praziquantel is central to efforts to eliminate Schistosomiasis. However, regions which respond poorly to MDA ("persistent hotspots") have been reported in many regions of Africa, including in Western Kenya. One possible explanation for persistent hotspots is that these areas contain PZQ resistant schistosome parasites. Recent studies have shown that Sm.TRPMPZQ gene is the molecular target for PZQ in schistosome parasites and that mutations in this gene can result in PZQ resistance. This study characterized mutations within Sm.TRPMPZQ in 23,420 miracidia collected from both hotspot and non-hotspot villages in Siaya County, western Kenya. We collected triplicate pools of 780.67 (SD {+/-} 183.47) miracidia from 135 people in five hotspot villages, where S. mansoni prevalence remains high despite over 5 annual treatments, and from 62 people from 5 non-hotspots villages where annual treatment resulted in reduction in prevalence. We extracted DNA from each miracidia pool, amplified 15 amplicons covering 1,695bp of the Sm.TRPMPZQtransmembrane domain and sequenced these to high read depth (21,110x) using a Miseq at KEMRI-CGHR. We identified five high confidence (frequency [≥] 0.01) Sm.TRPMPZQ variants. These included four synonymous changes and a non-synonymous variant (p.L1476I). p.L1476I is found at similar frequency in non-hotspot (0.040 {+/-} 0.006) and hotspot villages (0.044 {+/-} 0.0050) (Mann Whitney U=18, p= 0.31) and does not impact PZQ-response in Ca2+ reporter assays. Our studies show that resistance variants in Sm.TRPMPZQ are rare or non-existent in the locations studied and do not explain the existence of hotspots in this region.

Malaria parasite virulence will be impacted by naturally occurring variation in multiplication rates. Clinical isolates of Plasmodium falciparum exhibit a range of multiplication rates under exponential growth conditions in culture, but it needs to be discovered if relative multiplication rates under competitive conditions are principally defined by this underlying multiplication rate potential. Relative multiplication rates of P. falciparum lines were investigated in 14-day competition assays against a standard competitor clone HB3, with other long-term laboratory-adapted clones showing similar or higher mean per-48-hour rates ranging from 0.98 to 1.51 relative to HB3. In contrast, thirteen Ghanaian clinical isolates cultured for close to two months prior to assay had lower rates, ranging from 0.46 to 0.76 relative to HB3. Analysing the single-genotype clinical isolates and laboratory-adapted clones, there was a highly significant correlation between the competitive rates and previously determined exponential rates (Spearmans rho = 0.95, P = 0.0003), which accounted for most of the observed variance (Pearsons r2 = 0.77, P = 0.004). Testing the effect of alternative nutritional supplementation with human serum or Albumax showed a majority of parasites had consistent competitive rates under either condition, indicating that intrinsic differences determine most variation among parasites.

Ivermectin is an important broad-spectrum anthelmintic used to treat nematode parasites including gastro-intestinal infections of humans and animals. The mode of action for Ivermectin is understood to involve activation of inhibitory glutamate-gated chloride channels (GluCls). Ivermectin has also been reported to inhibit the release of extracellular vesicles (EVs). We found that EVs are released from the whole intestine of the gastro-intestinal parasite, Ascaris suum. Proteomic analysis identified 1,574 proteins within these intestinal EVs, including 96 nematode proteins with putative immune-associated functions based on homology to proteins involved in host immune processes and 130 proteins with predicted digestive functions. Comparative analysis following ivermectin exposure revealed 38 differentially abundant proteins that included the putative immune-related proteins: transthyretin-like proteins, a small heat-shock antigen, a phospholipase A2, and the NF-{kappa}B subunit p105. Thus, ivermectin modulated the potential immune-related cargo of intestinal EVs. The ivermectin inhibition of intestinal EV release was concentration-dependent with an IC50 of 64 nM. We also identified the expression of GluCl subunit receptor genes in the Ascaris intestine. The potent inhibitory effect of ivermectin on the release of these EVs from the nematode intestine and the expression of GluCl channel subunits sheds further light on the site and mechanisms of action of this important anthelmintic.

In a previous screen for compounds that inhibit Plasmodium falciparum merozoite invasion of red blood cells, we identified the Medicines for Malaria Venture compound MMV006833. This compound inhibits PfSTART1, a protein implicated in the expansion of the nascent parasitophorous vacuole membrane following invasion, to accommodate the developing ring-stage parasite. Live-cell lattice light-sheet microscopy of invading merozoites revealed that mNeonGreen-tagged PfSTART1 is released from structures within the merozoite into the nascent parasitophorous vacuole approximately 109 seconds after invasion. Expansion microscopy of PfSTART1-HA merozoites further showed that these punctate PfSTART1-containing structures do not colocalise with known secretory organelles (rhoptries, micronemes and dense granules). Although analogues of MMV006833 are highly potent against P. falciparum, they were previously found to be ineffective against P. berghei parasites in the mouse malaria model. Here, we demonstrate that PbSTART1 is highly resistant to MMV006833 and its analogues when expressed in P. falciparum, indicating that structural differences between the orthologous proteins reduce inhibitor potency. The crystal structure of PfSTART1 in complex with WEHI-991 revealed the molecular basis for inhibition and provided a structural explanation for the reduced potency of this family of compounds against P. berghei. To sensitise P. berghei parasites to MMV006833 analogues, the parasites were engineered to express PfSTART1; however, these chimeric parasites remained insensitive to the compounds. This suggests that factors beyond target engagement, such as compound half-life or bioavailability, contribute to the lack of efficacy observed in the mouse malaria model.

Background Trypanosoma cruzi, the causative agent of Chagas disease, is subdivided into distinct genetic groups known as Discrete Typing Units (DTUs), each with distinct genetic traits that influence epidemiology and transmission dynamics. Several triatomine species serve as potential vectors of T. cruzi in the United States. However, despite the growing number of Chagas disease cases in the country, little is known about the genetic diversity and population structure of T. cruzi in natural vector populations. Methodology/Principal Findings We applied a multilocus metabarcoding approach to improve DTU resolution and characterize the genetic diversity and structure of T. cruzi in triatomines collected across five states of the southern United States. Five single-copy nuclear markers and one mitochondrial marker were amplified and processed by high-throughput sequencing to assess genetic diversity. We recovered 35 nuclear and 15 mitochondrial haplotypes from 70 infected specimens. Overall, genetic diversity was low ({pi} < 0.01 at all nuclear loci), with DTUs TcI and the North American lineage of TcIV detected, TcI being the most prevalent. Geographic structuring was particularly evident in TcI strains, which exhibited a distinctive haplotype profile in Florida populations, potentially linked to the recently revalidated vector species Triatoma ambigua. Mitochondrial introgression from TcIV into TcI suggests inter-DTU genetic exchange in these populations. Multiple haplotypes within individual insects detected across single-copy nuclear markers, support multiclonal infection as common feature of T. cruzi in natural vectors. Conclusions/Significance These findings provide new insights into the genetic landscape and evolution of T. cruzi in the United States. Evolutionary connectivity through mitochondrial introgression and frequent multiclonality highlights the importance of deep sequencing approaches for resolving T. cruzi genetic diversity, with direct implications for understanding for transmission dynamics, disease monitoring and control.

Artemisinin partial resistance (ARTR) in Plasmodium falciparum is mediated by Pfkelch13 mutations but has not emerged in West Africa. Using gene editing, we introduced four Pfkelch13 mutations into a recent Senegalese isolate, evaluating the receptivity of this genetic background to ARTR using the ring-stage survival assay (RSA), a proxy for clinical resistance. One allele conferred the RSA phenotype but was rapidly outcompeted, whereas three conferred stable resistance, revealing allele-specific permissiveness

In Germany, insecticide use is recommended during mosquito-borne disease outbreaks, yet nationwide data on insecticide resistance in key vector species are scarce. This is particularly relevant for Culex pipiens s.l., a vector of West Nile virus. Here, we provide the first broad-scale assessment of molecular insecticide resistance (kdr and ace-1 mutations) in Cx. pipiens f. pipiens across Germany. We associated insecticide resistances with environmental conditions, potential links with mitochondrial variation of the cytochrome c oxidase I (COI) gene as well as phenotypic resistance (CDC bottle assay with permethrin) of the Frankfurt population with elevated kdr frequencies. Kdr resistance alleles were detected in German Cx. pipiens f. pipiens reaching frequencies of up to 50% in the metropolitan areas of Berlin and Frankfurt am Main. Annual precipitation was negatively associated with kdr allele frequency. Interestingly, ace-1 resistance mutations were only present in the metropolitan area of Frankfurt am Main and in Muggensturm (Rhine valley, southwestern Germany). COI haplotype analyses revealed low genetic variability within German Cx. pipiens f. pipiens populations with no association to the insecticide resistance genotype. Phenotypic assays of the Frankfurt population showed early signs of permethrin resistance, but unmatched genotypic and phenotypic resistance state. This study provides the first broad-scale assessment of molecular insecticide resistance in Cx. pipiens f. pipiens across Germany, establishing a baseline for resistance surveillance and vector control. Further research is needed to identify factors shaping phenotypic resistance, as molecular markers alone may not reliably predict resistance. Since sampling was restricted to urban green spaces, broader sampling across environmental gradients is needed to identify additional environmental effectors.

Current insecticide toxicology still lacks an integrated understanding of how lethal and sublethal exposure shape organismal performance across biologically and environmentally relevant contexts. In particular, insecticide susceptibility may vary with physiological state, environmental conditions, and recent exposure history, factors that are only partially captured by standardized toxicity assessments. Here, using Drosophila melanogaster, we systematically compared responses to three insecticides with distinct modes of action - acetamiprid, chlorantraniliprole, and deltamethrin - across developmental stages, sexes, adult ages, and ambient temperatures. Insecticide sensitivity varied substantially across these conditions, with temperature modulating toxicity in a compound-dependent manner. Notably, deltamethrin caused substantial lethality at concentrations more than 10,000-fold below approved field application rates. Beyond these context-dependent differences in acute toxicity, sublethal pre-exposure to each insecticide rapidly reduced lethality during a subsequent challenge with the same compound, with increased tolerance already detectable after 24 h. Importantly, these exposure conditions that enhanced insecticide tolerance were associated with reduced resistance to environmental challenges, including heat and nutrient starvation, revealing a potential trade-off in organismal stress resilience. Acetamiprid exposure further altered reproductive performance in a concentration-dependent manner, with higher exposure reducing egg production while increasing developmental success among the resulting offspring, such that the number of offspring reaching adulthood remained largely unchanged. Together, these findings demonstrate that insecticide susceptibility is highly context dependent and can be rapidly modified by recent exposure history. We identify short-term, compound-specific, and potentially costly tolerance as an underappreciated consequence of insecticide exposure and highlight the importance of incorporating biological context, environmental conditions, and sublethal exposure history into pesticide risk assessment.

BackgroundResistance of the arbovirus vector Aedes albopictus to pyrethroid insecticides is an emerging threat to vector control programs in Europe. Three knock-down resistance (Kdr) mutations affecting the voltage-gated sodium channels targeted by pyrethroids are known to confer resistance: V1016G, I1532T and F1534C. As these Kdr mutations are actively circulating in Europe, their monitoring is crucial for resistance surveillance programs. However, current Kdr genotyping methods are labor-intensive and costly, limiting their applicability for large-scale high-throughput surveillance. MethodologyNovel digital droplet PCR (ddPCR) TaqMan assays were developed, allowing the quantification of these Kdr mutations from pooled mosquito samples from field populations. The specificity of these assays was validated by using mosquitoes of known genotypes together with synthetic DNA constructs carrying haplotype combinations previously unseen in the field. The assays accuracy was assessed by comparing Kdr frequencies measured from pooled mosquitoes to those derived from individual genotypes. These assays were then used in a pilot surveillance study in mainland France, integrating deltamethrin bioassays, pooled Kdr mutation tracking and vector control interventions data. FindingsThe developed ddPCR assays demonstrated high accuracy and specificity, with matching Kdr mutation frequencies between pooled samples and individual genotyping. The pilot surveillance study confirmed the low prevalence of Kdr V1016G and I1532T mutations in most French populations, though some populations exhibited a moderate decrease in susceptibility to deltamethrin. Deltamethrin spraying intensity was weakly correlated with Kdr I1532T mutation frequency while no correlation was observed with deltamethrin susceptibility, suggesting that insecticide selective pressure from curative vector control activities is at most a minor driver of resistance. ConclusionThese novel ddPCR assays provide a simple, cost-effective and high-throughput method for quantifying the frequency of Kdr mutations in Ae. albopictus populations. Their implementation in routine country-wide surveillance programs will enhance the detection of emerging resistance, and inform vector control strategies, preventing arboviral disease transmission. Author summaryAedes albopictus, the Asian tiger mosquito, is an emerging global threat due to its ability to transmit the dengue, chikungunya and Zika viruses among others. Recurrent use of insecticides to prevent infections selects resistant mosquitoes. Kdr (knock-down resistance) mutations confer resistance to pyrethroid insecticides, like deltamethrin. In order to track the arrival of these mutations in a population, individual PCR tests are routinely used, which is very inefficient and costly. The digital droplet PCR tests presented in this study can instead be performed on pools of mosquitoes from the field, greatly increasing the output while reducing the costs. This makes them ideal for use in the surveillance of these mutations in field populations. We first confirmed that the tests are as efficient in pools as they are on single mosquitoes, then we applied these tests in a pilot study on French field mosquitoes to demonstrate their applicability for use in surveillance. Finally, we explored the link between deltamethrin sprayings, bioassay mortality and Kdr mutations frequency, which is non-existent. This means the current management of mosquito populations via insecticide use is sensible. We believe these tests have a place in streamlining the future surveillance programs of these mutations in the field.

BackgroundThe ecological dynamics between Trypanosoma parasites and their wild mammalian hosts, such as bats and armadillos, are complex. Recent 18S rRNA metabarcoding studies have reported extraordinary levels of hidden parasite diversity and frequent multi-lineage coinfections within individual wild hosts. However, the boundary between genuine biological coinfection and methodological artifact remains difficult to establish. Based on Gauses principle of competitive exclusion, the mammalian bloodstream represents a highly constrained niche where stable coexistence of identical ecological competitors is theoretically rare. We hypothesize that previously reported hyper-diverse Trypanosoma coinfections are largely bioinformatic artifacts, and that true intra-host dynamics instead favor single-lineage dominance. MethodsTo test this hypothesis, we sequenced samples from 27 wild armadillos (Dasypus novemcinctus) and 26 bats from Ecuador. The 18S rRNA gene was amplified via nested PCR and sequenced using an Oxford Nanopore Technologies MinION platform. We developed a progressively stringent bioinformatics pipeline to evaluate coinfection hypotheses. Raw reads were processed through three alignment scenarios: Lenient, Moderate, and Conservative. These scenarios modulate sequence identity, mapping quality (MAPQ), and coverage thresholds to effectively isolate true biological signals from alignment ambiguity. ResultsUnder lenient alignment parameters, the resulting profiles mirrored previous literature, exhibiting massive apparent intra-host multi-lineage diversity. However, as bioinformatic stringency increased to conservative thresholds ([&ge;] 98% sequence identity, [&ge;] 99% coverage, and MAPQ [&ge;] 30), artifactual pseudo-coinfections collapsed. The highly restricted dataset demonstrated overwhelming single-lineage dominance, validating only three active mixed infections out of the retained samples. Furthermore, our rigorous pipeline isolated rare but genuine biological signals, including the detection of Trypanosoma cruzi marinkellei--historically considered a bat-restricted subgenus--within the terrestrial armadillo cohort. We also confirmed the presence of T. cruzi DTU III (TcIII) in Ecuadorian armadillos, representing a significant biogeographical record for the region. ConclusionsOnce methodological noise is computationally stripped away, active multi-strain Trypanosoma coinfections in the host bloodstream are revealed to be ecologically anomalous. Our findings strongly support the principle of competitive exclusion, suggesting established lineages actively suppress competitors. While Oxford Nanopore sequencing offers necessary resolution for wildlife parasitology, fine-tuning algorithmic parameters is critical to accurately represent host-parasite networks and prevent the artificial inflation of intra-host diversity metrics. Author summaryPrevious studies using DNA metabarcoding have reported that wild mammals, such as bats, frequently harbor complex communities of multiple Trypanosoma parasite lineages simultaneously. However, ecological principles suggest that identical competitors struggle to coexist stably within a constrained environment like the host bloodstream. To investigate whether these reported high coinfection rates reflect true biology or methodological artifacts, we sequenced the 18S rRNA gene of Trypanosoma from 26 bats and 27 armadillos in Ecuador. We processed the sequencing data through computational pipelines with progressively stricter filtering parameters. We observed that under lenient filtering, animals appeared to have highly diverse, mixed infections. Conversely, when strict parameters were applied to remove potential analytical noise, the artificial complexity collapsed, revealing that the vast majority of hosts were dominated by a single parasite lineage. We confirmed only three active mixed infections in our highly restricted dataset. Our findings indicate that active multi-strain Trypanosoma coinfections are rare, aligning with the principle of competitive exclusion. These results highlight the necessity of applying rigorous bioinformatic filters to accurately evaluate host-parasite interactions and avoid overestimating diversity metrics.

A native entomopathogenic nematode isolate, Heterorhabditis indica Che4.1, was recovered from soils in Valencia (Spain), and its symbiotic bacterium, Photorhabdus akhurstii Che4.1, was isolated and characterized. Nematode identification was confirmed by ITS and 28S rDNA sequencing, while genome-based analyses supported the taxonomic assignment of the bacterial symbiont. The nematode-bacterium complex exhibited insecticidal activity against Spodoptera exigua and Spodoptera littoralis. H. indica Che4.1 showed an LC of 53.7 infective juveniles (IJs)/mL against S. exigua, comparable to that of the commercial reference Larvanem (54.3 IJs/mL). Against S. littoralis, Che4.1 exhibited an approximately twofold lower LC than Larvanem (77.3 versus 155.9 IJs/mL), with non-overlapping 95% confidence intervals. The complete genome of P. akhurstii Che4.1 consists of a single circular chromosome of 5.66 Mb encoding a diverse repertoire of putative virulence factors, including toxin complex (Tc) components, YenTc-like proteins, Mcf toxins, App-family proteins, Pra/Prb proteins, and chitinases, together with several predicted secondary metabolite biosynthetic gene clusters. LC-MS/MS analyses of bacterial culture supernatants and infected insect cadavers detected multiple predicted virulence-associated proteins, including Tc-related, YenTc-like, App-family, and Pra/Prb proteins. In addition, bacterial cell-free supernatants caused 68.8-81.3% mortality in S. exigua neonates under laboratory conditions. Together, these findings reveal a diverse repertoire of potential virulence determinants in P. akhurstii Che4.1 and demonstrate the insecticidal potential of its native nematodebacterium complex. The performance of H. indica Che4.1 relative to a commercial reference supports further evaluation of this isolate for biological control of Spodoptera spp., particularly under greenhouse and field conditions.

Entamoeba encystation process is a crucial developmental process that support to parasite persistence and disease transmission, yet the molecular mechanisms governing this stage conversion remain poorly understood and represent an attractive opportunity for therapeutic intervention. The Encystation Regulatory Motif-Binding Protein (ERM-BP), a transcription factor and key regulator of encystation, therefore represents a promising target for anti-amoebic drug development to prevent disease transmission. In the present study, we applied a target-based drug discovery approach to screen small molecules that can block the activity of ERM-BP, with a particular focus on the Cys-198 residue of this protein, which is functionally important. A library comprising 100 compounds including broad-spectrum nicotinamidase inhibitors and cysteine-reactive electrophiles were screened against ERM-BP by Protein thermal shift assays (PTSA). Thirty-six compounds bound to wild-type ERM-BP while analysis using the C198A mutant narrowed the candidates to three compounds exhibiting Cys-198-dependent interactions. These three peptidyl protease inhibitors, PFMK (Z-Ala-Phe-FMK), LEK (Z-Leu-EK), and EBLL (Ethylbenzyl-Leu-Lys) binds to ERM-BP-WT and abolishes the DNA-binding activity of ERM-BP in a concentration-dependent way. Cellular evaluation further revealed distinct activity of PFMK, LEK and EBLL on trophozoite growth and PFMK and EBLL reduced encystation efficiency significantly, produced irregular, and structurally defective cysts, which fails to excyst to trophozoites, whereas LEK had no significant effects on encystation efficiency or cyst morphology. Altogether, these findings identify peptidyl protease inhibitors PFMK and EBLL as promising compounds to target ERM-BP and block the Entamoeba encystation, providing a framework for developing therapeutics targeting parasite differentiation and encystation.

Tick-borne relapsing fever (TBRF) spirochetes are maintained in nature through vector-associated transmission routes primarily in argasid ticks. However, the extent to which strain-level differences influence the maintenance of TBRF spirochetes remains unclear. We evaluated two Borrelia turicatae strains, Bt-SSK1 and Bt-FCB, in Ornithodoros turicata. Both strains were acquired by female ticks, and after mating we unexpectedly observed that they were hyperparasitized by uninfected and unfed male O. turicata. Bt- SSK1 was maintained in males while Bt-FCB was not. Transovarial transmission (TOT) of Bt-SSK1 and Bt-FCB was also evaluated and striking differences were observed between strains. Bt-SSK1 was vertically maintained in F1 progeny but we failed to detect TOT of Bt-FCB. TOT of Bt-SSK1 occurred inefficiently in early ovipositions but increasing in later or delayed reproductive events, indicating a timing-dependent barrier. Using a gfp-expressing Bt-FCB strain, we show that failure of vertical transmission is associated with a lack of persistent oocyte colonization despite dissemination to ovarian tissues. These findings demonstrate strain-dependent differences in oocyte colonization and represent a critical bottleneck governing vertical transmission and persistence of relapsing fever spirochetes in tick populations. ImportanceVector-borne pathogens rely on diverse transmission strategies to persist in nature, yet the biological factors that govern these processes remain poorly understood. In this study, we demonstrated that strains of Borrelia turicatae differ markedly in their ability to persist within Ornithodoros turicata ticks. We also discovered that male ticks frequently hyperparasitized engorged females, creating an unrecognized transmission route in which previously uninfected ticks can acquire, maintain, and transmitted the B. turicatae. We further showed phenotypic differences in vertical transmission and that it is temporally regulated and linked to successful colonization of developing oocytes, identifying a critical bottleneck in pathogen maintenance. Together, these findings revealed that strain-specific traits influence both horizontal maintenance within tick populations and vertical transmission to progeny. This work provides insight into the ecological and evolutionary processes that enable relapsing fever spirochetes to persist in nature.

Visceral leishmaniasis (VL) is a neglected tropical disease caused by the protozoan parasites Leishmania infantum (LI) and Leishmania donovani (LD). VL disproportionately affects populations that live in extreme poverty and those affected by climate change. Treatment options are limited, fiscally prohibitive, and toxic to some that receive treatment. In cases where VL remains untreated, death is inevitable. Although disease prevalence is increasing in affluent countries with robust research capabilities, there remains little economic incentive to search for new, more accessible non-toxic treatments, emphasizing the neglected tropical aspect of this disease. Our previous results indicate that sulforaphane (SFN) inhibits VL promastigote and amastigote growth through an unknown mechanism. The current study extends the anti-parasitic effects of SFN establishing SFN's efficacy against multiple Leishmania parasites including L. tropica, L. mexicana, L. panamensis and L. amazonesis. Moreover, SFN was an effective therapy post-infection as stably infected macrophages treated with SFN (10 M for 48 hours) had a significant reduction in the number of infected cells and the number of amastigotes per cell against Leishmania species that cause both VL and cutaneous leishmaniasis (CL). Moreover, SFN led to a reduction of motility within 6 hours of treatment indicating a direct interaction with the parasite. Alkynyl SFN (A-SFN) was subsequently synthesized to determine the specific binding target of SFN. Interestingly, A-SFN was more effective than SFN in decreasing parasite viability and inhibiting motility against VL and CL parasite species at equivalent concentrations. Immunofluorescence analysis utilizing click chemistry indicated A-SFN binds to cytosolic promastigote components and colocalized only minimally with KMP-11. The binding of SFN to KMP-11 could not be confirmed in vitro. In terms of intracellular host effects, SFN treatment increased the induction of nonselective macroautophagy by increased lysosomal acidification as quantified by DALGreen and FITC-dextran staining in infected macrophages. The induction of autophagy occurred simultaneously with the upregulation of expression of cytoprotective, antioxidant and autophagy-related genes via the NRF2 transcription factor. These data provide robust evidence that SFN is an excellent lead compound against Leishmania that initiates multiple innate immune pathways that enable enhanced parasite killing. Future studies will focus on the in vivo efficacy of SFN against CL.