International Journal for Parasitology: Drugs and Drug Resistance

Anthelmintic drugs are used to control soil-transmitted helminths that infect a third of the worlds human population. There is increasing concern about the development of resistance to anthelmintic drugs because of the limited number of compounds available and there is an unmet need for new resistance-busting drugs. Here we describe the presence of a previously unrecognized endogenous acetylcholine analogue, {beta}-alanine betaine, which may serve as an endogenous ligand for an alternate subfamily of nicotinic receptors (DEG-3/DES-2) that could be developed as novel drug targets because their analogues are not present in their human or animal hosts. We collected peri-enteric fluid from female Ascaris suum (a model for the human parasite, Ascaris lumbricoides) and subjected it to chromatography and MS/MS to reveal signals consistent with acetylcholine, choline, and {beta}-alanine betaine but we did not recover betaine. We injected betaine into female Ascaris suum which produced no effect. However, injection of {beta}-alanine betaine, produced characteristic pretzel coiling and injection of levamisole produced a rod-like spastic paralysis. The differences between {beta}-alanine betaine and levamisole suggested that they activate different nAChRs subfamilies. PCR showed that messages of the DEG-3 subfamily of nAChR channels, which are betaine targets and were present in the intestine and body wall of A. suum. Calcium signaling experiments showed that {beta}-alanine betaine increased intracellular calcium of the intestine enterocytes and electrophysiology of the body muscle cells demonstrated that {beta}-alanine betaine produced membrane potential depolarization. In N2 elegans, application of {beta}-alanine betaine produced gradual inhibition of motility, which was reduced in acr-20, acr-23, des-2, deg-3 and lgc-41 null-mutants. These observations suggest that, in addition to acetylcholine, {beta}-alanine betaine - an anaerobic analog of betaine - may function as an endogenous ligand in anaerobic nematodes such as A. suum. An expanded repertoire of nicotinic acetylcholine receptor subfamilies in nematodes relative to mammals may reflect a corresponding need for diversification of cholinergic endogenous ligands in these organisms. This repertoire could allow their simpler neuronal system to perform more complex controls and be exploited for development of different and novel subfamily selective cholinergic anthelmintics.

Over a quarter of the worlds population is at risk of infection by soil transmitted helminths (STH). Among the STHs Trichuris trichiura infects approximately 7% of people globally, causing a loss of 232,000 DALYS. The main strategy to combat T. trichiura infection focusses on mass drug administration with the benzimidazoles. Whilst albendazole and mebendazole have been effective at reducing the burden of other STHs, the cure rate for whipworm is less than 50% with resistance alleles rising. Glucose is the most studied nutrient in Trichuris spp, however we have no understanding, at the molecular level of the mechanism of uptake in Trichuris spp. We sought to identify putative glucose transporters in Trichuris and investigate how these can be inhibited with phloretin. Using the C. elegans Facilitated Glucose Transporter 1 (FGT) sequence we identified two potential homologs in T. muris (TmGLT) and T. trichiura (TtGLT). We should both proteins contained sequence similarity to FGT1 and contained multiple sequence domains associated with glucose and sugar transport. Further, using Alphafold and molecular docking we show glucose docking sites consistent with transport. To asses the ability of phloretin to inhibit glucose transport, we also performed molecular docking with phloretin, showing possible inhibition. To validate the potential inhibition in vitro we measured the 48h LC50 of phloretin which we showed to be 111 ug/ml against adult T. muris worms, around half that of mebendazole in the same conditions. In contrast phloretin exhibited no effect on worm burden or fecundity in vivo. Together these findings provide the first in silico characterisation of putative glucose transporters in Trichuris spp and have identified glucose transport inhibition as a promising avenue for anthelminthic drug discovery. Whilst further work is required to optimise in vivo efficacy, our results highlight parasite glucose acquisition pathways as potential druggable targets in whipworm.

Insecticide-based vector control remains the cornerstone of malaria prevention, averting approximately 1.2 billion cases between 2000 and 2025. These interventions primarily reduce transmission by killing mosquitoes; however, widespread reliance on a limited number of compounds has driven the emergence of insecticide resistance. This has prompted the development of new insecticides with novel modes of action. Notably, the pyrrole insecticide chlorfenapyr has been shown to affect both the mosquito vector and the malaria parasite, suggesting that compounds with dual activity could provide an additional strategy to suppress transmission. Here, we present a medium-throughput discovery pipeline that integrates in vitro Plasmodium sporozoite motility assays with machine-learning-based analysis, alongside in vivo exposure of infected Anopheles mosquitoes and quantification of parasite development. Screening 32 insecticidal chemistries identified five compounds that significantly impaired sporozoite motility, including three avermectin endectocides, the mitochondrial complex III inhibitor hydramethylnon, and tralopyril, the active form of chlorfenapyr. Several compounds transiently increased motility, indicating that parasite physiology is frequently influenced by insecticide exposure. In vivo exposure to abamectin reduced parasite numbers in both the haemolymph and salivary glands and impaired productive motility. Importantly, this inhibition was confirmed in Plasmodium falciparum-infected mosquitoes, where exposure significantly reduced salivary gland invasion. These findings reveal that parasite-directed activity among insecticides may be more common than previously appreciated and demonstrate a scalable approach to identify compounds capable of simultaneously killing mosquitoes and suppressing parasite transmission. Significance StatementVector control relies heavily on insecticides that kill mosquitoes, yet rising resistance threatens their effectiveness. Here we show that several insecticides also affect the malaria parasite itself. Using a scalable screening pipeline combining machine learning-assisted sporozoite motility analysis with mosquito infection assays, we found that 15% of tested insecticides significantly impaired parasite motility, including compounds with distinct modes of action. Among these hits, the avermectin abamectin reduced parasite dissemination in mosquitoes and limited salivary gland invasion in both Plasmodium berghei and the human malaria parasite P. falciparum. These findings reveal that parasite-directed activity among insecticides may be more widespread than expected and highlight the potential to develop vector control tools that simultaneously kill mosquitoes and block parasite transmission.

Fasciola hepatica is a trematode parasite responsible for fasciolosis, a liver disease that affects humans and livestock worldwide. Together with other food-borne trematode infections, fasciolosis is considered a neglected tropical disease. Further, it imposes substantial agricultural losses due to infections in ruminants. No vaccine is currently available, and control heavily relies on drug treatment, especially with triclabendazole (TCBZ). However, the intensive use of TCBZ over the past four decades has led to increasing rates of treatment failures and the emergence of drug-resistant parasites. Therefore, the identification of new treatment options is an urgent priority. The currently available toolset for drug screening, however, is limited. To address this need, we established a novel, semi-automated, standardized, and objective screening assay based on motility monitoring of newly excysted juveniles using microscopic live imaging. The assay was validated by testing a panel of ten compounds with known anthelmintic properties, amongst them TCBZ (IC50: 1.5 {micro}M) and the new activator of the F. hepatica transient receptor potential melastatin (TRPM) ion channel, benzamidoquinazolinone (IC50: 1.05 {micro}M). In addition to these two compounds with known activity against F. hepatica, three compounds were identified as particularly promising with a fast onset of action and IC50 values in the nanomolar range: the salicylanilides MMV665807 (IC50: 44 nM), niclosamide (IC50: 32 nM), and its ethanolamine salt, niclosamide ethanolamine (IC50: 9 nM). Complementary live/dead staining revealed that only TCBZ displayed parasiticidal activity, while the other compounds, although leading to parasite paralysis, did not lead to parasite death within 72 hours. Scanning electron microscopy of drug treated parasites did not reveal any significant damage at concentrations corresponding to the IC50s, but strong phenotypes were visible at 20 {micro}M. The presented motility assay provides a robust method for the discovery of novel anthelmintic compounds and facilitates the ongoing effort to combat fasciolosis. Author SummaryFasciola hepatica, the common liver fluke, is a parasitic platyhelminth that infects the liver and biliary ducts of humans and livestock, causing fasciolosis, a Neglected Tropical Disease as defined by the World Health Organization. Triclabendazole is the drug of choice to treat humans and animals. However, its intensive use has led to the emergence of drug resistance resulting in treatment failures worldwide. The identification of novel drugs is therefore urgent. Here, we present a semi-automated and objective method to assess the activity of compounds on one of the key life stages of the parasite: the newly excysted juveniles (NEJ). This stage is highly motile and motility assessment can be exploited to screen for bioactive compounds. Using time-lapse imaging, we quantified NEJ movement after drug exposure. From a panel of ten tested reference anthelmintics, two known fasciolicides (triclabendazole and benzamidoquinazolinone) and three additional compounds (MMV665807, niclosamide, and niclosamide ethanolamine) displayed particularly strong activity and were selected for further investigation. This method represents a robust tool for drug screening and facilitates the discovery of new compounds against F. hepatica.

Motility of Plasmodium sporozoites (SPZs) in the skin is a key determinant of successful host infection. Earlier studies have described rapid movement of both murine and human SPZs in skin following syringe inoculation. It is typical to classify SPZ trajectories into ``motile'' and ``immotile'' and restrict the analysis of movement patterns to motile SPZs. Because criteria to define motile SPZs are dependent on the study and are often qualitative, it remains unclear if sub-selection of motile tracks introduces biases in characterization of SPZ movement in vivo. We processed imaging data (22 movies) from a recent study of movement of P. falciparum (Pf) and P. yoelii (Py) SPZ in skin. We proposed a novel metric -- maximal spatial spread (MSS or S) --- that is the maximum Euclidean distance between any two recorded positions in a trajectory. We used MSS to classify SPZ trajectories as immotile (S<Sthreshold) or motile (S>Sthreshold) for a given threshold value Sthreshold. Larger Sthreshold values naturally resulted in a smaller fraction of tracks classified as motile, and subsequently, in an increased overall displacement, instantaneous and mean speeds, decreased mean turning angle, and higher initial slopes of the mean squared displacement (MSD) curves. We found that at intermediate values of Sthreshold Pf SPZs had a lower average speed than Py SPZs suggesting that host environment may impact SPZ movement. Both species exhibited a small but statistically significant decline in average speed with time after inoculation but this was also dependent on the Sthreshold value. Our analysis of MSD curves and turning angle distributions suggests that both Pf and Py SPZs undergo correlated random walks -- a type of Brownian walk with short-term superdiffusive displacement. By using a novel methodology of hidden Markov models (moveHMM package in R) we found that SPZ movement is best described by three movement states; however, none of these states corresponded to previously described circling gliding. Taking together, our results suggest that inference of SPZ movement patterns depends on the criteria used to define tracks as motile or immotile. Standardized preprocessing criteria are therefore important when comparing motility across Plasmodium species, experimental time points, or laboratories. Analysis of turning angle distributions and application of hidden Markov models provided additional metrics to quantify distinct modes of SPZ movement in vivo.

The virulence of Toxoplasma gondii and other apicomplexan parasites relies on a unique form of cellular motility driven by MyoA, an unconventional class XIV myosin motor protein. To identify new chemical probes for investigating the molecular mechanisms of parasite motility, we screened over 50,000 small molecules for inhibitors of T. gondii MyoA (TgMyoA). The top hit from the screen, UCB-9721, is almost 40-fold more potent as an inhibitor of TgMyoA actin-activated ATPase activity than the previously described TgMyoA inhibitor, KNX-002, and 45-fold more potent at inhibiting parasite motility, with no detectable toxicity towards mammalian cells. UCB-9721 also inhibited the motility and/or growth of the related apicomplexan parasites Plasmodium falciparum, Cryptosporidium parvum, and Babesia duncani, suggesting that this compound will be a useful new chemical probe for studying motility and MyoA function in apicomplexan parasites more broadly. While UCB-9721 and KNX-002 were identified independently, they share a similar chemical scaffold. To determine why UCB-9721 is so much more potent than KNX-002 and to inform future development of this inhibitor class, we undertook comparative molecular docking analyses, targeted TgMyoA mutagenesis, and a directed structure-activity relationship analysis. The results identified the sulfonamide group of UCB-9721 and its hydrogen bond interactions with R249, E275 and a stabilized water network within the TgMyoA binding pocket as key to the compounds increased potency. Further development of UCB-9721, informed by the results presented here, may transform this promising new chemical class into actionable drug development leads against this important group of human and animal pathogens.

Human African trypanosomiasis, caused by a protozoan parasite Trypanosoma brucei, is a neglected tropical disease for which well-tolerated, conveniently administered, and highly efficacious medicines are still missing. Previously, T. brucei Phosphofructokinase was targeted by small-molecule inhibitor development efforts. This approach has shown promise both in vitro and in vivo. In this study, we have used these wet-lab results, evaluated the compounds already characterised by Molecular Dynamics simulations, found relationships between in silico and wet-lab data and used these observations to evaluate compounds that we selected through several different approaches of virtual screens. We observed that inhibitor-ATP interactions are highly predictive of the inhibitory activity. Several compounds selected through virtual screens have outperformed previously characterised compounds.

Malaria, of which the most virulent form is caused by Plasmodium falciparum parasites, remains a major global health burden. The appearance of resistance to first line treatments artemisinin-based therapies, emphasizes the need to identify new parasite vulnerabilities to develop new therapeutics. Phosphoinositides are central regulators of membrane identity, vesicular trafficking, and signaling, and their synthesis depends on tightly controlled phosphatidylinositol transfer by Sec14-like phosphatidylinositol transfer proteins in many eukaryotes, yet their roles in P. falciparum remain poorly defined. Here, we analyzed six P. falciparum Sec14 domain-containing proteins: PfSec14-1 (PF3D7_0626400), PfSec14-2 (PF3D7_0629900), PfSec14-3 (PF3D7_0717100), PfSec14-4 (PF3D7_0920700), PfSec14-5 (PF3D7_1007200), and PfSec14-6 (PF3D7_1127600). Domain organization segregates these proteins into a BNIP-2 and Cdc42GAP homology (BCH) subfamily (PfSec14-3, PfSec14-5) and a canonical Sec14 subfamily (PfSec14-1, PfSec14-2, PfSec14-4, PfSec14-6). Yeast complementation assays showed that PfSec14-1, PfSec14-4, and PfSec14-6 partially rescue growth of a temperature-sensitive sec14 mutant, suggesting phosphatidylinositol and phosphatidylcholine transfer activity. Gene disruption revealed that PfSec14-1 is important for asexual blood-stage proliferation, whereas PfSec14-2 is dispensable under standard culture conditions. In contrast, mislocalization of PfSec14-1 and PfSec14-4 using a knock-sideways approach did not impair asexual growth. Subcellular localization indicates distinct distributions for PfSec14-1, PfSec14-2, and PfSec14-4. Together, these findings reveal functional and spatial diversification of Sec14-like phosphatidylinositol transfer proteins in P. falciparum.

Malaria remains a pressing global health challenge, with the continued emergence of resistance threatening the long-term efficacy of artemisinin-based combination therapies (ACTs). Piperaquine (PPQ), an important partner drug in artemisinin-based combination therapies exhibits a unique bimodal dose-response phenotype associated with reduced susceptibility, yet the biological mechanism underlying this phenotype remains unknown. This phenotype is strongly associated with mutations in pfcrt and copy number amplification of plasmepsin II/III (pm II/III). Given that plasmepsins play a central role in hemoglobin degradation within the blood stage parasite digestive vacuole, and that PPQ accumulates within this compartment and perturbs heme detoxification, this phenotype likely reflects alterations in fundamental biological processes alongside drug-specific effects. We used isogenic PPQ-resistant parasite clones differing only in pm II/III copy number to integrate phenotypes with metabolic changes, and transcriptional responses to ascertain the impact of genotype combinations on parasite response to PPQ. Across increasing PPQ concentrations, parasites with elevated pm II/III copy number exhibited distinct metabolic responses compared to single-copy parasites, specifically, an altered abundance of peptides derived from hemoglobin degradation, directly implicating a core biological pathway long associated with plasmepsin function. The combination of metabolic and transcriptional data with phenotypic measurements supports a model in which increased plasmepsin expression enhances the parasite's capacity to sustain hemoglobin digestion and associated metabolic activity under high PPQ concentrations. This points to a mechanistic basis for continued parasite survival, indicating that changes in hemoglobin processing within the digestive vacuole contribute to the bimodal response to PPQ. Molecular dynamics simulations further support a direct interaction between PPQ and PM II/III, as a mechanism by which these proteins impact PPQ response dynamics through both modulation of hemoglobin digestion and protein-drug interactions within the digestive vacuole.

BackgroundMatrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) is widely used for sand fly identification, but its potential to detect Leishmania infections in vectors remain underexplored. This pilot study evaluated whether MALDI-ToF MS protein profiles of lab-reared Lutzomyia longipalpis and Nyssomyia neivai can discriminate Leishmania infantum-infected from uninfected females. MethodologyColonies were experimentally infected with L. infantum using membrane feeding, and females were collected at different days post-blood meal. Thoraces and legs were processed individually for MALDI-ToF MS, and spectra were analysed using both Bruker software and custom R pipelines. Principal findingsUnsupervised approaches (MSP dendrograms, PCA) showed limited or inconsistent separation of infection status for Lu. longipalpis. In contrast, supervised machine-learning models built on peak-intensity matrices achieved excellent discrimination between infected and uninfected specimens for both species, with several algorithms reaching near-perfect performance on an external test set not used for training. Variable-importance analysis highlighted sets of m/z peaks, mainly showing decreased intensity in infected sand flies, as putative infection biomarkers. ConclusionThis proof-of-concept study highlights that L. infantum infection induces reproducible, species-specific alterations in sand-fly MALDI-TOF profiles, supporting further development of high-throughput, MS-based screening of infected vectors. Author summaryLeishmania infantum is a parasite responsible for visceral leishmaniasis, a severe neglected tropical disease. It is transmitted to humans by sandfly vectors. This study explored whether the MALDI-ToF mass spectrometry technique can detect infection by the L. infantum parasite in the two main sandfly vectors in Brazil: Lutzomyia longipalpis and Nyssomyia neivai. The method has already been tested to identify sandfly species, but its ability to detect infected insects had not been well studied. We infected laboratory-reared sandflies and analyzed their protein profiles to see whether infected and uninfected individuals could be distinguished. We found that infection changes the molecular fingerprints of both sandfly species. Machine-learning models were able to distinguish infected from uninfected specimens with very high accuracy. A small part of the most informative signal was shared between both species, while most of the peaks were species-specific, suggesting that infection affects each vector in a slightly different way. These results show that MALDI-ToF has promise as a rapid, low-cost tool for screening sandflies for Leishmania infection. With further validation, this approach could complement existing surveillance methods and help monitor disease transmission in endemic areas.

Wildlife hosts play important roles in the ecology and transmission of vector-borne parasites, yet information on host associations remains scarce in many biodiverse tropical regions. Within a One Health framework, characterizing parasite diversity in wildlife can improve understanding of ecosystem health and disease emergence. Road-killed animals provide a non-invasive opportunity to investigate host-parasite interactions while minimizing disturbance to natural habitats. We screened 127 liver and intestinal tissue samples obtained from 76 road-killed vertebrates collected near protected areas in two Ecuadorian biodiversity hotspots, the Tropical Andes and Choco-Darien, for trypanosomatids and other vector-borne microorganisms. Molecular analyses targeted the 18S rRNA and cytochrome b genes of trypanosomatids and included additional screening for Trypanosoma cruzi, Trypanosoma rangeli, Rickettsia spp., and piroplasmids. Twenty-nine samples were positive for kinetoplastids. We detected diverse trypanosomatids representing the genera Leishmania, Porcisia, Trypanosoma, Phytomonas, Blastocrithidia, and Obscuromonas, as well as free-living kinetoplastids of the order Neobodonida. The most frequently detected species was Leishmania amazonensis, identified in 17 samples from at least 13 species of birds, reptiles, and caecilians, predominantly in liver tissue, suggesting previously unrecognized host associations. We also identified a putatively novel species of Porcisia and three potentially undescribed avian trypanosomes belonging to the subgenus Ornithotrypanum. No evidence of T. cruzi, T. rangeli, Rickettsia spp., or piroplasmids was found. Our findings identify birds, reptiles, and caecilians as potential reservoir hosts of L. amazonensis. In addition, we substantially expanded current knowledge of kinetoplastid diversity in Ecuadorian wildlife. This study demonstrates the value of road-killed animals as a practical, non-invasive resource for wildlife pathogen surveillance and highlights the importance of integrating biodiversity research into One Health approaches to better understand parasite transmission dynamics in rapidly changing tropical ecosystems. Author summaryMany parasites that affect humans circulate naturally in wildlife, but identifying their animal hosts is often difficult in remote, biodiverse regions. We used road-killed animals as a non-invasive source of biological material to investigate parasites in wildlife from two biodiversity hotspots in Ecuador. By analyzing tissues from birds, reptiles, amphibians, and mammals, we found a remarkable diversity of kinetoplastid flagellates, a group that includes the agents of Chagas disease and leishmaniasis. Although we did not detect human-infective trypanosomes, we repeatedly identified Leishmania amazonensis (a species causing human disease) in birds, reptiles, and caecilians. These vertebrate groups have not previously been recognized as potential hosts of this parasite. We also discovered several undescribed trypanosomatid species, emphasizing how little is known about parasite diversity in tropical wildlife. Our results show that road-killed animals can provide valuable information on host-parasite interactions without disturbing living populations. Such surveillance contributes to One Health efforts by improving our understanding of how environmental change, wildlife, and human health are interconnected.

Background: Tuberculosis, especially drug-resistant tuberculosis (DR-TB) including multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains, remains a leading cause of infectious death worldwide. The rapid accumulation of whole-genome sequencing (WGS) data had spurred numerous computational methods for predicting antimicrobial resistance in Mycobacterium tuberculosis. However, heterogeneous datasets, preprocessing pipelines, and evaluation protocols have made fair comparisons impossible and have hindered clinical translation. A critical yet missing resource is a large-scale, unified benchmark to systematically assess and compare existing methods. Methods: We curated an integrated MTB WGS--phenotypic drug susceptibility testing (pDST) dataset from three sources: the CRyPTIC dataset (Comprehensive Resistance Prediction for Tuberculosis: an International Consortium), a published multi-study compilation, and newly curated literature-derived datasets. The final benchmark contains 54,364 paired WGS-pDST records with broad geographic, lineage, and drug coverage. After harmonizing phenotypes and generating standardized variant features, we evaluated seven models (including classical machine learning and deep learning architectures) across 18 drug-level and six clinical resistance category prediction tasks. Results: XGBoost achieved the highest mean drug-level AUPRC (0.674) and F1-score (0.620) and ranked first in AUPRC for 11 of 18 drugs, whereas WDNN achieved the highest mean AUROC. Random forest yielded the highest mean specificity (0.956) and accuracy (0.933), whereas logistic regression achieved the highest mean recall (0.774), highlighting distinct clinical trade-offs. Drug-level difficulty was highly heterogeneous: rifampicin and isoniazid were predicted robustly, whereas bedaquiline, delamanid, linezolid, and clofazimine remained persistently difficult. In clinical resistance category evaluation, RR-TB, MDR-TB, and pan-susceptibility were well predicted, but XDR-TB and other resistance categories constituted major bottlenecks. Conclusions: Under the largest unified benchmark to date, classical machine-learning methods, particularly XGBoost, provided the strongest precision--recall and F1 performance overall, while neural models remained competitive by AUROC. Emerging drugs (bedaquiline, delamanid, linezolid, clofazimine) and XDR cases remain persistently difficult to predict, identifying key bottlenecks for future method development. This benchmark can serve as a community standard for evaluating MTB resistance prediction and the provided evaluation pipeline offers an actionable baseline for regulatory qualification and clinical decision support system validation, accelerating the translation of WGS-based resistance prediction into practice.

Sex chromosomes are central to many ecological and evolutionary processes. Evidence has accumulated that sex chromosome systems vary extensively in age, turnover and transitions, motivating renewed efforts to study the diversity of sex chromosome systems across the tree of life. However, successful genomic detection of sex chromosomes depends on several factors, including the size and divergence time, background genetic diversity, and the number of sequenced females and males. In addition, technical challenges associated with sequencing and analysing the sex-limited Y/W chromosome remain. Here, we present PhaseWY, an automated Snakemake pipeline that uses whole-genome sequencing data from multiple female and male individuals to identify sex-chromosomal regions and extract the corresponding Y/W sequences. PhaseWY (i) detects sex differences in alignment depth, (ii) applies read-based and statistical haplotype phasing, (iii) identifies sex-linked regions using haplotype clustering, and (iv) subsets autosomal, X/Z- and Y/W-linked variants for downstream analyses. We applied PhaseWY to simulated data to benchmark factors influencing sex-linkage detection and successful extraction of Y/W-linked variants. To demonstrate its practical utility, we further applied PhaseWY to the neo-sex chromosome system in Alauda larks (Alaudidae) and performed a range of downstream analyses demonstrating the scope of applications of the PhaseWY output. We conclude that PhaseWY provides an easy-to-use and reproducible tool for population-genomic analyses in non-model organisms, with particular importance for advancing our understanding of sex-chromosome evolution.

BACKGROUNDThe soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is a pest of soybean in North America that can cause significant yield loss when outbreaks are not managed. Current management tactics primarily rely on inexpensive pyrethroids, but the sustainability of this option is threatened by insecticide-resistance in A. glycines populations across the Upper-Midwest United States. Field-evolved resistance is associated with mutations in the voltage-gated sodium channel subunit h1 (vgsc-h1) gene. RESULTSFour double-stranded RNA (dsRNA) molecules, each matching the sequence of a vgsc-h1 transcript variant ("Specific dsRNAs"), were topically applied to aphids with a genotype carrying the corresponding allele. The mortality of pyrethroid resistant aphids exposed to a Specific dsRNA increased in a dose-dependent manner when applied alone or with a constant concentration of lambda-cyhalothrin, plateauing at 1000 ng ul-1. Synergism was detected between two of four combinations of the Specific dsRNAs and lambda-cyhalothrin. These results were mirrored by the topical application of a single dsRNA with the consensus sequence of all vgsc-h1 variants ("Combined dsRNA"). Mortality was consistently higher in aphids treated with either Specific dsRNA or the Combined dsRNA, alone or with lambda-cyhalothrin, compared to insecticide alone. The number of nymphs produced per female treated with the Specific or Combined dsRNA alone decreased significantly compared to untreated controls. CONCLUSIONThis study demonstrates that the topical application of dsRNAs targeting vgsc-h1 increases the susceptibility and reduces the reproductive capacity of pyrethroid resistant soybean aphids, potentially providing a novel tool for the management of insecticide-resistant aphid populations.

Trichomonas vaginalis is the causative agent of trichomoniasis, the most common non-viral sexually transmitted infection (STI). Despite its prevalence, low levels of public knowledge and research funding and the absence of T. vaginalis screening or control programs have led to its categorization as a "neglected" STI. Unlike other STIs in the USA, prevalence increases with age, peaking among individuals in their 40s. Both a motile trophozoite stage and a non-motile pseudocyst state have been described for the parasite, although it is debated whether the latter is a quiescent stage or a degenerate form on its way to cell death. Here we characterize the T. vaginalis pseudocyst by flow cytometry, membrane integrity assays, transcriptomics, and reversion studies. Pseudocysts were induced by culturing trophozoites in acidic media or in iron depleted media, with a variety of resulting survival rates. Flow cytometry studies showed that pseudocysts have intact cell membranes and express phosphatidylserine on their cell surface. Fluorescence-activated cell sorting studies also identified distinct sub-populations of parasites, revealing the importance of using pure live pseudocyst cultures in reversion studies. Pseudocysts were transcriptionally active for several days and had consistent subsets of genes with increased expression compared to trophozoites, although decreased transcription of genes involved in metabolism. Comparative transcriptomics of pseudocysts and trophozoites of two T. vaginalis strains revealed distinct cell states. Combined, our results provide evidence that the pseudocyst cell state is a stress-induced quiescent stage of T. vaginalis that can remain viable for days, with implications for a role in persistent infections. Author SummaryThe sexually transmitted parasite Trichomonas vaginalis has two well-known cell forms: a free-swimming, flagellated trophozoite and an amoeboid form adhered to host epithelia. A third morphology, the pseudocyst, has been described, but it is unclear whether this is a quiescent stage capable of facilitating persistent infections, or a degenerate form indicating cell death. Here we describe experiments revealing that pseudocysts have overall decreased gene expression compared to trophozoites but exhibit stage-specific gene transcription and plasma membrane integrity for days. Moreover, pseudocysts maintain externalized phosphatidylserine for multiple days. Taken together, our results suggest that pseudocysts are a viable cell stage in T. vaginalis distinct from cell death. Further research into pseudocysts and particularly their interactions with host immune cells is needed to reveal what role they may play in T. vaginalis pathogenesis and persistent or asymptomatic infections.

Homing endonuclease genes spread by cleaving homologous chromosomes that lack the endonuclease cassette, after which repair from the endonuclease-containing chromosome converts the cut allele into a copy of the drive allele. This mechanism has provided a conceptual foundation for synthetic gene drive systems, including CRISPR-based drives, that represent promising strategies for the genetic control of insect pests. However gene drive performance depends critically on the repair pathways available in the germline of the target organism. Here, we report a set of transgenic assays originally developed as part of an attempt to establish gene targeting in the malaria mosquito Anopheles gambiae using an in vivo-generated linear targeting molecule. Although the intended FLP-mediated excision step was not achieved in the mosquito germline, analysis of the component strains revealed efficient germline activity of the rare-cutting homing endonuclease I-SceI and a striking bias towards homology-based repair of I-SceI-induced double-strand breaks. Across reporter and donor configurations, cleavage outcomes were dominated by single-strand annealing, microhomology-mediated repair, synthesis-dependent strand annealing and gene conversion-like events, with comparatively limited evidence for classical non-homologous end joining. In reciprocal crosses designed to distinguish gene conversion from gamete loss, I-SceI cleavage also produced inheritance distortion consistent with both conversion of the cleaved allele and reduced recovery of gametes carrying extensively damaged donor alleles. These findings indicate that the An. gambiae germline can strongly favour homology-dependent repair following homing endonuclease cleavage and that cleavage can also generate meiotic drive-like distortion through selective loss of damaged gametes. The results have direct relevance for the design and interpretation of homing endonuclease and CRISPR-based gene drives in malaria mosquitoes, where the balance between homology-directed repair, end joining and gamete viability will determine drive efficiency, resistance formation and transmission bias. Author summaryGene drives depend on a simple but demanding principle: a nuclease cuts one chromosome, and the cell repairs the break using the homologous chromosome as a template, copying the drive element in the process. Before CRISPR, this type of system was explored using naturally occurring homing endonucleases such as I-SceI. We attempted to develop a gene targeting system in Anopheles gambiae based on the Rong and Golic strategy, in which FLP recombinase would excise a donor molecule and I-SceI would linearise it to stimulate recombination. The full knockout technology did not work because FLP-mediated excision was not detected in the mosquito germline. However, the component tests revealed something more broadly important: I-SceI-induced breaks were repaired predominantly through homology-based pathways rather than simple end joining. We also observed inheritance distortion consistent with both gene conversion and loss of damaged gametes. These results help explain why homing-based systems can work in mosquitoes, while also highlighting why repair pathway choice and gamete viability need to be measured directly in any new drive configuration.

The Project to Accelerate New Treatments for Tuberculosis (PAN-TB) aims to accelerate development of shorter, simpler and safer pan-TB combinations. We previously identified 3 out of 25 first-generation novel PAN-TB 4-drug combinations, that cured 90% of mice in less than 3 months, at clinically relevant doses in the relapsing mouse model of TB. These regimens include BPa830Sut, BPa286Sut and BQSut286 (B: bedaquiline; Pa: pretomanid; 830: GSK3211830; 286: GSK2556286; Sut: sutezolid; Q: quabodepistat). Here, we assess the efficacy of these combinations where the original candidates are substituted next-generation or more advanced compounds (ganfeborole (656) for 830, sorfequiline, S for B, TBD09 for Sut and TBD11 for 286) and the individual contributions of specific agents. Six novel regimens demonstrated bactericidal activity more rapid than comparators PHMZ (Rifapentine P, Isoniazid H, Moxifloxacin M, Pyrazinamide Z) and BPaMZ. Modelled cure/relapse data showed that SPa286Sut, SPaSut and SPa656Sut cured 90% of mice in about 1 month, while SPa286, SPaQTBD11 and SPaTBD09 in less than 2 months, faster than PHMZ. Consistent with our previous findings, the fastest-curing regimens centered on a diarylquinoline (S), a nitroimidazole (Pa) and an oxazolidinone (TBD09 or Sut) together with an Rv1625c agonist (TBD11 or 286), DprE1 inhibitor (Q) or a LeuRS inhibitor (656). Notably, significant contributions to sterilizing efficacy were demonstrated for S in all combinations and for Pa, Sut, TBD09, Q and TBD11 or 286 in specific S-containing combinations. These findings suggest potential for these novel agents and combinations to improve treatment of both DS-and DR-TB.

BackgroundNeurulation is a fundamental process in the formation of the central nervous system (CNS). The process begins with the folding and fusion of the neural plate to form the neural tube which subsequently gives rise to the development of the brain and spinal cord. Environmental and genetic factors that disrupt neurulation can induce neural tube defects (NTDs) and consequently cause additional developmental complications, including motor impairments. Purinergic signaling is a conserved form of extracellular communication (i.e. paracrine, synaptic signaling) that plays a role in early development. This signaling is mediated by purine nucleotides and nucleosides, which activate metabotropic P2Y and ionotropic P2X purinoceptors, respectively. Distinct patterns of intracellular calcium dynamics are observed throughout vertebrate development, from fertilization through organogenesis, including neurulation. Among P2X receptors, P2X4 is an ATP-modulated, Ca2+-permeable, ligand-gated ion channel characterized by having the highest Ca2+ permeability and is known to be modulated by ivermectin (IVM). ObjectiveOur investigation focuses on assessing the effects of IVM treatment during neurulation and evaluating the impact of this drug on phenotype, motor behavior and neuromuscular junction (NMJ) structure at tadpole stage. These results were compared with those obtained following separate treatments with compounds that specifically block glycine, GABA(A) and nACh receptors, all which have been described as IVM targets. ResultsIn this study we demonstrate the transcriptional expression for both P2X and P2Y purinergic receptors during neurulation, as well as the expression of P2X4. Following IVM neurula-treatments, we observed neural tube defects (NTDs), pigmentation changes, motor paralysis and alterations in neuromuscular junction (NMJ) structure, particularly affecting axonal branching. In contrast, treatment with the blockers strychnine, bicuculline and -bungarotoxin, used to assess the involvement of GlyR, GABA(A)R and 7nAChR, respectively, failed to show similar outcomes. ConclusionsIn summary, our results highlight the critical role of purinergic signaling during early development, particularly P2X4 receptor mediated signaling during neurulation which may account for the pharmacological effects induced by the positive allosteric modulator ivermectin.

BackgroundGenomic prediction of antimicrobial resistance (AMR) relies on the accurate detection of resistance genes or allelic variants of core genes from raw or assembled genomes sequences. For several bacterial species and antibiotics, AMR genotype-phenotype discrepancies are common, indicating that important sources of error remain unresolved. For Enterococcus faecium, we focused on identifying the sources of discrepancies for tetracycline resistance, for which genotypic detection had shown particularly low accuracy. We investigated the effect of structural variation in antibiotic resistance genes (ARGs)--including gene duplications, truncations, interruptions, and mixed configurations of complete and partial gene copies-- as a source of genotype-phenotype discrepancies from short{square}read data. We conduct further extended investigations to other antibiotic families and into another bacterial species: Escherichia coli. MethodsWe analyzed collections of E. faecium and E. coli genomes, integrating high{square}quality complete assemblies, simulated Illumina short reads, and matched AMR phenotypic data. The integrity, copy number, and allelic diversity of ARGs were examined for multiple antibiotic classes, and their impact on ARG detection and accuracy of AMR determination was assessed using several commonly used bioinformatic tools (SRST2, ARIBA and AMRFinderPlus). ResultsFor E. faecium, after ruling out the effect of specific tet allelic variants on tetracycline susceptibility, we found that the integrity and copy number of tet(M) had a major effect on detection accuracy. Duplicated and incomplete ARGs are also common in E. faecium genomes, particularly for macrolides (erm(B)) and aminoglycosides (ant(6)-Ia and aph(3)-IIIa). In E. coli, similar patterns were observed for tet(A), erm(B) and aminoglycoside{square}associated genes (aph(3{square})-IIIa and ant(6)-Ia). Across ARGs in both species, short-read mapping methods wrongly reported interrupted genes as complete in some instances, while assembly{square}based methods often failed to resolve complete copies of duplicated genes. Detection accuracy improved when tools were adapted to account for gene integrity and when extended AMR databases incorporating species{square}specific alleles were included. ConclusionsOur findings reveal that bioinformatic limitations in dealing with ARG copy number and completeness, and in accounting for allelic variation, underly a substantial source of genotype-phenotype errors, highlighting the need for improved AMR databases and bioinformatic tools that consider these factors to achieve reliable genomic prediction of AMR.

Caenorhabditis nematodes are a powerful model clade for evolutionary genetics. Isogenic lines and panels of recombinant inbred lines (RILs) are among the most essential tools for genetic studies in these species. While most Caenorhabditis species are gonochoristic, large RIL panels have only been developed for self-fertilizing species. This gap biases our understanding and limits our ability to address questions related to the genetic architecture of traits in outbred populations, which have radically higher genetic diversity, heterozygosity, and effective recombination than selfers. Having previously identified Caenorhabditis becei as a tractable gonochoristic species due to its moderate inbreeding depression, we generated two panels of advanced-intercross RILs derived from three individual outbred C. becei worms collected from a single locality on Barro Colorado Island, Panama. One panel derives from a pair of worms sampled from a single rotting fig; the other derives from a cross between worms from two different figs. The panels share one founder in common, yielding two half-sib RIL panels. We sequenced and haplotyped the lines, identifying millions of variants and thousands of recombination breakpoints. Using simulations, we demonstrate the suitability of these lines for quantitative genetics studies and QTL mapping. In our single-fig panel, we observe abundant heritable variation in population growth rate, individual body size, and sexual dimorphism for body size. We detected four QTLs associated with population growth rate and show that estimated allelic effects are good predictors of selection that occurred during panel derivation.