Blowflies of the genus Lucilia (Insecta: Diptera: Calliphoridae) are important in forensic science, medical research, and agriculture, particularly due to their role as facultative ectoparasites of domesticated sheep. The Australian sheep blowfly, L. cuprina dorsalis, is a primary causative agent of cutaneous myiasis, leading to substantial economic losses to the wool industry. In contrast, the closely related subspecies L. cuprina cuprina does not induce myiasis in sheep, but functions as a necrophagous subspecies in urban regions of Australia. Differences in the physiology, behavior, and host interactions of these subspecies might be attributed to their bacterial community structures as demonstrated in other blowfly species. However, our understanding of the bacterial communities associated with these flies is limited. This study aimed to characterize the microbiomes of two geographically distinct subspecies of L. cuprina—L. c. dorsalis (rural) and L. c. cuprina (urban) using 16S rRNA gene sequencing. The findings indicate that, while both subspecies harbor broadly similar microbial compositions, their respective urban and rural environments influence the prevalence of specific bacterial genera. The bacterial communities of these flies were predominantly composed of members of the phyla Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria. The genus Ignatzschineria was highly abundant in L. c. dorsalis, whereas Pseudomonas was more prevalent in L. c. cuprina, highlighting distinct microbial compositions between the two subspecies. These findings provide fundamental insights into the microbial composition of Lucilia species, contributing to a broader understanding of their ecological and biological roles, with potential applications in forensic entomology, medical research, and agricultural pest management. • The microbial communities of Lucilia cuprina dorsalis and Lucilia cuprina cuprina have a broadly similar composition, but the abundance of particular bacterial genera is notably different. • Proteobacteria is the most abundant phylum in both subspecies. • The genus Ignatzschineria is notably abundant in Lucilia cuprina dorsalis, while Pseudomonas is predominant in Lucilia cuprina cuprina, emphasizing differences between the microbial community members between these subspecies.

Abstract Cryptosporidium spp. are protozoan parasites responsible for diarrheal diseases. In humans, cryptosporidiosis is predominantly caused by the human-specific Cryptosporidium hominis and by Cryptosporidium parvum. This second species has been classically reported as zoonotic, with a host preference for ruminants. However, the recently described subspecies C. parvum anthroponosum has been found to be restricted to humans. Here, we generated novel whole genome sequences from West African samples of C. p. anthroponosum , and analyzed them together with all those already available, originating from East Africa, Europe, North America and Asia. Phylogenomics showed that all C. p. anthroponosum isolates are strongly clustered together, forming the sister clade of the zoonotic C. parvum representatives. The phylogenetic variations within C. p. anthroponosum did not present a clear geographic structure, consistent with C. hominis , primarily transmitted in humans. To elucidate the evolution of host species adaptation in C. p. anthroponosum , we then investigated genetic exchanges with C. hominis , detecting an ancestral introgression present in all C. p. anthroponosum isolates. This introgression involved a single gene, encoding for an extracellular galectin-like protein, which we predicted with high confidence to form a protein complex with the human insulin-degrading enzyme, a key metabolic regulator. Considering the role of host insulin metabolism in the proliferation of parasites as well as its known intrinsic differences between humans and ruminants, this molecular interaction could represent a plausible mechanism for an important role of the galectin-like protein in host-parasite interactions and in the host specificity of C. p. anthroponosum .

Sexual recombination is a hallmark of eukaryotic evolution. Without recombination, asexual eukaryotes should succumb to deleterious mutations and more rapidly evolving pathogens. Giardia duodenalis, a parasitic protist, sits within one of the earliest-branching eukaryotic lineages and has no known sexual stage. Whether Giardia are 'ancient asexuals' has been long explored but is unresolved. Here, we find clear evidence of sex in Giardia and also discover an asexual sublineage that has a broader host range than its sexual ancestor. This asexual lineage is not ancient, and is accumulating deleterious mutations. Unlike its sexual counterparts, its genetic variation lacks the signatures of selection and Red Queen coevolution. We propose a new hypothesis that explains how a mutational meltdown during Muller's Ratchet might enable asexual pathogens to expand their host ranges transiently. Fittingly, our results suggest that Giardia is not the last exception to, but rather further evidence of, the essentiality of eukaryotic sex.

Giardia duodenalis (syn Giardia intestinalis, Giardia lamblia) is a neglected, microaerophilic gastrointestinal parasite reliant on broad spectrum anti-microaerophilic/-anaerobic nitroheterocyclic antibiotics (metronidazole) which have been in use for over 70 years. New drugs which avoid their predecessor's shortfalls of toxic and adverse effects, as well as circumvent its increasing treatment failure, are urgently required to lower global rates of up to 200 million symptomatic cases annually. Kinases are essential regulatory enzymes that primarily catalyse the phosphorylation post-translational modification involved in dynamic cellular processes. Kinases are well-validated and attractive drug targets, with many kinase inhibitors demonstrating great success in the clinic as anticancer therapeutics. In G. duodenalis, its intriguing set of minimal "core" protein kinases and the highly expanded Giardia-specific Never-in-Mitosis-A related kinases (Neks) emerge as a novel druggable space. We propose this kinome as an understudied and underutilised space to explore novel antigiardial targets. Intriguingly, despite over 15 years of advances in kinase biology and new annotation tools, there are limited functional evidence on the existence of 'Neks' in G. duodenalis. To incentivise new efforts, we provide an updated kinome reannotation and examination of the giardial core and specific sub-kinomes using novel bioinformatic tools, suggesting a nomenclature and providing insights in a drug-discovery context. Lastly, we have conducted a high-throughput screening of 430 compounds, covering 53 kinase targets and 51 chemical scaffolds, identifying 83/430 antigiardial kinase inhibitors of which 33 true positives could be validated in a subset subjected to drug-susceptibility testing, highlighting intriguing spaces for further development and molecular probes to further explore kinase regulatory pathways in this parasite.

Flystrike (cutaneous myiasis) is caused by blowfly larvae of the genus Lucilia. This disease is a major obstacle to sustainable global sheep and wool production. Flystrike control relies primarily on breech modification surgery (mulesing) and insecticidal treatment; however, control is constantly compromised by the emergence and spread of insecticide-resistance. Preventing the spread of resistance is severely hindered by a limited understanding of genetic variation, structure and gene flow within and among Lucilia populations. Australia is one of the world’s largest producers of sheep and wool products, where Lucilia cuprina dorsalis is the major cause of flystrike. Here, we collected 2,034 Lucilia cuprina dorsalis individuals among 86 populations from sheep-grazing regions across the continent. Each fly was genetically characterised at 20,000 loci using DArTseq, a reduced complexity genome sequencing strategy. Three genetically distinct population clusters (i.e., Western Australia, Eastern Australia and Tasmania) were revealed through population structure analyses. This investigation into population structure and gene flow yields significant insights into the genetic composition of diverse L. c. dorsalis populations throughout Australia. These findings will be essential for the sustainable management of flystrike on a global scale and for addressing the ongoing challenge of insecticide resistance.

Lucilia cuprina, a species of blowfly, consists of two recognized subspecies: L. cuprina cuprina and L. cuprina dorsalis. Although they are morphologically and molecularly similar to each other, they have very different ecological roles. In Australia, L. c. dorsalis is predominantly found in rural areas and is the primary causative agent of sheep myiasis (flystrike), while L. c. cuprina is necrophagous and not a significant pest of livestock in the Americas or elsewhere. Here, we present a chromosome-scale genome assembly for L. c. cuprina and an improved assembly for L. c. dorsalis, enabling comparative genomic analysis between these subspecies. While both genomes share a similar gene content, subspecies-specific genes were identified, which may contribute to their divergent ecological roles -necrophagy in L. c. cuprina and parasitism in L. c. dorsalis. Phylogenetic analyses across target genomic regions reaffirm the close relationship between L. c. cuprina and L. c. dorsalis and position L. sericata as their sister species. Gene mutations linked to diazinon resistance were exclusively observed in L. c. dorsalis, whereas malathion resistance was detected in both subspecies. Additionally, we identified genes with accelerated evolutionary rates in each subspecies, which may underlie their distinct feeding behaviours. We also conducted a detailed analysis of chemosensory genes, revealing that L. c. dorsalis possesses slightly larger repertoires of all four chemosensory gene families studied. In comparison to Drosophila melanogaster, both subspecies exhibit an expanded gustatory receptor clade. Our findings provide valuable insights into the genetic factors underpinning parasitism and insecticide resistance and provide a valuable genetic resource for future research endeavours, including the development of engineered strains aimed at genetic biocontrol strategies. This work enhances our understanding of the evolutionary adaptations for this important blowfly species.

Soil-transmitted helminths (STH) are prevalent in pigs, with the nodular worm, Oesophagostomum dentatum, causing a chronic infection that affects animal welfare and productivity. Unlike the two other dominant soil-transmitted helminths of pigs, Ascaris suum and Trichuris suis, O. dentatum promotes immune responses that deviate from the normal parasite-induced type 2 helper (Th2) response causing long-term infection with minimally acquired immunity. O. dentatum induces a proinflammatory Type 1 helper(Th1)-like response followed by a delayed non-protective Th2 response. The mechanism underlying this disparate response is unclear. Intestinal parasites interact with the host via excreted-secreted molecules and here we investigate the protein and microRNA content of the infectious L3 and adult stage of O. dentatum focusing particularly on identifying the content of extracellular vesicles (EVs). Using Cryo-EM, nanoparticle tracking analysis and silver staining, we confirmed the presence of EVs in adult worm ES. Proteomic analysis revealed prototypical ES proteins, including SCP-like proteins, transthyretin-like proteins, and peptidases, proteases and hydrolases. Interestingly, a DEAD-Box RNA helicase was found unique to EVs, while a Paz domain protein and a Piwi domain protein was found in the adult worm ES that shows sequence similarity to these domains in the exWAGO protein found in H. bakeri EVs. Pathway enrichment analyses revealed proteins involved in nutrient metabolism, and the proteasome. To understand which microRNAs O. dentatum parasites secrete, we annotated the microRNA complement from small RNA-seq libraries from the ES, EVs and EV-depleted SN using microRNA prediction tools (MirMachine and MirMiner). We identified 44 precursor microRNAs and 88 mature microRNAs in the O. dentatum ES microRNA complement. Quantification revealed 22, 44, and 48 mature microRNAs in L3 EVs, adult EVs and adult SN, respectively, with substantial overlap between L3 and adult EVs (n = 22, 37 % of shared set) and between adult EVs and SN (n = 58, 75 % of shared set). Differential analysis identified 11 microRNAs significantly differentially abundant between sample types. To explore potential conservation of EV microRNAs across nematode species, we here, for the first time, compare the microRNA family profiles of O. dentatum adult EVs with those of A. suum and T. suis. We found eight shared microRNA families that are known to be highly abundant in the worms themselves: LET-7, MIR-10, MIR-34, MIR-9, MIR-305, MIR-54, MIR-216, and MIR-750. Their diverging microRNA profiles might underlie their species-specific differences in parasitism. Taken together, this study expands the current knowledge of excretory-secretory molecules in non-model parasitic nematodes and confirms the presence of proteins involved in RNAi processing in the ES of the clade V nematode, O. dentatum. ABSTRACTS: Parasitic nematodes pose a significant challenge to animal health and productivity, particularly in livestock. Among these, Oesophagostomum dentatum establishes chronic infections in pigs with minimally acquired immunity, with an underlying mechanism that remains poorly understood. In this study, we uncovered the excretory-secretory (ES) molecules and extracellular vesicles (EVs) released by O. dentatum, which are thought to play a key role in host-parasite interactions at the gut interphase. Using advanced proteomic and RNA sequencing approaches, we characterized the proteins and microRNAs associated with these compartments. Our findings confirmed the presence of EVs in adult worm ES and identified an EV protein profile significantly enriched in nutrient metabolic and proteasomal pathways, as well as an RNA helicase in the EVs and Paz and Piwi domain proteins in the ES that might be involved in argonaute RNA-mediated silencing. We also annotated the first microRNA complement of O. dentatum ES, identifying 88 mature microRNAs, with a substantial overlap between L3 and adult worm EVs. Comparative analysis with other parasitic nematodes revealed eight conserved microRNA families also generally known as highly abundant microRNAs in nematodes. By characterizing the secreted proteins and microRNAs of this non-model parasitic nematode, our study lays the groundwork for future research into host immune modulation and survival strategies of parasitic nematodes.

ABSTRACT The future of the COVID pandemic and its public health and societal impact will be determined by the profile and spread of emerging variants and the timely identification and response to them. Wastewater surveillance of SARS-CoV-2 has been widely adopted in many countries across the globe and has played an important role in tracking infection levels and providing useful epidemiological information that cannot be adequately captured by clinical testing alone. However, novel variants can emerge rapidly, spread globally, and markedly alter the trajectory of the pandemic, as exemplified by the Delta and Omicron variants. Most mutations linked to the emergence of new SARS-CoV-2 variants are found within variable regions of the SARS-CoV-2 Spike protein. We have developed a duplex hemi-nested PCR method that, coupled with short amplicon sequencing, allows simultaneous typing of two of the most highly variable and informative regions of the Spike gene: the N-terminal domain and the receptor binding motif. Using this method in an operationalized public health program, we identified the first known incursion of Omicron BA.1 into Victoria, Australia and demonstrated how sensitive amplicon sequencing methods can be combined with wastewater surveillance as a relatively low-cost solution for early warning of variant incursion and spread. IMPORTANCE This study offers a rapid, cost-effective, and sensitive approach for monitoring SARS-CoV-2 variants in wastewater. The method’s flexibility permits timely modifications, enabling the integration of emerging variants and adaptations to evolving SARS-CoV-2 genetics. Of particular significance for low- and middle-income regions with limited surveillance capabilities, this technique can potentially be utilized to study a range of pathogens or viruses that possess diverse genetic sequences, similar to influenza.

Antibiotics may alter the gut microbiome, and this is one of the mechanisms by which antimicrobial resistance may be promoted. Suboptimal antimicrobial stewardship in Asia has been linked to antimicrobial resistance. We aim to examine the relationship between oral antibiotic use and composition and antimicrobial resistance in the gut microbiome in 1093 Bangladeshi infants. We leverage a trial of 8-month-old infants in rural Bangladesh: 61% of children were cumulatively exposed to antibiotics (most commonly cephalosporins and macrolides) over the 12-month study period, including 47% in the first 3 months of the study, usually for fever or respiratory infection. 16S rRNA amplicon sequencing in 11-month-old infants reveals that alpha diversity of the intestinal microbiome is reduced in children who received antibiotics within the previous 7 days; these samples also exhibit enrichment for Enterococcus and Escherichia/Shigella genera. No effect is seen in children who received antibiotics earlier. Using shotgun metagenomics, overall abundance of antimicrobial resistance genes declines over time. Enrichment for an Enterococcus-related antimicrobial resistance gene is observed in children receiving antibiotics within the previous 7 days, but not earlier. Presence of antimicrobial resistance genes is correlated to microbiome composition. In Bangladeshi children, community use of antibiotics transiently reprofiles the gut microbiome.

Anemia is highly prevalent globally, especially in young children in low-income countries, where it often overlaps with a high burden of diarrheal disease. Distribution of iron interventions (as supplements or iron-containing multiple micronutrient powders, MNPs) is a key anemia reduction strategy. Small studies in Africa indicate iron may reprofile the gut microbiome towards pathogenic species. We seek to evaluate the safety of iron and MNPs based on their effects on diversity, composition, and function of the gut microbiome in children in rural Bangladesh as part of a large placebo-controlled randomized controlled trial of iron or MNPs given for 3 months (ACTRN12617000660381). In 923 infants, we evaluate the microbiome before, immediately following, and nine months after interventions, using 16S rRNA gene sequencing and shotgun metagenomics in a subset. We identify no increase in diarrhea with either treatment. In our primary analysis, neither iron nor MNPs alter gut microbiome diversity or composition. However, when not adjusting for multiple comparisons, compared to placebo, children receiving iron and MNPs exhibit reductions in commensal species (e.g., Bifidobacterium, Lactobacillus) and increases in potential pathogens, including Clostridium. These increases are most evident in children with baseline iron repletion and are further supported by trend-based statistical analyses.

Algae and cyanobacteria are microorganisms found in almost all fresh and marine waters, where they can pose environmental and public health risks when they grow excessively and produce blooms. Accurate identification and quantification of these microorganisms are vital for ecological research, water quality monitoring, and public health safety. However, traditional methods of manually counting and morphologically identifying these microorganisms are time-consuming and prone to human error. Application of the machine learning-driven Fast Segment Anything Model (FastSAM), an image segmentation model, automates and potentially enhances the accuracy and efficiency of cell identification and enumeration from microscopic images. We assessed FastSAM for algal cell image segmentation, and three clustering evaluation metrics. Segmentation of microscopic images of algal and cyanobacterial cells in water and treated wastewater samples using the FastSAM algorithm, which is a Vision Transformer (ViT) based algorithm that utilizes a compact ViT encoder, demonstrated benefits and challenges of this machine learning driven image processing. Notably, the pre-trained algorithm segmented entire elements in all microscopic images used in this study. Depending on the shape, 50-100% similarity was observed between machine-based segmentation and manual validation of all segmented elements, with 100% of single cells being correctly segmented by FastSAM. The performance of clustering metrics varied between 57-94% with the Spectral Angle Mapper achieving the most accurate performance, 84-94%, compared to the manually chosen clustering benchmarks. Cyanobacterial and algal communities are biologically diverse and have ecological significance. The application of image clustering techniques in studying their cell shapes marks an important advancement in microbial ecology and environmental monitoring. As technology progresses, these methods will become increasingly utilised to decipher the complex roles that algae and cyanobacteria play in our ecosystems supporting mitigation and public health protection measures.

Antimicrobial resistance (AMR) is a complex challenge that poses a critical threat to food and water safety and security as well as to human, animal and environmental health. It is projected to cost the global economy US$100 trillion by 2050. Australia’s new Cooperative Research Centre (CRC) for Solving Antimicrobial Resistance in Agribusiness, Food and Environments (SAAFE) is part of Australia’s One Health approach to mitigating AMR. SAAFE’s 10-year, A$150-million industry-led program will help protect Australia’s food and agribusiness industries, and the environments in which they operate, from the growing threat of AMR. Through its research programs, CRC SAAFE uses a partner-based approach to assist industries to monitor, analyse and mitigate AMR, with projects spanning horticulture, viticulture, aquaculture, animal industries, water and waste.

A new study in this issue of Cell Host & Microbe from Huang et al. provides important insights into the global epidemiology of human-infectious Cryptosporidium and mechanisms leading to the rapid emergence and rise to dominance of new, possibly more virulent, parasite strains.

Midichloria spp. are intracellular bacterial symbionts of ticks. Representatives of this genus colonise mitochondria in the cells of their hosts. To shed light on this unique interaction we evaluated the presence of an intramitochondrial localization for three Midichloria in the respective tick host species and generated eight high-quality draft genomes and one closed genome, showing that this trait is non-monophyletic, either due to losses or multiple acquisitions. Comparative genomics supports the first hypothesis, as the genomes of non-mitochondrial symbionts are reduced subsets of those capable of colonising the organelles. We detect genomic signatures of mitochondrial tropism, including the differential presence of type IV secretion system and flagellum, which could allow the secretion of unique effectors and/or direct interaction with mitochondria. Other genes, including adhesion molecules, proteins involved in actin polymerisation, cell wall and outer membrane proteins, are only present in mitochondrial symbionts. The bacteria could use these to manipulate host structures, including mitochondrial membranes, to fuse with the organelles or manipulate the mitochondrial network.

Trichomonas vaginalis is the most prevalent, non-viral sexually transmitted human infection, causing 170 million cases of trichomoniasis annually. Since the 1950s, treatment has relied on 5-nitroimidazoles (5NIs), leading to increasing drug resistance. A similar drug resistance problem is present in the veterinary pathogen, Tritrichomonas foetus. There are currently no agreed standards for defining 5NI resistance, due in part to two distinct oxygen-dependent (“aerobic”) and oxygen-independent (“anaerobic”) resistance phenotypes. Diagnostic tools to detect 5NI resistance are lacking, and current assays used to phenotypically assess 5NI resistance in vitro are complicated by these two resistance phenotypes. We demonstrate that microaerophilic conditions support sufficient parasite growth to interrogate oxygen-dependent resistance of 5NIs against known resistant and susceptible isolates of T. vaginalis and T. foetus. We further demonstrate that microaerophilic conditions allow sufficient growth for compatibility with existing growth assays, including our TriTOX assay. Adopting microaerophilic conditions eliminates traditional ‘by-eye’ estimates of minimum inhibitory concentrations and opens up options for increased throughput and automation, scalable to higher-throughput analyses of 5NI resistance. This would further allow the development of quantitative phenotypic standards to benchmark oxygen-dependent or oxygen-independent trichomonad 5NI resistance towards standardised surveillance programs to combat drug resistance.

Abstract Extracellular vesicles (EVs) recently emerged as important players in the pathophysiology of parasitic infections. While the protist parasite Giardia duodenalis can produce EVs, their role in giardiasis remains obscure. Giardia can disrupt gut microbiota biofilms and transform commensal bacteria into invasive pathobionts at sites devoid of colonizing trophozoites via unknown mechanisms. We hypothesized that Giardia EVs could modify gut bacterial behaviour via a novel mode of trans‐kingdom communication. Our findings indicate that Giardia EVs exert bacteriostatic effects on Escherichia coli HB101 and Enterobacter cloacae TW1, increasing their swimming motility. Giardia EVs also decreased the biofilm‐forming ability of E. coli HB101 but not by E. cloacae TW1, supporting the hypothesis that these effects are, at least in part, bacteria‐selective. E. coli HB101 and E. cloacae TW1 exhibited increased adhesion/invasion onto small intestine epithelial cells when exposed to Giardia EVs. EVs labelled with PKH67 revealed colocalization with E. coli HB101 and E. cloacae TW1 bacterial cells. Small RNA sequencing revealed a high abundance of ribosomal RNA (rRNA)‐ and transfer RNA (tRNA)‐derived small RNAs, short‐interfering RNAs (siRNAs) and micro‐RNAs (miRNAs) within Giardia EVs. Proteomic analysis of EVs uncovered the presence of RNA chaperones and heat shock proteins that can facilitate the thermal stability of EVs and its sRNA cargo, as well as protein‐modifying enzymes. In vitro, RNase heat‐treatment assays showed that total RNAs in EVs, but not proteins, are responsible for modulating bacterial swimming motility and biofilm formation. G. duodenalis small RNAs of EVs, but not proteins, were responsible for the increased bacterial adhesion to intestinal epithelial cells induced upon exposure to Giardia EVs. Together, the findings indicate that Giardia EVs contain a heat‐stable, RNase‐sensitive cargo that can trigger the development of pathobiont characteristics in Enterobacteria, depicting a novel trans‐kingdom cross‐talk in the gut.

The emergence of large language models (LLMs) and assisted artificial intelligence (AI) technologies have revolutionized the way in which we interact with technology. A recent symposium at the Walter and Eliza Hall Institute explored the current practical applications of LLMs in medical research and canvassed the emerging ethical, legal and social implications for the use of AI-assisted technologies in the sciences. This paper provides an overview of the symposium's key themes and discussions delivered by diverse speakers, including early career researchers, group leaders, educators and policy-makers highlighting the opportunities and challenges that lie ahead for scientific researchers and educators as we continue to explore the potential of this cutting-edge and emerging technology.

Cryptosporidium parvum is a zoonotic apicomplexan parasite and a common cause of diarrheal disease worldwide. The development of vaccines to prevent or limit infection remains an important goal for tackling cryptosporidiosis. At present, the only approved vaccine against any apicomplexan parasite targets a conserved adhesin possessing a thrombospondin repeat domain. C. parvum possesses 12 orthologous thrombospondin repeat domain-containing proteins known as CpTSP1-12, though little is known about these potentially important antigens. Here, we explore the architecture and conservation of the CpTSP protein family, as well as their abundance at the protein level within the sporozoite stage of the life cycle. We examine the glycosylation states of these proteins using a combination of glycopeptide enrichment techniques to demonstrate that these proteins are modified with C-, O-, and N-linked glycans. Using expansion microscopy, and an antibody against the C-linked mannose that is unique to the CpTSP protein family within C. parvum, we show that these proteins are found both on the cell surface and in structures that resemble the secretory pathway of C. parvum sporozoites. Finally, we generated a polyclonal antibody against CpTSP1 to show that it is found at the cell surface and within micronemes, in a pattern reminiscent of other apicomplexan motility-associated adhesins, and is present both in sporozoites and meronts. This work sheds new light on an undervaccines against cryptosporidiosis.

The impact of the host immune environment on parasite transcription and fitness is currently unknown. It is widely held that hookworm infections have an immunomodulatory impact on the host, but whether the converse is true remains unclear. Immunity against adult-stage hookworms is largely mediated by Type 2 immune responses driven by the transcription factor Signal Transducer and Activator of Transcription 6 (STAT6). This study investigated whether serial passage of the rodent hookworm Nippostrongylus brasiliensis in STAT6-deficient mice (STAT6 KO) caused changes in parasites over time. After adaptation to STAT6 KO hosts, N. brasiliensis increased their reproductive output, feeding capacity, energy content, and body size. Using an improved N. brasiliensis genome, we found that these physiological changes corresponded with a dramatic shift in the transcriptional landscape, including increased expression of gene pathways associated with egg production, but a decrease in genes encoding neuropeptides, proteases, SCP/TAPS proteins, and transthyretin-like proteins; the latter three categories have been repeatedly observed in hookworm excreted/secreted proteins (ESPs) implicated in immunosuppression. Although transcriptional changes started to appear in the first generation of passage in STAT6 KO hosts for both immature and mature adult stages, downregulation of the genes putatively involved in immunosuppression was only observed after multiple generations in this immunodeficient environment. When STAT6 KO-adapted N. brasiliensis were reintroduced to a naive WT host after up to 26 generations, this progressive change in host-adaptation corresponded to increased production of inflammatory cytokines by the WT host. Surprisingly, however, this single exposure of STAT6 KO-adapted N. brasiliensis to WT hosts resulted in worms that were morphologically and transcriptionally indistinguishable from WT-adapted parasites. This work uncovers remarkable plasticity in the ability of hookworms to adapt to their hosts, which may present a general feature of parasitic nematodes.

Benzimidazole-2-carbamates (BZ, e.g., albendazole; ALB), which bind β-tubulin to disrupt microtubule polymerization, are one of two primary compound classes used to treat giardiasis. In most parasitic nematodes and fungi, BZ-resistance is caused by β-tubulin mutations and its molecular mode of action (MOA) is well studied. In contrast, in Giardia duodenalis BZ MOA or resistance is less well understood, may involve target-specific and broader impacts including cellular damage and oxidative stress, and its underlying cause is not clearly determined. Previously, we identified acquisition of a single nucleotide polymorphism, E198K, in β-tubulin in ALB-resistant (ALB-R) G. duodenalis WB-1B relative to ALB-sensitive (ALB-S) parental controls. E198K is linked to BZ-resistance in fungi and its allelic frequency correlated with the magnitude of BZ-resistance in G. duodenalis WB-1B. Here, we undertook detailed transcriptomic comparisons of these ALB-S and ALB-R G. duodenalis WB-1B cultures. The primary transcriptional changes with ALB-R in G. duodenalis WB-1B indicated increased protein degradation and turnover, and up-regulation of tubulin, and related genes, associated with the adhesive disc and basal bodies. These findings are consistent with previous observations noting focused disintegration of the disc and associated structures in Giardia duodenalis upon ALB exposure. We also saw transcriptional changes with ALB-R in G. duodenalis WB-1B consistent with prior observations of a shift from glycolysis to arginine metabolism for ATP production and possible changes to aspects of the vesicular trafficking system that require further investigation. Finally, we saw mixed transcriptional changes associated with DNA repair and oxidative stress responses in the G. duodenalis WB-1B line. These changes may be indicative of a role for H2O2 degradation in ALB-R, as has been observed in other G. duodenalis cell cultures. However, they were below the transcriptional fold-change threshold (log2FC > 1) typically employed in transcriptomic analyses and appear to be contradicted in ALB-R G. duodenalis WB-1B by down-regulation of the NAD scavenging and conversion pathways required to support these stress pathways and up-regulation of many highly oxidation sensitive iron-sulphur (FeS) cluster based metabolic enzymes.