ABSTRACT
BACKGROUND: Neutrophils are granulocytes with essential antimicrobial effector functions and short lifespans. During infection or sterile inflammation, emergency granulopoiesis leads to release of immature neutrophils from the bone marrow, serving to boost circulating neutrophil counts. Steady state and emergency granulopoiesis are incompletely understood, partly due to a lack of genetically amenable models of neutrophil development. METHODS: We optimised a method for ex vivo production of human neutrophils from CD34+ haematopoietic progenitors. Using flow cytometry, we phenotypically compared cultured neutrophils with native neutrophils from donors experiencing emergency granulopoiesis, and steady state neutrophils from non-challenged donors. We carry out functional and proteomic characterisation of cultured neutrophils and establish genome editing of progenitors. RESULTS: We obtain high yields of ex vivo cultured neutrophils, which phenotypically resemble immature neutrophils released into the circulation during emergency granulopoiesis. Cultured neutrophils have similar rates of ROS production and bacterial killing but altered degranulation, cytokine release and antifungal activity compared to mature neutrophils isolated from peripheral blood. These differences are likely due to incomplete synthesis of granule proteins, as demonstrated by proteomic analysis. CONCLUSION: Ex vivo cultured neutrophils are genetically tractable via genome editing of precursors and provide a powerful model system for investigating the properties and behaviour of immature neutrophils.
Subject(s)
Antigens, CD34 , Neutrophils , Humans , Neutrophils/metabolism , Neutrophils/cytology , Antigens, CD34/metabolism , Cells, Cultured , Reactive Oxygen Species/metabolism , Proteomics , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/cytology , Gene Editing , Cell Degranulation , Stem Cells/metabolism , Stem Cells/cytology , Cytokines/metabolism , PhenotypeABSTRACT
Free-living amoebae (FLA) serve as hosts for a variety of endosymbionts, which are microorganisms that reside and multiply within the FLA. Some of these endosymbionts pose a pathogenic threat to humans, animals, or both. The symbiotic relationship with FLA not only offers these microorganisms protection but also enhances their survival outside their hosts and assists in their dispersal across diverse habitats, thereby escalating disease transmission. This review is intended to offer an exhaustive overview of the existing mathematical models that have been applied to understand the dynamics of FLA, especially concerning their interactions with bacteria. An extensive literature review was conducted across Google Scholar, PubMed, and Scopus databases to identify mathematical models that describe the dynamics of interactions between FLA and bacteria, as published in peer-reviewed scientific journals. The literature search revealed several FLA-bacteria model systems, including Pseudomonas aeruginosa, Pasteurella multocida, and Legionella spp. Although the published mathematical models account for significant system dynamics such as predator-prey relationships and non-linear growth rates, they generally overlook spatial and temporal heterogeneity in environmental conditions, such as temperature, and population diversity. Future mathematical models will need to incorporate these factors to enhance our understanding of FLA-bacteria dynamics and to provide valuable insights for future risk assessment and disease control measures.
Subject(s)
Amoeba , Bacteria , Symbiosis , Amoeba/microbiology , Models, Biological , Bacterial Physiological Phenomena , Models, Theoretical , AnimalsABSTRACT
Pathogenic free-living amoebae (pFLA) are single-celled eukaryotes responsible for causing intractable infections with high morbidity and mortality in humans and animals. Current therapeutic approaches include cocktails of antibiotic, antifungal, and antimicrobial compounds. Unfortunately, the efficacy of these can be limited, driving the need for the discovery of new treatments. Pan anti-amebic agents would be ideal; however, identifying these agents has been a challenge, likely due to the limited evolutionary relatedness of the different pFLA. Here, we discuss the potential of targeting amoebae glucose metabolic pathways as the differences between pFLA and humans suggest specific inhibitors could be developed as leads for new therapeutics.
Subject(s)
Amoeba , Animals , Humans , Antifungal AgentsABSTRACT
[This corrects the article DOI: 10.3389/fmicb.2023.1149145.].
ABSTRACT
Pathogenic free-living amoebae (pFLA) can cause life-threatening central nervous system (CNS) infections and warrant the investigation of new chemical agents to combat the rise of infection from these pathogens. Naegleria fowleri glucokinase (NfGlck), a key metabolic enzyme involved in generating glucose-6-phosphate, was previously identified as a potential target due to its limited sequence similarity with human Glck (HsGlck). Herein, we used our previously demonstrated multifragment kinetic target-guided synthesis (KTGS) screening strategy to identify inhibitors against pFLA glucokinases. Unlike the majority of previous KTGS reports, our current study implements a "shotgun" approach, where fragments were not biased by predetermined binding potentials. The study resulted in the identification of 12 inhibitors against 3 pFLA glucokinase enzymesâNfGlck, Balamuthia mandrillaris Glck (BmGlck), and Acanthamoeba castellanii Glck (AcGlck). This work demonstrates the utility of KTGS to identify small-molecule binders for biological targets where resolved X-ray crystal structures are not readily accessible.
Subject(s)
Acanthamoeba castellanii , Amoeba , Balamuthia mandrillaris , Naegleria fowleri , Humans , GlucokinaseABSTRACT
BACKGROUND: Despite clinically demonstrated accuracy in next generation sequencing (NGS) data, many clinical laboratories continue to confirm variants with Sanger sequencing, which increases cost of testing and turnaround time. Several studies have assessed the accuracy of NGS in detecting single nucleotide variants; however, less has been reported about insertion, deletion, and deletion-insertion variants (indels). METHODS: We performed a retrospective analysis from 2015-2022 of indel results from a subset of NGS targeted gene panel tests offered through the Mayo Clinic Genomics Laboratories. We compared results from NGS and Sanger sequencing of indels observed in clinical runs and during the intra-assay validation of the tests. RESULTS: Results demonstrated 100% concordance between NGS and Sanger sequencing for over 490 indels (217 unique), ranging in size from 1 to 68 basepairs (bp). The majority of indels were deletions (77%) and 1 to 5 bp in length (90%). Variant frequencies ranged from 11.4% to 67.4% and 85.1% to 100% for heterozygous and homozygous variants, respectively, with a median depth of coverage of 2562×. A subset of indels (7%) were located in complex regions of the genome, and these were accurately detected by NGS. We also demonstrated 100% reproducibility of indel detection (n = 179) during intra-assay validation. CONCLUSIONS: Together this data demonstrates that reportable indel variants up to 68 bp can be accurately assessed using NGS, even when they occur in complex regions. Depending on the complexity of the region or variant, Sanger sequence confirmation of indels is usually not necessary if the variants meet appropriate coverage and allele frequency thresholds.
Subject(s)
Genome , High-Throughput Nucleotide Sequencing , Humans , Reproducibility of Results , Retrospective Studies , High-Throughput Nucleotide Sequencing/methods , Gene FrequencyABSTRACT
Antimicrobial host defense is dependent on the rapid recruitment of inflammatory cells to the site of infection, the elimination of invading pathogens, and the efficient resolution of inflammation that minimizes damage to the host. The peritoneal cavity provides an accessible and physiologically relevant system where the delicate balance of these processes may be studied. Here, we describe murine models of peritoneal inflammation that enable studies of competent antimicrobial immunity and inflammation-associated tissue damage as a consequence of recurrent bacterial challenge. The inflammatory hallmarks of these models reflect the clinical and molecular features of peritonitis seen in renal failure patients on peritoneal dialysis. The development of these models relies on the preparation of a cell-free supernatant derived from an isolate of Staphylococcus epidermidis (termed SES). Intraperitoneal administration of SES induces a Toll-like receptor 2-driven acute inflammatory response that is characterized by an initial transient influx of neutrophils that are replaced by a more sustained recruitment of mononuclear cells and lymphocytes. Adaptation of this model using a repeated administration of SES allows investigations into the development of adaptive immunity and the hallmarks associated with tissue remodelling and fibrosis. These models are therefore clinically relevant and provide exciting opportunities to study innate and adaptive immunity and the response of the stromal tissue compartment to bacterial infection and the ensuing inflammatory reaction.
Subject(s)
Peritoneal Dialysis , Peritonitis , Humans , Mice , Animals , Peritoneum , Inflammation , Peritoneal Cavity , Peritoneal Dialysis/adverse effectsABSTRACT
Cytokines that signal via STAT1 and STAT3 transcription factors instruct decisions affecting tissue homeostasis, antimicrobial host defense, and inflammation-induced tissue injury. To understand the coordination of these activities, we applied RNA sequencing, chromatin immunoprecipitation sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing to identify the transcriptional output of STAT1 and STAT3 in peritoneal tissues from mice during acute resolving inflammation and inflammation primed to drive fibrosis. Bioinformatics focused on the transcriptional signature of the immunomodulatory cytokine IL-6 in both settings and examined how profibrotic IFN-γ-secreting CD4+ T cells altered the interpretation of STAT1 and STAT3 cytokine cues. In resolving inflammation, STAT1 and STAT3 cooperated to drive stromal gene expression affecting antimicrobial immunity and tissue homeostasis. The introduction of IFN-γ-secreting CD4+ T cells altered this transcriptional program and channeled STAT1 and STAT3 to a previously latent IFN-γ activation site motif in Alu-like elements. STAT1 and STAT3 binding to this conserved sequence revealed evidence of reciprocal cross-regulation and gene signatures relevant to pathophysiology. Thus, we propose that effector T cells retune the transcriptional output of IL-6 by shaping a regulatory interplay between STAT1 and STAT3 in inflammation.
Subject(s)
Interleukin-6 , Th1 Cells , Animals , Mice , Cytokines/metabolism , Inflammation/metabolism , Interleukin-6/metabolism , Retroelements , STAT Transcription Factors/metabolism , STAT1 Transcription Factor/metabolism , STAT3 Transcription Factor/metabolism , Th1 Cells/metabolismABSTRACT
Acanthamoeba species, Naegleria fowleri, and Balamuthia mandrillaris are opportunistic pathogens that cause a range of brain, skin, eye, and disseminated diseases in humans and animals. These pathogenic free-living amoebae (pFLA) are commonly misdiagnosed and have sub-optimal treatment regimens which contribute to the extremely high mortality rates (>90%) when they infect the central nervous system. To address the unmet medical need for effective therapeutics, we screened kinase inhibitor chemotypes against three pFLA using phenotypic drug assays involving CellTiter-Glo 2.0. Herein, we report the activity of the compounds against the trophozoite stage of each of the three amoebae, ranging from nanomolar to low micromolar potency. The most potent compounds that were identified from this screening effort were: 2d (A. castellanii EC50: 0.92 ± 0.3 µM; and N. fowleri EC50: 0.43 ± 0.13 µM), 1c and 2b (N. fowleri EC50s: <0.63 µM, and 0.3 ± 0.21 µM), and 4b and 7b (B. mandrillaris EC50s: 1.0 ± 0.12 µM, and 1.4 ± 0.17 µM, respectively). With several of these pharmacophores already possessing blood-brain barrier (BBB) permeability properties, or are predicted to penetrate the BBB, these hits present novel starting points for optimization as future treatments for pFLA-caused diseases.
ABSTRACT
OBJECTIVE: Men with systemic lupus erythematosus (SLE) are an understudied population. The present study characterized differences between men and women with SLE. METHODS: We examined cross-sectionally participants with SLE in the All of Us Research Program, a US cohort with a participant survey at enrollment (May 2018 to June 2022) and linked electronic health record (EHR) data. We described and compared characteristics of men and women with SLE encompassing disease manifestations and prescribed medications from EHR data and socioeconomic factors, including health literacy and health care access and utilization, from surveys. We reported racial variations stratified by sex. RESULTS: Of 1,462 participants with SLE, 126 (9%) were male. Men reported lower educational attainment and less fatigue than women. Myocardial infarction was significantly more common in men. Men had significantly less confidence in completing medical forms than women and exhibited a trend toward requiring more help in reading health-related materials. Barriers to health care access and utilization were common in both men and women (40% versus 47%, respectively, reporting some reason for delay in care; P = 0.35). Women of race other than Black or African American or White more often reported delaying care due to cultural differences between patient and provider. CONCLUSION: Our study demonstrated major clinical and health literacy differences in men and women with SLE. Socioeconomic factors were significant barriers to health care in both sexes. Our study suggests men have disproportionately poorer health literacy, which may exacerbate preexisting disparities. Further large prospective studies, focusing on recruiting men, are needed to better characterize racial differences in men with SLE.
Subject(s)
Lupus Erythematosus, Systemic , Population Health , Adult , Humans , Male , Female , Race Factors , Prospective Studies , Lupus Erythematosus, Systemic/diagnosis , Lupus Erythematosus, Systemic/epidemiology , WhiteABSTRACT
Neutrophils are vital in defence against pathogens, but excessive neutrophil activity can lead to tissue damage and promote acute respiratory distress syndrome. COVID-19 is associated with systemic expansion of immature neutrophils, but the functional consequences of this shift to immaturity are not understood. We used flow cytometry to investigate activity and phenotypic diversity of circulating neutrophils in acute and convalescent COVID-19 patients. First, we demonstrate hyperactivation of immature CD10- subpopulations in severe disease, with elevated markers of secondary granule release. Partially activated immature neutrophils were detectable 12 wk post-hospitalisation, indicating long term myeloid dysregulation in convalescent COVID-19 patients. Second, we demonstrate that neutrophils from moderately ill patients down-regulate the chemokine receptor CXCR2, whereas neutrophils from severely ill individuals fail to do so, suggesting an altered ability for organ trafficking and a potential mechanism for induction of disease tolerance. CD10- and CXCR2hi neutrophil subpopulations were enriched in severe disease and may represent prognostic biomarkers for the identification of individuals at high risk of progressing to severe COVID-19.
Subject(s)
COVID-19 , Neutrophils , Receptors, Interleukin-8B , Humans , COVID-19/immunology , Flow Cytometry , Neutrophils/immunology , Receptors, Interleukin-8B/metabolismABSTRACT
A tunable diode laser absorption spectroscopy (TDLAS) device has been developed to study long-path atmospheric transmission near diode pumped alkali laser (DPAL) emission wavelengths. By employing a single aperture and retro reflector in a mono-static configuration, the noise associated with atmospheric and platform jitter were reduced by a factor of â¼30 and the open-air path length was extended to 4.4 km and over a very broad spectral range, up to 120 cm-1. Water vapor absorption lines near the rubidium (Rb) and cesium (Cs) variants of the DPAL near 795 and 894 nm, oxygen lines near the potassium (K) DPAL near 770 nm, and water vapor absorption in the vicinity of the neodymium-doped yttrium aluminum garnet (Nd:YAG) laser 1.064 µm and chemical oxygen iodine laser (COIL) 1.3 µm lines were studied. The detection limit for path absorbance increases from ΔA = 0.0017 at 100 m path length to 0.085 for the 4.4 km path. Comparison with meteorological instruments for maritime and desert environments yields agreement for the 2.032 km path to within 1.5% for temperature, 4.5% for pressure, and 5.1% for concentration, while agreements for the 4.4 km path are within 1.4% for temperature, 7.7% for pressure, and 23.5% for concentration. An intra cavity output spectroscopy (ICOS) device was also used as a spectral reference to verify location of atmospheric lines. Implications of TDLAS collection system design on signal-to-noise (S/N) are discussed as well as the effect of path turbulence on baseline noise and inform the selection of the DPAL variant least affected by molecular absorption.
ABSTRACT
Infection with pathogenic free-living amoebae, including Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris, can lead to life-threatening illnesses, primarily because of catastrophic central nervous system involvement. Efficacious treatment options for these infections are lacking, and the mortality rate due to infection is high. Previously, we evaluated the N. fowleri glucokinase (NfGlck) as a potential target for therapeutic intervention, as glucose metabolism is critical for in vitro viability. Here, we extended these studies to the glucokinases from two other pathogenic free-living amoebae, including Acanthamoeba castellanii (AcGlck) and B. mandrillaris (BmGlck). While these enzymes are similar (49.3% identical at the amino acid level), they have distinct kinetic properties that distinguish them from each other. For ATP, AcGlck and BmGlck have apparent Km values of 472.5 and 41.0 µM, while Homo sapiens Glck (HsGlck) has a value of 310 µM. Both parasite enzymes also have a higher apparent affinity for glucose than the human counterpart, with apparent Km values of 45.9 µM (AcGlck) and 124 µM (BmGlck) compared to ~8 mM for HsGlck. Additionally, AcGlck and BmGlck differ from each other and other Glcks in their sensitivity to small molecule inhibitors, suggesting that inhibitors with pan-amoebic activity could be challenging to generate.
Subject(s)
Acanthamoeba , Amebiasis , Amoeba , Balamuthia mandrillaris , Naegleria fowleri , Amebiasis/drug therapy , Amebiasis/parasitology , Glucokinase , HumansABSTRACT
Studies on an organic extract of a marine fungus, Periconia sp. (strain G1144), led to the isolation of three halogenated cyclopentenes along with the known and recently reported rhytidhyester D; a series of spectrometric and spectroscopic techniques were used to elucidate these structures. Interestingly, two of these compounds represent tri-halogenated cyclopentene derivatives, which have been observed only rarely from Nature. The relative and absolute configurations of the compounds were established via mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, Mosher's esters method, optical rotation and GIAO NMR calculations, including correlation coefficient calculations and the use of both DP4+ and dJ DP4 analyses. Several of the isolated compounds were tested for activity in anti-parasitic, antimicrobial, quorum sensing inhibition, and cytotoxicity assays and were shown to be inactive.
Subject(s)
Anti-Infective Agents , Ascomycota , Anti-Bacterial Agents/pharmacology , Ascomycota/chemistry , Cyclopentanes/pharmacology , Magnetic Resonance Spectroscopy , Molecular StructureABSTRACT
Introduction: Acanthamoeba keratitis is often caused when Acanthamoeba contaminate contact lenses and infect the cornea. Acanthamoeba is pervasive in the environment as a motile, foraging trophozoite or biocide-resistant and persistent cyst. As contact lens contamination is a potential first step in infection, we studied Acanthamoeba's behavior and interactions on different contact lens materials. We hypothesized that contact lenses may induce aggregation, which is a precursor to encystment, and that aggregated encystment would be more difficult to disinfect than motile trophozoites. Methods: Six clinically and/or scientifically relevant strains of Acanthamoeba (ATCC 30010, ATCC 30461, ATCC 50370, ATCC 50702, ATCC 50703, and ATCC PRA-115) were investigated on seven different common silicone hydrogel contact lenses, and a no-lens control, for aggregation and encystment for 72 h. Cell count and size were used to determine aggregation, and fluorescent staining was used to understand encystment. RNA seq was performed to describe the genome of Acanthamoeba which was individually motile or aggregated on different lens materials. Disinfection efficacy using three common multi-purpose solutions was calculated to describe the potential disinfection resistance of trophozoites, individual cysts, or spheroids. Results: Acanthamoeba trophozoites of all strains examined demonstrated significantly more aggregation on specific contact lens materials than others, or the no-lens control. Fluorescent staining demonstrated encystment in as little as 4 hours on contact lens materials, which is substantially faster than previously reported in natural or laboratory settings. Gene expression profiles corroborated encystment, with significantly differentially expressed pathways involving actin arrangement and membrane complexes. High disinfection resistance of cysts and spheroids with multi-purpose solutions was observed. Discussion: Aggregation/encystment is a protective mechanism which may enable Acanthamoeba to be more disinfection resistant than individual trophozoites. This study demonstrates that some contact lens materials promote Acanthamoeba aggregation and encystment, and Acanthamoeba spheroids obstruct multi-purpose solutions from disinfecting Acanthamoeba.
ABSTRACT
Peptidylarginine deiminase 4 (PAD4) is a key regulator of inflammation but its function in infections remains incompletely understood. We investigate PAD4 in the context of malaria and demonstrate a role in regulation of immune cell trafficking and chemokine production. PAD4 regulates liver immunopathology by promoting neutrophil trafficking in a Plasmodium chabaudi mouse malaria model. In human macrophages, PAD4 regulates expression of CXCL chemokines in response to stimulation with TLR ligands and P. falciparum. Using patient samples, we show that CXCL1 may be a biomarker for severe malaria. PAD4 inhibition promotes disease tolerance and may represent a therapeutic avenue in malaria.
Subject(s)
Malaria , Neutrophils , Animals , Chemotactic Factors , Disease Models, Animal , Humans , Malaria/metabolism , Mice , Protein-Arginine Deiminase Type 4ABSTRACT
Balamuthia mandrillaris, a pathogenic free-living amoeba, causes cutaneous skin lesions as well as granulomatous amoebic encephalitis, a 'brain-eating' disease. As with the other known pathogenic free-living amoebas (Naegleria fowleri and Acanthamoeba species), drug discovery efforts to combat Balamuthia infections of the central nervous system are sparse; few targets have been validated or characterized at the molecular level, and little is known about the biochemical pathways necessary for parasite survival. Current treatments of encephalitis due to B. mandrillaris lack efficacy, leading to case fatality rates above 90%. Using our recently published methodology to discover potential drugs against pathogenic amoebas, we screened a collection of 85 compounds with known antiparasitic activity and identified 59 compounds that impacted the growth of Balamuthia trophozoites at concentrations below 220 µM. Since there is no fully annotated genome or proteome of B. mandrillaris, we sequenced and assembled its transcriptome from a high-throughput RNA-sequencing (RNA-Seq) experiment and located the coding sequences of the genes potentially targeted by the growth inhibitors from our compound screens. We determined the sequence of 17 of these target genes and obtained expression clones for 15 that we validated by direct sequencing. These will be used in the future in combination with the identified hits in structure guided drug discovery campaigns to develop new approaches for the treatment of Balamuthia infections.
Subject(s)
Balamuthia mandrillaris/genetics , Drug Design/methods , Trophozoites/genetics , Acanthamoeba/genetics , Amebiasis/drug therapy , Amoeba/genetics , Balamuthia mandrillaris/drug effects , Balamuthia mandrillaris/growth & development , Base Sequence , Brain/pathology , Drug Discovery/methods , Encephalitis/pathology , Gene Expression/genetics , Naegleria fowleri/genetics , Transcriptome/genetics , Trophozoites/drug effectsABSTRACT
Noncanonical inflammasome activation by cytosolic lipopolysaccharide (LPS) is a critical component of the host response to Gram-negative bacteria. Cytosolic LPS recognition in macrophages is preceded by a Toll-like receptor (TLR) priming signal required to induce transcription of inflammasome components and facilitate the metabolic reprograming that fuels the inflammatory response. Using a genome-scale arrayed siRNA screen to find inflammasome regulators in mouse macrophages, we identified the mitochondrial enzyme nucleoside diphosphate kinase D (NDPK-D) as a regulator of both noncanonical and canonical inflammasomes. NDPK-D was required for both mitochondrial DNA synthesis and cardiolipin exposure on the mitochondrial surface in response to inflammasome priming signals mediated by TLRs, and macrophages deficient in NDPK-D had multiple defects in LPS-induced inflammasome activation. In addition, NDPK-D was required for the recruitment of TNF receptor-associated factor 6 (TRAF6) to mitochondria, which was critical for reactive oxygen species (ROS) production and the metabolic reprogramming that supported the TLR-induced gene program. NDPK-D knockout mice were protected from LPS-induced shock, consistent with decreased ROS production and attenuated glycolytic commitment during priming. Our findings suggest that, in response to microbial challenge, NDPK-D-dependent TRAF6 mitochondrial recruitment triggers an energetic fitness checkpoint required to engage and maintain the transcriptional program necessary for inflammasome activation.
Subject(s)
Inflammasomes , Nucleoside Diphosphate Kinase D , Animals , Inflammasomes/genetics , Inflammasomes/metabolism , Lipopolysaccharides/metabolism , Macrophages/metabolism , Mice , Mitochondria/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Nucleoside Diphosphate Kinase D/metabolism , Reactive Oxygen Species/metabolismABSTRACT
Ecologists require standardized, ecologically relevant information on the thermal ecology of aquatic ectotherms to address growing concerns related to changing climates, altered habitats, and introduced species. We measured multiple thermal endpoints to investigate potential for establishment of the invasive Ringed Crayfish (Faxonius neglectus) in thermally heterogeneous habitat of the narrowly distributed endemic Coldwater Crayfish (Faxonius eupunctus). For each species, we examined the relationships between thermal endpoints at the cellular and organismal levels. We then compared results between the two species to gain insight as to the generality of linkages between cellular and organismal-level endpoints, as well as the potential for thermal niche separation between the native and potential invader. At the cellular level, we found no differences in the temperature for maximum activity of electron transport system enzymes (ETSmax) between species. At the organismal level, F. neglectus preferred significantly warmer temperatures than F. eupunctus, but this difference was small (1.3 °C) and likely to have only limited biological significance. The critical thermal maximum (CTM) did not differ between species. For both species, the thermal performance curve for ETS enzyme activity served as a useful framework to link thermal endpoints and estimate the transition from optimal to stressful temperatures - organismal thermal preference and optimal temperature estimates consistently fell below ETSmax whereas CTM estimates fell above ETSmax. Taken together, the strong similarities in thermal endpoint patterns between the two species suggest habitats thermally suitable for the native F. eupunctus will also be thermally available to expanding populations of F. neglectus, thereby increasing the opportunity for negative interactions and population effects if F. neglectus invades one of the few remaining, uninvaded, critical habitats of F. eupunctus.