Scholarly Works, Biological Sciences

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A framework for developing a real-time lake phytoplankton forecasting system to support water quality management in the face of global change
Carey, Cayelan C.; Calder, Ryan S. D.; Figueiredo, Renato J.; Gramacy, Robert B.; Lofton, Mary E.; Schreiber, Madeline E.; Thomas, R. Quinn (Springer, 2024-09-20)
Phytoplankton blooms create harmful toxins, scums, and taste and odor compounds and thus pose a major risk to drinking water safety. Climate and land use change are increasing the frequency and severity of blooms, motivating the development of new approaches for preemptive, rather than reactive, water management. While several real-time phytoplankton forecasts have been developed to date, none are both automated and quantify uncertainty in their predictions, which is critical for manager use. In response to this need, we outline a framework for developing the first automated, real-time lake phytoplankton forecasting system that quantifies uncertainty, thereby enabling managers to adapt operations and mitigate blooms. Implementation of this system calls for new, integrated ecosystem and statistical models; automated cyberinfrastructure; effective decision support tools; and training for forecasters and decision makers. We provide a research agenda for the creation of this system, as well as recommendations for developing real-time phytoplankton forecasts to support management.
Atmospheric Deposition of Microplastics in South Central Appalachia in the United States
Elnahas, Adam; Gray, Austin; Lee, Jennie; AlAmiri, Noora; Pokhrel, Nishan; Allen, Steve; Foroutan, Hosein (American Chemical Society, 2024-12-26)
Due to the increased prevalence of plastic pollution globally, atmospheric deposition of microplastics (MPs) is a significant issue that needs to be better understood to identify potential consequences for human health. This study is the first to quantify and characterize atmospheric MP deposition in the Eastern United States. Passive sampling was conducted at two locations within the Eastern United States, specifically in remote South Central Appalachia, from March to September 2023. Each location had five sampling periods, with collections over a 21 day period. Samples were processed to remove biological material, and the presence of MPs was confirmed using Raman spectroscopy to match particles based on polymer similarity. The relative average atmospheric MP deposition in South Central Appalachia was determined to be 68 MPs m^-2 d^-1. Most verified MPs were fibers, and the most abundant polymer type identified was poly(ethylene terephthalate) PETE. This study's average MP deposition rate is qualitatively comparable to rates reported in other studies that employed a similar methodology in a similar landscape. Scaling up our measured deposition rate to all of South Central Appalachia, an area of over 94,000 km² and home to five million people, suggests a yearly MP deposition of approximately 321 metric tonnes. Our study highlights the prevalence of MP deposition in the Eastern United States, providing baseline data for future work to further assess routes of MP introduction.
Aquatic and Wetland Plants of Northeastern North America, Second Edition [Book review]
Metzgar, Jordan (Torrey Botanical Society, 2024-05-30)
Colder temperatures augment viability of an indirectly transmitted songbird pathogen on bird feeders
Teemer, Sara R.; Tulman, Edan R.; Arneson, Alicia G.; Geary, Steven J.; Hawley, Dana M. (Wiley, 2024-12)
Inanimate surfaces that are contaminated with infectious pathogens are common sources of spread for many communicable diseases. Understanding how ambient temperature alters the ability of pathogens to remain viable on these surfaces is critical for understanding how fomites can contribute to seasonal patterns of disease outbreaks. House finches (Haemorhous mexicanus) experience fall and winter outbreaks of mycoplasmal conjunctivitis, caused by the bacterial pathogen Mycoplasma gallisepticum (MG). Although bird feeder surfaces serve as an indirect route of MG transmission between sick and healthy individuals, the contributions of feeders to MG transmission in the wild will depend on how ambient temperature affects viability and pathogenicity of MG on feeder surfaces over time. Here, we used two experiments, with identical initial design, to assess such temperature effects. For both experiments, we pipetted equal amounts of MG onto replicate feeder ports held at night-day temperatures representing summer (22–27°C) or winter (4–9°C). We allowed MG to incubate on feeders at either temperature and swabbed remaining inocula from surfaces at 0, 1, 2, 4, or 7 days post-inoculation of the feeder, with each replicate feeder port only swabbed at a single time point. In the first study, we analyzed swabs using a culture-based assay and found that MG incubated at colder versus warmer temperatures maintained higher viability on feeder surfaces over time. In the second study, we replicated the same experimental design but used MG swabs from feeder surfaces to inoculate wild-caught, pathogen-naïve birds and measured resulting disease severity and pathogen loads to determine pathogenicity. We found that MG remained pathogenic on feeder surfaces at cold ambient temperatures for up to one week, much longer than previously documented. Further, MG was significantly more pathogenic when incubated on feeders in colder versus warmer temperatures, with the strongest effects of temperature present after at least four days of incubation on feeder surfaces. Overall, cold ambient temperatures appear to alter the role of fomites in the MG transmission process, and temperature likely contributes to seasonal disease dynamics in this system and many others.
Proteomic insights into breast cancer response to brain cell-secreted factors
Ahuja, Shreya; Lazar, Iuliana M. (Springer, 2024-08-21)
The most devastating feature of cancer cells is their ability to metastasize to distant sites in the body. HER2 + and TN breast cancers frequently metastasize to the brain and stay potentially dormant for years until favorable conditions support their proliferation. The sheltered and delicate nature of the brain prevents, however, early disease detection and effective delivery of therapeutic drugs. Moreover, the challenges associated with the acquisition of brain biopsies add compounding difficulties to exploring the mechanistic aspects of tumor development. To provide insights into the determinants of cancer cell behavior at the brain metastatic site, this study was aimed at exploring the early response of HER2 + breast cancer cells (SKBR3) to factors present in the brain perivascular niche. The neural microenvironment was simulated by using the secretome of a set of brain cells that come first in contact with the cancer cells upon crossing the blood brain barrier, i.e., endothelial cells, astrocytes, and microglia. Cytokine microarrays were used to investigate the secretome mediators of intercellular communication, and proteomic technologies for assessing the changes in the behavior of cancer cells upon exposure to the brain cell-secreted factors. The cytokines detected in the brain secretomes were supportive of inflammatory conditions, while the SKBR3 cells secreted numerous cancer-promoting growth factors that were either absent or present in lower abundance in the brain cell cultures, indicating that upon exposure the SKBR3 cells may have been deprived of favorable conditions for optimal growth. Altogether, the results suggest that the exposure of SKBR3 cells to the brain cell-secreted factors altered their growth potential and drove them toward a state of quiescence, with broader overall outcomes that affected cellular metabolism, adhesion and immune response processes. The findings of this study underscore the key role played by the neural niche in shaping the behavior of metastasized cancer cells, provide insights into the cellular cross-talk that may lead cancer cells into dormancy, and highlight novel opportunities for the development of metastatic breast cancer therapeutic strategies.
Proteomic assessment of SKBR3/HER2+ breast cancer cellular response to Lapatinib and investigational Ipatasertib kinase inhibitors
Karcini, Arba; Mercier, Nicole R.; Lazar, Iuliana M. (Frontiers, 2024-08-29)
Introduction: Modern cancer treatment strategies aim at achieving cancer remission by using targeted and personalized therapies, as well as harnessing the power of the immune system to recognize and eradicate the cancer cells. To overcome a relatively short-lived response due to resistance to the administered drugs, combination therapies have been pursued. Objective: The objective of this study was to use high-throughput data generation technologies such as mass spectrometry and proteomics to investigate the broader implications, and to expand the outlook, of such therapeutic approaches. Specifically, we investigated the systems-level response of a breast cancer cell line model to a mixture of kinase inhibitors that has not been adopted yet as a standard therapeutic regime. Methods: Two critical pathways that sustain the growth and survival of cancer cells, EGFR and PI3K/AKT, were inhibited in SKBR3/HER2+ breast cancer cells with Lapatinib (Tyr kinase inhibitor) and Ipatasertib (Ser/Thr kinase inhibitor), and the landscape of the affected biological processes was investigated with proteomic technologies. Results: Over 800 proteins matched by three unique peptide sequences were affected by exposing the cells to the drugs. The work corroborated the anti-proliferative activity of Lapatinib and Ipatasertib and uncovered a range of impacted cancer-supportive hallmark processes, among which immune response, adhesion, and migration emerged as particularly relevant to the ability of drugs to effectively suppress the proliferation and dissemination of cancer cells. Changes in the expression of key cancer drivers such as oncogenes, tumor suppressors, EMT and angiogenesis regulators underscored the inhibitory effectiveness of drugs on cancer proliferation. The supplementation of Lapatinib with Ipatasertib further affected additional transcription factors and proteins involved in gene expression, trafficking, DNA repair, and development of multidrug resistance. Furthermore, over fifty of the impacted proteins represent approved or investigational targets in the DrugBank database, which through their protein-protein interaction networks can inform the selection of effective therapeutic partners. Conclusion: Altogether, the exposure of SKBR3/HER2+ cells to Lapatinib and Ipatasertib kinase inhibitors uncovered a broad plethora of yet untapped opportunities that can be further explored for enhancing the anti-cancer effects of each drug as well as of many other multi-drug therapies that target the EGFR/ERBB2 and PI3K/AKT pathways.
Circadian clock gene polymorphisms implicated in human pathologies
Janoski, Jesse R.; Aiello, Ignacio; Lundberg, Clayton W.; Finkielstein, Carla V. (Cell Press, 2024-06-12)
Circadian rhythms, ~24 h cycles of physiological and behavioral processes, can be synchronized by external signals (e.g., light) and persist even in their absence. Consequently, dysregulation of circadian rhythms adversely affects the well-being of the organism. This timekeeping system is generated and sustained by a genetically encoded endogenous mechanism composed of interlocking transcriptional/translational feedback loops that generate rhythmic expression of core clock genes. Genome-wide association studies (GWAS) and forward genetic studies show that SNPs in clock genes influence gene regulation and correlate with the risk of developing various conditions. We discuss genetic variations in core clock genes that are associated with various phenotypes, their implications for human health, and stress the need for thorough studies in this domain of circadian regulation.
The importance of peripheral populations in the face of novel environmental change
Hoff, Samantha; Hoyt, Joseph R.; Langwig, Kate E.; Johnson, Luanne; Olson, Elizabeth; O'Dell, Danielle; Pendergast, Casey J.; Herzog, Carl J.; Parise, Katy L.; Foster, Jeffrey T.; Turner, Wendy C. (Royal Society, 2025-01-08)
Anthropogenically driven environmental change has imposed substantial threats on biodiversity, including the emergence of infectious diseases that have resulted in declines of wildlife globally. In response to pathogen invasion, maintaining diversity within host populations across heterogenous environments is essential to facilitating species persistence. White-nose syndrome is an emerging fungal pathogen that has caused mass mortalities of hibernating bats across North America. However, in the northeast, peripheral island populations of the endangered northern myotis (Myotis septentrionalis) appear to be persisting despite infection while mainland populations in the core of the species range have experienced sharp declines. Thus, this study investigated host and environmental factors that may contribute to divergent population responses. We compared patterns of pathogen exposure and infection intensity between populations and documented the environmental conditions and host activity patterns that may promote survival despite disease invasion. For island populations, we found lower prevalence and less severe infections, possibly due to a shorter hibernation duration compared to the mainland, which may reduce the time for disease progression. The coastal region of the northern myotis range may serve as habitat refugia that enables this species to persist despite pathogen exposure; however, conservation efforts could be critical to supporting species survival in the long term.
ASCB statement of commitment to diversity, equity, and inclusion
Murray, Sandra A.; Holzbaur, Erika L. F.; Munson, Mary; Cimini, Daniela; Lane, Timothy F.; Alvania, Rebecca; Applewhite, Derek A.; Chang, Fred; Chen, Elizabeth H.; Earnshaw, William C.; Evans, Chantell S.; Li, Rong; Mierzwa, Beata E.; Oliver, Tiffany; Segarra, Veronica A.; Skop, Ahna R.; Weaver, Lesley N.; Asai, David J.; Boyce, Michael; Zavala, Maria Elena; Hammonds-Odie, Latanya; Vigoreaux, Jim (American Society for Cell Biology, 2024-07-22)
Transcriptomic resources for Bagrada hilaris (Burmeister), a widespread invasive pest of Brassicales
Sparks, Michael E.; Nelson, David R.; Harrison, Robert L.; Larson, Nicholas R.; Kuhar, Daniel; Haber, Ariela I.; Heraghty, Sam D.; Rebholz, Zarley; Tholl, Dorothea; Grettenberger, Ian M.; Weber, Donald C.; Gundersen-Rindal, Dawn E. (Public Library of Science, 2024-12-27)
The bagrada bug, Bagrada hilaris (Burmeister), is an emerging agricultural pest in the Americas, threatening agricultural production in the southwestern United States, Mexico and Chile, as well as in the Old World (including Africa, South Asia and, more recently, Mediterranean areas of Europe). Substantive transcriptomic sequence resources for this damaging species would be beneficial towards understanding its capacity for developing insecticide resistance, identifying viruses that may be present throughout its population and identifying genes differentially expressed across life stages that could be exploited for biomolecular pesticide formulations. This study establishes B. hilaris transcriptomic resources for eggs, 2nd and 4th larval instars, as well as male and female adults. Three gene families involved in xenobiotic detoxification—glutathione S-transferases, carboxylesterases and cytochrome P450 monooxygenases—were phylogenetically characterized. These data were also qualitatively compared with previously published results for two closely related pentatomid species—the brown marmorated stink bug, Halyomorpha halys (Stål), and the harlequin bug, Murgantia histrionica (Hahn)—to elucidate shared enzymatic components of terpene-based sex pheromone biosynthetic pathways. Lastly, the sequence data were screened for potential RNAi- and virus-related content and for genes implicated in insect growth and development.
Identifying Barriers and Bridging Gaps Between Researchers and Decision Makers in Water Quality Modeling
Chowdhury, Mahabub; Carey, Cayelan C.; Figueiredo, Renato; Gramacy, Robert; Hoffman, Kathryn; Lofton, Mary; Patil, Parul; Schreiber, Madeline; Thomas, R. Quinn; Calder, Ryan S. D. (2024-12-12)
Diversity of unique, nonmycorrhizal endophytic fungi in cultivated Phalaenopsis orchids: A pilot study
Watkinson, Jonathan I.; Winkel, Brenda S. J. (Wiley, 2024-05-17)
Orchids comprise one of the largest, most diverse, and most broadly distributed families of flowering plants and contribute significantly to habitat biodiversity. One key aspect of orchid growth and development is the formation of mycorrhizal symbioses with compatible endophytic fungi, which are maintained throughout the life of the plant. Substantial efforts to identify the fungi that form mycorrhizal symbioses across a range of orchid species have often also uncovered numerous nonmycorrhizal, endophytic fungi. These fungi could also have significant effects on orchid growth and development and are beginning to be analyzed more closely, particularly in wild species. The role of endophytic fungi in the production, distribution, and continued growth by the hobbyist of orchids is not known. As an initial step toward characterizing nonmycorrhizal endophytic fungi associated with cultivated orchids, we undertook a survey of fungi residing within roots of Phalaenopsis plants growing in home environments. Sequence analysis of ITS regions amplified from total DNA isolated from roots allowed rapid identification of endophytic fungi to the class level and may offer a useful initial screening method for beneficial species, for example, in horticultural settings. ITS-PCR sequences subsequently obtained from individual fungi cultured from surface-sterilized orchid roots corroborated the findings of the initial screen, while also providing a more complete characterization of the array of fungal taxa that were present. Although lower in diversity than has been reported for orchids growing in the wild, these endophytes have the potential to substantially enhance the growth and disease resistance of horticultural orchids.
Aurora B inhibition induces hyper-polyploidy and loss of long-term proliferative potential in RB and p53 defective cells
Vora, Shivam; Chatterjee, Saptarshi; Andrew, Ariel; Kumar, Ramyashree Prasanna; Proctor, Martina; Zeng, Zhen; Bhatt, Rituparna; Nazareth, Deborah; Fernando, Madushan; Jones, Mathew J. K.; He, Yaowu; Hooper, John D.; McMillan, Nigel A. J.; Urosevic, Jelena; Saeh, Jamal; Travers, Jon; Cimini, Daniela; Chen, Jing; Gabrielli, Brian (Springer Nature, 2025-01-08)
Polyploidy is a common outcome of chemotherapies, but there is conflicting evidence as to whether polyploidy is an adverse, benign or even favourable outcome. We show Aurora B kinase inhibitors efficiently promote polyploidy in many cell types, resulting in the cell cycle exit in RB and p53 functional cells, but hyper-polyploidy in cells with loss of RB and p53 function. These hyper-polyploid cells (>8n DNA content) are viable but have lost long-term proliferative potential in vitro and fail to form tumours in vivo. Investigation of mitosis in these cells revealed high numbers of centrosomes that were capable of supporting functional mitotic spindle poles, but these failed to progress to anaphase/telophase structures even when AURKB inhibitor was removed after 2–3 days. However, when AURKB inhibitor was removed after 1 day and cells had failed a single cytokinesis to become tetraploid, they retained colony forming ability and long-term proliferative potential. Mathematical modelling of the potential for polyploid cells to produce viable daughter cells demonstrated that cells with >8n DNA and >4 functional spindle poles approach zero probability of a viable daughter, supporting our experimental observations. These findings demonstrate that tetraploidy is tolerated by tumour cells, but higher ploidy states are incompatible with long-term proliferative potential.
Demographic feedbacks during evolutionary rescue can slow or speed adaptive evolution
Draghi, Jeremy A.; McGlothlin, Joel W.; Kindsvater, Holly K. (The Royal Society, 2024-02-14)
Populations declining toward extinction can persist via genetic adaptation in a process called evolutionary rescue. Predicting evolutionary rescue has applications ranging from conservation biology to medicine, but requires understanding and integrating the multiple effects of a stressful environmental change on population processes. Here we derive a simple expression for how generation time, a key determinant of the rate of evolution, varies with population size during evolutionary rescue. Change in generation time is quantitatively predicted by comparing how intraspecific competition and the source of maladaptation each affect the rates of births and deaths in the population. Depending on the difference between two parameters quantifying these effects, the model predicts that populations may experience substantial changes in their rate of adaptation in both positive and negative directions, or adapt consistently despite severe stress. These predictions were then tested by comparison to the results of individual-based simulations of evolutionary rescue, which validated that the tolerable rate of environmental change varied considerably as described by analytical results. We discuss how these results inform efforts to understand wildlife disease and adaptation to climate change, evolution in managed populations and treatment resistance in pathogens.
Spatial clustering of hosts can favor specialist parasites
Draghi, Jeremy A.; Zook, Evan (Wiley, 2024-11-17)
Generalist parasites seem to enjoy the clear ecological advantage of a greater chance to find a host, and genetic trade-offs are therefore often invoked to explain why specialists can coexist with or outcompete generalists. Here we develop an alternative perspective based on optimal foraging theory to explain why spatial clustering can favor specialists even without genetic trade-offs. Using analytical and simulation models inspired by bacteriophage, we examine the optimal use of two hosts, one yielding greater reproductive success for the parasite than the other. We find that a phage may optimally ignore the worse host when the two hosts are clustered together in dense, ephemeral patches. We model conditions that enhance or reduce this selective benefit to a specialist parasite and show that it is eliminated entirely when the hosts occur only in separate patches. These results show that specialists can be favored even when trade-offs are weak or absent and emphasize the importance of spatiotemporal heterogeneity in models of optimal niche breadth.
Data assimilation experiments inform monitoring needs for near-term ecological forecasts in a eutrophic reservoir
Wander, Heather L.; Thomas, R. Quinn; Moore, Tadhg N.; Lofton, Mary E.; Breef-Pilz, Adrienne; Carey, Cayelan C. (Wiley, 2024-02-13)
Ecosystems around the globe are experiencing changes in both the magnitude and fluctuations of environmental conditions due to land use and climate change. In response, ecologists are increasingly using near-term, iterative ecological forecasts to predict how ecosystems will change in the future. To date, many near-term, iterative forecasting systems have been developed using high temporal frequency (minute to hourly resolution) data streams for assimilation. However, this approach may be cost-prohibitive or impossible for forecasting ecological variables that lack high-frequency sensors or have high data latency (i.e., a delay before data are available for modeling after collection). To explore the effects of data assimilation frequency on forecast skill, we developed water temperature forecasts for a eutrophic drinking water reservoir and conducted data assimilation experiments by selectively withholding observations to examine the effect of data availability on forecast accuracy. We used in situ sensors, manually collected data, and a calibrated water quality ecosystem model driven by forecasted weather data to generate future water temperature forecasts using Forecasting Lake and Reservoir Ecosystems (FLARE), an open source water quality forecasting system. We tested the effect of daily, weekly, fortnightly, and monthly data assimilation on the skill of 1- to 35-day-ahead water temperature forecasts. We found that forecast skill varied depending on the season, forecast horizon, depth, and data assimilation frequency, but overall forecast performance was high, with a mean 1-day-ahead forecast root mean square error (RMSE) of 0.81°C, mean 7-day RMSE of 1.15°C, and mean 35-day RMSE of 1.94°C. Aggregated across the year, daily data assimilation yielded the most skillful forecasts at 1- to 7-day-ahead horizons, but weekly data assimilation resulted in the most skillful forecasts at 8- to 35-day-ahead horizons. Within a year, forecasts with weekly data assimilation consistently outperformed forecasts with daily data assimilation after the 8-day forecast horizon during mixed spring/autumn periods and 5- to 14-day-ahead horizons during the summer-stratified period, depending on depth. Our results suggest that lower frequency data (i.e., weekly) may be adequate for developing accurate forecasts in some applications, further enabling the development of forecasts broadly across ecosystems and ecological variables without high-frequency sensor data.
Translational Edge and Cloud Computing to Advance Lake Water Quality Forecasting
Figueiredo, Renato J.; Carey, Cayelan C.; Thomas, R. Quinn (IEEE, 2024-11-20)
In this article, we report on our experiences with interdisciplinary projects at the intersection of freshwater ecology, data science, and computer science. The translational research process has progressively led to the development of distributed systems that apply both edge computing and function-as-a-service (FaaS) cloud computing to support end-to-end water quality forecasting workflows across the edge-to-cloud continuum.
Can you predict the future? A tutorial for the National Ecological Observatory Network Ecological Forecasting Challenge
Olsson, Freya; Boettiger, Carl; Carey, Cayelan C.; Lofton, Mary E.; Thomas, R. Quinn (The Open Journal, 2024-12)
This tutorial introduces participants to key concepts in ecological forecasting and provides hands-on materials for submitting forecasts to the National Ecological Observatory Network (NEON) Forecasting Challenge (hereafter, Challenge), hosted by the Ecological Forecasting Initiative Research Coordination Network. The tutorial has been developed and used with >300 participants and provides the ecological understanding, workflows, and tools to enable ecologists with minimal forecasting experience to participate in the Challenge via a hands-on R-based tutorial. This tutorial introduces participants to a near-term, iterative forecasting workflow that includes obtaining observations from NEON, developing a simple forecasting model, generating a forecast, and submitting the forecast to the Challenge, as well as evaluating forecast performance once new observations become available. The overarching aim of this tutorial is to lower the barrier to ecological forecasting and empower participants to develop their own ecological forecasts.
Regional variation in growth and survival responses to atmospheric nitrogen and sulfur deposition for 140 tree species across the United States
Dalton, Rebecca M.; Miller, Jesse N.; Greaver, Tara; Sabo, Robert D.; Austin, Kemen G.; Phelan, Jennifer N.; Thomas, R. Quinn; Clark, Christopher M. (Frontiers, 2024-11-11)
Atmospheric deposition of nitrogen (N) and sulfur (S) alter tree demographic processes via changes in nutrient pools, soil acidification, and biotic interactions. Previous work established tree growth and survival response to atmospheric N and S deposition in the conterminous United States (CONUS) data by species; however, it was not possible to evaluate regional variation in response using that approach. In this study, we develop species- and region-specific relationships for growth and survival responses to N and S deposition for roughly 140 species within spatially demarcated regions of the U.S. We calculated responses to N and S deposition separately for 11 United States Forest Service (USFS) Divisions resulting in a total of 241 and 268 species × Division combinations for growth and survival, respectively. We then assigned these relationships into broad categories of vulnerability and used ordinal logistic regressions to explore the covariates associated with vulnerability in growth and survival to N and S deposition. As with earlier studies, we found growth and survival responses to air pollution differed by species; but new to this study, we found 45%−70% of species responses also varied spatially across regions. The regional variation in species responses was not simply related to atmospheric N and S deposition, but was also associated with regional effects from precipitation, soil pH, mycorrhizal association, and deciduousness. A large amount of the variance remained unexplained (total variation explained ranged from 6.8%−13.8%), suggesting that these or additional factors may operate at finer spatial scales. Taken together, our results demonstrate that regional variation in tree species' response has significant implications for setting critical load targets, as critical loads can now be tailored for specific species at management-relevant scales.
Strategies for genetic manipulation of the halotolerant black yeast Hortaea werneckii: ectopic DNA integration and marker-free CRISPR/Cas9 transformation
Hernandez-Rodriguez, Yainitza; Bullard, A. Makenzie; Busch, Rebecca J.; Marshall, Aidan; Vargas-Muñiz, José M. (American Society for Microbiology, 2024-12-10)
Hortaea werneckii is a halotolerant black yeast commonly found in hypersaline environments. This yeast is also the causative agent of tinea nigra, a superficial mycosis of the palm of the hand and soles of the feet of humans. In addition to their remarkable halotolerance, this black yeast exhibits an unconventional cell division cycle, alternating between fission and budding cell division. Cell density and the salt concentration in their environment regulate which cell division cycle H. werneckii uses. Although H. werneckii have been extensively studied due to their unique physiology and cell biology, deciphering the underlying mechanisms behind these remarkable phenotypes has been limited due to the lack of genetic tools available. Here, we report a new ectopic integration protocol for H. werneckii using polyethylene glycol-CaCl2 mediated protoplast transformation. This approach relies on a drug (hygromycin B) resistance gene to select for successful integration of the genetic construct. The same construct was used to express cytosolic green fluorescent protein. Finally, we developed a marker-free CRISPR/Cas9 protocol for targeted gene deletion using the melanin synthesis pathway as a visual reporter of successful transformation. These transformation strategies will allow testing hypotheses related to H. werneckii cell biology and physiology. IMPORTANCE Hortaea werneckii is a remarkable yeast capable of growing in high salt concentration, and its cell division cycle alternates between fission-like and budding. For these unique attributes, H. werneckii has gathered interest in research programs studying extremophile fungi and cell division. Most of our understanding of H. werneckii biology comes from genomic analyses, the usage of drugs to target a particular pathway, or the heterologous expression of its genes in S. cerevisiae. Nonetheless, H. werneckii has remained genetically intractable. Here, we report on two strategies to transform H. werneckii: ectopic integration of a plasmid and gene deletion using CRISPR/Cas9. These approaches will be fundamental to expanding the experimental techniques available to study H. werneckii, including live-cell imaging of cellular processes and reverse genetic approaches.

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