Biology Commons^™

Global Stabilization Of The Mitochondrial Proteome Is Associated With Extreme Anoxia Tolerance In Austrofundulus Limnaeus Ws40ne Cells, Chelsea Hughes, Meranda M. Corona, Dietmar Kültz, Jason E. Podrabsky

Biology Faculty Publications and Presentations

Background: Austrofundulus limnaeus is an extremophile vertebrate native to small temporary ponds of Venezuela. Embryos of A. limnaeus must survive variable and often extreme conditions, including long periods of anoxia. Neuroepithelial cells derived from these embryos, WS40NE cells, provide a unique tool to understand how the proteome changes in response to anoxic stress. Results Using label-free proteomics, 19,604 peptides and 3487 proteins were quantified in normoxic, 4d anoxic, and 24 h recovery WS40NE cells. Of these, 2612 proteins (74.9%) were statistically significantly differentially abundant in at least one comparison: 1988 comparing normoxia to 4 days anoxia (57.0%), 923 comparing 4 …

Comparative Analysis Of Bacterial Recbcd And Sbcc-Sbcd Systems And The Yeast Mre11-Rad50 Complex During Dna Replication, Tannos Seyedjavadi

Dissertations and Theses

Accurate completion of DNA replication when forks converge is essential for genome stability. In Escherichia coli, several proteins have been identified including RecG, ExoI, RecBCD, and SbcCD, that contribute to completing replication, and loss of these factors causes genetic instability. Eukaryotes encode proteins with homology to several E. coli completion enzymes. Mre11–Rad50 complex is structurally and functionally conserved with bacterial SbcCD, and Rrm3 shares homologous motifs with bacterial RecBCD. These proteins are often described as being involved in homologous double strand break repair. Whether they are also conserved and play a similar role as their E. coli counterparts in …

Bryophyte Community Composition In A Second-Order Tropical Stream At The Luquillo Experimental Forest, Puerto Rico, Cecilia Sahnow, Jean P. González-Crespo, Amelia Merced

Biology Faculty Publications and Presentations

Streams are important ecosystems that harbor a high diversity of plant species including bryophytes. Studies suggest streams can function as refugia and/or corridors that buffer against the effects of disturbances. In this study, we sampled five sites along a second-order tropical stream in the Luquillo Experimental Forest. We described the riparian bryophyte community composition in conjunction with the physical structure of the stream to understand what environmental factors influence bryophyte composition in the stream. We identified 56 bryophytes, 33 liverworts and 23 mosses; and we recorded cyanobacteria associated with over half of the species of bryophytes. Rocks were the most …

Assessing The Evolving The Culture Of Biology Program Through The Lens Of The Teacher-Centered Systemic Reform Model, Diyala S. Shihadih, Elizabeth Runkel Baez, Mitra Asgari, Justin Berl, Adam J. Chouinard, Stephanie J. Gutzler, Kaleb K. Heinrich, Eden Riordan, Star W. Lee, Erin E. Shortlidge

Biology Faculty Publications and Presentations

There are persistent national calls to advance evidence-based instructional practices to increase the number of students graduating with science degrees. Evolving the Culture of Biology is a program designed to improve undergraduate education by prioritizing graduate teaching assistant teaching professional development (TA-TPD). Guided by the Teacher-Centered Systemic Reform (TCSR) model, this study examined how TA TPD practitioners’ (participants) attitudes, personal factors, institutional contexts, and instructional practices shape TA-TPD reform outcomes. Using a pre-program survey and end-of-year semi-structured interviews, we gained insight into participants’ experiences as they developed and implemented reformed TA-TPD. We investigated how TCSR components differentially influenced participant progress, …

Complete Genome Sequence Of Strain Dk8, A Thermoacidophilic Archaeon From Lassen Volcanic National Park, Jonathan Abshier, Kenneth Stedman

Biology Faculty Publications and Presentations

Here, we report the genome sequence of strain DK8, a thermoacidophilic archaeal isolate from the Devil's Kitchen geothermal area of Lassen Volcanic National Park, California, USA. The genome is 2,783,064 bp, with a guanine-cytosine content of 35.02%. Strain DK8 is most closely related to L.S.2.15.

Integrative Evidence For Cryptic Diversity In The Thomomys Mazama Species Complex: Phylogenetics, Cranial Morphometrics, And Ecological Niche Modeling, Jessica Kate St. Michael

Dissertations and Theses

The population genetic structure and geographic distribution of the pocket gopher genus Thomomys Wied-Neuwied, 1839, lends itself to speciation events. Conserved morphology, ecological specialization, and high rates of molecular evolution may increase the probability of allopatric speciation, thereby contributing to the presence of morphologically cryptic taxa. In light of these patterns of evolution, we assessed genetic divergence and phylogenetic relationships, cranial morphometric differentiation, and ecological divergence among taxa in the Thomomys mazama species complex to test the hypothesis that T. mazama is a single species. Species delimitation analysis using Bayesian Poisson Tree Processes (bPTP) and data from the mitochondrial cytochrome …

Thermal Sensitivity Of Native And Introduced Crabs: Implications For Reproductive Success, Keria Moritsugu-Vandehey

Dissertations and Theses

Temperature is a major driver of physiological and developmental processes in marine organisms, and continued climate warming is expected to affect reproduction and recruitment in estuarine species. Early life stages are often particularly sensitive to environmental stress, yet relatively little is known about how warming influences embryonic development, hatching success, and larval survival across co-occurring native and introduced crab species.

This study examined the effects of temperature on embryonic development, hatching success, and early larval survival in four estuarine crab species from the Pacific Northwest: the introduced European green crab, Carcinus maenas, and the native shore crabs Hemigrapsus nudus …

Toxic Relationships: Characterization Of A Putative Virally Encoded Toxin In The Thermophilic Archaeal Fusellovirus Ssv1, Jonathan C. Abshier, Patrizia L. Alpapara, Guasåli Tomokane, Kenneth M. Stedman

Center for Life in Extreme Environments Publications

Mechanisms for the maintenance of chronic viruses are poorly understood, particularly for archaeal viruses. Here, we identify the product of Sulfolobus spindle-shaped virus 1 (SSV1) ORF a291 as a putative virally encoded toxin required for growth inhibition but dispensable for viral replication and virion production. Viruses lacking ORF a291 replicated their genomes and formed morphologically normal spindle-shaped particles, yet failed to inhibit the growth of uninfected Saccharolobus solfataricus. Substitution of residues at a predicted N-terminal signal peptide cleavage site abolished growth suppression without affecting replication, suggesting that secretion is essential for toxin function. Despite primary sequence divergence among fusellovirus …

Anoxia Tolerant Dna Replication Is Supported By Atr Kinase In The Annual Killifish Austrofundulus Limnaeus, Riley Roth-Carter, Erin Helms, Joshua C. Saldivar, Jason Podrabsky

Biology Faculty Publications and Presentations

Hypoxia and anoxia suppress cell proliferation due to an increase in replication stress and activation of DNA damage checkpoints. Embryos of the annual killifish Austrofundulus limnaeus tolerate prolonged anoxia, indicating improved genomic stability under oxygen starvation. We investigated the cell cycle regulation of the anoxia tolerant killifish embryonic cell line PSU-AL-WS40NE during anoxia. Live-cell imaging confirms continued proliferation of WS40NE cells for the first 24 h of anoxia with minimal cell death. Fluorescence imaging shows that cells accumulate in G1 after the first day in anoxia with a rapid entry into S phase upon reoxygenation. Pharmacological inhibition shows a reliance …

Elucidating The Function Of Yedk In Response To Dna Damage, Hank Munro

University Honors Theses

Apurinic/Apyrimidinic (AP) sites are one of the most prevalent forms of DNA damage. These lesions arise spontaneously or during base excision repair of modified DNA bases and are potentially mutagenic in both mammalian and bacterial cells. 5-hydroxymethylcytosine binding, ES cell specific (HMCES), a protein found in eukaryotic cells, has been shown to form a stable DNA-protein crosslink with AP sites in single stranded DNA and to reduce the number of AP sites occurring either spontaneously or after alkylative DNA damage, resulting in increased genome stability. YedK, the Escherichia coli ortholog of HMCES exhibits some of the same biochemical properties as …

Growing Pains: The Loss Of Plasticity To Extreme Anoxia In Developing Embryos Of Austrofundulus Limnaeus, Patrick Ryan Clouser

Dissertations and Theses

Oxygen plays a pivotal role for most animal life on Earth, so much so that even short exposures to no oxygen, anoxia, can have severe effects on the animal's wellbeing. Conversely, aerobic recovery after anoxic insult can be just as detrimental. Some animals are the exception to this rule and demonstrate survival through anoxia and recovery. There has been a great deal of research around anoxia tolerant animals, but what remains unclear are the genetic underpinnings that determine the anoxia tolerant phenotype. RNA sequencing allows for the quantification of RNA transcripts within a sample, which can indicate if a gene …

Life Without Oxygen: Cellular Mechanisms Enabling Genome Maintenance And Proliferation In Cells From An Anoxia-Tolerant Vertebrate, Riley Asa Roth-Carter

Dissertations and Theses

When exposed to the complete loss of oxygen (anoxia) most organisms undergo rapid cellular deterioration driven by a range of metabolic dysfunctions associated with the loss of the terminal electron acceptor for aerobic respiration. This loss of ability to create and maintain necessary levels of cellular ATP through oxidative phosphorylation contributes to loss of membrane potentials, increase in the accumulation of acidic by-products of anaerobic metabolism, and a decrease in the ability to support other molecular processes that rely on a steady supply of ATP. While this extreme oxygen stress is lethal to a majority of vertebrate organisms there are …

Ecological Drift And Host Filtering Jointly Structure Foliar Endophytes During Ecosystem Development., Caio César Pires De Paula, Petr Macek, Milan Varsadiya, Jakub Borovec, Mehmet A. Balkan, Brett S. Younginger, Daniel J. Ballhorn, Tomáš Picek, Tomáš Hájek, Jan Frouz, Jiří Bárta, Dagmara Sirová

Biology Faculty Publications and Presentations

Foliar endophytes contribute to plant nutrient acquisition, stress tolerance, and pathogen resistance, yet their responses to ecosystem-level processes remain poorly understood. Using a space-for-time substitution design, we investigated bacterial and fungal community dynamics in the foliar endosphere of four phylogenetically distinct plant hosts across a well-characterized successional chronosequence.

Phased-Genome Assemblies Of Saltgrass Reveal Structural Dynamics And Genomic Basis For Extreme Salt Tolerance And Dioecy., Kashif Nawaz, Izamar Olivas Orduna, Dal-Hoe Koo, Francisco Molina-Freaner, Hester L. Bell, Mara Miculan, Victor Llaca, Judith Harrington, Sarah M. Eppley, Jesse Poland

Biology Faculty Publications and Presentations

The climate crisis poses a critical challenge to agriculture, with freshwater scarcity becoming a major constraint on crop production. Harnessing halophytic adaptations for growth in saline environments offers a promising path for neodomestication of salt-tolerant crops. Distichlis spp. (saltgrass)-a genus of dioecious, halophytic C4 grasses in the PACMAD clade-thrives in extreme saline conditions, making it a compelling species to understand salinity tolerance and adaptation. Here, we present high-quality phased genome assemblies of four Distichlis genets, revealing an allotetraploid genome (576-610 Mb) with two highly syntenic but degenerate subgenomes, each exhibiting over 30% gene loss across orthologous pairs. Comparative genomic analyses …

Crispr Spacers Reveal Diverse And Abundant Thermococcales Viruses In Hydrothermal Vents, Ryan Catchpole, Justin Mclean, Emily St John, Anna-Louise Reysenbach, Mart Krupovic, Michael P. Terns

Biology Faculty Publications and Presentations

Viruses are the most pervasive biological entities on Earth and they profoundly shape host ecology and evolution. However, for many microbial lineages, knowledge of their viromes remains limited, especially for those inhabiting remote environments, including deep-sea ecosystems. Here, we leverage one of the most extensively cultivated and genomically characterized archaeal lineages, the Thermococcales, to identify novel viral genomes. By utilizing CRISPR spacers from isolates and spacer arrays reconstructed from metagenomes, we mined mobile genetic elements (MGEs) in 1,172 publicly available and newly sequenced hydrothermal vent metagenomic datasets. Comparative genomics and identification of viral hallmark proteins revealed 620 viral genomes across …

Coexisting Salps Exhibit Distinct Feeding Selectivity On Microorganisms In The North Pacific Subtropical Gyre, Anne W. Thompson, Kylee Lamberson, Kelly R. Sutherland

Biology Faculty Publications and Presentations

Mortality mechanisms play an important role in how oceanic microorganisms contribute to global biogeochemical cycles. Salps are widespread pelagic tunicates known to remove phytoplankton from coastal and high-latitude waters, but their interaction with microorganisms in the vast tropical and subtropical gyres is not well quantified. Using quantitative measurements of six major marine microorganisms in the guts of six distinct but co-occurring salp species from the North Pacific Subtropical Gyre, we examined the impact and dynamics of salp feeding on marine microorganisms in a vast open ocean region. All salps preferentially removed prey greater than 1 μm in diameter, including marine …

Global Deep-Sea Hydrothermal Deposit Metagenomes And Metagenome-Assembled Genomes Over Time And Space, Emily St John, Anna-Louise Reysenbach

Center for Life in Extreme Environments Publications

Actively venting high temperature deep-sea hydrothermal vent deposits along tectonic spreading centers and in backarc basins harbor a rich diversity of thermophilic Bacteria and Archaea, many of which have no representatives in cultivation nor any genomic representation in databases. Here, in order to produce a global-scale time series metagenomic resource for studying the microbial functional and genomic diversity in these high temperature ecosystems, we obtained 70 metagenomes from collections across spatial and temporal gradients from 21 different vent fields spanning 16 years (1993-2009). The dataset (Deep-Sea Hydrothermal Vent dataset (DSV70)) includes 3.56 Tbp of raw DNA sequence reads, that have …

Decomposition Processes And Insect Indicators In Forensic Investigations, Eli Taylor

University Honors Theses

Insects play an essential biological and ecological role in human life, and their predictable relationship with decomposing remains has positioned them as a cornerstone of modern forensic science. Since the early 20th century, forensic entomology has provided critical evidence for estimating the postmortem interval (PMI), relying on patterns of colonization, species succession, and insect developmental timelines. Accurate PMI estimations are vital to medicolegal investigations, influencing the interpretation of biological evidence, guiding resource allocation, and informing determinations of cause and manner of death. However, insect activity is profoundly shaped by environmental and ecological variables, including temperature, humidity, seasonal shifts, and geographic …

Population Observations And Microhabitat Use Of Isthmohyla Tica (Anura: Hylidae) In San Ramón, Costa Rica, Pablo Marin Pacheco, Jerson Santamaria Martinez, Jose Manuel Mora, Jennifer L. Stynoski, Randall Arguedas

Biology Faculty Publications and Presentations

Isthmohyla tica, the Tica Frog, is a critically endangered species endemic to Costa Rica and supposedly Panama that has only been seen in a few sites in recent decades. To aid in its conservation, we assessed the population status and microhabitat preferences of this frog in a section of the Danta stream at Rancho Alegre farm, San Ramón, Costa Rica. We conducted 18 nocturnal surveys and collected data on perch types, vegetation associations, stream characteristics, and environmental variables for each sighting of the Tica Frog. To identify potential microhabitat preferences, we compared these features between sites with or without frogs …

Uncovering The Diversity Of Bats In The Mbam Minkom Massif, Cameroon: Insights Into The Importance Of Tropical Inselbergs, Amanda L. Grunwald, Terrence C. Demos, Patrick J. Atagana, Courtney M. Volk, Ara Monadjem, Franck P. Meyo Konoo, Yvette Ngueagni, R. Travis Merrill, Daniel J. Ballhorn, Luis A. Ruedas, Multiple Additional Authors

Biology Faculty Publications and Presentations

We report the results of two expeditions (2019 and 2022) to the Mbam Minkom Massif (Centre Region, Cameroon), focused on an assessment of the montane area's bat fauna. We describe bat species' richness and highlight the incredible diversity and ecological importance of this highly threatened ecosystem. Across both years, we captured a total of 36 bat species representing 21 genera and six families on Mbam Minkom Massif and in the surrounding lowland forest. Our findings include two new species records for Cameroon, and two additional records of the recently described bat, Pseudoromicia mbamminkom. We provide accounts for all captured species, …

A Geothermal Amoeba Sets A New Upper Temperature Limit For Eukaryotes, H Beryl Rappaport, Natalie A. Petek-Seoane, Tomáš Tyml, Felix Mikus, Kurt Labutti, Godwin Ani, Jessica K. Niblo, Ethan Macvicar, Rachel M. Shepherd, Ignacio De La Higuera, Samuel J. Lord, Kenneth M. Stedman, Multiple Additional Authors

Center for Life in Extreme Environments Publications

The study of temperature limits has transformed our knowledge of the boundaries of life but has been largely focused on bacteria and archaea. We isolated a novel geothermal amoeba, , that divides at 63°C (145.4°F), establishing a new record for the upper temperature limit across all eukaryotes. We demonstrated cellular proliferation with growth experiments and visualized mitosis expansion microscopy. Using high-temperature live-cell imaging, we quantified movement up to 64°C. We assembled the genome of and using comparative genomics found an enrichment of genes related to proteostasis, genome stability, and sensing the external environment. Taken together, our findings challenge the current …

The Web Of Tpd Reform: A Reflective Tool For Analyzing And Improving Teaching Professional Development Programs For Teaching Assistants, Adam J. Chouinard, Diyala S. Shihadih, Star W. Lee, Stephanie J. Gutzler, Kaleb K. Heinrich, Mitra Asgari, Erin Shortlidge

Biology Faculty Publications and Presentations

Teaching assistants (TAs) play a pivotal role in the teaching mission of higher education, but too often they are asked to do this important job without adequate teaching professional development (TPD). By receiving training in inclusive and evidence-based teaching practices, TAs can in turn help disseminate effective teaching methods for the benefit of students throughout the nation; as future faculty, TPD also better prepares TAs for their careers, helps solve longstanding problems of inclusion and retention in STEM, and reforms disciplinary educational norms through cultural evolution. Thanks to prior research and advocacy efforts, there is a growing awareness that STEM …

Biodiversity Conservation Depends On The Expansion Of Taxonomy And Systematics Research, Luis A. Ruedas, Scott L. Gardner

Biology Faculty Publications and Presentations

Despite mammals constituting fewer than 0.3% of all described species, their conservation is compelling for a number of reasons. They contribute to biodiversity and are important in maintaining and regulating ecological communities. Many mammals have significant cultural and economic value, serving as sources of food, medicine, and tourism revenue. Furthermore, the conservation of mammals often leads to the protection of entire habitats, benefiting numerous other species and preserving ecosystem services that are critical for human well- being. Humans share a unique evolutionary history with other mammal species, making their preservation important for scientific research, education, and understanding of our own …

Cul3 Postnatal Homozygous Deletion In Forebrain Results In Behavioral Differences, Prathibha Sekar, Qiang‐Qiang Xia, Alex C. Stokes, Feiyu Quan, Jeffrey Singer, Craig M. Powell

Biology Faculty Publications and Presentations

Large-scale human genetic studies implicate multiple genes that regulate protein ubiquitination in autism spectrum disorder (ASD). De novo loss-of-function mutations in the gene CULLIN3 (CUL3) are implicated in autism and intellectual disability (ID). CUL3 is an essential component of an E3 ubiquitin ligase complex required for ubiquitination of substrates, often a signal for proteasomal degradation. Homozygous deletion of Cul3 is embryonically lethal. Recent studies show heterozygous deletion of Cul3 results in phenotypes with some face validity for autism in constitutive and conditional Cul3 heterozygotes. To understand the function of Cul3 in late postnatal development and function in the brain, we …

Systematics And Phylogeography Of Mundochthonius Chamberlin, 1929, In The Pacific Northwest (Chelicerata, Pseudoscorpiones), Amanda Rai Montoya-Cowan

Dissertations and Theses

Understanding the processes that drive genetic divergence and species formation is central to evolutionary biology. Invertebrates with low dispersal ability, such as pseudoscorpions, are especially prone to cryptic diversification due to their occupancy of niche microhabitats and sensitivity to landscape barriers. Despite their ecological roles as predators and bioindicators, pseudoscorpions remain one of the least studied arachnid groups, with limited taxonomic resolution and few integrative studies. This research focuses on the genus Mundochthonius in the Pacific Northwest, particularly M. pacificus, a morphologically conserved species whose true diversity and distribution remain poorly understood.

Field surveys guided by a GIS-based habitat …

Evaluating Gains In Student Self-Efficacy In Scientific Literacy Associated With A Brief Curricular Intervention, Radhika Reddy, Olivia M. Ainsworth, Mackenzie J. Gray, Andrea Hildebrand, Jazzmine Waugh, Jennifer Lindwall, Javier Huerta, Thomas E. Keller

Biology Faculty Publications and Presentations

Undergraduate research experiences (UREs) are high-impact practices that enhance retention, motivation, and self-efficacy in science, particularly for students with limited prior exposure to scientific environments. However, many students-especially those from underrepresented or marginalized backgrounds-enter these experiences with low research self-efficacy, stemming from limited familiarity with academic language, scientific literature, and research communication. The Research Induction Curriculum (RIC) was designed to address this challenge by providing early, structured training in scientific literacy through scaffolded journal club discussions. The RIC introduces students to progressively more complex scientific articles, supports peer discussion, and builds confidence in reading, interpreting, and presenting scientific information. This …

Roadkill Scavenging Behavior Of White-Throated Magpie-Jay (Cyanocorax Formosus) In Northwestern Costa Ricacomportamiento De Carroñeo En Carretera De La Urraca Copetona (Cyanocorax Formosus) En El Noroeste De Costa Rica, Juan De Dios Astorga-Acuña, José Manuel Mora

Biology Faculty Publications and Presentations

Corvids are opportunistic omnivores and facultative scavengers, but there are no direct records of Cyanocorax species scavenging on carrion. We documented opportunistic feeding behavior of the White-throated Magpie-Jay (Cyanocorax formosus) on a road-killed Common Pauraque (Nyctidromus albicollis) in northwestern Costa Rica. On 25 August 2023, we observed three White-throated Magpie-Jays feeding on the carcass of a Common Pauraque, likely killed by collision with a vehicle, along a gravel road near the Taboga Forestry Reserve. This provides de first documented evidence of the White-throated Magpie-Jay scavenging on roadkill, and of carrion consumption by a Central American corvid. Given the White-throated Magpie-Jay's …

Functional Ecology Of Antarctic Flora, Zoie C. Yetley

Dissertations and Theses

Antarctic mosses play foundational roles in terrestrial ecosystem processes, yet their functional ecology remains poorly understood. This study developed a novel functional trait-based framework for dominant moss species across glacier retreat gradients in the South Shetland Islands, addressing a critical gap in Antarctic bryophyte ecology. Nutrient- and water-related traits were measured for four moss species--Polytrichastrum alpinum, Bryum pseudotriquetrum, Sanionia uncinata, and Syntrichia saxicola--along with Deschampsia antarctica, across nine sites spanning early to late successional stages. Results revealed strong interspecific trait differentiation: P. alpinum exhibited conservative nutrient-use and rapid water-uptake strategies; S. uncinata and B. pseudotriquetrum …

Predation By The Brown Vine Snake, Oxybelis Koehleri, On The Autotomized Tail Of An Escaping Common Anole, Anolis Humilis, Jose M. Mora, Myzell Kenneth Amaro

Biology Faculty Publications and Presentations

During a natural history hike at La Selva Biological Station in Costa Rica, we observed a Brown Vine Snake, Oxybelis koehleri, consuming the autotomized tail of a Common Humble Anole, Anolis humilis, which had escaped predation. This event represents a rare documented instance of a vine snake feeding exclusively on a detached lizard tail. Tail autotomy is a widespread anti-predator strategy among lizards, with the shed tail continuing to writhe post-detachment, potentially attracting predators. Lizard tails are lipid-rich and offer a convenient energy source, especially in resource-scarce environments. Although such behavior has been noted in other reptiles, it is seldom …

Small Molecule Inhibitor 3i-1000 Dosed During Bouts Of Anoxia Increases Survivability Of Austrofundulus Limnaeus Embryos, Connor Kime

University Honors Theses

The annual killifish Austrofundulus limnaeus possesses exceptional tolerance to prolonged anoxia, offering a unique vertebrate model for investigating cardiac resilience and survival under metabolic stress. This study tested whether inhibition of the GATA4–NKX2-5 transcriptional axis with the small molecule 3i-1000 enhances post-anoxic survival. WS40 embryos were exposed to 50 µM 3i-1000 during anoxia and early aerobic recovery. Outcomes were assessed across two independent experimental cohorts. Treated embryos exhibited higher survivability during reoxygenation: in the October cohort, survival increased from 76.0% in vehicle controls (n = 25) to 91.7% in treated embryos (n = 24); in the November cohort, survival improved …