Biology Commons^™

The Exploration Of Nanotoxicological Copper And Interspecific Saccharomyces Hybrids, Matthew Joseph Winans Phd

Graduate Theses, Dissertations, and Problem Reports (ETD)

Nanotechnology takes advantage of cellular biology’s natural nanoscale operations by interacting with biomolecules differently than soluble or bulk materials, often altering normal cellular processes such as metabolism or growth. To gain a better understanding of how copper nanoparticles hybridized on cellulose fibers called carboxymethyl cellulose (CMC) affected growth of Saccharomyces cerevisiae, the mechanisms of toxicity were explored. Multiple methodologies covering genetics, proteomics, metallomics, and metabolomics were used during this investigation. The work that lead to this dissertation discovered that these cellulosic copper nanoparticles had a unique toxicity compared to copper. Further investigation suggested a possible ionic or molecular mimicry …

Development And Testing Of Artificial Membranes For Rearing Of Rhipicephalus Microplus, The Southern Cattle Fever Tick, Ann T. Vacek, John A. Goolsby, Rupesh R. Kariyat

School of Integrative Biological & Chemical Sciences (Formerly Dept. of Biology)

The southern cattle fever tick, Rhipicephalus microplus, is a livestock pest worldwide in tropical and subtropical climates, including South Texas, and can vector Babesia spp., the causal agents of bovine babesiosis. Artificial rearing methods for R. microplus are needed, especially for rearing specialist tick parasitoids that are under evaluation for classical biological control. In this study, we tested the efficiency of artificial feeding of R. microplus larvae, nymphs, and adults on a siliconized substrate (goldbeater’s membrane, lens paper, or Hemotek), or on nonsiliconized goldbeater’s membrane or Hemotek. Other variables tested were a warm water bath, incubator, positioning blood above or …

Survival Of Off-Host Unfed Rhipicephalus (Boophilus) Annulatus (Acari: Ixodidae) Larvae In Study Arenas In Relation To Climatic Factors And Habitats In South Texas, Usa, Emily J. Zamora, Brenda Leal, Donald B. Thomas, Robert K. Dearth

School of Integrative Biological & Chemical Sciences (Formerly Dept. of Biology)

The cattle fever tick, Rhipicephalus annulatus (Say), is an economically destructive arthropod because of its ability to vector bovine babesiosis. Cattle fever ticks can spend more than 90% of their life cycle as questing larvae, but the effect of climatic factors on their off-host behavior and survival is unclear. The goal of this study was to measure the effects of specific ecological factors on off-host larvae in nature. The study was conducted in a south Texas pasture over a 20-mo period, during which time larval populations were surveyed and ambient weather variables - relative humidity and temperatures - were recorded. …

Host Relationships And Geographic Distribution Of Species Of Blanchard, 1848 (Onchoproteocephalidea, Onchobothriidae) In Elasmobranchs: A Metadata Analysis, Francisco Zaragoza-Tapia, Griselda Pulido-Flores, Scott L. Gardner, Scott Monks

Scott L. Gardner Publications

Species of Acanthobothrium have been documented as parasites of the spiral intestine of elasmobranchs. Results of a metadata analysis indicate that 114 species of elasmobranchs have been reported as hosts of 200 species of Acanthobothrium. The metadata analysis revealed that 3.7% of species of sharks and 14.9% of species of rays that have been reported as hosts to date; some species are parasitized by more than one species of Acanthobothrium. This work provides a Category designation, as proposed by Ghoshroy and Caira (2001), for each species of Acanthobothrium. These Category designations are a tool to facilitate comparisons …

Obituary: Sydney Anderson (1927–2018), Scott Lyell Gardner, Robert M. Timm, Nancy Olds, Hugh H. Genoways

Scott L. Gardner Publications

On August 12, 2018, the American Society of Mammalogists (ASM) lost one of its giants of the latter half of the 20th century when Sydney Anderson quietly passed away at his home in Birmingham, Alabama, at the age of 91. “Syd,” to his many friends and colleagues, was born on January 11, 1927 in Topeka, Kansas, to Robert Grant and Evelyn Fern (Hunt) Anderson.

Anderson began his professional career at the University of Kansas, where for four years (1955–1959) he served as Assistant Curator in charge of mammals in the Museum of Natural History, and Instructor in the Department of …

Copper-Rich “Halo” Off Lake Superior's Keweenaw Peninsula And How Mass Mill Tailings Dispersed Onto Tribal Lands, W. Charles Kerfoot, Noel Urban, Jaebong Jeong, Carol Maclennan, Sophia Ford

Michigan Tech Publications, Part 1

Over a century ago, shoreline copper mills sluiced more than 64 million metric tonnes of tailings into Lake Superior, creating a “halo” around the Keweenaw Peninsula with a buried copper peak. Here we examine how tailings from one of the smaller mills (Mass Mill, 1902–1919) spread as a dual pulse across southern Keweenaw Bay and onto tribal L'Anse Indian Reservation lands. The fine (“slime clay”) fraction dispersed early and widely, whereas the coarse fraction (stamp sands) moved more slowly southward as a black sand beach deposit, leaving scattered residual patches. Beach stamp sands followed the path of sand eroding from …

Biocide Resistance As A Result Of Exposure To Biocides Used In Hydraulic Fracturing, Lindsey Schenten

Dissertations, Master's Theses and Master's Reports

Microbial resistance to antimicrobials is an important topic to investigate not only for our health but for the environment. There is a growing concern about expanding microbial resistance to both antibiotics and other antimicrobials such as biocides. We wanted to know how bacteria previously exposed to hydraulic fracturing fluids, including biocides, would react to biocide exposure and what the mechanism of resistance looked like. In order to test this hypothesis bacterial isolates were obtained from water and sediment from a stream that had been previously exposed to a spill of hydraulic fracturing water. Thirty bacterial isolates were obtained from these …

Bromocriptine Improves Glucose Tolerance Independent Of Circadian Timing, Prolactin, Or The Melanocortin-4 Receptor, Sarah N. Framnes-Deboer, Ellen Bakke, Suma Yalamanchili, Hannah Peterson, Darleen A. Sandoval, Randy J. Seeley, Deanna M. Arble

Biological Sciences Faculty Research and Publications

Bromocriptine, a dopamine D2 receptor agonist originally used for the treatment of hyperprolactinemia, is largely successful in reducing hyperglycemia and improving glucose tolerance in type 2 diabetics. However, the mechanism behind bromocriptine’s effect on glucose intolerance is unclear. Here, we tested three hypotheses, that bromocriptine may exert its effects on glucose metabolism by 1) decreasing prolactin secretion, 2) indirectly increasing activity of key melanocortin receptors in the central nervous system, or 3) improving/restoring circadian rhythms. Using a diet-induced obese (DIO) mouse model, we established that a 2-wk treatment of bromocriptine is robustly effective at improving glucose tolerance. …

Distinct Patterns Of Pigment Development Underlie Convergent Hyperpigmentation Between Nocturnal And Diurnal Geckos (Squamata: Gekkota), Aaron H. Griffing, Tony Gamble, Aaron M. Bauer

Biological Sciences Faculty Research and Publications

Background

Evolutionary transitions in temporal niche necessitates specialized morphology, physiology, and behaviors. Diurnal, heliothermic squamates (lizards and snakes) that bask require protection from ultraviolet radiation (UV) that can damage internal organs such as the brain, viscera, and gonads. Many smaller squamates have accomplished this protection by hyperpigmentation of the peritoneum and subcutaneous dorsum. Typically, nocturnal species do not require these protections from ultraviolet light. However, some nocturnal species that exhibit extreme crypsis may be exposed to sunlight and UV and require some means of mediating that damage. One such species is Gekko (Ptychozoon) kuhli, a nocturnal, arboreal …

Agricultural And Environmental Weeds Of South Texas And Their Management, Pushpa Soti, John A. Goolsby, Alexis Racelis

School of Integrative Biological & Chemical Sciences (Formerly Dept. of Biology)

The Lower Rio Grande Valley (LRGV) in south Texas is one of the most productive agricultural regions in southern United States. With subtropical climate and highly fertile soils, this region provides a year-round growing condition for crops. Along with citrus, major crops grown in the region are sorghum cotton and corn in the summer and vegetables in winter. Thus, a fallow period of 3-6 months between successive crops is common in the region. Growers in this region report weeds as their number one economic and agronomic problem affecting crop yield and quality and increasing the cost of production and weeds …

A Tale Of Two Cities: Aedes Mosquito Surveillance Across The Texas-Mexico Border, Teresa Patricia Feria-Arroyo, Consuelo Aguilar, Cuauhtémoc Quintero Vazquez, Rene Santos-Luna, Susana Roman-Perez, Tamer Oraby, Gustavo Sanchez Tejeda, Fabian Correa Morales, Víctor M. Salazar Bueyes, Pascual Camacho Guevara

School of Integrative Biological & Chemical Sciences (Formerly Dept. of Biology)

Cross border situations complicate epidemiologic risk assessments in transboundary regions such as the US-Mexico border. Countries have different health policies, mosquito control policies, and mosquito surveillance systems. We established a binational Aedes mosquito surveillance program in Reynosa, Tamaulipas, and McAllen, Texas by replicating a part of the Mexican Integrated Vector Monitoring System (IVMS) across the international border. The entomologic surveillance of the IVMSs is based on ova collection cups (ovitraps) and for the binational project, the surveillance protocol was modified to include an Autocidal Gravid Ovitrap (AGO) in the center of every city-block (100 m2) distribution of four ovitraps. We …

Review Of Major Crop And Animal Arthropod Pests Of South Texas, Holly N. Davis, John A. Goolsby, Donald B. Thomas, Ismael Badillo, Rupesh R. Kariyat, Christopher Vitek, Danielle Sekula

School of Integrative Biological & Chemical Sciences (Formerly Dept. of Biology)

The Lower Rio Grande Valley is an area in Texas that consists of the four southern-most counties. This area contains a diverse range of agriculture and land-use including vegetable, row-crop and livestock production. The year-around cool to hot subtropical climate means that green vegetation is continually present, including many crops. Geographically, it shares an international border, making it a region vulnerable to new invasive species and the re-introduction of pests that have been previously eliminated in the United States. These combined factors lead to an array of arthropod pests that may have serious impacts on the crops, animals, and people …

Detection Of Mitochondrial Dna (Mtdna) Mutations, Ali Naini, Robert Gilkerson, Sara Shanske, Jiuhong Pang

School of Integrative Biological & Chemical Sciences (Formerly Dept. of Biology)

The maternally inherited mitochondrial DNA (mtDNA) is a circular 16,569-bp double stranded DNA that encodes 37 genes, twenty-four of which (2 rRNA and 22 tRNA) are necessary for transcription and translation of 13 polypeptides that are all subunits of respiratory chain. Pathogenic mutations of mtDNA cause respiratory chain dysfunction, and are the underlying defect in an ever-increasing number of mtDNA-related encephalomyopathies with distinct phenotypes. In this chapter, we present an overview of mtDNA mutations and describe the molecular techniques currently employed in our laboratory to detect two types of mtDNA mutations: Single-large scale rearrangements and point mutations.

Terrestrial Snail Communities Of The Lower Rio Grande Valley Are Affected By Human Disturbance And Correlate With Vegetation Community Composition, Briante S. Lewis Najev, Alison Schofield, Raziel I. Flores, Benjamin T. Hutchins, J. Andrew Mcdonald, Kathryn E. Perez

School of Integrative Biological & Chemical Sciences (Formerly Dept. of Biology)

The lower Rio Grande Valley of Texas (LRGV) contains a unique, subtropical, semiarid floodplain with most of the few remaining Tamaulipan thorn forests in the United States. Less than 2% of Tamaulipan thorn forest remains, with urban and agricultural conversion potentially threatening diverse plant and animal communities native to the habitat. We performed vegetative community surveys and conducted a comprehensive survey of terrestrial snail communities in intact (refugia sites, minimally altered in recent times) and altered (previously cleared and regrown or restored) Tamaulipan thorn forest habitats of the LRGV. In a comparison of intact and altered sites that have comparable …

Characterizing The Transport Of Parasitoid Wasp Genaspis Hookeri Venom Serca Into Host Drosophila Hemocytes, Christopher Lark

Theses and Dissertations

The female endoparasitoid wasps introduce venom accompanied by an egg into the larval hosts’ body cavity during oviposition. The venom contains a mixture of proteins that exploit the host’s biological processes, such as, regulating gene expression, disrupting immune signaling pathways, or inducing immune cell death to give a decided advantage to the incipient wasp to develop unchallenged. Drosophila possesses a highly conserved innate immune response that recognizes and neutralizes foreign microbes and macroparasites. The venom of Ganaspis hookeri (strain G1) endoparasitoid wasp has co-evolved to suppresses the humoral phase Drosophila’s innate immune response by inhibiting Ca2+ bursts needed to induce …

Mitochondrial Dna Variation And Selfish Propagation Following Experimental Bottlenecking In Two Distantly Related Caenorhabditis Briggsae Isolates, Josiah Tad Wagner, Dana K. Howe, Suzanne Estes, Dee R. Denver

Biology Faculty Publications and Presentations

Understanding mitochondrial DNA (mtDNA) evolution and inheritance has broad implications for animal speciation and human disease models. However, few natural models exist that can simultaneously represent mtDNA transmission bias, mutation, and copy number variation. Certain isolates of the nematode Caenorhabditis briggsae harbor large, naturally-occurring mtDNA deletions of several hundred base pairs affecting the NADH dehydrogenase subunit 5 (nduo-5) gene that can be functionally detrimental. These deletion variants can behave as selfish DNA elements under genetic drift conditions, but whether all of these large deletion variants are transmitted in the same preferential manner remains unclear. In addition, the degree to which …

Genotypic Diversity And Host-Specificity Of Frankia Bacteria Associated With Sympatric Populations Of Alnus Rubra And Alnus Rhombifolia In Oregon, Mehmet Ali Balkan, Nathan U. Stewart, Emily S. Kauffman, Emily R. Wolfe, Daniel J. Ballhorn

Biology Faculty Publications and Presentations

Biological nitrogen fixation is one of the most critical processes contributing to ecosystem productivity and stability on a global scale. In temperate climates of the northern hemisphere, plant-root associated bacteria of the genus Frankia are the major nitrogen fixers in forest environments. Trees belonging to the genus Alnus are the most widespread hosts of Frankia in the Pacific Northwest, and a myriad of biotic and abiotic factors can influence the robustness of this symbiosis. Host identity and bacterial strain are important features that can impact Alnus-Frankia association, but little is known about the interplay of intrageneric hosts that co-occur in …

Roadmap For Naming Uncultivated Archaea And Bacteria, Alison Murray, John Freudenstein, Simonetta Gribaldo, Roland Hatzenpichler, Philip Hugenholtz, Peter Kämpfer, Konstantinos T. Konstantinidis, Christopher E. Lane, R. Thane Papke, Anna-Louise Reysenbach, Multiple Additional Authors

Biology Faculty Publications and Presentations

The assembly of single-amplified genomes (SAGs) and metagenome-assembled genomes (MAGs) has led to a surge in genome-based discoveries of members affiliated with Archaea and Bacteria, bringing with it a need to develop guidelines for nomenclature of uncultivated microorganisms. The International Code of Nomenclature of Prokaryotes (ICNP) only recognizes cultures as ‘type material’, thereby preventing the naming of uncultivated organisms. In this Consensus Statement, we propose two potential paths to solve this nomenclatural conundrum. One option is the adoption of previously proposed modifications to the ICNP to recognize DNA sequences as acceptable type material; the other option creates a nomenclatural code …

Prevalence Of Dirofilaria Immitis In Southern California’S Invasive Aedes Species And Native Culiseta Incidens, Zaina Chaban

University of the Pacific Theses and Dissertations

Dirofilaria immitis is a mosquito-borne parasite that causes dog heartworm disease, effecting over 100,000 dogs in the United States each year (CAPC - Parasite Prevalence Maps., 2021). Increased disease reports are commonly attributed to an increase in the local mosquito vector population. Since 2013, dog heartworm disease cases have increased by approximately 21% in the United States as reported by the American Heartworm Society. During the same time, invasive mosquitoes belonging to the genus Aedes have been found in Southern California and spread to other areas. The purpose of our research was to determine the prevalence of Dirofilaria …

Analysis Of Adult Neural Retina Extracellular Vesicle Release, Rna Transport And Proteomic Cargo, Jason Mighty, Jing Zhou, Alberto Benito-Martin, Sami Sauma, Samer Hanna, Onyekwere Onwumere, Cui Shi, Martin Muntzel, Moira Sauane, Michael Young, Henrik Molina, Dianne Cox, Stephen Redenti

Publications and Research

PURPOSE. Extracellular vesicles (EVs) contain RNA and protein cargo reflective of the genotype and phenotype of the releasing cell of origin. Adult neural retina EV release, RNA transfer, and proteomic cargo are the focus of this study.

METHODS. Adult wild-type mouse retinae were cultured and released EV diameters and concentrations quantified using Nanosight. Immunogold transmission electron microscopy (TEM) was used to image EV ultrastructure and marker protein localization. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze retinal cell transcripts present in EVs. Super-resolution microscopy was used to image fluorescent (green) RNA and (red) lipid membrane labeled EVs, released …

Human Impact Overrides Bioclimatic Drivers Of Red Fox Home Range Size Globally, Michael Main, Robert Davis, David Blake, Harriet Mills, Tim Doherty

Research Datasets

Aim Identifying the variables that influence animal home range size is important for understanding the biological requirements of individuals and their social interactions. Given their often broad distributions, carnivores are model organisms for studying range-wide determinants of home range size. Here we test predictions about environmental determinants of home range size for one of the world’s most widely distributed carnivores, the red fox (Vulpes vulpes).

Location Global

Methods We compiled a database of 70 mean home range estimates from 62 studies and four continents, which we analysed according to site-based temperature, precipitation, environmental productivity and human influence …

Hybrid Zone Barriers Comparative Data Set, David L. Field

Research Datasets

Many recent studies have addressed the mechanisms operating during the early stages of speciation, but surprisingly few studies have tested theoretical predictions on the evolution of strong reproductive isolation (RI). To help address this gap, we first undertook a quantitative review of the hybrid zone literature for flowering plants in relation to reproductive barriers. Then, using Populus as an exemplary model group, we analysed genome-wide variation for phylogenetic tree topologies in both early- and late-stage speciation taxa to determine how these patterns may be related to the genomic architecture of RI. Our plant literature survey revealed variation in barrier complexity …

Modelling Mycorrhizal Fungi Dispersal By The Mycophagous Swamp Wallaby (Wallabia Bicolor) [Dataset], Melissa Danks, Natalie Simpson, Todd F. Elliot, C.E. Timothy Paine, Karl Vernes

Research Datasets

Despite the importance of mammal-fungal interactions, tools to estimate the mammal-assisted dispersal distances of fungi are lacking. Many mammals actively consume fungal fruiting bodies, the spores of which remain viable after passage through their digestive tract. Many of these fungi form symbiotic relationships with trees and provide an array of other key ecosystem functions. We present a flexible, general model to predict the distance a mycophagous mammal would disperse fungal spores. We modelled the probability of spore dispersal by combining animal movement data from GPS-telemetry with data on spore gut-retention time. We test this model using an exemplar generalist mycophagist, …

Breastfeeding Practices 2008–2009 Among Chinese Mothers Living In Ireland: A Mixed Methods Study, Qianling Zhou, Katherine Younger, Tanya M. Cassidey, Wenyi Wang, John Kearney

Articles

Background

Migration to another country has a potential influence on breastfeeding practices. A significant difference in breastfeeding rates between Irish nationals and non-nationals has been reported. This study was conducted to explore breastfeeding practices of the Chinese in Ireland, one of the largest Irish ethnic groups, and to explore the influence of living in Ireland on breastfeeding practices. This is the first and the only migration study so far on breastfeeding practices among the Chinese in Ireland.

Methods

A sequential explanatory mixed methods approach was adopted. The first phase was a cross-sectional self-administered retrospective mailed survey, to explore breastfeeding practices …

Deriving Statistical Inference From The Application Of Artificial Neural Networks To Clinical Metabolomics Data, Kevin M. Mendez

Theses: Doctorates and Masters

Metabolomics data are complex with a high degree of multicollinearity. As such, multivariate linear projection methods, such as partial least squares discriminant analysis (PLS-DA) have become standard. Non-linear projections methods, typified by Artificial Neural Networks (ANNs) may be more appropriate to model potential nonlinear latent covariance; however, they are not widely used due to difficulty in deriving statistical inference, and thus biological interpretation. Herein, we illustrate the utility of ANNs for clinical metabolomics using publicly available data sets and develop an open framework for deriving and visualising statistical inference from ANNs equivalent to standard PLS-DA methods.

Analysis Of The Impact Of Professional Learning Teams On Teaching Practices And Student Achievement In Biology Using Dufour’S Model, Rowena K. Dawkins

Doctor of Education Dissertations

This mixed-methods study addresses the perceived impact of working in Professional Learning Teams (PLTs) on teaching practices and student achievement in biology. The study replicates Roberts’s (2010) study. Success in biology is important to the nation because it aligns with national efforts to prepare students to compete in global markets. Educators use PLTs to support development of educators and address student educational needs. PLTs share basic functions. Basic functions were defined using DuFour’s (2004) three big ideas of PLCs: ensuring students learn at high levels, promoting a collaborative culture, and focusing on academic results. Biology teachers completed an anonymous online …

First Report Of Molecular Characterization And Phylogeny Of Trichuris Fossor Hall, 1916 (Nematoda: Trichuridae), Malorri R. Hughes, Deborah A. Duffield, Dana K. Howe, Dee Denver

Biology Faculty Publications and Presentations

Because species of Trichuris are morphologically similar and ranges of host preference are variable, using molecular data to evaluate species delineations is essential for properly quantifying biodiversity of and relationships within Trichuridae. Trichuris fossor has been reported from Thomomys spp. (Rodentia: Geomyidae, ‘pocket gophers’) hosts based on morphological features alone. Partial 18S rRNA sequences for specimens identified as T. fossor based on morphology, along with sequences from 26 additional taxa, were used for a phylogenetic analysis. Evolutionary histories were constructed using maximum likelihood and Bayesian inference. In both analyses, the specimens fell within the Trichuris clade with 100% support and …

Reemergence Of Guadalupe Fur Seals In The U.S. Pacific Northwest: The Epidemiology Of Tranding Events During 2005–2016, Erin D'Agnese, Dyanna M. Lambourn, James Rice, Debbie Duffield, Jessica L. Huggins, Terry Spraker, Stephen A. Raverty, Tetiana Kuzmina, Micheal E. Grigg, Kristin Wilkinson, Multiple Additional Authors

Biology Faculty Publications and Presentations

Guadalupe fur seals (GFS), Arctocephalus philippii townsendi, an U.S. Endangered Species Act threatened pinniped, have recently reappeared in their historic range along the western seaboard of the continental United States. Starting 2005 through 2016, 169 GFSs stranded in Washington and Oregon, involving two designated unusual mortality events. The circumstances surrounding GFS strandings, mortality, and their increased presence in Oregon and Washington were analyzed during this study. Detailed necropsies, histopathology (n = 93), and epidemiological analysis found three main causes of death (COD): emaciation (44%), trauma (29%), and infectious disease (19%) and the factors associated with overall strandings and …

Crossing Boundaries: Steps Toward Measuring Undergraduates' Interdisciplinary Science Understanding, Brie Tripp, Sophia A. Voronoff, Erin Shortlidge

Biology Faculty Publications and Presentations

A desired outcome of education reform efforts is for undergraduates to effectively integrate knowledge across disciplines in order to evaluate and address real-world issues. Yet there are few assessments designed to measure if and how students think interdisciplinarily. Here, a sample of science faculty were surveyed to understand how they currently assess students’ interdisciplinary science understanding. Results indicate that individual writing-intensive activities are the most frequently used assessment type (69%). To understand how writing assignments can accurately assess students’ ability to think interdisciplinarily, we used a preexisting rubric, designed to measure social science students’ interdisciplinary understanding, to assess writing assignments …

Metabolic And Cardiovascular Marker Alterations During Critical Training In Wildland Firefighters, Shae Gurney

Graduate Student Theses, Dissertations, & Professional Papers

Introduction: Wildland firefighters (WLFFs) are confronted with numerous physical and mental stressors. Pre-fire season includes an intense 2-week critical training (CT) period; a preparatory phase of multiple activities that can result in injury, illness, and rhabdomyolysis. The purpose of this study was to identify physiologic changes in metabolic, inflammatory, and oxidative stress biomarkers during 2 weeks of CT in WLFFs.

Methods: Eighteen male (29.4 ± 1.1 yr, 182.1 ± 1.6 cm) and three female (26.7  2.6 yr, 169.5  4.2 cm) participants were recruited from a Type I interagency hotshot crew and monitored over their 2-week CT. Fitness was …