Explorations the Journal of Undergraduate Research and Creative Activities for the State of North Carolina

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Volume X 2015 Explorations The Journal of Undergraduate Research and Creative Activities for the State of North Carolina

http://uncw.edu/csurf/Explorations/explorations.html

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Center for the Support of Undergraduate Research and Fellowships UNCW Honors College Randall Library, room 2007 University of North Carolina Wilmington Wilmington, NC 28403 copyright © 2015 by the University of North Carolina Wilmington

Cover photographs: Beach: Logan P. Prochaska Piedmont: Logan P. Prochaska Mountain: Logan P. Prochaska

ISBN: 978-0-9908-932-2-6

Original Design by The Publishing Laboratory Department of Creative Writing University of North Carolina Wilmington 601 South College Road Wilmington, North Carolina 28403 www.uncw.edu/writers

Produced by Center for the Support of Undergraduate Research and Fellowships Honors College University of North Carolina Wilmington 601 South College Road Wilmington, North Carolina 28403 http://uncw.edu/csurf/Explorations/explorations.html Staff

Editor-in-Chief Katherine E. Bruce, PhD Director, Honors College and Center for the Support of Undergraduate Research and Fellowships Professor of Pyschology University of North Carolina Wilmington

Visual Arts Editor Edward Irvine, MPD Associate Professor of Studio Art University of North Carolina Wilmington

Consultant Jennifer Horan, PhD Associate Professor of Political Science Associate Director, Honors College University of North Carolina Wilmington

Copy Editor Catharine Nealley Psychology Graduate Student CSURF Graduate Assistant University of North Carolina Wilmington

Layout Designer Logan P. Prochaska Assistant Editor Pre-Communication Studies Major University of North Carolina Wilmington 2015 Board of Reviewers

Acchia Albury, PhD Glen Harris, PhD Assistant Professor Associate Professor Department of Biology Department of History Wingate University University of North Carolina Wilmington Catherine Bush, PhD Jennifer Horan, PhD Adjunct Assistant Professor Associate Professor Department of Biology Department of Public and International Elon University Affairs Associate Director, Honors College Tuan Cao, PhD University of North Carolina Wilmington Lecturer Department of Biological Sciences Courtney Johnson, MFA University of North Carolina at Charlotte Assistant Professor Department of Art and Art History Michael Deckard, PhD University of North Carolina Wilmington Assistant Professor Department of Philosophy Rosalie Kern, PhD Lenoir–Rhyne University Associate Professor Department of Psychology Wade Dudley, PhD Pfeiffer University Teaching Associate Professor Department of History Melanie Lee-Brown, PhD East Carolina University Associate Professor Department of Biology Elizabeth Fournier, PhD Director of Undergraduate Research & Professor Creative Endeavors Department of History, Government and Guilford College Public Policy Saint Augustine’s University Yaqin Li, PhD Assistant Professor Donald Furst, MFA Department of History and Political Professor Science Department of Art and Art History Meredith College University of North Carolina Wilmington James I. Martin, Sr., PhD Stephanie George, PhD Associate Professor Assistant Professor Department of History College of Engineering Campbell University East Carolina University John Myers, PhD Orestes Gooden, MAS Associate Professor emeritus Lecturer Department of Art and Art History Aviation Science Program Honors College Elizabeth City State University University of North Carolina Wilmington Kate Nooner, PhD With special thanks and appreciation to the Associate Professor faculty mentors and volunteer reviewers. Department of Psychology University of North Carolina Wilmington Joseph Pawlik, PhD Professor Department of Biology and Marine Biology University of North Carolina Wilmington Charles V. Reed, PhD Assistant Professor Department of History Elizabeth City State University Mark Smith, PhD Professor Department of Psychology Davidson College Jaclyn Stanke, PhD Associate Professor Department of History Campbell University Ceiléssia Williams, PhD Assistant Professor Department of Natural and Physical Sciences Saint Augustine’s University Shan Yan, PhD Assistant Professor Department of Biology University of North Carolina at Charlotte Taek You, PhD Associate Professor Department of Biological Sciences Campbell University

Table of Contents

1 Letter from the Editor Humanities 5 Without Due Process: Lynching in North Carolina 1880-1900 Sarah Burke, East Carolina University Creative Work 18 Weathered Aleah Michelle Howard, University of North Carolina at Chapel Hill Biological Sciences

22 To Fly or Not to Fly? An Observation of the Effects of Simulated Microgravity on the Flight Muscles of the House Cricket, Acheta domesticus Kaitlyn C. Leonard, Wingate University

28 Altered Immune Function in Space: Implications of a Gravity Sensitive Cytoskeleton Sejiro Littleton and Dylan Ludwick, Davidson College

46 Together is Better: The Importance of Heterotrophy and Photoautotrophic Symbiosis for Growth of the Sea Anemone Aiptasia pallida Jack Cushman Koch, University of North Carolina Wilmington Creative Work 62 Drowning Courtney Hockett, University of North Carolina at Pembroke Engineering

68 Miniaturizing Photoplethysmography for Use in a Multifunctional Health Monitoring Device with Applications for Asthma Analysis Brinnae Bent, North Carolina State University Creative Work 76 A Self-Fulfilling Prophecy Jack Twiddy, University of North Carolina at Chapel Hill Social Sciences

80 Sensory Sensitivities of Young Adults with High-Functioning Autism Spectrum Disorders Kendyl L. Cole, Meredith College

92 Rapid Acquisition of Preference in Concurrent Chains: Effects of Morphine on Sensitivity to Reinforcement Amount William D. Fetzner, University of North Carolina Wilmington 112 About the Student Authors

114 About the Faculty Mentors

116 Submission Process Letter from the Editor

e are pleased to present volume me to move Explorations to UNCW in time WX of Explorations, the Journal of to produce the 2009 volume, and we have Undergraduate Research and Creative now published seven volumes at UNCW. At Activities for the State of North Carolina. UNCW we are fortunate to have a very suc- For the fourth year, we have both scholarly cessful Publishing Laboratory that devel- articles and creative works. Again this year, oped the current look of Explorations, plus I appreciate the assistance of Ned Irvine as I have a great team of students who have Visual Arts Editor. His contributions and ex- done the layout for recent issues. This year, pertise are invaluable. I am indebted to Ms. Logan Prochaska, our associate editor and layout designer and This year we include seven papers and a member of the UNCW Honors College three creative pieces, selected from 19 sub- Media Board; she has done an amazing job missions from students at eleven different with layout and keeping me on schedule. I colleges or universities in North Carolina. also thank Jennifer Horan, Peggy Styes and Topics range from North Carolina history Morgan Alexander for day-to-day support to engineering, from symbiotic relation- in the Honors College office. ships to effects of space on physiological functioning, from autism to impulsive be- I am also very appreciative of the effort havior. Our creative works include several that the 25 ad hoc reviewers spent provid- different media, as well as an accompanying timely and thoughtful reviews of the ing flash fiction piece. Eight different pub- submissions this summer. These are the lic and private schools are represented. folks who ensure the continued quality of Explorations. They offer very positive con- It is useful each year to provide a bit structive feedback to the student authors. of background about Explorations. I can hardly believe the journal has been a re- So in the spirit of the excitement that ality for ten years! In 2005, the State of fresh approaches to research and dis- North Carolina Undergraduate Research covery bring, we offer you volume X of and Creativity Symposium, affectionately Explorations. known as SNCURCS (pronounced “Snick- ers”) was first held, and educators from all colleges, universities, community colleges, and high schools were invited to participate. Now in its 11th year, SNCURCS offers a venue for undergraduates to present their Katherine Bruce, PhD research and creative efforts. Explorations was the brainchild of the 2005 meeting, and we are thankful that Michael Bassman and East Carolina University organized and published the first three volumes. In 2008, the late George Barthalamus, former Di- rector of Undergraduate Research at NCSU and the visionary behind our state-wide undergraduate research efforts, convinced

1 2 Humanities

Sarah Burke

Without Due Process: Lynching in North Carolina 1880-1900

Sarah Burke East Carolina University Faculty Mentor: Wade Dudley East Carolina University

ABSTRACT In North Carolina, the last two decades of the nineteenth century were marked by significant sociopolitical changes. Attempts for improvement in areas like education and agriculture were challenged by the never-ending struggle for power between the state’s various political parties. As North Carolinians attempted to reconcile traditional values with the shifting political climate, lynching emerged as a brutally visible symbol of the tumultuous internal struggle. Between 1880 and 1900, North Carolina recorded fifty-eight lynchings. Of the victims, forty- five were black and thirteen were white. Although race certainly played a significant role in lynchings, it was not the only deciding factor. Many North Carolinians were not only skeptical of the state’s legal system, but also deeply vested in the ideas of self-governance, honor, and communal justice. Coupled with the rising popularity of white supremacists in the 1896 and 1898 elections, the final years of the nineteenth century were marked as some of the most violent in North Carolina’s history. n an 1893 speech, North Carolina Governor of equal protection under law and advancing IElias Carr declared “the South…the best social institutions, was far more fiction than governed, the most contented, the least dis- fact. The real South of the late nineteenth turbed and in truth the most prosperous and century was much harsher. Antebellum racial inviting section of this country.” As the first attitudes hardened, rather than softened, fol- president of the North Carolina Farmers’ lowing emancipation. The Civil War brought Alliance and an advocate for progressive an end to slavery, but it did not bring an end policy, Carr spent his term as governor work- to cultural dogmas created by the institution. ing to improve education and transportation The period between Reconstruction and within the state. He published several articles the introduction of de jure segregation is of- highlighting the “Resources & Advantages of ten eclipsed by the volatile nature of the war North Carolina,” and genuinely believed his years and the sweeping impact of Jim Crow state was among the most forward-looking laws in the twentieth century. To be sure, the in the South.1 In some ways, it was. Under introduction of the Thirteenth, Fourteenth, Carr’s administration, public school facili- and Fifteenth amendments were landmark ties were available for both races; tobacco 1 Elias Carr Papers (#160), East Carolina Manuscript Collection, J. and textile mills brought industrialization Y. Joyner Library, East Carolina University, Greenville, North Carolina, to the region; and in 1894, more than one USA. 2 North Carolina State Conference of the NAACP v. Patrick Lloyd thousand black men were elected to public McCrory, Civil Action No. 1:13-cv-658, U.S. District Court for the Middle 2 District of North Carolina. Expert Report of James L. Leloudis II, Ph.D., offices. But the South Elias Carr imagined “Fragile Democracy: Race and Voting Rights in North Carolina, 1860- in his speeches did not exist. His South, one 2013.” http://b.3cdn.net/advancement/c42ea59118ecaaa39c_ybm623yv9. pdf

5 Explorations |Humanities events. The same can be said for the rapid examination of the rise of extralegal violence disenfranchisement of African-Americans in the post-Civil War years include the war and the stringent tightening of segregation itself, the culture of militaristic justice cre- standards across the South at the start of the ated by the conflict, and the mistreatment twentieth century. Yet the twenty-three-year of blacks during slavery.5 In recent decades, gap between the Civil War and the introduc- historical analysis of lynchings has expanded tion of legal segregation is critically important to include those lynchings that were not ra- for understanding the quickly changed socio- cially motivated. As Waldrep points out in his political status of African-Americans. In less study of how African Americans confronted than one quarter of a century, black men went the reality of lynching, “white people have from holding office to barred from political quite often lynched other white people, we participation of any kind. There was no faster cannot say that lynching only means racial downhill slide of African-Americans’ rights violence.”6 than that found in the South. Across the re- Perhaps more than any other state in the gion, the shifting political climate coincided South, North Carolina offers diverse exam- with a staggering rise in extraordinary acts of ples of lynching cases involving both black vigilantism and extralegal violence. Simply and white victims. North Carolina did not by virtue of its sheer brutality, lynching be- lead the nation in terms of the number indi- came the defining symbol of race relations in vidually lynched – in fact, North Carolina the post-slavery, pre-Jim Crow South. trailed behind nearly every other state in the Much has been written about lynching, its region. The Chicago Tribune began tallying role as a form of vigilante social justice, and national lynchings in 1882. The 1886 report its notorious place in American history as a credited North Carolina with five lynchings distinguishing characteristic of the former – thirty-eight less than Texas.7 The African- Confederate states. But lynching cannot be American activist Ida B. Wells conducted singled out as a solely southern phenomenon. a similar study of lynching in 1893. North In 1879, the New York Times offered a justifi- Carolina ranked 13 out of 26 for the num- cation for racially motivated violence. Blacks ber of recorded lynchings in 1892. With 5 in “were overeager for their rights, walking 1892, the Tar Heel state had 24 fewer lynch- around with chips on their shoulders, while ings than Louisiana, the state with the most whites felt powerless and outnumbered.”3 during that year.8 Despite the seemingly low Yet, although it was not a solely southern number, lynching was a prominent issue be- event, lynchings in states like Mississippi, tween 1880 and 1900 in North Carolina. Both Alabama, Georgia and North Carolina were blacks and whites were lynched, and public far more frequent, and often far more brutal, reaction was well documented. The act was than those occurring in the North. Historians sensationalized, and though never fully ac- like W. Fitzhugh Brundage and Christopher cepted by Tar Heel society, the act provoked Waldrep point towards the long-valued ideas incredible responses by supporters and op- of southern honor, self-governance, and composition alike. As North Carolina continued munal justice as the reason for the preva- to progress in the closing years of the nine- lence of lynching in the southern states.4 teenth century, lynching became the reac- Other factors commonly referenced in an tionary mark of a deeply rooted, regressive

3 As quoted in Christopher Waldrep, African Americans Confront 6 Waldrep, p. xiii. Lynching: Strategies of Resistance from the Civil War to the Civil Rights 7 Yuri Han and Deborah L. Shelton, “A Laundry List of Lynchings,” Era (Lanham, MD: Rowman & Littlefield Publishers, Inc., 2009), p. 17. Chicago Reporter, February 12, 2015. http://chicagoreporter.com/a- 4 Waldrep, p. 1; W. Fitzhugh Brundage, “’Introduction’ in Under laundry-list-of-lynchings/ (accessed August 6, 2015). Sentence of Death: Lynching in the South, ed. W. Fitzhugh Brundage, 8 Ida B. Wells, “Lynch Law,” in The Reason Why the Colored Ameri- 1-20 (Chapel Hill: University of North Carolina Press, 1997), 3. can is Not in the World’s Columbian Exposition: The African American’s 5 Claude Clegg III, Troubled Ground: A Tale of Murder, Lynching, Contribution to Columbian Literature, ed. Ida B. Wells (1893). http:// and Reckoning in the New South (Chicago: University of Illinois Press, digital.library.upenn.edu/women/wells/exposition/exposition.html#IV 2010), 25. (accessed August 6, 2015).

6 Sarah Burke struggle between traditional values and shift- left the storeowner “literally riddled” with ing period politics. buckshot.12 Cox died instantly. Frazier was In North Carolina, as in other states in detained immediately after the shooting by the South, white lynchings were not nearly Cox’s brother and tied to a nearby mill to as common as black lynchings. Between await transportation to the county jail. In the 1880 and 1900, fifty-eight individuals were early hours before dawn on the morning of lynched. Forty-five of the victims were black May 6, a mob of masked men slipped past – the other thirteen were white.9 Although watchmen, “took possession of Frazier,” and the difference between forty-five and thirteen dragged him “near the exact spot where he seems great, the fact remains that out of ap- stood when he fired the first shot at hisin- proximately every four lynchings, one victim nocent victim. Here a volley of gun and pis- was white. The role of race cannot be dis- tol shots was emptied into his body, instantly missed in any examination of lynching, but killing and mutilating him almost beyond in North Carolina, such numbers suggest the recognition; and then the lynchers dispersed, motivations for lynching ran much deeper having done their work in much less time than than skin color. it does to tell the story.”13 Three days later, The primary drivers behind white-on- the local newspaper published a detailed ac- white lynchings are deeply rooted in the count of the incident, describing Frazier as a same individualistic, self-governing, honor “desperate character” who had confessed to driven mentality Waldrep references in his “several other murders.” The lynchers were study of African-American resistance to mob praised for “adroitly evading” Frazier’s tem- violence.10 A lynching, especially when the porary guard, and the community of Blount’s victim was white, exposed a legitimate fear Creek was left “greatly excited” by the entire among the populace that the legal system ordeal.14 would fail them. Because the death penalty Frazier’s death was the first in a series of was an integral part of North Carolina law, white lynchings between 1888 and 1894. and had been since the colonial period, white During that six-year period, nine other white lynchings reflected a genuine belief that the men were lynched. All were alleged mur- state’s judicial system was soft on crime and derers. Like Frazier, most of the men were slow to dispense justice. Fearful of delayed lynched before they had the opportunity to judgment, many North Carolinians chose to stand before a judge. The due process guar- opt for a quicker solution.11 anteed by the Fifth Amendment was largely The lynching case of Thomas Frazier in ignored. Such disregard was deeply reflec- May of 1885 fits well within typical parame- tive of North Carolinians’ discontent with ters for white-on-white lynchings. On the af- the state’s legal system. Perpetrators of an ternoon of May 5, 1888, Thomas Frazier shot earlier 1887 lynching in Tarboro summarized and killed Joshua A. Cox of Blount’s Creek, their feelings on North Carolina’s criminal North Carolina. Frazier entered Cox’s store laws in a note attached to the victim’s body: and demanded liquor. Cox refused. Angered, We hang this man not in passion, but Frazier “seized a gun, loaded with buckshot, calmly and deliberately with a due sense of from the hands of one of his friends standing responsibility we assume. We take executive near,” and opened fire on Cox. The shooting power in this case and hang this man because

9 Vann Newkirk, Lynching in North Carolina: A History 1865-1941 12 “A Murderer Lynched,” Chatham Record, May 10, 1888. http:// (Jefferson, NC: McFarland & Company, 2009), 167-169. newspapers.digitalnc.org/lccn/sn85042115/1888-05-10/ed-1/seq-2/# 10 Waldrep, p. 1. (accessed August 6, 2015). 11 Patrick Huber, “Caught Up in the Violent Whirlwind of Lynching: 13 Walter L. Daniels Papers (#96), East Carolina Manuscript Collec- The 1885 Quadruple Lynching in Chatham County, North Carolina,” The tion, J. Y. Joyner Library, East Carolina University, Greenville, North North Carolina Historical Review 75 (April 1998): 135. Carolina, USA. 14 “A Murderer Lynched,” Chatham Record.

7 Explorations | Humanities the written law provided no adequate penalty overpowered the jailer and forced him to re- to the crime. And be it understood, we have lease Harriet Finch, Jerry Finch, Lee Tyson, done this act and will repeat it under similar and John Pattishall from their cells. They circumstance. 15 were dragged one mile outside of Pittsboro For white lynching victims in North and found the “next morning hanging to a Carolina, it was simple enough to acknowl- tree near a public road.”18 Spectators of the edge that their capital offenses were met with lynching recalled later that Jerry Finch and an extralegal sort of capital punishment. For Tyson were hanged first. Ten minutes passed black victims of lynching, the crimes com- before the men succumbed to death. Pattishall mitted were not always so severe. Between was more fortunate. When a lyncher jerked 1880 and 1900, no white man was lynched for Pattishall’s rope, his neck snapped and killed an offense other than murder. Furthermore, him instantly. Harriet Finch’s arms and legs there is no record of a white woman ever were bound before she was hanged, and the being attacked by a mob and strung from a act was later interpreted as merciful on the tree to die. The same cannot be said for black part of the lynchers. Like her husband, she lynching victims. In North Carolina, black too struggled against the noose for several lynchings recognized no gender restrictions minutes before dying.19 The Roanoke News and no crime requirements. Though less called the event “a terrible sequel to the triple likely to be lynched than black men, black murder.”20 No one was ever convicted for the women often fell victim to being guilty by as- crime. sociation.16 And unlike white victims, blacks Henry London, editor of the local newspa- were lynched both for serious offenses and per, The Chatham Record, penned a scathing for minor infractions, including political ac- condemnation of the lynching in the follow- tivity and breaking the unspoken social code ing week’s edition. London felt the lynch- of behavior. In the autumn of 1885, Chatham ing was “the most horrible…that [had] ever County residents demonstrated the astonish- disgraced the State of North Carolina.”21 His ing brutality of coed lynching events with a editorial provided a detailed account of the quadruple hanging of four black residents lynching and focused heavily on the public’s – three male and one female. Various news- reaction to the event: paper accounts questioned the motivations This terrible tragedy is to be deeply de- behind the quadruple lynching. Unlike other plored, and we are pleased to know that it is lynchings, the four victims had been impris- condemned by every person of whom we have oned for months. John Pattishall, a young heard speak of it. The Record has so often and black tenant farmer, was awaiting trial for so strongly condemned lynch law…. All good an 1883 double murder of two local white citizens regret it now, and we doubt not that women. The three other accused, Lee Tyson, most of the misguided men who participated Harriet Finch, and Jerry Finch had already in the lynching will themselves soon regret been found guilty of a triple axe murder on it…. the lynching of a woman was especially September 7, 1885. A date was set during the horrible, and we doubt not that her piercing court’s next term for sentencing. None of the screams and piteous appeals for mercy…will accused lived to face the judge.17 often be recalled with a shudder of remorse On the night of September 29, three weeks by those who so cruelly put her to death.22 after the guilty verdict, twenty masked men

15 “A Negro Lynched,” Raleigh State Chronicle, May 12, 1887.7 18 State News,” Roanoke News, October 8, 1885. http://newspapers. 16 Patrick Huber, “Caught Up in the Violent Whirlwind of Lynching: digitalnc.org/lccn/sn84026524/1885-10-08/ed-1/seq-2.pdf (accessed The 1885 Quadruple Lynching in Chatham County, North Carolina,” The August 6, 2015). North Carolina Historical Review 75 (1998): 135-160. http://www.jstor. 19 Huber, p. 155. org/stable/23522619 (accessed March 1, 2015), p. 155. 20 “State News,” Roanoke News. 17 Chatham Record, September 10, 1885. 21 “Lynch Law!,” Chatham Record, October 1, 1885, http://newspapers.digitalnc.org/lccn/sn85042115/1885-10-01/ed-1/seq-1. pdf (accessed April 2, 2015). 22 Ibid.

8 Sarah Burke

In some ways, the 1885 Chatham County society, white women were sheltered and quadruple murder was a deviation from typi- fiercely guarded. Protection of their inno- cal North Carolina lynchings. Unlike most cence was directly linked to the honor of their victims, black or white, the victims had been menfolk; and in culture divided by race, their arrested and successfully provided, at least to white skin became a symbol of purity. Their some extent, their right to due process under virtue was starkly contrasted with the sullied the Fifth Amendment. For most lynching vic- darkness of black men. Since the early days tims, such was not the case. Black individuals of the Trans-Atlantic slave trade, black men specifically were often denied basic constitu- had been portrayed as overly sexualized.27 tional rights. Like Harriet Finch, who was tied Depictions placed them on the same level as only to the murders by a pair of her husband’s livestock, and white women were taught to be trousers, most blacks were arrested with little cautious of their proximity. or no proof to support their involvement in a In terms of lynch-worthy crimes, of- crime.23 fenses against women were second only to On the morning of June 9, 1899, the tiny murder. The National Association for the village of Bogue, North Carolina was rattled Advancement of Colored People published when local white merchant Elijah B. Weeks an examination of lynching in 1919. The re- was found dead in his store. Officials deter- port, titled “Thirty Years of Lynching in the mined Weeks died from strangulation, and United States, 1889-1913,” included statistics less than twenty-four hours later, a young on the offenses that appeared to cause lynch- black man was arrested and charged with ings. Of all black lynching victims during murder. When Lewis Patrick was detained, the 30-year period (2,522 individuals), 28.4 a lone piece of pork in his possession be- percent were lynched for rape or other “at- came the evidence needed to corroborate his tacks upon women.”28 Although rape is cer- guilt.24 He was taken to the county jail in tainly not a slight offense, most of the alleged Beaufort to await trial. On the night of June transgressions were flimsily supported. Often, 14, Patrick was taken from his cell and shot. evidence was no stronger than “entering the Local newspapers reported that Patrick “im- room of a woman or brushing against her.”29 plicated other parties” in an effort to save Hearsay generally provided enough proof to himself, “but the lynching party thought he justify lynching a black man accused of as- was lying and gave no weight to his words.”25 saulting a white woman. More than one week passed before Patrick’s The NAACP’s publication was a national body was “found by the roadside, riddled with report, but in North Carolina, as in the rest of bullets.”26 Members of the lynch mob from the country, untoward behavior in the pres- Bogue were never caught. ence of a white woman was a primary cause In today’s legal system, a slice of pork for many lynchings. The South may have lost would hardly be incriminating enough to sen- the war, but antebellum values had not been tence an individual to death. Yet in the late surrendered at Appomattox. During the 1896 nineteenth century, some blacks were lynched and 1898 election cycles, Democrats in the without any evidence to confirm their role in Tar Heel state fabricated an “imminent” rape an alleged crime. This was especially true in threat that capitalized on traditional values cases involving black men and white women. and fears. In an 1897 editorial piece printed In the patriarchal structure of Southern on the front page of the Goldsboro Headlight,

23 Huber, 155 27 Donald Matthews, “The Southern Rite of Human Sacrifice: 24 Vann Newkirk, Lynching in North Carolina: A History 1865-1941 Lynching in the American South,” Mississippi Quarterly: The Journal of (Jefferson, NC: McFarland & Company, 2009), p. 162. Southern Cultures 61 (2008): p. 40-41. 25 “A Nation’s Doings: The News From Everywhere Gathered and 28 National Association for the Advancement of Colored People, Condensed,” The Goldsboro Headlight, June 22 1899. http://newspapers. Thirty Years of Lynching in the United States, 1889-1913.(New York: digitalnc.org/lccn/sn91068337/1899-06-22/ed-1/seq-1.pdf (accessed Negro Universities Press, 1969), p. 10. August 6, 2015). 29 Ibid. 26 State News,” County Union, June 21, 1899, http://newspapers.digitalnc.org/lccn/sn91068061/1899-06-21/ed-1/ (accessed April 2, 2015).

9 Explorations |Humanities

W.C. Brann decried the depravity of black struggle between the state’s three political men: parties – the Republicans, the Populists, and The fact that a negro has been hanged, or the Democrats. even burned for ravishing a white woman, When Elias Carr ran for governor in 1892, makes others fearful, but it also suggests to he was the last in a string of plantation-based their foul minds the crime itself. To a negro leaders. His personal politics reflected the a white woman is as Dian [sic] to Satyr or Populist platform, and his primary goal was Athena to old Silenus. That one of these su- to improve the plight of North Carolina farm- perior creatures has actually been enjoyed ers. Unlike the Democrats running for office by a lustful black sets them all adreaming in the 1896 and 1898 elections, Carr often and makes them dangerous. A white woman collaborated with black leaders, like Baptist is found unprotected; all visions of the rope minister Walter Patillo, to bolster the success and the stake vanish, drowned in the hell of of the NC Colored Farmers Alliance.31 desire and Judge Lynch claims another vic- The 1896 election cycle brought a new col- tim…. Have we the moral right to apply such laboration to North Carolina politics, when a drastic remedy? I answer yes – that we the Republican and Populist parties aligned would be amply justified in slaughtering ev- to defeat the Democrats. This fusion of po- ery Ethiop on the earth to preserve unsullied litical parties was a great success. Together, the honor of one Caucasian home.30 the two groups swept the 1896 election. For the first time since Reconstruction, North Such deeply rooted social fears, however Carolina did not elect a Democrat for gov- ill founded, were instrumental in the ernor. More than 1,000 black men entered introduction of legalized segregation in the office, and black voter turnout peaked at 87 last decade of the nineteenth century. Brann’s percent.32 The Fusionist legislature contin- dream of eradicating all blacks from the earth ued to push for improvement of education was unrealistic – but the turn of the century and charitable institutions, as well as focus- witnessed blacks pushed even further to the ing heavily on agriculture in the state. More fringe of North Carolina society. As in all importantly, Fusionist politicians strove to southern states, the North Carolina govern- protect black and poor white voters from ment fought a fierce battle to ensure blacks discrimination or disenfranchisement at the lost all political rights during the 1890s. The polls. Emphasis was placed on prioritizing introduction of new limitations slowed lynch- the localness of elections, especially those ings, but not before racial violence reached a deciding county offices.33 fever pitch in 1898. If lynching was a reaction by whites to Between 1890 and 1900, North Carolina their perceived loss of power during the post- reported 21 lynchings – 15 fewer than the war years, then the introduction of Jim Crow previous decade. The decrease was largely legislation and the rise of the Democrats dur- due to strengthened legal sanctions against ing the last four years of the nineteenth cen- blacks and a spike in the influence of white tury marked a significant restoration of white supremacist politics. The sociopolitical power. Unwilling to succumb to the obscen- changes that came to define North Carolina in ity of a biracial government, the Democratic the last years of the nineteenth century were Party, notably Furnifold Simmons, Josephus the result of an aggressive, and often violent Daniels, and Charles Aycock, launched

30 W.C. Brann, “The Rape Fiend Remedy,” Goldsboro Headlight, 31 Nikki Brown and Barry Stentiford, eds., The Jim Crow Encyclope- September 2, 1897. dia: Greenwood Milestones in African American History (Westport, CT: Greenwood Press, 2008), Google Books, http://books.google.com/bo oks?id=oLjYbzkGWk8C&lpg=PP1&pg=PR4#v=onepage&q&f=false (accessed April 9, 2015). 32 North Carolina State Conference of the NAACP v. Patrick Lloyd McCrory 33 Ibid.

10 Sarah Burke a campaign to topple Fusionist politics. blacks fell victim to night raids and mass Guided by harsh white supremacist values, intimidation. People came by the thousands the Democratic Party flooded the state with to attend political rallies and witness the racist propaganda prior to the 1898 election. “rough-and-tumble” lawless men dressed in Daniels, the editor of the Raleigh News & crisp red shirts and mounted on horseback.36 Observer, printed cartoons depicting blacks Some four hundred Red Shirts gathered as flying satanic monkeys. Phrases like at a rally near Laurinburg in November of “Negro Rule” and “Negro Domination” were 1898, forming a “procession nearly a mile splashed across editions of the newspaper.34 long.”37 By day, the Red Shirts were truly Paired with other inflammatory advertising, a sight to behold. They exuded strength and the Democratic Party struck fear in both dominance, and no person could question the the white and black communities of North hold white supremacy had over the state. Yet Carolina. As the chair of the Democratic by night, they terrorized black settlements Party in the state, Simmons spoke frequently with “staged torchlight processions” and on the dangers of a politically active black whippings.38 populace, and often invited fellow white North Carolina’s outgoing governor, supremacists to speak at political rallies. Daniel Russell, condemned their actions. He In November of 1898, Dr. J.D. Hufham, a feared that the deliberate agitation of racial Baptist reverend, penned an opening prayer tensions would ignite full-scale riots, and for the State Democratic Convention. It en- went so far as to contact President McKinley capsulated the core belief of the party: “for to ask for the support of federal troops.39 the good of both races, white men and white News of Russell’s worries only further en- men alone must rule in North Carolina.”35 couraged the Democrats. Although the elec- Rhetoric was enough to garner white sup- tion was a landslide for the Democratic Party, port, and enough to deter some blacks from insecurities persisted – and on November 10, voting. Yet for the Democrats to be trium- 1898, the standoff between white suprema- phant in the 1898 election, black voters cists and the rest of North Carolina society had to be barred from the polls. Legislation flared violently in Wilmington. would later be passed to disenfranchise black Two days prior, on Election Day, “perfect voters, but Democrats quickly found more peace seemed to pervade” in the Port City. effective methods to deter political participa- The governor assured the city’s voters that so tion. White supremacists formed paramilitary long as black voters “cast their ballots qui- groups aimed at terrorizing and intimidating etly” and returned home, there would be no black voters – essentially lynch mobs without great disturbance.40 Unfortunately, the gov- the ropes. Known as Red Shirts, the groups ernor’s promise held for less than twenty-four were most active in counties with large black hours. The following night, Alex Manly, the populations. Lynching was not a common- proprietor and editor of the state’s only daily place tactic for Red Shirt groups, but they African-American newspaper, became the were notorious for violence. Republicans, target of a gang of angry white men. Some Fusionists, and especially politically active months prior, according to whom “a climax

34 The North Carolina Experience: An Interpretive and Documentary 36 H. Leon Prather, “The Red Shirt Movement in North Carolina: History, ed. Lindley Butler and Alan Watson (Chapel Hill: University of 1898-1900,” The Journal of Negro History 62, no. 2 (1977), http://www. North Carolina Press, 1984), 333. jstor.org/stable/2717177 (accessed April 21, 2015), 178. 35 Chatham Record, November 3, 1898. 37 Ibid. 38 Ibid. 39 Charles David Phillips, “Exploring Relations among Forms of So- cial Control: The Lynching and Execution of Blacks in North Carolina, 1889-1918,” Law & Society Review 21 (1987), http://www.jstor.org/ stable/3053375, 368. 40 J. Allen Kirk, “A Statement of Facts Concerning the Bloody Riot in Wilmington, N.C. of Interest to Every Citizen of the United States. (Wilmington, NC: 1898), p. 3. http://docsouth.unc.edu/nc/kirk/kirk.html (accessed August 6, 2015).

11 Explorations |Humanities

[had been] reached when the Negro paper government. Additionally, Jim Crow laws, published an article so vile and slanderous which restricted black rights and segregated that it would in most communities have re- the races, were well established by Charles sulted in the lynching of the editor.”41 The Aycock’s term as governor. In 1896, the city’s white population had not forgotten his Supreme Court case of Plessy v. Ferguson social misstep. Manly’s office was set afire, upheld state laws requiring separation of the and though demands were made for Manly’s races in public facilities. North Carolina’s capture, he managed to evade the mob and transportation facilities were segregated by slip from the city. 42 1899. The state legislature even went so far The mob, which numbered more than 500 as to bar black medical students from dissect- Democrats by morning, paraded through the ing white cadavers.44 Such legislation slowed streets, sending “fear and terror to the hearts lynchings by the early years of the twentieth of the Negroes in the City of Wilmington.” century. Yet even though whites appeared When night fell, the firing began. Kirk satisfied with their regained power, the “old reported: demon” of lynching occasionally reared its The shrieks and screams of children, of ugly head and reminded North Carolina’s mothers, of wives were heard, such as caused blacks that they were always just one misstep the blood of the most inhuman person to away from the mob’s noose.45 creep. Thousands of women, children and It would be remiss to say all North men rushed to the swamps and there lay upon Carolinians supported mob violence, vigi- the earth in the cold to freeze and starve. lantism, and lynching in the closing decades The woods were filled with colored people. of the nineteenth century. Although most The streets were dotted with their dead bod- citizens certainly did not favor integration ies. A white gentleman said that he saw ten or racial equality, most communities were bodies lying in the undertakers office at one appalled by the unrestrained brutality of ex- time. Some of their bodies were left lying in tralegal justice. Many newspapers followed the streets until up in the next day following the pattern of Henry London, editor of the the riot. Some were found by the stench and Goldsboro Headlight, and openly criticized miasma that came forth from their decaying lynchings. Even staunch white supremacists bodies under their houses.43 and politicians were tiring of lynch law by Most black residents never returned to the 1890s. In an 1891 edition of the Wilson Wilmington, permanently changing the Advance, a man who believed all blacks city’s sociopolitical dynamics. The mas- should be sent to Africa expressed his frustra- sacre of the city’s blacks was reflective of tion with lynching. The act, he said, did “not the spectacle-lynchings elsewhere in the reform or intimidate.”46 Instead, the act only state. After the riot, Democrats scaled back served to further exacerbate relations, stir intimidation tactics. They adopted legisla- tension, and incite more violence. In 1895, tive means of social control instead of vio- state representative J.D. McCall introduced lent strategies. Poll taxes, literacy tests, and a bill to prevent lynching to the House. The the grandfather clause prevented blacks from act called for the governor to “thoroughly participating politically and ensured whites investigate the circumstances” and ascertain remained in control of the North Carolina “whether or not the sheriff or…other officers

41 Ibid., p. 6. 44 Clegg, 32. 42 “Jim Crow Stories: Wilmington Riot 1898,” The Rise and Fall of 45 Clegg, 24. Jim Crow, Public Broadcasting Service, http://www.pbs.org/wnet/jim- 46 “Bill Arp’s Letter: The Rising Boy Watching the Struggles of crow/stories_events_riot.html (accessed April 17, 2015).. Genius,” Wilson Advance, October 15, 1891. 43 J. Allen Kirk, p. 9-10. 47 “To Prevent Lynching,” The Charlotte Democrat, January 25, 1895. http://chroniclingamerica.loc.gov/lccn/sn91068247/1895-01-25/ed-1/ seq-3.pdf (accessed August 6, 2015). 48 North Carolina General Assembly, House of Representatives, Jour- nal of the House of Representatives of the General Assembly of the State of North Carolina at its Session of 1895.http://digital.ncdcr.gov/cdm/ compoundobject/collection/p249901coll22/id/516749/rec/2 (accessed August 6, 2015).

12 Sarah Burke of the law…have done their duty as required and criminal lines. As traditional values and by law to prevent such lynching.”47 The bill shifting politics jostled for dominance at the was met with an “unfavorable recommenda- dawn of a new century, lynching became the tion” by a House committee. 48 most brutally visible symbol of a tumultuous Given the horrendous status of race re- internal struggle. Elias Carr’s claim that “the lations in other southern states – Georgia laws of the state are enacted and enforced lynched 423 people to North Carolina’s 75 impartially, without distinction as to class during a 50 year period – Tar Heels could or color,” was contradicted by nearly every be loosely classified as moderates.49 Yet in lynching that occurred in the last two decades a society defined by generational traditions of the nineteenth century.51 Most lynching of white superiority and black inferiority, victims, black and white, were already “poor, there was no easy solution to curtail mob- illiterate, and marginalized” by society. They style justice. White men who chose to par- died in “virtual anonymity,” and newspa- ticipate in lynching did so because they felt per accounts of their deaths would refer to it was their southern-born rite. The act came them only as a “Negro” or by their alleged to symbolize “elemental justice, a necessary, crimes.52 Future generations would strive to even moral, act in a drama of punishment that humanize such an inhumane act by match- portrayed good and evil in a way that could ing names to events, but at the close of the sacralize white supremacy….”50 nineteenth century, North Carolinians tragi- In North Carolina, the justifications for cally swapped due process for a “due sense social sanctioning crossed gender, racial, of responsibility.”53

49 Donald Matthews, “The Southern Rite of Human Sacrifice: Lynch- ing in the American South,” The Mississippi Quarterly 61 (2008), 32.. 50 Ibid. 51 Elias Carr Papers. 52 Huber, 141. 53 “A Negro Lynched,” Raleigh State Chronicle, May 12, 1887.

13 Explorations |Humanities

References

Primary Sources

“Bill Arp’s Letter: The Rising Boy Watching the Struggles of Genius.” Wilson Advance, October 15, 1891.

Brann, W.C. “The Rape Fiend Remedy.” Goldsboro Headlight. September 2, 1897.

Chatham Record. November 3, 1898. http://newspapers.digitalnc.org/lccn/sn85042115/1898-11-03/ed-1/ (accessed August 6, 2015).

Chatham Record. September 10, 1885. http://newspapers.digitalnc.org/lccn/sn85042115/1885-09-10/ed-1/ (accessed August 6, 2015).

Elias Carr Papers (#160), East Carolina Manuscript Collection, J. Y. Joyner Library, East Carolina University, Greenville, North Carolina, USA.

Ida B. Wells. “’Lynch Law.” In The Reason Why the Colored American is Not in the World’s Columbian Exposition: The African American’s Contribution to Columbian Literature, edited by Ida. B Wells, 1893. http://digital.library.upenn.edu/women/ wells/exposition/exposition.html#IV (accessed August 6, 2015).

Kirk, Allen J. “A Statement of Facts Concerning the Bloody Riot in Wilmington N.C. of Interest to Every Citizen of the United States. (Wilmington, NC: 1898). http://docsouth.unc.edu/nc/kirk/kirk.html (accessed August 6, 2015).

“A Murderer Lynched,” Chatham Record, May 10, 1888. http://newspapers.digitalnc.org/lccn/sn85042115/1888-05-10/ed-1/seq-2/# (accessed August 6, 2015).

“A Nation’s Doings: The News From Everywhere Gathered and Condensed.” The Goldsboro Headlight, June 22, 1899. http://newspapers.digitalnc.org/lccn/sn91068337/1899-06-22/ed-1/seq-1.pdf (ac cessed August 6, 2015).

“A Negro Lynched.” Raleigh State Chronicle. May 12, 1887.

North Carolina General Assembly, House of Representatives. Journal of the House of Representatives of the General Assembly of the State of North Carolina at its Session of 1895. http://digital.ncdcr.gov/cdm/compoundobject/collection/p249901coll22/id/5 16749/rec/2 (accessed August 6, 2015).

“State News.” County Union. June 21, 1899. http://newspapers.digitalnc.org/lccn/sn91068061/1899-06-21/ed-1/ (accessed April 2, 2015)

14 Sarah Burke

“State News.” Roanoke News. October 8, 1885. http://newspapers.digitalnc.org/lccn/sn84026524/1885-10-08/ed-1/seq-2.pdf (ac cessed August 6, 2015).

“To Prevent Lynching.” The Charlotte Democrat, January 25, 1895. http://chroniclingamerica.loc.gov/lccn/sn91068247/1895-01-25/ed-1/seq-3.pdf (ac cessed August 6, 2015).

Walter L. Daniels Papers (#96), East Carolina Manuscript Collection, J. Y. Joyner Library, East Carolina University, Greenville, North Carolina, USA.

Secondary Sources

Clegg, Claude A. III. Troubled Ground: A Tale of Murder, Lynching, and Reckoning in the New South. Chicago: University of Illinois Press, 2010.

Han, Yuri and Deborah L. Shelton. “A Laundry List of Lynchings.” Chicago Reporter, February 12, 2015. http://chicagoreporter.com/a-laundry-list-of-lynchings/ (accessed August 6, 2015).

Huber, Patrick. “Caught Up in the Violent Whirlwind of Lynching: The 1885 Quadruple Lynching in Chatham County, North Carolina.” The North Carolina Historical Review 75 (1998): 135-160. http://www.jstor.org/stable/23522619 (accessed March 1, 2015).

The Jim Crow Encyclopedia: Greenwood Milestones in African American History. Edited by Nikki Brown and Barry Stentiford. Westport, CT: Greenwood Press, 2005. Google Books.http://books.google.com/books?id=oLjYbzkGWk8C&lpg=PP1&pg=P R4#v=onepage&q&f=false (accessed April 9, 2015).

“Jim Crow Stories: Wilmington Riot 1898,” The Rise and Fall of Jim Crow. Public Broadcasting Service. http://www.pbs.org/wnet/jimcrow/stories_events_riot.html (accessed April 17, 2015).

Matthews, Donald. “The Southern Rite of Human Sacrifice: Lynching in the American South.” The Mississippi Quarterly 61 (2008): 20-47. http://search.proquest.com. jproxy.lib.ecu.edu/docview/233501926?accountid=10639 (accessed March 1, 2015).

Newkirk, Vann. Lynching in North Carolina: A History 1865-1941. Jefferson, NC: McFarland and Company, 2008.

The North Carolina Experience: An Interpretive and Documentary History. Edited by Lindley S. Butler and Alan D. Watson. Chapel Hill: University of North Carolina Press, 1984.

North Carolina State Conference of the NAACP v. Patrick Lloyd McCrory, Civil Action No. 1:13-cv-658, U.S. District Court for the Middle District of North Carolina. Expert Report of James L. Leloudis II, Ph.D., “Fragile Democracy: Race and Voting

15 Explorations |Humanities

Rights in North Carolina, 1860-2013.” http://b.3cdn.net/advancement/c42ea59118e caaa39c_ybm623yv9.pdf

Phillips, Charles. “Exploring Relations among Forms of Social Control: The Lynching and Execution of Blacks in North Carolina, 1889-1918.” Law & Society Review 21 (1987): 361-374: http://www.jstor.org/stable/3053375 (accessed March 1, 2015).

Prather, H. “The Red Shirt Movement in North Carolina: 1898-1900.” The Journal of Negro History 62 (1977): 174-184. http://www.jstor.org/stable/2717177 (accessed April 21, 2015), 178.

Under Sentence of Death: Lynching in the South. Edited by W. Fitzhugh Brundage. Chapel Hill: University of North Carolina Press, 1997.

Waldrep, Christopher. African Americans Confront Lynching: Strategies of Resistance from the Civil War to the Civil Rights Era. Lanham, MD: Rowman & Littlefield Publishers, Inc., 2009.

16 17 Explorations | Creative Work

Creative Work: Weathered

Artist: Aleah Michelle Howell Faculty Mentor: Beth Grabowski Affiliation: University of North Carolina at Chapel Hill Dimensions: 9” x 14” Medium: Cyanotype print

ARTIST STATEMENT: This piece is one in a series of prints and photographs called “Walking.” On my walks, I notice beautiful, found compositions that are created through my awareness and the repetition of walking. Line and texture create composition. I focused on the abstract beauty of roads and the way nature and man-made materials can combine into one. The walks started as being informed only by sight. With frottage techniques, I recorded the cracked pavement and other objects I passed with my hands. The blue of cyanotype printing can be abrasive, but I believe it only intensifies this sense of touch in my prints. Exploring age, decay, and memory, these prints represent a moment in time as well as the history of an object.

18 Aleah Michelle Howell

Weathered, Aleah Michelle Howell, 2015

19 20 Biological Sciences Explorations |Biological Sciences To Fly or Not to Fly? An Observation of the Effects of Simulated Microgravity on the Flight Muscles of the House Cricket, Acheta domesticus Kaitlyn C. Leonard Wingate University Faculty Mentor: Acchia N. J. Albury Wingate University ABSTRACT Microgravity is an environmental factor known to induce muscular atrophy. Muscular atrophy is characterized by a degradation of muscle proteins and the loss of muscle mass. In the house cricket, Acheta domesticus, the flight muscles degrade naturally through an endocrine regulated process known as flight muscle histolysis. Flight muscle histolysis in the house cricket begins on Day 2 and is complete by Day 3. Environmental factors have been known to alter the normal time course of histolysis; therefore, we proposed to investigate the effects of simulated microgravity on flight muscle histolysis within the house cricket. Results indicated that during simulated microgravity there was a significant loss in muscle mass and enzymatic function by Day 1. Therefore, these findings suggest that under simulated microgravity conditions, the time course of flight muscle histolysis in the house cricket, Acheta domesticus deviates from the normal course observed within the control group and as reported by Oliver et al. (2007).

echanical stimuli, such as gravitational Exposure to microgravity results in sub- Mforce, are essential for the normal de- stantial changes in muscle structure and velopment and maintenance of cellular struc- function, including muscle atrophy, altered ture and function (Benavides Damm et al., muscle phenotype and reduced metabolic 2013). Microgravity is defined as less than capacity. Muscle atrophy is characterized 1-G, which is comparable to the force of grav- by significant loss in muscle mass, protein ity experienced on Earth, but less than zero-G content, fiber diameter, force production and that is associated with complete weightless- fatigue resistance (Fitts et al., 2000; Jackman ness (Freed and Vunjak-Novakovic, 2002). and Kandarian, 2004). During actual and Studies have shown that microgravity may simulated microgravity, muscle atrophy directly or indirectly lead to alterations in is due to an imbalance between the rate of cellular morphology, metabolism and func- protein synthesis and degradation. Studies tion of tissues within an organism (Freed and have provided consistent evidence that pro- Vunjak-Novakovic, 2002). Gravitational grammed cell death (PCD) pathways are ac- force is essential for the development and tivated or accelerated during skeletal muscle normal function of skeletal muscles; hence, atrophy induced by microgravity (Kandarian the neuromuscular system is directly im- and Jackman, 2006), which may account for pacted during actual (space flight) and simu- the increase in protein degradation. lated (grounded-based models) microgravity The disruption of the neuromuscular sys- (Di Prampero and Narici 2003; Benavides tem during microgravity has continued to Damm et al., 2013). be studied with an emphasis placed on the

22 Kaitlyn Leonard alterations/adaptations of the skeletal mus- isolated they were designated as Day 0 cles, especially muscle atrophy. Due to their (Oliver et al., 2007). structural similarities, several model systems, including rodents and crustaceans have been Treatment Groups: Adult crickets were proposed as alternatives to large mammalian maintained in smaller colonies by age, Day 1 models. Hence, we proposed to investigate being 24 hours after adult emergence. Day 2, the effects of simulated microgravity on the 3 and 4 were 48, 72 and 96 hours respectively house cricket, Acheta domesticus as model to after adult emergence. Crickets designated study the impact of microgravity on muscles as controls were maintained in small colonies predestined to die. The house cricket exhib- and reared under the ideal conditions de- its age-dependent flight muscle histolysis, scribed above. Clinorotation was the method which is induced by juvenile hormone and is utilized to simulate microgravity within the as an example of PCD (Oliver et al., 2007). experimental group. Clinorotation has been Flight muscle histolysis occurs within the first used as an effective method to simulate mi- few days after adult emergence and accounts crogravity, as it eliminates the directional for the loss of flight in winged-adults. The cues associated with gravity; therefore, al- normal time course of flight muscle histolysis tering the organism’s perception of gravity has been established; it begins 48 hours after (Di Prampero and Narici, 2003). A benchtop adult emergence (Day 2) and is complete by clinostat was used to stimulate microgravity Day 3 (Oliver et al., 2007). (simulated microgravity is measured at <30 Despite being an endocrine regulated event rpm; the benchtop clinostat was recorded at initiated by juvenile hormone, studies have 17 rpm). Day 0 crickets were placed in 50 indicated that environmental factors such as mL conical tubes with Fluker’s Orange Cube climate, photoperiod and availability of re- Complete Cricket food (Fluker Farms, Port sources may disrupt the normal time course Allen, LA) to supply both food and water. of histolysis (Edwards, 1969). The influence Crickets were subjected to clinorotation for of environmental factors on the time course 24, 48, 72 or 96 hours and then removed of histolysis suggests that under micrograv- to obtain treatments for the respective ages ity conditions deviations from the normal (Day 1, 2, 3 and 4). time course of histolysis may be observed. Therefore, we postulate that the normal time Lactate Dehydrogenase (LDH) Assay: course of flight muscle histolysis in the house LDH activity was used to assess whether the cricket, A. domesticus may be altered during muscle tissue was functional. Frozen DLMs simulated microgravity. were homogenized in potassium phosphate buffer (50 mM; pH 7.5); homogenates were METHODS separated by centrifugation and supernatant retained. Total protein concentration was Insects: Juvenile crickets were obtained determined for paired DLMs by the bicin- from the commercial breeder (Armstrong choninic acid method (BCA Assay; Pierce, Cricket Farm, Glenville, GA). Crickets were Rockport, IL). LDH activity was measured reared at 30°C with a 14:10 (light:dark) pho- for 10 µg total protein from DLMs (n = 4) us- toperiod. Crickets were provided with dis- ing β-NADH as described by Bergmeyer et al. tilled water and fed Fluker’s High Calcium (1965). The reaction mixture (total volume Cricket Food (Fluker Farms, Port Allen, LA). 1 mL) contained 100 µL of NADH (1.2 mg/ Crickets were maintained in a large colony mL), 800 µL of potassium phosphate buffer and adult crickets were isolated upon adult and 100 µL of sample (10 µg of protein from emergence; adult female crickets were re- DLM homogenates). LDH activity was initi- tained for experimentation and male organ- ated by adding 25 µL sodium pyruvate (0.35 isms were discarded. Once the adults were mg/mL) to 250 µL of the reaction mixture.

23 Explorations |Biological Sciences

The oxidation rate of the NADH was mea- the control group aligns with the normal pat- sured at 340 nm at 1 minute intervals for 16 tern of histolysis (see Figure 2). There was minutes by the PowerWave 340i plate reader no significant loss in muscle mass from Day (Bio-Tek Instruments, Inc., Winooski, VT). 0 to Day 1 (p = 0.817). There was a 27% loss One unit of enzymatic activity is defined as in muscle mass from Day 1 to Day 2 (p = the amount of enzyme which changes the 0.011), and a 21% loss in muscle mass from O.D. of NADH, by 0.001 in 1 minute. Day 2 to Day 3 (p = 0.010). There was no significant loss in muscle mass from Day 3 to Tissue Wet Weight Measurements: Oliver Day 4 (p = 0.799). et al., (2007) described that there was a loss in muscle mass associated with flight muscle An ANOVA confirmed differences in mus- histolysis; hence, we collected wet weight cles loss for the experimental group [F (4, measurements to obtain the mass of the dor- 40) = 20.228, p<.0001]. The experimental solongitudinal muscles (DLMs) to determine group exhibited a different pattern of muscle histolysis. Crickets were anesthetized on ice mass loss compared to the control group (see prior to experimental manipulation. DLMs Figure 2). There was a 23% loss in muscle were excised from crickets by ventral dis- mass from Day 0 to Day 1 (p = 0.001). No section. Paired muscles were rinsed with significant loss in muscle mass was observed cricket ringer (154 mM NaCl, 8 mM KCl, from Day 1 to Day 2 (p = 0.099), Day 2 to 8 mM CaCl, 4 mM MgCl2) and blotted dry Day 3 (p = 0.149), or Day 3 to Day 4 (p = to remove adherent fat body. Muscle mass 0.169). was measured for paired DLMs (n = 9) using an analytical balance (Sartorius, Elk Grove, IL) and reported in mg/paired DLMs. DLMs CONCLUSIONS were then frozen and stored at -50°C for biochemical assays. Flight muscle histolysis is an endocrine regulated event, which results in the degrada- Data Analysis: Results were reported tion of the flight muscles (Oliver et al., 2007). as means ± SE. Data were analyzed using Studies have indicated that the normal time ANOVA and SNK test in XL Stat, a compo- course of histolysis may be interrupted nent of Excel. Statistical significance was by environmental and/or external factors determined if p < 0.05. (Edwards, 1969). Our results demonstrate that under simulated microgravity conditions RESULTS the normal time course of flight muscle histolysis in the house cricket, A. domesticus LDH Assay: Preliminary data collected appears to be accelerated. show that the control and experimental Microgravity has been shown to induce groups experienced a decline in enzymatic muscle atrophy in healthy skeletal muscles activity from Day 1 to Day 3 (see Figure 1). not predestined to die by activating and ac- The LDH levels exhibited by Day 1 experi- celerating PCD pathways (Kandarian and mental were similar to the levels exhibited by Jackman, 2006). Day 1 muscles subjected to Day 3 control. simulated microgravity (experimental group) expressed similar levels of LDH activity as Time Course of Histolysis: An ANOVA Day 3 control, muscles that had undergone confirmed differences in muscles loss for the flight muscle histolysis. These preliminary control group [F (4, 40) = 15.81, p<.0001]. results led to the examination of the time Flight muscle histolysis is characterized by course of flight muscle histolysis under simu- a significant loss in muscle mass by Day3 lated microgravity conditions. During flight (Oliver et al., 2007). The results obtained for muscle histolysis there was a 27% loss in

24 Kaitlyn Leonard

LDH Ac'vity 60

protein) 20

LDH Ac'vity (Units of ac'vity/10µg LDH Ac'vity C -‐ 1 C -‐ 3 E -‐ 1 E -‐ 3 Treatments

Figure 1: LDH Assay Results; means are presented.

Loss in Muscle Mass 6.000

5.000

4.000

3.000 Control

2.000 Experimental

1.000 Muscle Mass (mg/paired DLMs) (mg/paired Muscle Mass

0.000 Day 0 Day 1 Day 2 Day 3 Day 4 Day (Age)

Figure 2: DLM Wet Weights of the Experimental and Control Groups from Day 0 to Day 4; means and standard errors are presented.

25 Explorations |Biological Sciences muscle mass from Day 1 to Day 2, indicating to gain insight on how environmental pres- the initiation of PCD. There was a 21% loss sures may impact the underlying mechanisms in muscle mass from Day 2 to Day 3 with of muscular degenerative diseases. no significant loss in muscle mass after Day 3 indicating that the process of flight muscle FUTURE WORK histolysis was complete by Day 3. These findings coincide with the Oliver et al. (2007) Previous studies have identified the loss of study. However, during simulated micro- muscle mass as an indicator of flight muscle gravity there was a 23% loss in muscle mass histolysis in the house cricket, A. domes- from Day 0 to Day 1, with no subsequent loss ticus. However, we plan to follow through in muscle mass thereafter. These results sug- with biochemical analyses to confirm flight gest that flight muscle histolysis is complete muscle histolysis. We are currently conduct- by Day 1 under simulated microgravity con- ing acid phosphatase assays to measure lytic ditions. The results from the LDH assay indi- activity. In addition, flight muscle histolysis cate that there was a 72% difference in LDH is an endocrine regulated event; therefore, activity between the Day 1 control group and evaluating the relationship between the envi- Day 1 experimental group. ronmental stress of microgravity and the po- In summary, flight muscle histolysis in the tential impact on hormone regulation during house cricket, Acheta domesticus is an exam- flight muscle histolysis may prove essential ple of endocrine regulated PCD that begins to better understand the alterations seen in on Day 2 and is complete by Day 3 and ac- the time course of histolysis. counts for the loss of flight in winged-adults. Hence, this system can be an excellent model ACKNOWLEDGMENTS to study the impact of microgravity on muscles predestined to die. During simulated mi- We would like express our gratitude to crogravity, there is significant loss in muscle Wingate University for the support of our mass and decline of enzymatic activity that project through the Creative Investigative occurs on Day 1, with no significant reduc- Partnership Symposium (CIPS). We would tion thereafter. These findings suggest that also like to offer special thanks to Dr. Rush the normal time course of flight muscle his- Oliver (Benedict College) for all of his sup- tolysis in the house cricket is altered during port and expertise, as well as Dr. Debra simulated microgravity. While the findings Davis for her contributions throughout the of this research provide insight on the effects duration of our project. Finally, we would of simulated microgravity on flight muscle like to thank Brooklyn Boston, Maria Ortiz, histolysis, the implications are farther reach- Samantha Bourn and Walter Loza who were ing. The house cricket may prove to be an extremely instrumental in animal care and ideal model system to study the impact of en- data collection. vironmental pressures such as microgravity

26 Kaitlyn Leonard

REFERENCES

Bergmeyer, H., Bernt, E. and Hess, B. Lactic Dehydrogenase. In: Methods of Enzymatic Analysis. New York: Academic Press 1965; 736 – 743.

Benavides Damm, T., Richard, S., Tanner, S., Wyss, F., Egli, M. and Franco-Obregón, A. Calcium-dependent deceleration of the cell cycle in muscle cells by simulated micro gravity. The FASEB J. 2013; 27: 2045 – 2054.

Di Prampero, P. and Narici, M. Muscles in Microgravity: from fiber to human motion. J. Biomech 2003; 36(3): 403 – 412.

Edwards, F. Environmental control of flight muscle histolysis in the bugDysdercus interme dius. J. Insect Physiol 1969; 15(1): 2013 – 2020.

Fitts, R., Riley, D. and Widrick J. Physiology of a microgravity environment invited review: Microgravity and skeletal muscle. J. Appl Physiol 2000; 89: 823 – 839.

Freed, L. and Vunjak-Novakovic, G. Spaceflight bioreactor studies of cells and tissues. Adv Space Biol Med 2002; 8: 177 – 195. Review.

Jackman, R. and Kandarian, S. The molecular basis of skeletal muscle atrophy. Am J Physiol. 2004; 287: C834 – C843.

Kandarian, S. and Jackman, R. Intracellular signaling during skeletal muscle atrophy. Muscle Nerve 2006; 33: 155 – 165.

Oliver, R., Albury, A. and Mousseau, T. Programmed cell death in flight muscle histolysis of the house cricket. J Insect Physiol 2007; 53: 30 – 39.

27 Explorations |Biological Sciences

Altered Immune Function in Space: Implications of a Gravity Sensitive Cytoskeleton

Sejiro Littleton and Dylan Ludwick Davidson College Faculty Mentor: Sophia Sarafova Davidson College

ABSTRACT Future plans in space exploration have outlined missions to both asteroids and planets such as Mars. Extended spaceflight has long been associated with immunosuppressive effects. A growing body of evidence suggests that microgravity plays an important role in astronaut immune dysfunction through its interactions with the cytoskeleton. This review summarizes the current understanding of the effects of microgravity on both adaptive and innate immunity, while dis- cussing the implications of cytoskeletal alterations in the search of a primary mechanism for gravity sensing. We conclude by addressing the need for more thorough investigation in order to fully understand the long-term consequences of cellular adaptation to low gravity conditions during extended spaceflight.

ince the beginning of time humans have Muscle atrophy and loss of bone density are Sgazed unto the heavens with curiosity among the topics best known by the public and awe. In ancient times the stars guided and scientists. However, serious concerns farmers to the seasons and sailors across have been raised about impaired eyesight, oceans. More recently, we have been inspired psychological and physical stress, ionizing to take the first steps in exploration of the fi- radiation, as well as latent viral reactivation nal frontier. We have sent a rover to Mars, and alterations to the immune system. The landed the Philae probe on an asteroid, and first immunologically relevant concerns were for the past 15 years, through international documented by the Apollo missions, which cooperation, have maintained the orbital re- reported symptoms of “infectious illness” in search lab known as the International Space 50% - 60% of crewmembers (1). To mini- Station (ISS). mize this health risk NASA implemented The technical limitations of launch, navi- the Health Stabilization Program starting gation and safe landing are not the only with the Apollo 14 mission. This program obstacles for placing humans in orbit or on significantly reduced the frequency of symp- celestial bodies. Research on space-faring hu- toms by enforcing light quarantine on crew- mans, cell cultures, and model organisms has members prior to launch. A summary of post shown that there are a great number of physi- flight debriefs in 106 shuttle missions reports ological changes that accompany this event. 29 incidents of infectious disease out of 742

28 Sejiro Littleton and Dylan Ludwick crewmembers. Among those reported were knowledge for application in treating disease fever and chills, fungal infections, flu-like on Earth. symptoms, urinary tract infections, and viral diseases (1). Despite improvements, consis- ADAPTIVE IMMUNITY tent reports of infection still remain, suggesting that there might be a separate effect of T Lymphocytes space flight directly on immune function. If humankind is to conduct further missions The inhibition of T lymphocyte activation and extended manned flights such as those by microgravity is a widely documented phe- required to place a human on Mars, we must nomenon dating back to the 1970s as initial understand the changes and risks imposed by observations from US and Soviet space mis- microgravity and extended spaceflight. The sions noted a reduced response to mitogen focus of this paper is on the changes seen stimulation in the T cells of crew members in the immune system under these circum- upon returning to Earth (2). Subsequent ex- stances. We begin by laying out the relevant perimentation over the next two decades in changes found in the T and B lymphocytes of Spacelab D1, SLS-1 (Space Launch System), the adaptive immune system. We move on to and IML-2 (International Microgravity discuss the innate immune system and altera- Laboratory) would go on to independently tions that occur in Natural Killer (NK) cells, confirm the consistent and substantial disrup- the third lymphocyte lineage, and monocytes tion of T cell activation in microgravity with and macrophages of the myeloid lineage. 1g-onboard centrifuge controls (3, 4, 5). The mechanism by which microgravity The non-responsiveness of T lymphocytes alters immune cell function remains unclear. may be attributed to disruption at one or We hypothesize that the cytoskeleton and its more of the 3 signals involved in full activa- associated proteins are gravity sensors of the tion. The first of these, signal 1, is initiated cell, and changes to their regulation induced through the specific binding of the T Cell by microgravity disrupt processes such as Receptor (TCR) with a unique antigenic pep- motility, signaling, gene expression, and cell tide presented on a Major Histocompatibility proliferation. Given that cells of the immune Complex (MHC) molecule by an Antigen system use their cytoskeleton to undergo cell Presenting Cell (APC). Signal 2, also known division during proliferation, to alter their as costimulation, involves the triggering of shape and migrate more than any other cell in CD28 on the T cell by CD80/86 on the APC. the body through the processes of extravasa- Signal 3 is a cytokine signal usually deliv- tion, and to differentiate in response to vari- ered by Interleukin-2 (IL-2) in an autocrine ous environmental stimuli, it stands to reason fashion, binding to IL-2 Receptor (IL-2R) that the loss of Earth’s gravity could lead to on the cell surface of T cells. If a cell should dysfunction in the innate and adaptive im- receive only signal 1 through its TCR, in mune responses. other words there is no costimulation through Understanding these changes and the signals 2 or 3, the cell will be rendered non- mechanisms which lead to altered immune responsive to antigen. This process is known function will be important to define the as anergy and it plays an important role in health risk of missions to Mars, other manned maintaining tolerance to self-antigen (6). missions seeking to explore our solar system, However, when the signaling cascades initi- and any prolonged time spent inhabiting an ated by signals 2 and 3 are disrupted it can altered gravity environment such as the ISS. lead to tolerance to pathogens, which leaves Additionally, this knowledge will bring a the host immunocompromised. greater understanding to the possibility and Because both signals 1 and 2 involve cell- nature of life on other planets, the structure cell interaction, it was important to con- and role of the cytoskeleton, and impart new firm these processes were functional under

29 Explorations |Biological Sciences microgravity conditions. In a series of experi- displayed significant reduction in CD69 ex- ments performed onboard MASER flights 3 pression, and unchanged production of IFN-γ. and 4, as well as the MAXUS 2 flight, the Both groups showed a decrease in IL-2 pro- binding of mitogen Con A to glycoproteins duction, as well as a decrease in INF- γ:IL-10 on the cell membrane was used as a mimic ratio, suggesting a detectable T-helper type 2 of signal 1 in order to assess the effects of (Th2) cytokine shift (11). microgravity on T cell functionality (7, 8, 3). To start revealing the molecular mecha- Although the patching of Con A glycopro- nism behind the dysregulation of T cell teins after binding is significantly inhibited function, experiments on board the ISS in- in microgravity in comparison to 1g-onboard vestigated patterns of early gene expression and ground centrifuge controls, researchers between microgravity exposed T cells and 1g concluded that the overall influence in such controls after 1.5 and 4 hours of stimulation a rapid process was small, and unlikely to ac- with Con A and anti-CD28 (12). Researchers count for the documented large scale inhibi- found 47 significantly down-regulated genes tion of T cell activation (9). In another exper- in the microgravity group at the 1.5 hour iment human peripheral blood lymphocytes, mark, including down-regulated key imme- which include both T cells and APCs, formed diate early genes and their targets, such as progressively larger aggregate clusters 12-78 transcription factors Rel/NF-kB, CREB, and hours after stimulation with Con A indicating Serum Response Factor (SRF). This confirms that physical contact between cells can occur earlier reports of NF-kB and CREB pathway under microgravity conditions (10). dysregulation, which was attributed to down- Experiments modeling microgravity in the regulation of protein kinase A but not protein Random Positioning Machine (RPM) have kinase C (13). The transcription of cREL and found a significant dysfunction in the tran- its gene targets were significantly reduced as scription of IL-2 and IL-2R alpha genes in well, and downstream Tumor Necrosis Factor lymphocytes stimulated with Con A (9). This (TNF) effector functions were identified as confirms previous reports of decreased IL-2 particularly gravity sensitive after genetic and IL-2R in peripheral blood lymphocytes pathway analysis (12). Induction of NF-kB is exposed to Con A in real microgravity on a result of the costimulatory signals 2 and 3, board Spacelab SLS-1 (4). As IL-2R and IL-2 and in the absence of this signaling cascade production are the primary targets of signal the cell can become anergic. Therefore, the 1 and 2 signaling cascades, their decrease alterations observed in the gene expression of could indicate incomplete or inefficient func- T cells activated in microgravity is concern- tion of signals 1 and 2. Regardless of the ing because it might lead to immunosuppres- cause-and-effect relationship, the outcome is sion in astronauts. CREB plays an important interference with signal 3 followed by T cell role in the production of IL-2, IFN-γ, and the inactivation as a result of gravity unloading. induction of T cell proliferation (14). The Comparisons of peripheral leukocyte sub- dysregulation of this pathway might there- sets in crewmembers after short and long fore explain the reduced IL-2 levels and the duration spaceflight missions have been per- Th2 cytokine shift observed in previous ex- formed, revealing time spent in microgravity periments. Even with a transcriptionally per- to be an important factor in the analysis of missive methylation of the IFN-γ promoter, immune adaptation to spaceflight (11). CD69 if CREB activity is reduced the production cell surface expression, a marker of early T of IFN-γ might not be sufficient to promote a cell activation, was elevated after stimulation Th1 response, resulting in a cytokine shift and with mitogens in short duration shuttle crew- immunosuppression (15). Analysis at 4 hours members after landing, however production identified 98 other genes down-regulated in of Interferon-gamma (IFN-γ) was decreased. microgravity, illustrating the interaction be- In contrast, long duration ISS crewmembers tween primary and secondary microgravity

30 Sejiro Littleton and Dylan Ludwick

effects (12). Authors note in particular that Interestingly, a significant difference in mo- SRF binding sites are present in the promot- tility was also noted between controls, most ers of a majority of genes inhibited in T cells likely due to the fact that inflight 1g samples activated in microgravity (12). Given that were exposed to microgravity during ascent Ras/MAPK and Rho/actin pathways activate and transfer to centrifuge. These results re- SRF, these cytoskeleton-regulating small inforce earlier studies in J-111 monocytes GTPases could be disrupted in microgravity exposed to 24 hours simulated microgravity explaining the reduced T cell activation (16). in the RPM, which showed similar impair- If cytoskeletal regulatory elements are ments in cell motility (18). It is conceivable impaired and lead to alterations in T cell ac- that diminished motility in monocytes or tivation, then cell motility becomes an im- other APCs may be substantial enough to in- portant consideration as the cytoskeleton is terfere with proper migration into lymphoid the primary effector of this process. In vivo, organs, leading to decreased stimulation and APCs must migrate to the lymph nodes in increased susceptibility to pathogens a po- order to present antigen to T cells (signal tential source for the observed latent viral 1) and deliver a costimulatory signal 2 like- reactivation and symptoms of pathogenic wise T cells must enter lymph nodes through infection. the processes of extravasation. Should cells lack normal motility then the immune sys- B Lymphocytes tem could be impaired. In both the presence and absence of mitogen stimulation, cells in Experiments investigating the humoral re- single suspension were shown to be signifi- sponse and the effects of microgravity on the cantly more motile than 1g ground controls. B cell populations are more limited than their This may not hold true for all cell types, as T cell counterparts (see Table 1 for compari- J-111 human monocytes exposed to 24 hours son. Fortunately, antibody binding is not dis- of microgravity aboard the ISS displayed a rupted in microgravity and Enzyme-Linked substantial reduction in motility in compari- Immunosorbent Assay (ELISA) tests can be son to onboard 1g and ground controls (17). conducted aboard the ISS (19). Serum levels

TableTable 1. Summary 1. Summary of the of theRelation Relation between between Microgravity Microgravity and andthe Adaptivethe Adaptive Immune Immune System System

Cell type Effects References ● disruption of activation Konstantinova et al 1993 (2) , (long term exposure) Cogoli et al 1988 (3), Cogoli et al 1993 (4), Pipia et al 1996 (5) T lymphocytes ● increase in activation (short term exposure), Crucian et al 2008 (11) short-term decrease in INF-ɤ, decrease in INF- ɤ:IL-10 ratio

● increase in motility Cogoli-Greuter et al 1996 (10) ● decrease in transcription Cogoli-Greuter 2004 (9) of IL-2 and IL-2R alpha

● down-regulation of 47 Chang et al 2012 (12), early gene targets, Boonyaratanakronkit et al 2005 including Rel/NF-kB, (13) CREB, SRF, PKA, and cREL

B lymphocytes ● some increase in IgM and Rykova et al 2008 (21), IgA levels Konstantinova et al 1993 (2)

31 Explorations |Biological Sciences of the immunoglobulin isotypes taken from headed by Namni Goel should help to es- many astronauts suggest little clinical risk of tablish risk factors, useful biomarkers, and a a dysfunctional humoral response of the im- standard for comparison when measuring the mune system. Given no change to antibody effects of stress. These results indicate that binding in microgravity and no significant humoral immune response is not likely to be change in serum levels for each of the im- a prime risk factor for exploration class mis- munoglobulins IgG, IgM, IgA, IgD, & IgE sions, such as a Mars mission, however any the risk in short term missions lasting around unexpected complications that arise could be ten days is negligible (2, 20, 21). untreatable and place the crew and mission at It is less clear what the clinical risks are severe risk (1). for longer missions such as those lasting 125 days or longer. The trend seems to indicate INNATE IMMUNITY a maintenance of Ig within standard levels; however, cases have been documented where Natural Killer Cells cosmonauts experienced an increase in both their IgM and IgA levels during long term NK cells comprise the third lymphocyte flight (2, 21). Interestingly after 5 months lineage and play an important role in the in space, the newt, Pleurodeles waltl, one of defense of the host against viruses and intra- the few vertebrate animals to be immunized cellular bacteria (28). These cells are named in space, demonstrated a 3.5 fold increase in “natural killer” because unlike the other IgY heavy chain transcription, the equivalent cells of the lymphocyte lineage they require of human IgA (22). It is unclear whether mi- no APC for activation. NK cells are able to crogravity is the direct factor responsible for recognize ligands on the surface of infected this as the researchers were unable to con- cells, stressed cells, and those lacking self- trol for radiation and stress responses (22). peptides presented on MHC-I to quickly However, the ability to class switch to IgY identify threats and respond by lysing target is consistent with the described Th2 cytokine cells as a primary defense against invaders shift. (28). This innate defense provided by the Valeri Vladimirovich Polyakov holds NK cell allows time for the adaptive immune the current record for longest spaceflight, a response to clonally expand and respond to consecutive 438 days (23). No major com- the infection should it not be eradicated by plications due to altered immune response the innate immune response (28). With the developed during his time in space. Viral observed viral reactivation among many as- reactivation has been observed in a number tronauts and the unknown virulence of many of subjects; however, this has not led to any bacterial strains in microgravity, it becomes a significant health threat (24). It is possible priority to determine the ability of NK cells this reactivation can be attributed to stress to maintain functional cytolytic activity. induced suppression of the cell mediated Data analysis of NK cells extracted from branch of the immune system as tests con- the peripheral blood of 88 cosmonauts after ducted in the Arctic Winter Over missions returning from long term missions revealed and the Haughton Mars Project (conducted two phenotypes. The first of these phenotypes on Devon Island in the Canadian high arc- was characterized by a static or increased tic) demonstrated similar viral reactivation percentage of CD3-/CD56+/CD16+ lympho- with reduced T cell activity and function (1, cytes compared to preflight, with reduced 25, 26, 27). The Human Research Roadmap cytotoxic activity of the NK population. The tasks “Individualized Stress Detection second phenotype demonstrated significantly System” headed by Daniel Mollicone and reduced fraction of CD3-/CD56+/CD16+ “Biomarkers as Predictors of Resiliency lymphocytes but significantly higher NK cell and Susceptibility to Stress in Spaceflight” cytotoxic activity (29). A previous study on

32 Sejiro Littleton and Dylan Ludwick

ISS crewmembers and shuttle members, long specifically due to each of the factors of mi- and short duration missions respectively, crogravity, stress, and radiation. However, found a significant reduction in NK cell should the risk of reduced NK cell function numbers which persisted at least three days be sufficient for clinical intervention the ad- post flight, unfortunately no functional ac- ministration of IL-12 and IL-15 presents as tivity was measured in this experiment (11). one possible solution (31). Further studies should seek to understand the mechanism behind these two distinct pheno- Monocytes & Macrophages types. Below we discuss some in vitro studies that addressed these issues. Precursors to macrophages and dendritic Initial studies using two-dimensional (2D) cells, monocytes are large, granular, mono- clinostat and RPM seemed to indicate that nuclear cells that circulate the blood until tis- simulated microgravity had no effect on the sue damage or inflammatory signals recruit cytolytic activity of NK cells (30, 31). This them to extravasate and differentiate into ef- would seem to suggest that the primary cause fector progeny (32). Macrophages are tissue of the changes seen in human NK cells from resident and long lasting, they engulf patho- peripheral blood of astronauts is more likely gens to present processed peptides on their due to a combination of radiation and stress surfaces which can be recognized to activate responses. However, these initial studies of- the adaptive immune system (28). The gran- ten only tested incubation periods up to 24 ules present in these cells can also be used to hours. When the time was extended to 48 activate oxidative burst reactions to disrupt hours there was a significant reduction in and destroy pathogens (28). It is essential that the ability of NK cells to lyse target MHC- the processes involved in cell motility and negative K562 cells and this inhibition was signaling remain unimpaired for a functional even more significant at 72 hours (31). When innate immune system, unfortunately results human NK cells were incubated under these seem to indicate microgravity significantly same conditions, expression of IFN-γ, perfo- impairs both of these aspects of monocyte rin, and the receptors NKG2D and NKG2A and macrophage function. were significantly reduced after 48 hours Monocytes obtained from blood sam- (31). Additionally human NK cells exposed ples of healthy volunteers were activated to simulated microgravity for 48 hours were with LPS containing solution at preset time found to have an increased rate of early and points, at 1g and 0gs; however, only 31 of 96 late apoptosis in comparison with 1g and planned samples were suited for analysis due vertical rotational controls after Annexin V/ to technical failures (33). Results obtained Propidium Iodide flow cytometry (31). In indicated an increasing trend but no signifi- contrast, Granzyme-B levels and the recep- cant change in p38 MAP Kinase activity, as tors NKp30 and NKp44 were unaffected (31). determined by the differences in mean fluo- This decrease in receptor, IFN-γ and perforin rescent intensity, between experimental and levels and the increased rate of apoptosis is control conditions (33). In contrast Jun-N- indicative of a lowered activity state of the terminal kinase (JNK) activation is signifi- NK population, and might explain the reduc- cantly reduced under spaceflight micrograv- tion in cytolytic ability after incubation in ity conditions, though no changes in nuclear simulated microgravity. Further experimen- localization were noted (33). Interestingly, tation revealed IL-15 alone or in combination coactivation of p38 MAPK and JNK by in- with IL-12 can rescue this suppressed cyto- flammatory signals occurs through distinct toxicity (31). upstream kinases, MKK3/6 and MKK4/7 Despite these developments it still remains respectively which could explain the differ- unclear to what extent the changes observed ential effect of microgravity on these kinases in the NK cell population of astronauts are (33). ERK, extracellular regulated kinase,

33 Explorations |Biological Sciences was also inhibited by microgravity albeit to a transport components for Syk phosphoryla- lesser degree than JNK, this was corroborated tion and ROS production are disrupted in by results from another study which showed microgravity (38). While not a cause in the that microgravity affected phosphorylation in experiments conducted in simulated micro- Jurkat T cells (33, 34). One should note the gravity, ionizing radiation has been shown to p38 MAPK pathway is implicated in early activate NF-kB through a Poly(ADP-ribose) bacterial response whereas JNK has a role Polymerase (PARP-1) (39). in the chronic inflammatory response and An outcome of macrophage activation is the mediation of Reactive Oxygen Species the production of TNF-α and other pro-in- (ROS) to induce apoptosis and necrosis (33, flammatory cytokines, yet another function 35). These results illustrate the differential that has been reported to be altered by mi- effect of microgravity on kinases within cell crogravity. Mouse macrophages stimulated populations and raises the question of what with LPS and exposed to simulated gravity mechanism leads to these opposing effects. via Rotary Cell Culture System-1 (RCCS-1) Another important feature of macrophages for 24 hours resulted in significant depression affected by microgravity is their ability to TNF-α expression (40). Consistent with the initiate oxidative bursts in response to patho- results of Brungs et al., no effect was seen gens. The production of ROS in NR8383 rat on translocation or DNA binding of NF-kB; alveolar macrophages was shown to be sig- however, no differences were observed in nificantly affected by changes in micrograv- JNK or IKK phosphorylation. Researchers ity, specifically reduced in microgravity and did note that heat shock factor 1 was acti- enhanced in hypergravity. These changes ap- vated under these conditions and it is possible pear to be both rapid and reversible within that stress responses could offer an alternate seconds (36). Studies were conducted in explanation to the reduced TNF-α expression parabolic flight and 2D clinostats, thus de- (40). spite the significance of the rapidly revers- Results from experiments analyzing the ible changes, more studies are needed about Toll-Like Receptors (TLR) of 14 ISS mem- the long term effects of microgravity expe- bers demonstrate opposing and variable rienced during exploration class missions on trends but also implicate heat shock factors tissue resident macrophages. in immune system shifts (29). In five cosmo- The steps in generation of ROS most likely nauts the relative number TLR-expressing to be gravity sensitive are those involved in monocytes and neutrophilic granulocytes in the initial activation of a burst reaction. The peripheral blood on the 1st day after return spleen tyrosine kinase, Syk, plays an impor- from spaceflight was found to be signifi- tant role in the signaling pathway initiated by cantly increased. In the remaining nine cos- the binding of Pattern Recognition Receptors monauts the opposite trend was observed by (PRRs) leading to oxidative burst and release decreased representation of these cells. An of ROS. Phosphorylation of ITAMs by Src increase in heat shock proteins was shown family kinases and recruitment and activation to be significant on the first day of landing. of Syk relies on adaptor proteins and occurs Ligands of the TLRs 2 and 4, these proteins early in the pathway initiating oxidative burst are known to undergo significant increase in (37, 38). Because the changes to ROS pro- production and release from necrotic cells duction are rapid and reversible, and down- when an organism is exposed to biological, stream NF-kB translocation and binding to psychological or physical stressors, all of DNA in the nucleus is largely unaffected, which an astronaut is known to experience it is plausible that NF-kB can be compen- (41). This protein production could have a sated through some other signaling pathway, protective effect on the innate immune cells whereas the early signaling steps near the cell expressing the proper receptors (29). The loss surface involving integrins and cytoskeletal of bone density, thereby bone marrow and

34 Sejiro Littleton and Dylan Ludwick the production of hematopoietic cells, might THE CYTOSKELETON explain this decreases in the monocyte and neutrophil repertoire in the second group of The cytoskeleton is an active and dy- cosmonauts (29). namic intracellular network composed of Another study by Chongzen Wang further three main types of polymer: actin filaments, explored the changes induced by micrograv- microtubules, and intermediate filaments. ity in mouse macrophages by incubation with Collectively, these proteins are responsible IL-4 in a RCCS-1 for 24 hours. They noted for the structural organization of cellular induction of the p38 MAPK pathway activ- contents in space, physical and biochemical ity downstream of the transcription factor C/ connections to the external environment, as EBPβ and significantly enhanced levels of well as generating the forces and contractions arginase mRNA and protein (42). This in- required for movement and change of shape crease of MAPK activity was confirmed in (44). Important cellular processes such as monocytes as well (33). Consistent with the signal transduction, growth, gene expression, maintenance of a Th2 shift observed in other and proliferation are all influenced by cyto- experiments IL-6 production increased and skeletal organization (45). Evidence mount- IL-12B was depressed (11, 42). ing over the years suggests that the cytoskel- To determine the effect of microgravity eton plays a key role in sensing changes in on macrophage differentiation one study gravity as well (46, see Table 3, Figure 1). analyzed the transcriptional profiles of mac- In order for gravity to influence the cell, rophages derived from bone marrow of a mechanical signal must be converted to C57BL/6 mice after 14 days differentiation a chemical signal in a process known as in the presence of rmM-CSF (recombinant mechanotransduction. Mediating this con- mouse Macrophage Colony Stimulating version are mechanosensitive structures, Factor) (43). Phenotypically a significant several of which have been associated with increase of activated macrophages and in- the cytoskeleton (47). Focal adhesions, sites creased expression levels of Mac2 and c- of attachment between actin stress fibers and Fms markers were found in the space flight the cellular membrane, are known to house sample in comparison to the ground control. tension sensitive integrins and vinculin (48). Researchers interpreted this finding as an in- Cadherins prominent in cellular junctions dicator of a population of macrophages that show mechanosensitivity as well. Even actin is alternatively differentiated after spaceflight filaments themselves have been shown to be but not necessarily inhibited. Microarray tension sensitive (49). In searching for the analysis from this experiment found genes initial mechanism(s) of gravity sensing, the associated with the coagulation system, Fc- cytoskeleton and its associated elements ap- gamma receptor-mediated phagocytosis, pear to be the most likely candidates. endoplasmic reticulum stress and growth Changes in the cytoskeleton in response to hormone signaling pathways to be most sig- gravity unloading are widely reported for all nificantly reduced under the effects of micro- three types of filaments amongst many differ- gravity in comparison to the ground control. ent cell types (50, see Table 3) Human J-111 Specifically, CCR5 and the kinases mTOR, monocytes exposed to real microgravity p38 MAPK and FLT3 were significantly re- aboard the ISS showed a significant decrease duced (43). in the density of filamentous biopolymers of A summary of the innate immune system F-actin, with filaments localizing close to the and the alterations that occur under the ef- plasma membrane as opposed to being orga- fects of microgravity in NK cells, monocytes nized within the complex cytosolic network and macrophages are included in Table 2. (17). This reinforces earlier observations of identical changes seen in cells undergoing simulated microgravity in the RPM (18).

35 Explorations |Biological Sciences

TableTable 2. 2.Summary Summary of theof theRelation Relation between between Microgravity Microgravity and the and Innate the ImmuneInnate Immune System System

Cell type Effects References

Natural killer cells ● phenotypic Rykova 2013 (29) differentiation featuring both reduced and enhanced functional activity levels

● reduction in cell numbers Crucian et al 2008 (11) (long term exposure)

● decrease in expression of Li et al 2013 (31) IFN-y, perforin, and the receptors NKG2D and NKG2A

● reduction in motility Meloni et al 2011 (17), 2006 (18) Monocytes ● disruption in Jun-N- terminal kinase activation

● activation of p38 MAPK Verhaar et al 2014 (33) pathway

● rapid and reversible Adrian et al 2013 (36) reduction in ROS production

Macrophages ● decrease in Syk Brungs et al 2015 (38) phosphorylation

● depression of TNF-α Wang et al 2014 (40) expression, activation of heat shock factor 1

● Activation of p38 MAPK Wang et al 2015 (42) pathway, increased IL-6 production, decreased IL- 12B production

Actin filaments in osteoblasts experiencing 4 space flight mission, however, demonstrated days of microgravity aboard the space trans- significantly increased beta-tubulin expres- port system STS-56 shuttle flight also dem- sion during the microgravity phase, which onstrated notable disorganization, as well as a did not occur in the on-board 1g control (34). reduction in the number of stress fibers (51). Meloni’s experiment with J-111 cells in real Lewis et al. first reported disruptions of the microgravity revealed highly disorganized microtubule organizing center in nonadherent b-tubulin architecture, with thickening in a Jurkat cells within four hours of micrograv- perinuclear position which was not seen in ity (52). These changes reverted within 48 on-board 1g or ground controls as well (16). hours. Researchers noted that this effect was Additionally, self-organization of microtu- also seen in the on-board 1g control, and thus bules in vitro largely failed to occur in sam- attributed it to vibrations from launch as op- ples exposed to 13 minutes of weightlessness posed to a real microgravity effect. Human T (53). lymphocytes flown on MASER-12 suborbital Studies focusing on intermediate filaments

36 Sejiro Littleton and Dylan Ludwick

TableTable 3. Summary 3. Summary of the of Relation the Relation between between Microgravity Microgravity and the andCytoskeleton the Cytoskeleton

Cytoskeletal Effects Cell Types References Component

Actin ● decrease in density of F- J-111 monocytes Meloni et al 2011 (17), actin filaments Crucian et al 2008 (11)

● disorganization of actin Osteoblasts Hughes-Fulford and Lewis 1996 filaments (51) ● reduction in number of actin stress fibers

Tubulin ● disorganization of b- J-111 monocytes Meloni et al 2011 (17), tubulin architecture Crucian et al 2008 (11)

● increase in b-tubulin T lymphocytes Tauber et al 2013 (34) expression

Intermediate ● thickening of vinculin, J-111 monocytes Meloni et al 2011 (17), Filaments reorganized parallel to cell Crucian et al 2008 (11) membrane

● reorganization of vimentin Jurkat cells, Cogoli et al 1988 (3), into thick bundles Breast cancer cells Masiello et al 2014 (54)

● increase in integrin protein Mesenchymal stem Meyers et al 2004 (56) subunit expression cells

● increase in Fibronectin and WB-F344 cells Qu et al 2006 (57) E-cadherin mRNA levels

Functional ● disorganization of complex J-111 monocytes Meloni et al 2011 (17), Structures cytosolic network Crucian et al 2008 (11) ● reduction in number and size of focal adhesions

● reduction in number size of focal adhesions Osteoblasts Guignandon et al 1997 (55)

● reversible disruption of microtubule organizing Jurkat cells Lewis et al 1998 (52) center

● reduction in phosphorylation of focal Mesenchymal stem Meyers et al 2004 (56) adhesion-associated cells kinases are limited, however an experiment per- cytoskeletal rearrangements. formed with Jurkat cells in real microgravity Mechanosensitive structures associated aboard MAXUS 2 recorded a striking reorga- with the cytoskeleton are likely key compo- nization of the intermediate filament vimen- nents in cellular adaptation to microgravity, tin into thick bundles within 30 seconds of however more work needs to be done in order exposure, in comparison to the thin networks to fully understand the process and players seen in Earth gravity controls (3). Breast can- involved. Meloni’s J-111 monocytes dem- cer cells undergoing simulated microgravity onstrated thickening of the focal adhesion in the RPM showed similar rearrangement anchor protein vinculin in real microgravity, of vimentin (54). Interestingly, these cells di- which reorganized parallel to the cell mem- verged into two nearly equal phenotypes of brane (17). The size and number of focal ad- adherent cells and free floating cell clumps, hesions per cell was also reduced, in accor- each with disorganized but notably different dance with the reduction of actin stress fibers.

37 Explorations |Biological Sciences

Figure 1. A working model of the cytoskeleton at zero gravity and its relationship to immune Figuredysfunction. 1. A working Alterations model to ofthe the cytoskeleton cytoskeleton cause at disruption zero gravity to a varietyand its of relation cell functions to immune in- dysfunction.cluding motility, signal transduction, cell proliferation and gene expression. Due to the require- ment of immune cells to hone to a site of infection and respond according to specific stimulus it is suggested the changes in the cytoskeleton lead to impaired immune function in zero gravity environments.

38 Sejiro Littleton and Dylan Ludwick

This is consistent with earlier parabolic flight observations of spaceflight-associated im- data regarding similar changes in osteoblast mune dysfunction. Simulated low gravity focal adhesions (55). Human mesenchymal techniques in the 2D-clinostat and RPM have stem cells cultured for 7 days in simulated allowed for detailed comparison to more microgravity showed increases in integrin complicated studies in real microgravity protein subunit expression, but significant aboard parabolic flights or the ISS. Decades reduction in phosphorylation of downstream of research into the effects of microgravity focal adhesion-associated kinases (56). suggest a dynamic cellular adaptation process Additionally, rat WB-F344 cells under composed of primary and secondary down- simulated microgravity displayed signifi- stream responses. The cytoskeleton holds a cantly increased levels of Fibronectin and substantial influence over signal transduc- E-cadherin mRNA (57). These effects may tion, gene expression, and proliferation, and relate to observations by Levenberg et al., its early disruption seems to precede many of which revealed an autoregulatory system the observed changes in both adaptive and in- whereby stimulation through cadherins or nate immunity (see Figure 1). That being said, integrins specifically and preferentially en- the picture is far from clear. An exact mecha- hanced the formation of its associated adhe- nism of gravity sensing remains obscure. The sive structure. Essentially, when cell-cell ad- notable variations in results reflects the expo- herence junctions are increased, cell-matrix sure-dependent nature of this microgravity- focal adhesions are decreased and vice versa mediated process, as well as the wide-scale (58). This dichotomy could be a possible variances in experimental methodology. source of variation seen in cellular adaptation Short timescales must be used in searching to microgravity, such as the dual cytoskeletal for the presence of an initial gravity sensor, phenotypes seen in the breast cancer cell line, while longer timescales will be useful in dis- or the motility differences between T lym- tinguishing between short term and long term phocytes and monocytes. adaptational effects. RhoGTPases appear to Finally, a recent review paper by Louis et be a promising candidate for upstream, grav- al. described how nearly all of the cytoskel- ity-sensitive regulators of the cytoskeleton, etal changes associated with microgravity and Louis et al. further predict the role of could be attributed to the molecular workings GAP (GTPase Activating Protein) and GEF of Rho GTPases. The authors hypothesized (Guanine Exchange Factor) RhoGTPase that cells exposed to microgravity respond regulators (inhibitors and activators, respec- with depressed RhoA activity and increased tively) in this cellular adaptation process Rac1 activity initially, which changes to de- (59). Future experiments in real micrograv- pressed activity of both proteins in the long ity which monitor these specific activities at term (59). Additionally, Guignandon et al. onset of exposure will be a critical next step have found that silencing of Rho GTPases in developing our understanding of gravity- Rac1 and the protein Cdc42, but not RhoA, sensing by non-specialized cells. effectively counteracted the reduction in While the disruptive impacts of micrograv- number of focal adhesions and F-actin fibers ity on the immune system are apparent, more seen in osteoblasts exposed to real micro- work needs to be done in order to determine gravity (60). Although the initial mechanisms the relative risk it poses for astronauts engag- of gravity sensing have yet to be fully illu- ing in extended spaceflight. Clinical studies minated, it appears that we may be one step assessing immune functioning of astronauts closer to the source. at regular intervals during and after longer duration missions aboard the ISS will be CONCLUSION helpful in this regard. An ongoing research mission, the NASA Twins Study, is working We have come a long way from our initial to address some of these issues by comparing

39 Explorations |Biological Sciences

the physiological changes that occur over the real and simulated low gravity conditions course of one year spent on the ISS (61). The demonstrated altered gene expression and researchers will compare complete genome enhanced virulence (62). Far more research sequencing, metabolic markers, and a variety in both real and simulated microgravity will of other data generated from experiments on be necessary to properly address the potential Mikhail Kornienko and Scott Kelly during effects of microgravity on microbial patho- their time in microgravity with Scott’s mono- genicity. With factors such as stress, sleep zygotic twin brother retired astronaut Mark deprivation, as well as solar and cosmic ra- Kelly who will remain on Earth as a control. diation largely unaccounted for, microgravity At least one study suggests that micrograv- will be but one of many potential hurdles as ity may affect pathogens as well, as bacte- humankind pushes ever deeper into the beck- rium Salmonella typhimurium grown in both oning cosmos.

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Together is Better: The Importance of Heterotrophy and Photoautotrophic Symbiosis for Growth of the Sea Anemone Aiptasia pallida Jack Cushman Koch University of North Carolina Wilmington Faculty Mentor: Joe Pawlik University of North Carolina Wilmington ABSTRACT Scleractinian corals are important because they are the foundation species that build coral reefs, but they are in decline worldwide. The sea anemone Aiptasia pallida is a possible model system for studying the nutrition of reef-building corals because both have the same algal symbionts (Symbiodinium spp.). The relative importance of exogenous nutrition (NX) versus symbiont-derived nutrition (Ns) for A. pallida is largely unknown. When stressed, both anemones and corals bleach (expel symbionts) and have to rely solely on NX. I measured changes in wet mass after 60 days for anemones divided into four treatments that manipulated the presence and absence of NX and Ns. The treatments were: symbionts present and food provided (S+/F+), symbionts absent, but food provided (S-/F+), symbionts present, but no food provided (S+/F-), and neither symbionts present nor food provided (S-/F-). TetraMin flake food was used for exogenous food. Anemones were rendered aposymbiotic using a combination of cold shocks and treatments with a photosynthesis-inhibiting compound, and subsequently maintained in darkness. Anemones with S+/F+ gained significantly more mass than other treatments (p<0.001). All other treatments lost mass and there were no significant differences among them. Under the experimental conditions used in this study, A. pallida needed both NX and Ns to grow, suggesting that some component of each nutritional source is necessary for the health of this sea anemone.

ymbiotic relationships are ubiquitous in 1.1 Symbiosis Snature and can be classified into three sub-categories: mutualism, parasitism, and Symbiotic relationships often involve ad- commensalism. These sub-categories are aptations that enable the success of the asso- complex and many change over time on a ciation (Furla et al., 2005). In cnidarian (e.g. continuum from parasitism to mutualism de- corals, jellyfish, and sea anemones) symbi- pending on environmental conditions, and otic relationships with algae, the association possible interactions among host and sym- can be facultative or obligate and usually biont genotypes (Muller-Parker et al., 1990; consists of the transfer of nutritionally use- Neuhauser & Fargione, 2004; Thrall et al., ful compounds (photosynthetically-derived 2007; Lesser et al., 2013). carbon and metabolic waste products) from

46 Jack Koch one partner to the other (Muller-Parker & bleaching include increased light intensity, Davy, 2001; Buhl-Mortensen & Mortensen, decreased pH, changes in seawater tempera- 2004; Burriesci et al., 2012). The stability of ture and chemistry, and pollution. However, symbiotic relationships between cnidarians the conditions under which bleaching occurs and their algal symbionts are under threat is a species-specific phenomenon (Gates et by anthropogenic and climate-related factors al., 1992; Edmunds, 1994; Fitt et al., 2001). (Hoegh-Guldberg et al., 2007; Lesser et al., In some cases, bleaching may lead to death of 2013). Global climate change (e.g., changes the host due to starvation, although in other in sea surface temperature and ocean acidi- cases, recovery of the host is possible through fication), disease, and other negative influ- increased heterotrophic feeding or reinfec- ences (e.g., pollution and sedimentation) tion with symbionts (Steen, 1987; McAuley may cause the host to expel its symbionts & Cook, 1994; Anthony & Fabricius, 2000; (Burriesci et al., 2012). Fitt & Cook, 2001; Knowlton, 2001; Borell Reef-building coral symbioses are the et al., 2008). Coral growth, and therefore reef best-known example of marine symbioses formation, is dependent upon nutrition pro- and contribute to the importance of coral vided by zooxanthellae (Muscatine, 1967; reefs in global marine health and to the abil- Muscatine & Cernichiare, 1969; Burriesci et ity of coral reefs to provide a range of eco- al., 2012; Lehnert et al., 2014). logical services. These services include food, After a bleaching event, the host must find recreation, coastal protection, habitat, and additional sources of exogenous nutrition nutrient cycling (Moberg & Folke, 1999). (NX) to compensate for the loss of symbiont- Unfortunately, corals are difficult to study derived nutrition (Ns). Anemones such as in the lab because they grow and reproduce Anthopleura elegantissima and Aiptasia pal- slowly and they have an endoskeleton. The lida have the ability to derive 100% of their calcareous endoskeleton makes it difficult daily nutritional needs from NX and use the to measure their tissue mass and even more symbiotic association to compensate for the difficult to maintain the animal, because of ATP required to capture food (Engebretson the requirements for very specific seawater & Muller-Parker, 1999; Bergschneider & chemistry (Lehnert et al., 2012). In addition, Muller-Parker, 2008; Weis et al., 2008; regulations restricting coral collection and Lehnert et al., 2012). Other organisms, like manipulation make it difficult to study - cor corals, can increase heterotrophic feeding als in the laboratory and field (Lehnert et al., efforts when Ns is inadequate (Anthony & 2012). Fabricius, 2000; Ferrier-Pagès et al., 2010; Hoogenboom et al., 2010). 1.2 The Bleaching Response 1.3 The Sea Anemone Aiptasia and The The cnidarian-zooxanthellae symbi- Dinoflagellate Symbiodinium otic relationship undergoes a phenomenon called bleaching (Davy et al., 2012). This Sea anemone species in the genus Aiptasia phenomenon is becoming more prevalent are found worldwide in temperate and tropi- on coral reefs world-wide as environmen- cal waters (Sunagawa et al., 2009; Leal et tal conditions become increasingly stressful al., 2012). The temperate species A. pallida (Hoegh-Guldberg, 1999). Bleaching is the (Agassiz in Verrill, 1864) and Symbiodinium process in which corals eject their symbionts (see Figure 1) engage in a symbiosis that is or their symbionts cease to synthesize their similar to corals and their Symbiodinium. photosynthetic pigments due to environmen- A. pallida are mixotrophs, feeding on mi- tal, chemical, or physical stressors (Gates croplankton from the water column and re- et al., 1992; Brown & Ogden, 1993; Glynn, ceiving photosynthate from Symbiodinium 1996; Weis et al., 2008). Factors that cause (Muscatine, 1967; Muscatine & Cernichiare,

47 Explorations |Biological Sciences

Figure 1. Tentacle squash from temperate symbiotic sea anemone Anthopleura elegantissima at 40x power oil immersion under differential interference contrast microscope. Symbiodinium are the golden-brown circles. Although this is not the genus of sea anemone in this study, the Symbiodinium are similar. Dinoflagellate species assigned to Symbiodinium are found worldwide in temperate and tropical waters as both endosymbionts and as free-living microalgae (Lajeunesse & Trench, 2000). The dominant vegetative form is a single, spherical or broadly ellipsoidal pigmented cell ranging from 5 – 15 µm in diameter (Freudenthal, 1962). A motile phase is present, but only when the alga is free-living. Scale bar is 10 µm.

1969; Muscatine, 1980a; Burriesci et al., 1.4 The Symbiotic Relationship Between 2012). Compared to most corals, A. pallida Aiptasia and Symbiodinium survives under a wider array of conditions in the lab and field, grows and reproduces rap- The symbiosis between cnidarians and idly, and is easy to collect and manipulate, Symbiodinium is highly specific. Anemones, making this anemone and its symbionts an for example, may initially take up many ideal model system for studying the ecology clades of Symbiodinium, but preferential and physiology of reef-building corals (Weis clades outcompete others over time (Belda- et al., 2008). Baillie et al., 2002). Even when there is The relationship between A. pallida and not competition among clades within hosts, Symbiodinium boosts the overall fitness of the some anemones are unable to host particular anemone when compared to aposymbiotic in- Symbiodinium clades; this incompatibility is dividuals (Fitt & Pardy, 1981). Previous stud- reportedly caused by the inability of the host ies indicate that starved symbiotic A. pallida and symbiont to recognize each other (Belda- use zooxanthellae-derived photosynthate to Baillie et al., 2002). The symbiosis between fulfill their daily nutritional needs whereas A. pallida and Symbiodinium is facultative starved aposymbiotic anemones use host- and not required for the survival of the host. derived lipids to fulfill their daily nutritional Symbionts inhabit a host-derived vacuole needs (Muscatine, 1961; Fitt & Pardy, 1981). called a symbiosome that is located within Reliance on host-derived lipids contributes to gastrodermal cells of the host (Wakefield et an increased rate of mass loss when compared al., 2000; Kazandjian et al., 2008). The sym- to starved symbiotic anemones (Muscatine, biosome protects the algal cell and enables 1961; Fitt & Pardy, 1981). the transfer of inorganic nutrients from host

48 Jack Koch to symbiont and organic nutrition from sym- nutritional needs of the anemone. The anem- biont to host (Rahav et al., 1989; Cook et al., ones with neither symbionts present nor food 1992; Yellowlees et al., 2008; Davy et al., provided (S-/F-) would lose the most mass; 2012; Detournay et al., 2012). The host de- this treatment served as the negative control rives these inorganic nutrients from metabolic because anemones would get no nutritional by-products and dissolved particles from input. the surrounding environment (Muscatine, 1980b; Szmant-Froelich & Pilson, 1984; Materials and Methods Steen, 1986). In turn, Symbiodinium can provide the host with up to 95% of their photo- 2.1. Animal Collection and Maintenance synthate which can account for 100% of the hosts daily carbon needs depending upon the A. pallida in association with season, symbiont, and host (Davies, 1984; Symbiodinium were collected from float- Falkowski et al., 1984; Muscatine et al., ing docks at Sea Path Marina, Wrightsville 1984; Davies, 1991; Phillips & Gregg, 2003). Beach, North Carolina (34º 12’ 46.436” N, 77º 48’ 20.283” W), during January 2014 1.5 Purpose and Predictions and September 2014. Anemones were maintained in an aquarium in filtered (5 µm) and Understanding the roles of NX and Ns in UV treated natural seawater (FUVSW) at 25 overall host nutrition may allow scientists to - 32 psu at room temperature for a minimum better understand how corals and other mixo- of two weeks until acclimated. During this trophic symbiotic organisms respond to the time anemones were fed crushed TetraMin loss of one or both sources of nutrition. The Tropical Flake (Tetra GMBH, Melle, purpose of this study was to investigate the Germany) fish food three times per week. importance of NX and Ns for A. pallida by One hundred and fifty 15 mm x 60 mm measuring changes in wet mass under dif- plastic petri dishes were numbered and three ferent feeding regimes in combination with - 1/8” holes were drilled into each petri dish different states of symbiosis under laboratory to allow water flow through them. Each petri conditions. I predicted that anemones with dish was initially weighed on a Mettler Toledo symbionts present and food provided (S+/ AJ100 Analytical Balance (Mettler-Toledo, F+) would gain the most mass and this treat- Columbus, OH), and conditioned by soak- ment served as the positive control in this ing them in freshwater for 10 hrs, and then in experiment because both nutrition sources FUVSW overnight to remove any chemical are available to the anemone. The treatment residue. One hundred and fifty medium-sized containing symbionts absent, but food pro- (>1 cm oral disk diameter) to large-sized (>2 vided (S-/F+) would grow a small amount or cm oral disk diameter) A. pallida were indi- show no change in mass. If A. pallida was vidually placed onto conditioned petri dishes. able to derive 100% of its daily nutritional Anemones that were selected for experimen- needs from NX then the anemone mass would tal treatment ranged in size from 0.06 to 1.11 remain the same but, if the anemone was able g wet mass (x̅ ± SE=0.26 ± 0.03 g, n=86). to derive more than 100% of its daily nutri- The petri dishes and anemones were placed tional needs from NX then the anemone mass into shallow trays of FUVSW for up to 12 hrs would increase. Anemones with symbionts to allow anemones to attach. Once attached, present, but no food provided (S+/F-) would the anemones were placed in a maintenance lose some mass because the symbionts would aquarium and starved for one week before be unable to produce enough photosynthate starting the experiment. to sustain both themselves and the anemone. In other words, the symbionts would provide 2.2. Preparation of Experimental Chambers the anemone with less than 100% of the daily and Incubator Set-up

49 Explorations |Biological Sciences

Experimental chambers consisted of 300 diluted to a final concentration of 50 µM with - 450 mL beakers. Fifty beakers were com- 4 ºC MSFW. (Burriesci et al., 2012; Lehnert pletely wrapped with black vinyl electrical et al., 2012). tape to exclude light. A Precision Scientific low temperature illuminated incubator 818 2.4. Cleaning and Feeding Protocol (Jouan; Winchester, VA) was used to maintain temperature (23ºC), photosynthetically Anemone beakers were cleaned five days active radiation (PAR), and photoperiod per week (Sun, M, W, F, Sat). The petri (12:12 h light:dark cycle) during the course dishes were cleaned with a cotton Q-tip to of the experiments. A Coralife Trichromatic remove any food particles or algal growth. A 6500K/Full Spectrum (Coralife, Franklin, turkey baster was used to rinse the petri dish WI) and a GE Plant & Aquarium Wide with FUVSW and the petri dish was returned Spectrum (General Electric, Cleveland, OH) to the beaker. light were attached to the door of the incu- After the beakers were cleaned, the anemo- bator to provide PAR. The PAR was mea- nes were fed. During experimentation, food sured using a LiCor LI-193SA Underwater was prepared from TetraMin Large Tropical Spherical Quantum Sensor attached to a Flake fish food. Food was standardized in LiCor LI-1400 data logger (Lincoln, NE). size by making round flakes (6 mm -diam The PAR underwater inside the dark beaker eter) using a hand-held paper hole punch measured 0.02 µmol m-2 sec-1 PAR and un- to produce food discs. The total area of 51 derwater inside the light beaker measured food discs was calculated from digital im- 61.91 µmol m-2 sec-1 PAR (average of two ages of them using the image analysis pro- 5-second averages). gram ImageJ v2.00-rc-19. The average area of each food disc was 0.33 cm2 (±0.01 SE, 2.3. Artificial Bleaching and Maintenance of n=51). Anemones were fed one food disc five Aposymbiotic Anemones days per week for 60 days. Anemone feeding was observed until the food disc was fully in- Fifty symbiotic anemones were randomly gested (Fig. 2). chosen from the petri dish plated anemones and placed into the dark beakers. These 2.5. Estimation and Quantification of anemones were maintained for several days Zooxanthellae Density in the incubator and FUVSW was chilled to 4 ºC in another incubator (Burriesci et al., Two methods were used to estimate the 2012; Lehnert et al., 2012). Approximately density of zooxanthellae and therefore the 150 mL of 4 ºC FUVSW was placed into each symbiotic state of anemones during and at beaker and the anemones were cold shocked the end of the experiment. During the experi- in the dark for 4 hours (Lehnert et al., 2012). ment an estimation of zooxanthellae density This process was repeated two more times per tentacle was examined for all anemones, over the course of 2 weeks. which were observed under a dissecting mi- To speed up the process and en- croscope. Additionally, before and after cold sure that anemones were aposymbi- shocks, and four times during the experimen- otic, the photosynthesis inhibitor Diuron tal period (days 15, 30, 45, and 60) an estima- [3-(3,4-dichlorophenyl)-1,1-dimethylurea] tion of zooxanthellae density per unit tentacle (DCMU; Sigma-Aldrich, St. Louis, MO) slurry was quantified for five representative was added and the cold shocks were repeated anemones using a haemocytometer. Scissors an additional seven times over the course of were used to clip a tentacle from the specified seven weeks (ten total cold shocks). 0.0313 individuals in the aposymbiotic treatments. M DCMU (0.73 g of powder DCMU was Tentacle clipping does not significantly ef- dissolved into 100 mL of 100% ethanol) was fect the weight of the anemones, and is a

50 Jack Koch

Figure 2. Time lapse pictures of Aiptasia pallida eating a TetraMin food disc.

0.3

0.25

0.2

0.15 Final Wet Mass (g) Final Wet Mass 0.1

0.05

0 0 0.002 0.004 0.006 0.008 0.01 0.012 0.014 0.016 Final Dry Mass (g)

Figure 3. Variations in final dry mass versus final wet mass in polyps of the anemone Aiptasia pallida from Wrightsville Beach, North Carolina, USA (n=20; y=15.75x + 0.03; R2=0.88; p<0.001).

51 Explorations |Biological Sciences

non-lethal and repeatable method to measure different treatments. The Tukey Honest zooxanthellae densities (Levitis & Goldstein, Significant Difference (HSD) statistical test 2013). Each tentacle was crushed with a pair at the 5% family-wise error rate was used to of forceps in a 1.5 mL microcentrifuge tube determine which means were significantly in 0.1 mL of MSFW. Three subsamples of the different. RStudio v0.98.977 was used for the resulting slurry were counted on a Bright- paired t-test, ANOVA analysis and the Tukey Line haemocytometer (Sigma, St. Louis, (HSD) multiple comparisons of means statis- MO) and an Olympus 100 compound mi- tical test. croscope (40x objective; Japan). Anemones were considered to be aposymbiotic when Results the average number of Symbiodinium cells per 0.1 mm3 tentacle slurry was ≤ 20 cells. 3.1 Accuracy of Wet Mass Measurements

2.6. Massing Protocol The wet mass of the anemones and the dry mass of the anemones were positively corre- The wet mass of the anemones was mea- lated (n=20, R2=0.88, p<0.001; see Figure 3). sured on day 0 and day 60 of experimental treatment using a standardized protocol. The 3.2 Artificial Bleaching and its Effect on petri dish and anemone were removed from Anemone Mass the beaker and blotted dry, then the anemone was touched with a pair of forceps, causing There was a significant (p<0.001) differ- it to expel most of the water that was held ence in the average wet mass of the anem- within its gastrovascular cavity. The petri ones (n=41) before and after the artificial dish and anemone was then weighed on a bleaching treatments (t=8.08; d.f.=40). The Mettler Toledo AJ100 Analytical Balance. maximum initial wet mass of anemones was After the experiment was complete, anemo- 0.32 g and the maximum final wet mass of nes were frozen, freeze-dried, and weighed the anemones was 0.23 g. The minimum ini- in the same manner to determine dry mass. tial wet mass of the anemones was 0.06 g and Twenty anemones were randomly selected to the minimum final wet mass of the anemo- determine the variation in dry and wet mass. nes was 0.02 g. Two anemones experienced The final wet mass was plotted against the positive growth (+0.01 g) during the artifi- dry mass of the corresponding individual and cial bleaching, but this positive increase in a line of best fit was plotted to determine the mass is likely due to error by the scale. All R2 value, and linear equation (Fig 3). other anemones lost mass during the artificial bleaching treatments. 2.7. Statistical Analyses A single tentacle was clipped from five anemones before and during the artificial All data were entered into Microsoft Excel, bleaching treatments to track the expulsion which was used to calculate Symbiodinium of zooxanthellae and to determine the den- per unit tentacle slurry, mass change of the sity of zooxanthellae in the anemones. By the anemones, and percentage mass change. A end of the artificial bleaching treatment, all paired two-tail t-test at the alpha = 0.05 sig- anemones were aposymbiotic according to nificance level was used to determine if there the minimum zooxanthellae density thresh- was any difference in the starting mass of old (<20 cells per 0.1 mm3 anemone tissue the anemones before (symbiotic) and after slurry). Anemones continued to be aposym- (aposymbiotic) cold shock treatments. The biotic throughout the experiment and at the ANOVA analysis was used to determine if end, zooxanthellae density counts were zero there was a significant difference in the mean cells per 0.1 mm3 anemone tissue slurry (Fig. percentage mass change of anemones among 4).

52 Jack Koch

Additionally, climate change is likely to af- 3.3 Effect of Feeding Regime on Anemone fect the way that Aiptasia interacts with Mass its symbiotic microalgae, Symbiodinium, thereby changing the trophic interaction be- There was a significant (p<0.001) differ- tween the two organisms (Fitt & Cook, 2001; ence in the average change of mass of the Borell et al., 2008; Ferrier-Pagès et al., 2010). anemones among all treatments (see Table These changes may lead to a lack of food and 1). The Tukey (HSD) test indicated that increased stress for Aiptasia and other symbi- there was a significant (p<0.001) difference otic planktivores. Therefore, it is important to between the average change of mass of the study how changes in heterotrophic and pho- anemones in the S+/F+ treatment and all toautotrophic feeding will affect Aiptasia and other treatments. There was not a significant its symbionts, a relationship used as a model difference between the average changes of system for other symbiotic planktivores such mass of the anemones in any of the other as corals. The findings of this experiment treatments though (see Table 2). show that changing nutrition availability for The anemones in the S+/F+ treatment on Aiptasia with and without symbionts is likely average gained mass. The average percent- to affect the relationship and the host in a age change in mass over 60 days was 57.96% negative way. increase (± 12.52 SE; see Figure 5). The A. pallida with S+/F- lost a significant maximum percentage change over 60 days amount of mass when compared to A. pallida was 238.29% increase and the minimum was with S+/F+. A. pallida with S+/F+ showed 34.99% decrease. The anemones in the S-/F+ a significant increase in mass over 60 days treatment on average lost mass. The average compared to the other three treatments. These percentage change in mass over 60 days was results suggest that photosynthate provided 35.85% decrease (± 16.74 SE; see Figure 5). by zooxanthellae in A. pallida under the The maximum percentage change over 60 experimental conditions used in this study days was 246.90% increase and the mini- are unable to provide sufficient nutrition mum was 91.51% decrease. The anemones (<100%) to maintain anemone mass or allow in the S+/F- treatment on average lost mass. for growth. The average percentage change in mass over Under natural conditions, all non-symbi- 60 days was 62.75% decrease (± 4.17 SE; otic and some symbiotic corals and anemo- see Figure 5). The maximum percentage nes such as Anthopleura elegantissima and change over 60 days was 20.24% decrease A. pallida are able to survive solely on het- and the minimum was 90.70% decrease. The erotrophic feeding (Engebretson & Muller- anemones in the S-/F+ treatment on average Parker, 1999; Bergschneider & Muller- lost mass. The average percentage change in Parker, 2008). The results of this study mass over 60 days was 67.00% decrease (± suggest that the exogenous food used in this 5.22 SE; see Figure 5). The maximum per- study is an inadequate source of nutrition centage change over 60 days was 0.90% in- for A. pallida when it is the sole source of crease and the minimum was 100% decrease. nutrition. Anemones with S-/F+ lost a significant amount of mass when compared to Discussion anemones with S+/F+. This is an interesting result because aposymbiotic A. pallida have Climate change will affect food availabil- been found in nature, indicating that there is ity for planktivores, such as Aiptasia, due to enough exogenous food for them to maintain changes in the timing of plankton blooms, themselves at a minimum. changes in reproductive efforts of the plank- All photosynthetic organisms have an op- ton community, and community shifts within timal lighting intensity at which they photo- these plankton blooms (Walther et al., 2002). synthesize maximally. Muller-Parker (1984)

53 Explorations |Biological Sciences

Fig 4. Graph of the effect of cold shocks on the average densities of Symbiodinium within Aiptasia pallida tentacles (n=5). When the representative anemones had average Symbiodinium densities below 20 cells per 0.1 mm3 tentacle slurry, they were considered aposymbiotic. Snowflakes indicate cold shock and snowflakes with chemical symbols indicate cold shock with DCMU (CS = cold shock; DCMU/CS = DCMU cold shock).

54 Jack Koch

80 80

60 60

40 40

20 20

0 0

-‐20 -‐20

-‐40 -‐40

-‐60

Average Percent Change of Wet Mass (%) Wet Average Percent Change of -‐60 Average Percent Change of Wet Mass (%) Wet Average Percent Change of -‐80 -‐80 Aposymbio.c Fed Symbio.c Starved Aposymbio.c Starved Symbio.c Fed Aposymbio.c Fed Symbio.c Starved Aposymbio.c Starved Symbio.c Fed

Fig 5. Graph of average 60 day percentage change of wet mass between the four treatment groups. Error bars are standard error.

Table 1. ANOVA statistics from the means of the change in mass over 60 days of the four treatment groups in the experiment.

Source D.F. Sum sq Mean Sq F value Pr (>F) Treatment 3 238710 79570 31.33 1.36E-‐13 Residuals 82 208253 2540

Table 2. Tukey HSD stats from the comparison of multiple mean percentage change in mass after 60 days of treatment in four experimental groups. Adjusted p-values are tested at the alpha = 0.05 significance level.

Difference of Lower difference Upper difference Adjusted+p7 Source mean of mean of mean value Dark starved -‐ Dark fed -‐30.15 -‐72.49 12.19 0.25 Light fed -‐ Dark fed 93.81 53.58 134.03 <0.001 Light starved -‐ Dark fed -‐26.91 -‐68.30 14.48 0.33 Light fed -‐ Dark starved 123.95 84.31 163.60 <0.001 Light starved -‐ Dark starved 3.24 -‐37.59 44.07 >0.99 Light starved -‐ Light fed -‐120.71 -‐159.35 -‐82.08 <0.001

55 Explorations |Biological Sciences found that zooxanthellae in association with and correlated to the change in mass of each A. pulchella have a maximum photosynthetic anemone. Furthermore, wild anemones do efficiency around 500 µE -2m sec-1 PAR. In not feed just once a day. In future experi- this study the symbiotic anemones were pro- ments, feeding anemones to satiation several vided with 61 µmol m-2 sec-1 PAR (1 µE m-2 times per day might enhance the effect of sec-1 = 1 µmol m-2 sec-1), which is well below exogenous food, allowing for a better deter- the optimal light intensity for maximal pho- mination of the minimum exogenous food tosynthesis of zooxanthellae. Under optimal requirements for maintenance, and those that conditions, the zooxanthellae can produce result in anemone growth, with and without the maximum amount of photosynthate and symbionts. therefore provide the most photosynthate to The high variance observed in the experi- their symbiotic hosts. Under the lighting con- mental treatment S-/F+ might be explained ditions of this study, it seems that the symbi- by incomplete feeding by the anemones (Fig. onts present were unable to provide enough 5). Despite the fact that anemones were ob- photosynthate to feed both themselves and served until the food disc was ingested, it is the host. Future research should examine possible that the anemones egested the food how much nutrition is needed to maintain A. before the food disc had been digested com- pallida, if A. pallida is able to induce their pletely or that the food disc was only partially symbionts to release this amount of nutrition, digested. It is also possible that switching and at what light level zooxanthellae are able to a more natural food source such as live to produce such levels of nutrition. Artemia would have produced more success- Many experiments have been performed ful feedings. Although, the nutritional value with aposymbiotic Aiptasia that have been of TetraMin, Artemia, and supplemented forced to expel their symbionts. Authors have Artemia should be examined to determine noted that aposymbiotic anemones have been which food source provides the most com- maintained for several months (Weis, 1991; plete and consistent nutritional array for the Kuo et al., 2004; Kuo et al., 2010; Lehnert anemones. et al., 2012). When stated, the food source In order for a model system such as Aiptasia has always been brine shrimp (Artemia) fed to effectively represent the target system, on a daily basis. No survivorship informa- control laboratory experiments manipulating tion or indication that aposymbiotic indi- factors such as food availability and compo- viduals lose mass during this time has been sition, lighting, as well as other environmen- reported. In this experiment, the amount of tal conditions must be kept as close to natural food fed to each anemone might have con- as possible. For example, it is possible that tributed to the observed results. Feeding each anemones and corals did not receive optimal anemone to satiation would have given the light intensity at all times of the day or ever greatest likelihood that each anemone was during the day so that the regular light inten- receiving enough nutrition to at least main- sity received by the study organisms needed tain body mass if not increase body mass. to be measured in the field and provided in In the wild, anemones would consume dif- the lab. A better experimental protocol would ferent amounts of food, but that amount of model the light intensity of an average day food would be proportional to the body size and provide similar nutritional content and of the anemone. The body size of the anem- similar food amounts to model the target sys- one is proportional to the amount of food in- tem as closely as possible. Model systems gested (Sebens & Koehl, 1984). By feeding such as Aiptasia are essential to the effective the anemones TetraMin, the amount of food study of target systems such as scleractinian given to each anemone can easily be recorded corals.

56 Jack Koch

Acknowledgements

I would like to thank my Dr. Joseph Pawlik for his support and feedback, comments, and funding for this project. I would also like to thank Dr. Nathaniel Grove, Dr. J Wilson White, Dr. Allison Taylor, Mark Gay, Kathy Bennett-Carter, and Nathan Gavin for their support, comments, and assistance. And finally, thank you to my friends and family for their support and guidance. This project was funded in part by a UNCW CSURF supplies grant.

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Leal, M. C., Nunes, C., Engrola, S., Dinis, M. T., & Calado, R. (2012). Optimization of mono clonal production of the glass anemone Aiptasia pallida (Agassiz in Verrill, 1864). Aquaculture, 354, 91-96.

Lehnert, E. M., Burriesci, M. S., & Pringle, J. R. (2012). Developing the anemone Aiptasia as a tractable model for cnidarian-dinoflagellate symbiosis: The transcriptome of apo symbiotic A. pallida. BMC Genomics, 13, 271.

Lehnert, E. M., Mouchka, M. E., Burriesci, M. S., Gallo, N. D., Schwarz, J. A., & Pringle, J. R. (2014). Extensive differences in gene expression between symbiotic and aposym biotic cnidarians. G3 (Bethesda), 4(2), 277-295.

Lesser, M. P., Stat, M., & Gates, R. D. (2013). The endosymbiotic dinoflagellates (Symbiodinium sp.) of corals are parasites and mutualists. Coral Reefs, 32(3), 603-611.

Levitis, D. A., & Goldstein, J. (2013). The consistent, non-destructive measurement of small proteiform aquatic animals, with application to the size and growth of hydra. Marine and Freshwater Research, 64(4), 332-339.

59 Explorations |Biological Sciences

McAuley, P. J., & Cook, C. B. (1994). Effects of host feeding and dissolved ammonium on cell division and nitrogen status of zooxanthellae in the hydroid Myrionema amboi nense. Marine Biology, 121(2), 343-348.

Moberg, F., & Folke, C. (1999). Ecological goods and services of coral reef ecosystems. Ecological Economics, 29(2), 215-233.

Muller-Parker, G., Cook, C. B., & D’Elia, C. F. (1990). Feeding affects phosphate fluxes in the symbiotic sea anemone Aiptasia pallida. Marine Ecology Progress Series, 60, 283-290

Muller-Parker, G., & Davy, S. K. (2001). Temperate and tropical algal-sea anemone symbioses. Invertebrate Biology, 120(2), 104-123.

Muscatine, L. (1961). Symbiosis in marine and freshwater coelenterates. In H. M. Lenhoff & W. F. Loomis (Eds.), The biology of hydra and of some other coelenterates (pp. 255-268). Miami, FL: University of Miami Press.

Muscatine, L. (1967). Glycerol excretion by symbiotic algae from corals and Tridacna and its control by the host. Science, 156(3774), 516-519.

Muscatine, L. (1980a). Productivity of zooxanthellae. In P. G. Falkowski (Ed.), Primary pro ductivity in the sea (Vol. 19, pp. 381-402): Springer US.

Muscatine, L. (1980b). Uptake, retention and release of dissolved inorganic nutrients by marine alga-invertebrate associations. In C. B. Cook, P. W. Pappas, & E. D. Rudolph (Eds.), Cellular interactions in symbiosis and parasitism (pp. 229-244). Columbus, Ohio: Ohio State University Press.

Muscatine, L., & Cernichiare, E. (1969). Assimilation of photosynthetic products of zooxan thellae by a reef coral. The Biological Bulletin, 137(3), 506-524.

Muscatine, L., Falkowski, P. G., Porter, J. W., & Dubinsky, Z. (1984). Fate of photosynthetic fixed carbon in light- and shade-adapted colonies of the symbiotic coralStylophora pistillata. Proceedings of the Royal Society of London. Series B, Biological Sciences, sssssssss222(1227), 181-202.

Neuhauser, C., & Fargione, J. E. (2004). A mutualism–parasitism continuum model and its ap plication to plant–mycorrhizae interactions. Ecological Modelling, 177(3-4), 337-352.

Phillips, D. L., & Gregg, J. W. (2003). Source partitioning using stable isotopes: Coping with too many sources. Oecologia, 136(2), 261-269.

Rahav, O., Dubinsky, Z., Achituv, Y., & Falkowski, P. G. (1989). Ammonium metabolism in the zooxanthellate coral, Stylophora pistillata. Proceedings of the Royal Society of London. Series B, Biological Sciences, 236(1284), 325-337.

60 Jack Koch

Sebens, K. P., & Koehl, M. A. R. (1984). Predation on zooplankton by the benthic anthozoans Alcyonium siderium (Alcyonacea) and Metridium senile (Actiniaria) in the new-england subtidal. Marine Biology, 81(3), 255-271.

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Steen, R. G. (1987). Evidence for facultative heterotrophy in cultured zooxanthellae. Marine Biology, 95(1), 15-23.

Sunagawa, S., Wilson, E. C., Thaler, M., Smith, M. L., Caruso, C., Pringle, J. R., et al. (2009). Generation and analysis of transcriptomic resources for a model system on the rise: The sea anemone Aiptasia pallida and its dinoflagellate endosymbiont.BMC Genomics, 10, 258.

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Wakefield, T., Farmer, M., & Kempf, S. (2000). Revised description of the fine structure of in situ “zooxanthellae” genus Symbiodinium. The Biological Bulletin, 199(1), 76-84.

Weis, V. M. (1991). The induction of carbonic-anhydrase in the symbiotic sea anemone Aiptasia pulchella. The Biological Bulletin, 180(3), 496-504.

Weis, V. M., Davy, S. K., Hoegh-Guldberg, O., Rodriguez-Lanetty, M., & Pringle, J. R. (2008). Cell biology in model systems as the key to understanding corals. Trends in Ecological Evolution, 23(7), 369-376.

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61 Explorations |Creative Work

Creative Work: Drowning

Artist: Courtney Hockett Faculty Mentor: Brandon Sanderson Affiliation: University of North Carolina at Pembroke Dimensions: 9”x12” Medium: Zinc Etching, Aquatint, Watercolor

Artist Statement: Drowning, flash fiction and print, was created as a result of researching how to incorporate creative literacy in visual arts using a cross curricular approach. During my studies, I experimented with the writing technique of flash fiction and focused on creating a story that included aspects of magic realism that worked cohesively with the use of extreme detail. I then analyzed the story’s contents and chose a memorable scene to illustrate. I used my previous knowledge in the field of printmaking to create a print that acted as a narrative producing an image that portrayed a magical feel through movement and color. The story and print was created in a way that allows each piece to stand alone and the reader/viewer can still understand the content.

That Saturday night marked one week since I grabbed the keys out of the storage cabinet taking on the responsibility of opening and and headed out of the office. closing the museum alone. The facility could My first stop was the turtle room. I crawled only afford to hire around half a dozen em- into the interactive sand dune exhibit and ployees at the time, so when one of them quit squeezed in the children’s play room to make I was eager to step up in the intern world and sure the baby sea turtle video hadn’t been take on more important responsibilities (at left on and wasn’t still playing – that bubble- least until a replacement was found). screened television was notorious for turning Two endless hours of trying to balance the on and off when it pleased. It was just as I had weekly finance sheet had passed and I was left it earlier, so I popped out the other side of about to go insane if I had to sit through an- the faux dune and into Swamp Hall. I slowly other second of trying to decipher that calcu- walked towards the entrance way and listened lating machine. Even though all the museum’s for the sound. electronic devices were outdated they all still Woosh, swoosh, whoosh. worked, so the staff opted each year to not up- I had already blown it twice earlier that grade in hopes to save some money. It also week. One night I didn’t shut the safe all the didn’t help that ever since flipping the closed way leaving the zipper bag of cash register sign earlier that night a low rumble had been money out in plain view. Then, two mornings echoing throughout the museum, ricocheting later I forgot to clean up the left over shrimp from room to room. after the tank feeding and a group of kids de- Woosh, swoosh, whoosh. cided it would be a great idea to rip apart their Flustered about the whole situation, I de- meat filled shells and stick their lifeless bodies cided to take a break from the mind-numb- to the surrounding glass displays. Determined ing task of counting numbers and re-do my to just get the week over with and finally get nightly shut-down routine. I needed to reas- some rest, I continued down the hall passing sure myself that I had turned off every possi- the vintage taxidermied swamp animals glar- ble light and device there was within sight. So ing back with their marble eyes, and entered

62 Courtney Hockett

the saltwater touch tank. The spotlights illuminated the room allow- I grabbed the railing beside me and used ing me to see just enough to walk around yet my other hand to caress the wall as I felt for a not disturb the mischievous horseshoe crabs light switch. Then I cautiously walked down who were finally settled in their beds for the the ramp and listened for the wave cassette in night. The faint sound of hermit crabs tap- case a volunteer had forgot to turn it off af- tapping with their feet lingered throughout ter leaving for the night. Only the filtration the entrance hall as they marched around the pump could be heard, buzzing vigorously as tank scrounging for food. I walked down to it rotated gallons of salt water throughout the the bottom of the platform and hung the front aquatic ecosystem. half of my body over the marble ledge – pink

63 Explorations |Creative Work and purple ripples fled the surface as the water I began to run; jotting through the halls, reflected my dress. leaping from exhibit to exhibit. I kept imag- Glued to the center of the coral rocks was ining the duct tape melting away as the ma- a stranded sea star. I stretched my arm out as chine’s loose chord wires entangled them- far as it would go to pluck him off to safety. selves together creating an electrical circuit. Just as I grabbed his leg, a passing puffer fish I ran past the Dry Aquarium sign, towards accidentally brushed against the submerged the passageway, and ripped open the entrance sleeves of my dress and burst through the sur- drapes. Then, I tripped over a loose board on face. My muscles twisted into a pretzel and a the land bridge and toppled off the side fifty wave a bumps covered my arms from the icy feet above the bottom of the indoor sea. water which now soaked everything from my The same rumble that had been taunting elbows down. me all night was now louder than ever as I I could hear the filtration pump buzzing as glided backwards, watching the lights on the it forced more water back into the touch tank, simulator machine go up and down with the and then it hit me. I was so caught up in the rhythm of the waves. The bodies of fiberglass bureaucracy of my new title that I had ne- fish swam around me like marionette dolls, glected a room in the museum when complet- translucent strings suspending them in their ing the shut-down routine. The Dry Aquarium eternal home. was the easiest to close considering it was just I reached for help from a nearby manta an oversized, walk-in diorama. There was ray as I fell past him feet first, but he swayed only one task for that exhibit – turn off the away as I toppled by. Tired of constantly feel- underwater simulator machine. ing as though I was drowning, I relaxed my Woosh, swoosh, whoosh. body and embraced the gentle giants as they I jogged back up the stairs and power- swam around me, their bodies morphing into walked down the main hallway. All I could a tie-dye swirl as I fell deeper into the abyss. think about was how long the underwater For the first time in days, I finally had a mo- simulator must’ve been playing the music and ment to relax. lights for – six, maybe seven hours.

64 65 66 Engineering

67 Explorations |Engineering Miniaturizing Photoplethysmography for Use in a Multifunctional Health Monitoring Device with Applications for Asthma Analysis Brinnae Bent North Carolina State University Faculty Mentor: Alper Bozkurt North Carolina State University ABSTRACT Current treatment plans for chronic respiratory diseases are limited to single point measurements obtained in the regulated environment of a clinic. Thus, the development of a continual health and environmental monitoring system would allow for a better understanding of how external factors are affecting the patient’s respiratory condition and would shift healthcare into a preventative rather than a reactive system that treats only the symptoms. In addition to the integrated electrocardiogram (ECG), ozone detector, three-axis accelerometer, and microphone, photoplethysmography (PPG) allows for a more complete physiological understanding of chronic respiratory conditions. Dual wavelength PPG allows for the measurement of respiratory rate, heart rate and arterial oxygenation (SpO2), and can be used in unison with ECG to provide an estimation of blood pressure via pulse transit time. Thus, the development of a robust, biologically compatible optical probe is crucial in achieving accurate results. In this paper, we offer a plan to develop such a probe. Flexible conformation to the tissue, time multi- plexing of the differing wavelengths, protective polymer coating, and overall power consumption are some of the main concerns in our design of the probe. The PPG probe is miniaturized and manufactured to increase accuracy while maintaining a flexible, lightweight, and energy efficient device component. With the ability to monitor both heart rate and respiratory rate, it greatly improves the versatility and accuracy of the overall multifunctional health monitoring device.

he fourth leading cause of death in the worsen these chronic conditions. For exam- TUnited States is obstructive lung disease. ple, asthma can be triggered by a variety of Obstructive lung disease is the only major environmental factors such as humidity, air disease that is rising in both prevalence and pollution, and temperature changes [6]. mortality [5]. Further, nearly 235 million Current chronic respiratory monitoring is individuals currently suffer from asthma (a done in a health care facility utilizing spi- common type of obstructive lung disease) rometry, which is limited to single point mea- worldwide, and this number is growing at an surements. These measurements are taken in alarming rate. In order to combat the rise of a regulated clinical environment without the these chronic diseases, monitoring systems variety of environmental factors that may have been proposed for long term individual trigger an asthma attack [1]. Thus, the cur- analysis. Long term individual analysis is rent monitoring system is inadequate to pro- important because a variety of triggers can vide a complete picture of the physiology

68 Brinnae Bent and environmental factors affecting chronic rate, respectively. The PPG signal contains respiratory illness. both components of cardiac contraction and Commercial monitoring systems for respiration as a result of the simultaneous asthma management are limited in sensor absorption of the near infrared light by the capabilities and in the large power require- arteries and the veins, which subsequently al- ments to power these devices that require lows for monitoring of both heart rate and re- frequent power charging by the user [1]. spiratory rate. The PPG on the wristband can Because of low capabilities and high power be used in conjunction with the ECG to pro- requirements, the current commercial moni- vide an estimation of the blood pressure via toring systems are inconvenient to the user. pulse transit time (PTT) [4]. The PPG on the In order to increase understanding of the chest patch determines a signal correspond- health and environmental factors that impact ing to the respiratory rate. chronic respiratory diseases, a system that The research described in this paper is an uses a variety of sensors to monitor in real- extension of the chronic respiratory disease time is essential. This system is required to monitoring system described by Dieffenderfer be energy efficient, portable, and provide the and colleagues [1, 2, 3]. Previously only a user with continuous, real-time data to better concept, in this research I describe the actual manage chronic respiratory illness. The pho- design of a miniaturized, flexible probe to toplethysmograph probe is one component of monitor heart and respiratory rate. The PPG a health monitoring system designed to provide these requirements to the user, enabling a preventative system of chronic respiratory disease health care [1]. The purpose of the research described here was to develop a photoplethysmograph probe that can be used as such a sensor.

Device Integration

Combined with integrated electrocardiogram (ECG), ozone detection, three-axis ac- celerometry, and microphone, the PPG probe can contribute to a health monitoring system that provides comprehensive monitoring of heart and respiratory function and enables improved management of chronic respiratory disease. The system is comprised of a wristband with humidity, temperature, and ozone sensors and a chest patch with integrated ECG, an accelerometer, and a microphone, as shown in Figure 1. Bluetooth Low Energy (BLE) enables the system to be synchronized between the wristband and chest patch. When combined with the PPG probe, this system allows for complete and continuous health and environmental monitoring. Figure 1. Complete chronic respiratory As seen in Figure 1, the PPG probe is inte- health monitoring system as described by grated into both the wristband and the chest Dieffenderfer and colleagues [1]. patch to monitor heart rate and respiratory

69 Explorations |Engineering

Figure 2. PPG probe and tissue interface as described by Dieffenderfer and colleagues [1].

Figure 3. PPG probe etching process (photo courtesy of author).

70 Brinnae Bent probe was designed, developed, and manu- the etching process is complete. factured in house to meet the design speci- The photodiode (SI1143, Silicon Labs) fications of the chronic respiratory disease and light emitting diodes (LED) with wave- monitoring system. lengths of 660nm and 940 nm were soldered onto the etched copper traces, as shown in Method Figure 4. These components were chosen based on availability and cost as well as over- PPG Development all effectiveness. A ribbon cable connects the probe to the device integrated circuit in either When developing the photoplethysmo- the wristband or the chest patch. graph probe, we considered several design A coating of dark liquid electrical tape features. In keeping with the design of the insulator followed by a layer of epoxy was overall health monitoring device, low power used to electrically insulate the probe and consumption is required. In order for the provide a cushion for the edges of the optical probe to provide user safety and comfort elements and an optical barrier between the while maximizing the efficiency of the de- photodiode and the LEDs. vice, the design provides both flexible con- Several prototypes were designed in the formation to the tissue and a protective poly- development of the PPG probe, with size re- mer coating. duction being the major influence in the rede- Both respiratory rate and heart rate are signs. The overall effectiveness of the probe evaluated through a measurement of the was determined by how well it addressed the changes in light absorption in an artery. Light design needs outlined above, including low emitting diodes (LED) shine light through power consumption, flexible conformation to the tissue into the artery, which reflects back the tissue, and a having a polymer protective and is measured using a photodiode, a com- coating to maximize safety and efficiency. ponent that turns light into current. This process is shown in Figure 2. The dual wavelength PPG with time mul- tiplexing (a combination of multiple signals) of the differing wavelengths allows for the additional measurement of arterial oxygenation (SpO2). Clear epoxy and Tegaderm, a thin, waterproof film, encase the circuit components.

PPG Fabrication

The PPG probe was manufactured in- house using a flexible copper covered polyimide substrate. Traces of the circuit were defined by printing wax ink as a resist and the copper is etched using an etching solution of hydrochloric acid (HCl) and hydrogen peroxide (H2O2). The etching process from resist printed on copper to the copper trace left on the polyimide substrate is shown in Figure 4. Soldered PPG probe (photo cour- Figure 3. For a complete etch, the process tesy of author). took approximately thirty minutes. Note the flexibility of the polyimide substrate when

71 Explorations |Engineering

Results and Analysis the probe was integrated into the chronic respiratory disease monitoring system. PPG measurements can be easily corrupted by noise due to motion. In order to remove Discussion and Significance this noise distortion from motion, the nor- malized least means square (NLMS) adap- The PPG probe is crucial to attaining accu- tive filtering algorithm was used. This filter- rate results in the chronic respiratory health ing method normalizes the power of the input monitoring system. The design components and decreases computational complexity. of flexibility, accuracy, and energy efficiency The simultaneous ECG signals from the in- were attained while miniaturizing the probe. tegrated electrocardiogram and both the heart The resulting simultaneous ECG and PPG rate PPG signal and respiratory rate PPG sig- signals exemplify the capabilities of both nal are illustrated in Figure (5). These actual heart rate monitoring and estimating blood signals were obtained during in-vivo prelimi- pressure via pulse transit time. Respiratory nary trials. In the preliminary trials, we com- rate from the PPG probe on the chest patch is pared the PPG probe to a commercial heart also illustrated and shows the significant ca- rate monitor (not pictured) and ECG from the pabilities of the PPG probe in the integrated chest patch. The direct correlation shown in chronic respiratory health monitoring system. Figure 5 between ECG and heart rate exem- Overall, the PPG probe greatly improves the plifies the PPG probe’s capabilities to pro- versatility and accuracy of the overall multi- duce accurate measurements of heart rate. As functional health monitoring device. discussed previously, the PPG probe on the In addition to current PPG probe capabili- wristband monitors heart rate and is capable ties, in future applications the probe would of estimating the blood pressure via pulse enable measurement of arterial oxygenation transit time when used in conjunction with (SpO2). This would provide a non-invasive the integrated ECG signal. The PPG probe on examination of lung function in real-time, the chest patch monitors respiration rate. which would further the physiological When accurate data were obtained utiliz- knowledge of chronic respiratory conditions. ing a PPG probe design that fitted within size The next steps in the development of a com- constraints and had the design components of plete probe include optimizing for successful flexibility, accuracy, and energy efficiency, arterial oxygenation (SpO2) measurements.

Figure 5. Simultaneous recordings of ECG and PPG signals (photo courtesy of author).

72 Brinnae Bent

As noted above, obstructive lung dis- Acknowledgements ease, the fourth leading cause of death in the United States, is rising in prevalence at For the generous support and funding from an alarming rate [5]. Continuous health and the NSF Nanosystems Engineering Research environment monitoring systems that work Center for Advanced Self-Powered Systems in real-time would increase understanding of Integrated Sensors and Technologies of the health and environmental impacts on (ASSIST) through the Undergraduate chronic respiratory disease and empower in- Research Fellowship. dividuals to utilize the data and take control For the continued encouragement from Dr. of their respiratory condition. Not only will Bozkurt and the iBionicS Lab team. this system provide a better understanding For the mentorship from James of how external factors are affecting the pa- Dieffenderfer on this project and its integra- tient’s respiratory condition, but it can also tion into the Chronic Respiratory Disease shift health care into a preventative system Monitoring project. rather than simply treating the symptoms.

73 Explorations |Engineering

References

[1] J. P. Dieffenderfer, H. Goodell, B. Bent, E. Beppler, R. Jayakumar, M. Yokus, J. S. Jur, A. Bozkurt, “Wearable Wireless Sensors for Chronic Respiratory Disease Monitoring, “ IEEE Body Sensor Netowrks (BSN) 2015, 2015, no. 76, pend ing publication.

[2] J. P. Dieffenderfer, E. Beppler, T. Novak, E. Whitmire, R. Jayakumar, W. Qu, R. Rajagopalan, A. Bozkurt, and I. Member, “Solar powered wrist worn acquisition sys tem for continuous photoplethysmogram monitoring,” in EMBC, 2014.

[3] J. P. Dieffenderfer, M. C. Bair, and A. Bozkurt, “Towards a smartbandage with func tional near infrared spectroscopy,” IEEE Biowireless 2013, 2013, no. 4, pp. 13–15.

[4] P. Fung, G. Dumont, C. Ries, C. Mott, and M. Ansermino, “Continuous Noninvasive Blood Pressure Measurement by Pulse Transit Time,” in IEEE EMBS, 2004, vol. 2, no. 4, pp. 738–741.

[5] D. M. Mannino, R. C. Gagnon, T. L. Petty, E. Lydick, “Obstructive Lung Disease and Low Lung Function in Adults in the United States: Data from the National Health and Nutrition Examination Survey,” Arch Intern Med. 2000; 160(11):1683-1689.

[6] “Environmental Asthma Triggers.” Environmental Asthma Triggers. New York State Department of Health, Oct. 2011. Web. 27 Sept. 2015.

74 75 Explorations | Creative Work

Creative Work: A Self-Fulfilling Prophecy Artist: Jack Twiddy Faculty Mentor: Beth Grabowski Affiliation: University of North Carolina at Chapel Hill Dimensions: 40” x 60” Medium: Inkjet-Printed Digital Render

Artist Statement: My work deals with the notion that our understanding of death – as an inevitable consequence of life – is rationally unjustified, and further that our assumption of our own mortality serves as a self-fulfilling prophecy preventing us from overcoming this existential challenge. I see a twofold task for myself here: Firstly, to reach the viewer and get them to take such an idea seriously, and secondly, to provide a detailed and scientific rationale for the pursuit of an indefinite lifespan, as well as a plan for how to accomplish this feat. In my various pursuits, I address both components of this idea. My written work primarily deals with the latter goal, and my artwork is geared specifically towards the former. The work that I’ve produced for my thesis deals with the notion of the self-fulfilling prophecy, and how this relates to our society’s treatment of death. It serves as an attempt to break through the defense mechanisms of the viewer, to get them to realistically consider the seriousness of their life in a world where mortality is as-of-yet guaranteed, and to hopefully open their mind – through desperation, if for no other reason – to the possibility of our societal pursuit of radical life extension. It is my hope that in doing so, I might be able to inspire the general public – one individual at a time – to seriously consider and accept the very real possibility of overcoming the current mortal status of our species, and thus lift the current barriers to the research and political and economic support necessary to unshackle ourselves from the chains binding us to our own mortal fate.

The sculpture itself depicts an allegorical scene from a not-too-distant future, in which a progression of shackled individuals are led forward into a grave in the process of being dug. These individuals are escorted and forced forwards by their captors, but the power dynamic between the different characters is different than the dynamic found within other oppressive situations. If the viewer looks closely, they will observe that the aggressive figures themselves are attached to the same chain as those offering resistance. This is intended to serve as a metaphor for the behavior of our species regarding death. In our attempts to rationalize and defend our collective psyche against looming and ever-approaching obliteration, we have duped ourselves into an acceptance of this fate that nullifies any emerging attempts to challenge the inevitability of this outcome.

76 Jack Twiddy

A Self-Fulfilling Prophecy (Front), Jack Twiddy, 2015

A Self-Fulfilling Prophecy (Back), Jack Twiddy, 2015

Social Sciences Sensory Sensitivities of Young Adults with High-Functioning Autism Spectrum Disorders

Kendyl L. Cole Meredith College Faculty Mentor: Cynthia Edwards Meredith College

ABSTRACT The present study examines sensory processing issues in young adult males (n = 11) and fe- males (n = 3) with High-Functioning Autism Spectrum Disorders. Participants were currently enrolled or planning to enroll in a southeastern college. This study used The Adolescent/ Adult Sensory Profile® to measure the range of sensory processing issues within foursen- sory processing domains (Low Registration, Sensory Seeking, Sensory Sensitivity, and Sensory Avoidance) and six sensory stimulus modalities (Taste/Smell, Movement, Visual, Touch, Activity Level, Auditory). Scores showed more sensory processing issues in the Low Registration and Sensory Avoidance Quadrants compared to neurotypical norms. Further analysis showed a significant difference between sensory stimuli modality scores within all four sensory quadrants. The findings of this study may provide a more concrete understanding of the complex sensory issues that young adults with Autism Spectrum Disorders (ASD) face every day.

he diagnostic criteria for individuals previously diagnosed with Autistic Disorder Tliving with autism has rapidly shifted who have a higher level of cognitive and so- and evolved over the past twenty years, as cial functioning compared to individuals with more awareness and understanding of the more severe autism. The diagnostic criteria disorder expands. In The Diagnostic and for Asperger’s Syndrome in the DSM-IV-TR Statistical Manual of Mental Disorders was similar to High-Functioning Autism, but (4th ed., text rev.; DSM–IV-TR; American differed in two main domains: No clinically Psychiatric Association, 2000), Autistic significant delay in language or in cognitive Disorder (Autism) and Asperger’s Disorder abilities in childhood (DSM-IV-TR, 2000). (Asperger’s Syndrome) were separate diag- In 2013, the DSM-V was released and con- noses within the larger category of Pervasive tained revised diagnostic criteria for disorders Developmental Disorders. While not a di- within the former Pervasive Developmental agnostic category, High-Functioning Autism Disorders category. According to the (HFA) is a term used to categorize individuals DSM-V, Autistic Disorder and Asperger’s

80 Kendyl Cole

Disorder, among other diagnoses, are all cat- and functional levels of the sample popula- egorized under one disorder with differing tion vary greatly and comparisons across levels of severity, known as Autism Spectrum studies are difficult to make due to the vari- Disorder (ASD). The main diagnostic crite- ety of methodological techniques. The maria include: deficits in social communication jority of research on this topic has focused and interactions, restricted and repetitive on younger children with lower functionality, behaviors, interests or activities (including due to the more visible and quantitative na- atypical sensory behaviors), present symp- ture of the sensory processing issues in this toms in childhood, and symptoms limit and population (Ben-Sasson et al., 2009). Of the impair functioning (DSM-V, 2013). There limited research that includes adolescents exists a large variability in the severity of and adults, the sensory processing issues symptoms, manifestation of diagnostic crite- are often discussed as less significant com- ria, and functioning level of individuals with pared to children, and studies are limited by this diagnosis, and it is therefore viewed as a variability in age or severity of autism. The spectrum disorder to represent this variabil- available research on individuals with Autism ity. The present study was conducted with Spectrum Disorders is abundant (Ben-Sasson young adults who were diagnosed before the et al., 2009), and although literature related introduction of The Diagnostic and Statistical to sensory processing issues is not lacking, Manual of Mental Disorders (5th ed.; DSM– there still exists a lack of research geared 5; American Psychiatric Association, 2013), particularly towards adolescents and young and all participants disclosed diagnoses ac- adults on the higher end of the autism spec- cording to the DSM-IV-TR criteria. trum. As new diagnosis methods and thera- In the DSM-IV-TR (2000), sensory ab- pies are developed and implemented within normalities were not included as diagnostic the population, the number of individuals criteria for Autistic Disorder or Asperger’s with ASD better able to function and con- Disorder, though research from that time tribute to society is growing (Hart, Grigal, period indicated that differences in sensory & Weir, 2010). The number of students in processing did occur in these individuals higher education with a diagnosis of Autism (Ben-Sasson et al., 2009). The recognition Spectrum Disorder may be increasing (Hart of sensory behaviors as diagnostic criteria et al., 2010), but there still lacks an under- for Autism Spectrum Disorders is one of standing of the sensory processing issues of the most significant additions to the DSM-V this subset of the population that might be (2013). Atypical sensory behaviors can range impacting functioning in an educational set- significantly depending on the individual and ting. The present study seeks to understand the severity of autism but generally include the unexplored sensory processing issues of hyper or hypo-reactivity to sensory input or young-adults on the higher end of the autism sensory stimuli in the environment (DSM-V, spectrum in advanced educational settings. 2013). Among individuals higher on the Foundational work in this field includes autism spectrum, difficulties with social in- a study by Ben-Sasson et al. (2009) that ex- teraction are most predominant, but sensory amined the sensory modulation symptoms in abnormalities still persist and can severely individuals with autism spectrum disorders interfere with functionality in school, work, in a comprehensive analysis. The inclu- and everyday life (Dunn, Myles, & Orr, sion criteria for this meta-analysis was as 2002; Crane, Goodard, & Pring, 2009). follows: parent-report measure that covered Research on sensory processing issues of sensory processing over multiple modalities, individuals with Autism Spectrum Disorders individuals with ASDs as a distinct popula- has expanded with increased awareness of tion, reported the findings in English, in- this complex disorder. Although there is cluded a neurotypical or other developmen- substantial sensory research, the age groups tal disorder comparison group, and provided

81 Explorations |Social Sciences enough statistical information to perform an autism. This study described individuals (n effect size test. Distinct moderators were = 104) with a childhood diagnosis of autism, used to categorize the studies: chronologi- between 3 to 56 years of age. The caregiv- cal age, percent of autism diagnosis, and ers of participants completed The Sensory type of control group. A total of 14 studies Profile (Dunn, 1999) to assess the frequency were analyzed for scores in total sensitivity, of particular responses to sensory experi- over-responsitivity, under-responsitivity, and ences and the Childhood Autism Rating Scale sensory seeking (Ben-Sasson et al., 2009). (CARS) to assess the severity of autism for The 14 studies comparing ASD with a neu- each individual. Participants were divided rotypical control group found that effect size into three age groups in order to analyze the across all studies was significant for all sen- correlation between age, severity of autism, sory scores ( d = .82 - 2.01). The analysis and multisensory dysfunction: 3 to 12 years also concluded that of the four sensory mo- of age (n1 = 37); 13 to 25 years of age (n2 = dalities, under-responsitivity occurred most 33); and 26 and over (n3 = 34). Correlational often, then over-sensitivity and sensory seek- analyses were performed to examine the pos- ing. Chronological age was found to be a sible relationship between abnormal sensory significant factor across all sensory modali- processing and severity of autism, as well as ties, and effect size tended to increase from change in age as a factor in the relationship birth until age nine, and decreased thereaf- between Sensory Profile and CARS scores. ter. The mean effect size for the chronic age No correlation was found between scores on groups was as follows: 0-3 years (d = 1.27), The Sensory Profile and CARS, indicating 3-6 years (d = 1.58), 6-9 years (d = 1.89), < 9 that overall sensory processing abnormali- years (d = 1.26). The meta-analysis showed ties may not be related to severity of autism. that individuals with ASD’s show heightened These findings indicated that sensory process- sensory modality symptoms when compared ing abnormalities are consistent in the autism to a neurotypical control group, but show population independent of functioning level, variability in magnitude when chronologi- although the extent of these issues was not cal age and severity of autism are considered indicated. Correlation analysis of age groups (Ben-Sasson et al., 2009). Findings report found a significant positive correlation be- that individuals on the autism spectrum show tween abnormal sensory processing issues substantial and significant sensory process- and CARS score in children, but no correla- ing issues when compared to a neurotypical tion was found within the adolescent/young group. This meta-analysis suggests that the adult or adult participants (Kern et al., 2007). next step for research in this field is to ad- The results of this study indicate a possible dress the following question: To what extent negative relationship between severity of do individuals with ASD show decreased sensory abnormalities and age, finding that as sensory processing issues after age nine? age increases, sensory processing abnormali- The research of Ben-Sasson et al. (2009) ties seem to lessen in severity. Although the served as the foundation for the present study findings of this study are significant and shed in regards to overall sensory processing is- light on the complex sensory processing ab- sues in individuals with ASD, but left gaps normalities of this population, there are still in areas relating to age and functioning level. questions in the research left unanswered. Ben-Sasson et al. (2009) provided statisti- The sensory sensitivities in adolescents and cal evidence that individuals with ASD expe- adults may be less severe than in children, rience abnormal sensory symptoms. In addi- but the extent of these issues is unclear. tion, Kern et al. (2007) examined the relation Research examining children with higher- between auditory, visual, touch, and oral sen- functioning Autism Spectrum Disorders was sory dysfunction in autism and the relation conducted by Dunn et al. (2002) and differed to multisensory dysfunction and severity of from the research of Kern et al. (2007) by

82 Kendyl Cole examining the specific sensory processing disorders manifest significant sensory pro- issues associated with Asperger’s syndrome cessing issues in childhood positively cor- in children when compared to a neurotypical related with severity of autism, but the in- group. The Sensory Profile was used to mea- tensity of these abnormalities decreases with sure the sensory processing issues of both par- age. The findings of Dunn et al. (2002) more ticipant groups. The profile consisted of 125 thoroughly examined the relation between questions that measured the degree in which individuals with less “severe” autism and children showed difficulties in sensory pro- sensory abnormalities, showing that children cessing, behavioral and emotional responses, with Asperger’s manifest substantially differ- responsiveness to sensory events, and modu- ent sensory processing when compared to a lation. The study found that children with neurotypical group. Crane et al. (2009) fo- Asperger’s syndrome were significantly dif- cused on a larger group of adults with ASD, ferent from the neurotypical control group on using a well-known and implemented clinical 22 of 23 items on the Sensory Profile. These questionnaire. The present study sought to results revealed that children with Asperger’s replicate the research of Crane et al. (2009), syndrome show a significantly different pat- but within the subgroup of young adults with tern of sensory processing issues from their High-Functioning Autism and Asperger’s peers without disabilities (Dunn et al., 2002). syndrome in a higher education setting. No Previous research provides a limited un- previous research has examined this group derstanding of how individuals with Autism specifically, and with a growing number of Spectrum Disorders (ASD) process and react young adults on the autism spectrum pursu- to sensory stimuli in the environment because ing further education after high school (Hart it does not examine all subgroups within the et al., 2010), the need to research this group broad spectrum, specifically adolescents and was apparent. adults on the higher-functioning end of the The hypotheses for the present study were: autism spectrum. Crane et al. (2009) provided 1) Young adults with higher-functioning more information about this subgroup by re- Autism Spectrum Disorders will manifest searching sensory processing in older adults more sensory processing issues in at least with ASD. The Adult/Adolescent Sensory two quadrants when compared to neurotypi- Profile (AASP) is a 60 item self-report ques- cal standards, and 2) Sensory modality scores tionnaire that describes everyday situations will differ significantly within all sensory where sensory problems are encountered, processing quadrants. and is divided into four quadrants: low registration, sensation seeking, sensory sensi- Method tivity, sensation avoiding. Crane et al. found a significant difference in overall scores on Participants the AASP between ASD and a comparison group; adults with ASD displayed signifi- Undergraduate male (n = 11, ages 18- cantly higher AASP scores in the low reg- 24) and female (n = 3, ages 18-24) college istration, sensory sensitivity, and sensation students diagnosed with High-Functioning avoidance quadrants, but scored lower in the Autism or Asperger’s Syndrome were re- sensation seeking quadrant. The results of cruited. Participants were currently enrolled Crane et al.’s research provided a foundation or planning to enroll in a higher education for the present study, confirming that older establishment in North Carolina. The un- individuals with high-functioning ASD dis- dergraduate students were recruited through play sensory processing issues. Disability Services at a small private liberal Previous research conducted by Ben- arts college, a large state university from the Sasson et al. (2009) and Kern et al. (2007) in- same city, and from several agencies associ- dicated that individuals with autism spectrum ated with the local autism advocacy groups.

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Diagnosis for participants associated with 60-question survey measuring the extent local advocacy groups was self-reported on of sensory processing issues among adults the consent form. The present study was and adolescents Participants answered how conducted with young adults who were often a certain sensory related situation ap- diagnosed before the introduction of The plied to them using a Likert scale of Almost Diagnostic and Statistical Manual of Mental Never, Seldom, Occasionally, Frequently, Disorders (5th ed.; DSM–5; American and Almost Always. The profile is divided Psychiatric Association, 2013), and all par- into six different sensory stimuli modali- ticipants disclosed diagnoses according to ties: Taste/Smell, Movement, Visual, Touch, the DSM-IV-TR criteria. The present study Activity Level, Auditory. Each modality respected the choice of some participants to contains questions related to four unique sen- identify diagnoses under the previous DSM, sory processing quadrants: Low Registration, but to remain consistent with the current Sensation Seeking, Sensory Sensitivity, and DSM-V, all participants in the study are la- Sensation Avoiding. The sensory quadrants beled as having “High-Functioning Autism are used to describe the response of an in- Spectrum Disorder.” The participants were dividual when processing sensory stimuli in representative of the autism population (1:4 the environment. Crane et al. (2009) defined female to male). Participation was voluntary each of the sensory quadrants as follows: and anonymous and participants were treated “Low registration: Responding slowly or in accordance with the “Ethical Principles of not noticing sensory stimuli; Sensation seek- Psychologists and Code of Conduct” (APA, ing: Actively pursuing sensory information; 2002). The Meredith College Internal Review Sensory sensitivity: Experiencing discom- Board approved the present study. fort in response to sensory stimuli; Sensation avoiding: Engaging in behaviors designed Comparison Group to reduce exposure to sensory stimuli.” (pp. 19) A Scale Information Sheet was provided Brown and Dunn (2002) developed a to quantify the Likert scale for individuals standardization study for the AASP using a who were not able to understand the qualita- sample of 950 adolescents and adults consid- tive nature of the scale. The AASP contains ered to represent the neurotypical population a Summary Score Sheet that sums items without disabilities. The neurotypical sample from each sensory quadrant to create the sen- was separated into three distinct age groups: sory profile. The Adolescent/Adult Sensory adolescents (n = 193, ages 11-17), adults (n = Profile also contains a Quadrant Summary 496, ages 18-64), and older adults (n = 261, Chart for three age groups, containing clas- ages ≥65). Sensory profiles and summary sifications based on an AASP standardiza- charts were created for each group. Mean tion study of neurotypical adolescents and age, standard deviation, and participant re- adults (Brown & Dunn, 2002). The Quadrant cruitment methods were not disclosed. The Summary Chart is divided into five classifi- present study used the results of Brown & cations: Much Less Than Most People, Less Dunn’s AASP standardization study as a Than Most People, Similar To Most People, comparison for the High-functioning ASD More Than Most People, and Much More group. Than Most People. Once participants were recruited, they Materials & Procedure were required to provide consent, and asked to complete a 60-question survey measuring The Adolescent/Adult Sensory Profile® individual range of sensory processing is- (Brown & Dunn, 2002), a Scale Information sues. Surveys were either mailed to a partici- Sheet, and a writing utensil were provided. pant’s home to be mailed back or they were The Adolescent/Adult Sensory Profile is a completed in person at a designated location.

84 Kendyl Cole

Raw scores for each quadrant were totaled of sensory stimuli: Taste/Smell, Movement, on the Summary Score Sheet. Each quadrant Visual, Touch, Activity Level, and Auditory. raw score was compared with the neurotypi- Scoring for the profile is based on the four cal norms included in the AASP [Brown and sensory quadrants and does not contain in- Dunn (2002)] and fell into a range of classifi- dividual scores for each sensory modality. cations (Much Less Than Most People, Less Previous research using this profile (Kern Than Most People, Similar To Most People, et al., 2007; Dunn et al., 2002; Crane et al., More Than Most People, Much More Than 2009) defines the extent of sensory pro- Most People). cessing issues based on how an individual responds to sensory stimuli, quantified by Results sensory quadrant scores (i.e. low registration, sensation avoidant, sensation seeking). By Inferential statistics were not appropriate only using quadrant scores to demonstrate when analyzing the sensory quadrant data sensory processing issues, previous research due to the small sample size (N = 14), thus generalized that individuals had only one descriptive statistics were used for analysis sensory response for each modality. Each of this mixed design. The AASP Quadrant sensory processing quadrant was analyzed Summary Chart was used to compare aver- through a univariate general linear model age quadrant scores of participants with the to examine trends among specific sensory comparison group of individuals without stimuli modalities. The present study found disabilities. On average, participants scored significant differences between mean sensory higher in the Low Registration Quadrant and processing modality scores within all four the Sensation Avoiding Quadrant compared sensory quadrants (Figure 1). to the neurotypical comparison group. The The Sensation Seeking quadrant had sig- neurotypical range in the Low Registration nificant differences between mean sensory Quadrant is a score between 24 and 35, plac- processing modality scores [F (5, 60) = 8.49, ing the participant average score ( M = 39.21, p = .000]. Post hoc comparisons using the SD = 7.44) outside of the range and into the LSD test indicated the following: the Taste/ More Than Most People (36 to 44) classifica- Smell modality was significantly lower than tion of the AASP summary chart. The neu- the Movement, Visual, and Auditory mo- rotypical range in the Sensation Avoiding dalities; the Movement modality was sig- Quadrant is a score between 27 and 41, plac- nificantly higher than the Touch and Activity ing the participant average score ( M = 44.64, level modalities; the Visual modality was sig- SD = 10.49) outside of the range and into the nificantly higher than the Touch and Activity More Than Most People (42 to 49) classifica- level modalities; the Touch modality was sig- tion of the AASP summary chart. The mean nificantly lower than the Auditory modality; score in the Sensory Sensitivity Quadrant was the Activity level modality was significantly 41. 43 (SD = 10.01) and Sensation Seeking lower than the Auditory modality. Quadrant was 44.86 (SD = 6.05), with par- The Sensory Sensitivity quadrant had ticipants scoring similarly to the neurotypi- significant differences between mean sen- cal comparison group and within the Similar sory processing modality scores [F (5, 60) = To Most People classification. These results 10.45, p = .000]. Post hoc comparisons us- indicated that participants in this study on av- ing the LSD test indicated the following: the erage score outside of the neurotypical range Movement modality was significantly lower and show more sensory processing issues in than the Touch modality; the Activity Level the Low Registration and Sensation Avoidant and Auditory modalities were significantly Quadrants than most people. higher than all other modalities. The Adult/Adolescent Sensory Profile con- The Sensation Avoidant quadrant had sig- tains six distinct modalities related to types nificant differences between mean sensory

85 Explorations |Social Sciences

Taste/Smell Movement Visual Taste/Smell Touch Movement Ac1vity Level Visual Auditory Touch Ac1vity Level Auditory 5.00 5.00

4.00 4.00

3.00 3.00

2.00 2.00 MeanLikert Scale Score MeanLikert Scale Score

1.00 1.00

0.00 0.00 Sensory Sensi1vity Sensory Sensa1on Sensi1vity Avoidant Sensa1on Sensa1on Seeking Avoidant Low Sensa1on Registra1on Seeking Low Registra1on Sensory Quadrant Sensory Quadrant

Figure 1. Mean Likert scale scores for sensory processing modalities by quadrant. Error bars represent standard deviations.

86 Kendyl Cole processing modality scores [F (5, 60) = present study, the unique sensory sensitivi- 17.50, p = .000]. Post hoc comparisons us- ties of the participants were made clear by the ing the LSD test indicated that the Movement abnormal sensory processing trends within sensory modality was significantly lower high-functioning ASD group compared with than all other modalities. a neurotypical group, and the further analy- The Low Registration quadrant had sig- sis of modalities. The meta-analysis pro- nificant differences between mean sensory vides empirical evidence that individuals processing modality scores [F (5, 60) = with ASD show heightened sensory modality 11.21, p = .000]. Post hoc comparisons us- symptoms when compared to a neurotypical ing the LSD test indicated the following: the control group, but show variability in mag- Visual modality was significantly lower than nitude when chronological age and severity all other modalities except for Touch; the of autism are considered (Ben-Sasson et al., Activity Level and Auditory modalities were 2009). The present study supported these significantly higher than the Taste/Smell, findings, and provided more concrete evi- Movement, and Touch modalities. dence for the variability in sensory modality These results revealed the complexity of symptoms within a small subset of the ASD sensory processing issues of participants, population (higher-functioning young adults) showing that individuals with ASD have spe- that was previously unexamined. cific sensory responses for each stimulus, and Similarly, Kern et al. (2007) examined the the sensory processing issues cannot be gen- relationship between multisensory dysfunc- eralized by sensory quadrant. tion and severity of autism in individuals (N = 104) with a childhood diagnosis of autism, Discussion representing multiple age groups and functioning levels. The findings of Kern et al. The hypotheses of the present study were: (2007) in regards to processing modalities 1) Young adults with higher-functioning were further validated by the present study, Autism Spectrum Disorders will manifest but current findings indicate sensory abnor- more sensory processing issues in at least malities exist in an older population with two quadrants than a neurotypical compari- higher-functioning ASD. The present study son group,and 2) Sensory modality scores found that participants with higher-func- will differ significantly within all sensory tioning ASD had unique sensory process- quadrants. Based on the results of the data, ing issues when compared to a neurotypical the hypotheses were supported. comparison group, regardless of their age The meta-analysis conducted by Ben- and severity of autism. It is not possible to Sasson et al. (2009) was further validated conclude that these sensory processing issues by the present study. The findings in the are less severe than individuals in a younger meta-analysis revealed that individuals with age group because the AASP standardiza- ASD show heightened sensory modality tion results for neurotypical adults were used symptoms when compared to a neurotypi- instead of a college-age comparison group. cal control group. The meta-analysis found However, the present study does show that that of the four sensory modalities, under- sensory processing issues still exist in indi- responsitivity occurred most often, followed viduals with ASD after childhood, indepen- by over-sensitivity and sensory seeking. The dent of their severity of autism. These differ- results of the present study validate the over- ences in findings might be due to the different all findings of the meta-analysis, finding that methods of measurement, but that they could in the particular subset of young adults with also be indicative of a difference in percep- higher-functioning ASD, Low Registration tion between parents and guardians versus and Sensation Avoiding quadrants showed the individual with ASD. the most sensory processing issues. In the Crane et al. (2009) expanded previous

87 Explorations |Social Sciences research on higher-functioning children with Sensory Profile. In regards to the hard copy ASD to adults with autism spectrum disor- version of the profile, the grading system ders and the sensory processing issues among only allows for results in relating to sensory this group. The present study validated the quadrants. The present study shows that dis- findings of the Crane et al.(2009) study. The playing sensory processing results only in studies are very similar, but the present study relation to how sensory information is pro- targeted young adults in a higher education cessed as a whole, and not how this informa- setting. Crane et al. (2009) found a signifi- tion is processed based on the sensory stimuli cant difference in overall scores on the AASP is limiting and does not fully reveal the extent between ASD and the comparison group. of an individuals sensory processing issues. Results specifically revealed that adults with The profile could be improved by including ASD displayed significantly higher AASP a more in depth analysis of sensory process- scores in the low registration, sensory sensi- ing issues in relation to each sensory modal- tivity, and sensation avoidance quadrants, but ity. By adding more depth to the analysis of scored lower in the sensation seeking quad- results, the profile would be able to not only rant (Crane et al., 2009). The present study display sensory quadrant scores as a whole, found similar results, showing that young but also the sensory modality score within adults with higher-functioning ASD had each of the sensory quadrants. This modifi- higher AASP scores in the low registration cation would make the profile more appropri- and sensation avoidant quadrant, but scored ate for a population where sensory process- within neurotypical range in the sensation ing issues are complex and variable. seeking and sensory sensitivity quadrant. While the results of the present study indi- Further investigation is needed to determine cate that there is a difference in the way that whether the difference in findings regarding young adults with higher-functioning ASD the sensory sensitivity quadrant is simply an process sensory information when com- artifact of the smaller sample size and limited pared to a neurotypical group, this prelimi- age range, or whether it represents a real dif- nary study is limited. The biggest limitation ference specific to the present population. to this study is the small sample size (N=14) Results of the present study indicate sub- and lack of control group, allowing for great stantial sensory processing issues in young variability in the results. A t-test was not con- adults with higher-functioning Autism ducted due to a lack of access to the norma- Spectrum Disorders. Further analysis of the tive data set used for the AASP control group. data showed that sensory stimuli modalities The particular sub-population of individuals within each quadrant are significantly differ- with ASD was small, but previous research ent. The present study brings to light very has been conducted with similar sized sam- unique and complex sensory sensitivities in a ples (Crane et al., 2009). The present study subset of individuals with ASD that have not relied on the normative data included in the been considered or researched previously. Adolescent/Adult Sensory Profile to compare The potential impact of this research spans ASD scores, and did not actively recruit a multiple fields, some of which include: oc- college-age comparison group. The distribu- cupational therapy, vocational rehabilitation, tion of age in the AASP neurotypical group and higher education. (ages 18-64) was much greater than the ASD Further analysis of sensory modalities group (ages 18-23) used in this study, possi- found that participants may have overall sen- bly limiting the validity of results. Sampling sory processing trends, but the way they pro- in the present study was subject to selection cessing sensory information depends greatly bias due to the use of existing groups (i.e., on the particular stimuli/sensory modality. the AASP neurotypical sample) and a con- This information could be used to further venience sample (i.e., the ASD group). The modify and redesign the Adolescent/Adult present study did not use any psychological

88 Kendyl Cole assessments to validate the self-disclosed di- their sensory processing issues, while faculty agnosis of participants, although Disability members would be able to further adapt and Services of local colleges and autism advo- modify their classrooms and teaching meth- cacy groups provided significant proof of ods to best accommodate the sensory pro- diagnoses. Participants were not asked to cessing needs of their students. For example, disclose any diagnoses besides ASD, and if a particular student scored abnormally high comorbidity could have affected the internal on the Sensory Sensitivity quadrant, particu- validity of the present study. larly in response to visual stimuli, a professor Participants in the present study were pur- might then be able to adjust the lighting in the suing or planning to pursue a degree from classroom to make it easier for the student to a higher educational institute. The applica- focus in class. Similarly, if a student scored tions of this study are most appropriately abnormally high in the Sensation Avoidant geared towards helping individuals with quadrant in regards to large groups of people, higher-functioning ASD adjust and succeed the faculty members might instead place the in a higher education setting. It is crucial that student in smaller class sizes. individuals with these diagnoses receive the In conclusion, young adults diagnosed with support necessary to grow and advance their higher-functioning ASD manifest sensory unique abilities and skills. Further research processing issues in the Low Registration should include a neurotypical comparison and Sensation Avoiding Quadrant outside of group and use inferential statistics to analyze neurotypical norms. Despite trends in the sen- sensory processing differences. Research sory processing quadrants as a whole, within in the future could examine the satisfaction each sensory quadrant there was substantial rates of individuals with higher-functioning variability between the sensory stimuli mo- ASD in a higher education institute where dalities. The data show that young-adults The Adolescent/Adult Sensory Profile® with Higher-Functioning Autism Spectrum is given to professors. By providing both Disorders can have different sensory process- students and faculty with the results of this ing experiences than the neurotypical popula- profile, individuals with higher-functioning tion, and their sensory processing issues as a ASD can better quantify and understand whole cannot be easily generalized.

References

American Psychological Association. (2002). Ethical principles of psychologists and code of conduct. American Psychologist, 57(12), 1060-1073.

American Psychiatric Association. (2013). Autism Spectrum Disorder. In Diagnostic and statistical manual of mental disorders (5th ed.)

American Psychiatric Association. (2000). Pervasive Developmental Disorder. In Diagnostic and statistical manual of mental disorders (4th ed., text rev.)

Ben-Sasson, A., Hen, L., Fluss, R., Cermak, S. A., Engel-Yeger, B., & Gal, E. (2009). A meta-analysis of sensory modulation symptoms in individuals with autism spec trum disorders. Journal of Autism and Developmental Disorders, 39(1), 1-11. doi: 10.1007/s10803-008-0593-3

Brown, C. & Dunn, W. (2002). Adolescent/Adult Sensory Profile [Measurement instrument]. San Antonio, TX: Pearson

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Crane, L., Goodard, L., & Pring, L. (2009). Sensory processing in adults with autism spectrum disorders. Autism, 13(1), 215. doi: 10.1177/1362361309103794

Dunn, W. (1999). The Sensory Profile [Measurement instrument]. San Antonio, TX: Pearson

Dunn, W., Myles, B. S., & Orr, S. (2002) Sensory processing issues associated with Asperger’s syndrome: A preliminary investigation. American Journal of Occupational Therapy, 56(1), 97-102. doi: 10.5014/ajot.56.1.97

Hart, D., Grigal, M., & Weir, C. (2010). Expanding the paradigm: Postsecondary education op tions of individuals with autism spectrum disorder and intellectual disabilities. Focus on Autism and Other Developmental Disabilities, 25(3), 134–150

Kern, J. K., Trivedi, M. H., Grannemann, B. D., Garver, C. R., Johnson, D. G., Andrews, A. A., & Schroeder, J. L. (2007). Sensory correlations in autism. Autism, 11(2), 123- 134. doi: 10.1177/1362361307075702

90 91 Explorations |Social Sciences

Rapid Acquisition of Preference in Concurrent Chains: Effects of Morphine on Sensitivity to Reinforcement Amount

William D. Fetzner University of North Carolina Wilmington Faculty Mentor: Raymond C. Pitts University of North Carolina Wilmington

ABSTRACT It previously has been reported that acute administration of opiates (e.g., morphine) increases “impulsive” choice (i.e., shifts preference toward a smaller, sooner reinforcer). In the present study, effects of morphine on sensitivity to reinforcement amount under a concurrent-chains schedule were examined using a rapid-acquisition procedure. Six pigeons were given the choice between two different reinforcement amounts. The side keys associated with the larger and smaller reinforcers changed pseudo-randomly across sessions. Under this procedure, relatively high sensitivities to reinforcement amount were obtained (0.59-1.53). Once preference consistently tracked the change in reinforcement amount across sessions, doses of morphine (0.3 – 3.0 mg/kg) were administered. Typically, at intermediate doses (in which response rates were not substantially reduced), a modest decrease in sensitivity to reinforcement amount was observed. These results suggest one potential behavioral mechanism of morphine’s effects on impulsive choice: a drug-induced decrease in sensitivity to reinforcement amount.

veryone is faced with choices in their past. Skinner (1963) described behavior that Edaily lives. These choices can be simple is controlled by its consequences as operant (e.g., deciding what to wear in the morning) behavior. In this view, choice can be consid- or complicated (e.g., deciding where to attend ered operant behavior (i.e., our choices are college). What causes us to make a certain controlled by their consequences). decision given the choice between the behav- Behavior analysts study operant behavior ioral alternatives is fundamentally important. to understand the basic principles that govern For example, when a boy is sitting quietly in our decisions. In the above example, the boy a classroom, it could be said that he is mak- has the choice between multiple behavioral ing the decision to remain quietly in his seat options (e.g., sitting and listening, talking while the teacher is talking, rather than do- with classmates, being disruptive). Through ing numerous other reproachable things, such many years of experimental analysis, it has as talking to his neighbor or acting out. The been found that critical variables influencing boy’s decision under these circumstances human choice can be studied in nonhuman likely is based upon the consequences of animals under controlled experimental con- those actions that he has experienced in the ditions (see McDowell, 1988). Considerable

92 William Fetzner evidence suggests that choices made by all important), then the individual is likely to animals (including humans) are governed by make an impulsive choice. Conversely, if similar principles (e.g., reinforcement and control by reinforcement amount is relatively punishment). high (i.e., the difference in the amounts is relatively important) and the degree of control “Impulsive” Choice by delay is relatively low (i.e., the difference in the delays is relatively unimportant), then A major topic of interest in the study of the individual is likely to make a self-control decision making is why suboptimal decisions choice. The degree of control that a given are made. A subject is said to make a subopti- parameter of reinforcement (amount or de- mal decision when they choose an alternative lay) has over the subject’s preference for one that may not produce the most reinforcement choice over the other is known as the sen- in the long run. One type of suboptimal be- sitivity to that parameter (see Baum, 1974). havior is impulsive choice. When an indi- Relatively high sensitivity to delay predicts vidual is presented with the choice between impulsive choice and relatively high sensitiv- a smaller, sooner reinforcer, or SSR, and a ity to amount predicts self-control choice. larger, later reinforcer, or LLR, the individual is said to make an impulsive choice if they Behavioral Pharmacology of Impulsive choose the SSR. Conversely, if the individ- Choice ual chooses the LLR, they are considered to make a self-controlled choice (e.g., Madden Recent attention has been directed to the & Johnson, 2010). In the above example, study of the effects of drugs on impulsive the boy in the classroom is said to make an choice. (e.g., de Wit & Mitchell, 2010). impulsive choice if he talks to his neighbor There are at least two reasons for this. First, or acts out during class while the teacher is there has been a long history with the use of talking, and is said to make a self-controlled drugs therapeutically to ameliorate problems choice if he sits quietly and listens. associated with impulsive behavior. For ex- A key factor that contributes to impulsive ample, stimulants such as methylphenidate choice is the diminishing effect of delay on (Ritalin®) and amphetamine (Adderall®) are the value of a reinforcer. This phenomenon commonly prescribed to individuals diag- is known as delay discounting. Data from nu- nosed with ADHD (Sibley, Kuriyan, Evans, merous studies have shown that the effective- Waxmonsky, & Smith, 2014). Therefore, ba- ness of a reinforcer is a decreasing, hyper- sic researchers have been interested in deter- bolic, function of its delay (e.g., Mazur, 1987, mining effects of stimulant drugs on impul- 1988; Richards, Mitchell, De Wit, & Seiden, sive choice in laboratory animal models (e.g., 1997). Another key factor is the size of the Pitts & McKinney, 2005; Pitts & Febbo, 2004; reinforcer. All else being equal (e.g., if there Richards, Sabol, & de Wit, 1999; Slezak & is no delay), an individual usually selects a Anderson, 2011). A goal in conducting basic larger over a smaller reinforcer. Thus, there research with these sorts of drugs is to deter- are two important variables at work to determine which drug classes decrease the likeli- mine impulsive choices: the difference in the hood of impulsive choice and, thus, to deter- sizes of the reinforcers and the difference in mine the behavioral mechanisms involved the delays to the reinforcers. The degree of in those effects (e.g., Maguire, Rodewald, control by both these variables can influence Hughes, & Pitts, 2009; Pitts & Febbo, 2004; the type of choice. If control by reinforce- Ta, Grace, McLean, Hughes, & Pitts, 2008). ment amount is relatively low (i.e., the differ- A second reason to study effects of drugs ence in the amounts is relatively unimport- on impulsive choice is the considerable evi- ant) and control by delay is relatively high dence showing an association between sub- (i.e., the difference in the delays is relatively stance abuse and impulsive behavior. (see

93 Explorations |Social Sciences

Perry & Carroll, 2008). For example, opi- reflect a reduced sensitivity to reinforcement ate addicts exhibit higher levels of impulsive amount; that is, the difference in the size of choice than do non-addicted control partici- the reinforcers is less important and, thus, the pants (e.g., Madden, Petry, Badger, & Bickel, individual is less likely to choose the larger 1997). Although data such as those reported reinforcer. Finally, both of these behavioral by Madden et al. (1997) do not allow for a mechanisms could operate simultaneously to causal inference (it also may be the case increase impulsive choice. that those who are more impulsive are more One approach to identifying the behavioral likely to abuse drugs), they do suggest the mechanisms associated with opiate effects possibility that exposure to opiates can in- on impulsive choice is to study each variable crease the likelihood of impulsive behavior, (delay and amount) separately. Cummings which would be an undesired side-effect of (2010) tested effects of morphine on the these drugs, even if used appropriately. sensitivity to reinforcement delay using a In a number of experiments with nonhu- rapid-acquisition, concurrent-chains proce- mans, opiates (usually morphine) consis- dure. This procedure is an effective and effi- tently have been shown to increase impulsive cient method for characterizing sensitivity to choice (Harvey-Lewis, Perdrizet, & Franklin, various parameters of reinforcement (Grace, 2012; Kieres et al., 2004; Pattij, Schetters, Brageson, & McLean 2003; Maguire, Janssen, Wiskerke, & Schoffelmeer, 2009; Hughes, & Pitts, 2007), and for testing effects Pitts & McKinney, 2005). That is, admin- of drugs on control of choice by these param- istration of morphine increases choice of an eters (Maguire et al., 2009; Ta et al., 2008). SSR. These findings are consistent with the In Cummings’ study, pigeons responded un- notion that opiate exposure can increase im- der a concurrent-chains procedure with vari- pulsive choice, and suggest that the findings able-interval (VI) 10-s initial links. During reported by Madden et al. (1997) may reflect the initial (choice) links, two response keys a direct effect of opiate exposure. were illuminated and the pigeon could peck either one. Pecks on each side key lead to Behavioral Mechanisms of Drug Action separate terminal link (the outcome link). One terminal link was a fixed-interval (FI) In addition to identifying classes of drugs 4-s (shorter delay) and the other was a FI 8-s that decrease impulsive choice and classes schedule (longer delay). That is, choice re- of drugs that increase impulsive choice, it sponding in the initial link was based upon also is important to identify the behavioral the delays to reinforcement provided in the mechanisms of those effects (Pitts, 2014; terminal links – one choice lead to a shorter Thompson, 1984). As noted above, two im- delay and one choice lead to a longer delay. portant behavioral variables determining In the rapid-acquisition feature of the proce- impulsive choice are sensitivity to reinforce- dure, the side keys associated with the shorter ment delay and sensitivity to reinforcement and longer delays alternated unpredictably amount. One aim of basic research is to de- across sessions; in some sessions the shorter termine if one, or both, of these behavioral delay was on the left and in other sessions, mechanisms is involved in the observed ef- the shorter delay was on the right. Therefore, fects on impulsive choice. For example, the within each session, the pigeons had to learn increase in impulsive choice produced by which side key had the shorter delay, and ad- opiates could reflect an increase the sensi- just their choices accordingly. After several tivity to reinforcement delay; that is, opiates sessions under this procedure, the pigeons could cause the individual to be hypersensi- learned to track the reinforcement delay; in tive to reinforcement delay and, thus, choose those sessions when the shorter delay was on the more immediate reinforcer. Alternatively, the left, the pigeons learned to prefer the left the increase in impulsive behavior could key and in those sessions when the shorter

94 William Fetzner delay was on the right, the pigeons learned to behavioral mechanism of morphine’s effects prefer the right key (i.e., the pigeons showed on impulsive choice. a relatively strong sensitivity to delay). After the pigeons learned to track the changing de- Method lays across sessions, effects of several doses of morphine were tested. Morphine tended Subjects to bring the allocation of responses made by the pigeon toward indifference. That is, mor- Six male racing homer pigeons were phine decreased the overall sensitivity to re- maintained at 85% of their free-feeding inforcement delay. This finding suggests that body weights with post-session feedings morphine lessens the control of reinforce- of Mazuri® Gamebird Maintenance diet. ment delay over choice. Pigeons were housed individually in a colony Interestingly, Cummings’ (2010) find- room operating under a 12/12-hr light/dark ings are inconsistent with those from previ- cycle (lights on at 7:00 am). Water and grit ous studies showing that morphine increases were provided for the pigeons continuously impulsive choice. All else being equal, a in their home cages. Pigeons 409 and 17560 decrease in the sensitivity to delay would had previously responded under the con- predict a decrease, rather than an increase, in current-chains procedure described below. impulsive choice. However, there also is an- Pigeon 17567 had previously responded un- other possibility. Perhaps the effects of opi- der a similar procedure involving reinforce- ates on impulsive choice are related mainly ment probability. Pigeons 4299, 4295, and to effects on sensitivity to reinforcement 27938 were experimentally naïve. amount. The current study investigated that possibility. Apparatus

Current Study Each pigeon was placed in its own operant chamber. The dimensions for chambers 1-4 Given the puzzling results of the were 32 cm x 25.4 cm x 32.6 cm; those for Cummings (2010) study, it is important to chambers 5 and 6 were 32 cm x 24.6 cm x determine the effects of opiates on sensitivity 33 cm (henceforth, when measurements dif- to reinforcement amount. Thus, the purpose fer, those for chambers 1-4 are listed first). of the present study was to investigate effects The side walls and ceiling were made of of morphine on sensitivity to reinforcement Plexiglas. The front and back walls were amount using a rapid-acquisition concurrent- made of aluminum. Each chamber was en- chains procedure. Reinforcement delay was closed in a light- and sound-attenuating cu- held constant, while reinforcement amount bicle which was equipped with an air-cir- was varied between the two options. As noted culation fan. White noise was broadcast by above, previous studies have shown that opi- speakers to mask extraneous noises. Pigeons ates increase impulsive choice (Kieres et al., received food from a feeder opening (6.0 cm 2004; Pattij et al., 2009; Pitts & McKinney, x 4.8 cm; 6.3 cm x 5.7 cm) which was cen- 2005). Based upon these findings, it could tered on the front wall of the chamber, 6.4 be predicted that morphine would decrease cm/5.5 cm above the floor. Located within sensitivity to reinforcement amount in the the opening was a small aperture from which current study (i.e., that morphine adminis- the pigeons received milo grain reinforce- tration would cause the subject to view the ment via a hopper. Three response keys, each difference in the reinforcer amounts as less 2.5 cm in diameter, were arranged horizon- important). Such an outcome in the current tally 12.8/11 cm above the feeding window. study would suggest that a decrease in sensi- The center key was 6.85 cm/5.15 cm be- tivity to reinforcement amount is a potential tween the left and right keys (measured from

95 Explorations |Social Sciences center-to-center). The force required to oper- key was not signaled to the pigeon. Entry ate the keys ranged from 0.12-0.42 N; within into the terminal link was provided accord- a chamber, the force required to operate each ing to a VI schedule - the first peck on the key was similar. The houselight (1.6 cm di- target key after a variable amount of time had ameter) in boxes 1-4 was located 1.1 cm elapsed led to its terminal link. For every 6 from the ceiling on the opposite wall from cycles, the right key paid off 3 times and the the keys. The houselight (1.1 cm diameter) in left key paid off 3 times (i.e., the pigeons had boxes 5 and 6 was located 7.0 cm above the to sample both keys during the initial links). center key. The houselight was illuminated A 2-s changeover delay (COD) was in effect throughout the session except when the hop- to prevent reinforcement of rapid switching per was presented. During the hopper presen- between the two keys. The delivery of rein- tations the houselight extinguished and the forcement ended each cycle. food window was illuminated. A Windows Upon entry into a terminal link, the target computer located in a nearby control room key changed to its associated color (red or controlled all events within each session and green) and the other key turned off. For three recorded the data. of the pigeons (409, 17567, 4299), the left and right terminal-link colors were green and Behavioral Procedure red, respectively; for the other three pigeons, this was reversed. Each terminal-link was a Initial training fixed-interval (FI) schedule of food presentation; the first peck on the illuminated side The three experienced pigeons (409, key after the FI had elapsed provided food. 17560, and 17567) were placed directly on Each terminal link was associated with a dif- the rapid-acquisition, concurrent-chains pro- ferent reinforcement amount. The larger re- cedure described below. The naïve pigeons inforcer amount was four consecutive 1.2-s (4299, 4295, and 27938) were acclimated to hopper presentations, and the smaller rein- the chamber and trained to eat from the hop- forcer amount was two consecutive 1.2-s per when it was raised. Once these pigeons hopper presentations. Thus, the amount ratio ate reliably from the hopper immediately (larger to smaller) was 2/1 (i.e., the larger upon presentation, key peck training was ini- reinforcer was twice the size of the smaller tiated. During these sessions, the center key reinforcer). Pigeons 17567 and 4295 had was illuminated white and key pecking was showed a relatively small preference for the shaped by differentially reinforcing succes- larger reinforcer at 2/1, so their amount ra- sive approximations. After center key peck- tio was increased to 3/1 (the larger reinforcer ing was acquired, the pigeons were trained to consisted of six 1.2-s hopper presentations). peck each of the side keys. Consecutive hopper presentations were separated by 0.5-s, during which the hopper light Concurrent-chains procedure remained on. The naïve pigeons (4299, 4295, and 27938) The concurrent-chains procedure was began the training on the concurrent-chains composed of cycles of initial (choice) and schedule with shortened VI and FI schedules. terminal (outcome) links. Each cycle began During this time the larger reinforcer re- with the initial links, during which the two mained on one side (left or right) until a clear side keys were illuminated white. At the be- preference formed for that side. Then the side ginning of each cycle either the left or right keys associated with the larger and smaller key was chosen randomly by the computer reinforcers were reversed, and remained in as the target key (the key that would pay off effect until the preference reversed accord- with access to its terminal link); the target ingly. The VI initial-link and FI terminal-link

96 William Fetzner schedules were then increased to 10 s and 5 by visually inspecting graphs of the log re- s, respectively, and the rapid-acquisition pro- sponse ratios (L/R) and the log amount ra- cedure began. tios across sessions. If the log response ratio consistently followed the variation in log Rapid-Acquisition Procedure amount, than this was considered to be consistent tracking. Doses of morphine (0.3-3.0 In this procedure, the side keys associ- mg/kg) then were administered intramuscu- ated with the larger and smaller reinforcers larly 15-min before the start of selected ex- changed across sessions unpredictably ac- perimental sessions. Saline vehicle injections cording to a 31-step pseudo-random binary also were given intermittently throughout the sequence (PRBS; Hunter & Davison, 1985). dosing regimen as a control for the injection That is, for some sessions, the larger and procedure. A dose was given only if there had smaller reinforcers were associated with the been 2 days since the previous injection and left and right terminal links, respectively; for only when the previous session’s data were the other sessions, this relation was reversed. consistent with the previous 10 non-injection This 31-step sequence was repeated through- control sessions. Unless otherwise indicated, out the study. Thus, for some sessions, the each dose and saline was administered a min- ratio (L/R) of reinforcer amounts was 2/1 imum of 4 times (twice for each session type: (or 3/1 for Pigeons 17567 & 4295), and for large left/small right and large right/small other sessions the ratio of reinforcer amounts left). Doses were tested in a haphazard order. was 1/2 (or 1/3). The PRBS ensures that, for For each pigeon, the highest dose of mor- any given session, the pigeons could not de- phine tested was one that decreased response termine the sides associated with the larger rates to the point that the pigeon no longer re- and smaller reinforcer beforehand. This is liably finished all of the cycles. These doses because there are no sequential dependencies were 1.0 mg/kg for 409 and 4295, 1.78 mg/ in the sequence. Within a given session, the kg for 17560 and 27938, and 3.0 mg/kg for sides associated with the larger and smaller 4299 and 17567. reinforcer remained constant; that is, for any given session, the pigeons had to learn Data Analysis which side was associated with the larger and smaller reinforcers and adjust their choice The dependent variable in this study was accordingly. Sessions ended after 72 initial- the allocation of responses to the left and right link/terminal-link cycles or 60 min, which- keys during the initial links. For each session, ever occurred first. A diagram illustrating the log10 response ratio (L/R) during the ini- the rapid-acquisition concurrent-chains pro- tial links was obtained. Log response ratios cedure is presented in Figure 1; this figure were then compared to log10 amount ratios provides an example for a pigeon with the (L/R). The degree to which preference was red key associated with the left terminal link controlled by reinforcer amount was numeri- and the green key associated with the right cally expressed as sensitivity (Baum, 1974). terminal link. Sensitivity was calculated by plotting log response ratios on the y-axis against the log Pharmacological Procedure amount ratios on the x-axis. A linear regression line was fit to these data points; slope Once the pigeons tracked the larger rein- of this line indicated the degree of sensitiv- forcer consistently as it changed randomly ity. A slope of 1.0 indicates that the change across sessions (i.e., they consistently showed in the log response ratio matched the change a preference for the larger reinforcer), the in the log amount ratio. Bias was reflected by morphine regimen began. Consistent track- the y-intercept of the line (a positive value ing of the larger reinforcer was determined indicates a bias for the left key and a negative

97 Explorations |Social Sciences ! ! !28! ! Large Left Sessions!

Initial Links! VI 10-!s! ! 2-!s! COD! ! VI 10-! s! !

Terminal Links!

FI 5-s FI 5-s

4 Hopper Presentations! 2 Hopper Presentations! (Large Reinforcer)! (Small Reinforcer)! !

FI 5-s FI 5-s

! Figure 1. Diagram of the rapid-acquisition concurrent-chains procedure. In the initial Figure(choice) 1. Diagram links, ofthe the two rapid white-acquisition side keys concurrentwere available-chains concurrently. procedure. Entries In the initialinto the (choice) terminal links , the twolinks white were side available keys wereunder available a VI 10-s concurrently. schedule. Each Entries terminal into link the consistedterminal linksof an wereFI 5-s avai schedlable- underule. a VIIn some10-s schedule. sessions (large Each left;terminal top panel), link consisted the left terminal of an FI link 5-s leadschedule. to 4 hopper In some presentations sessions (large left; (largetop panel), reinforcer) the left and terminal the right link terminal lead to link4 hopper lead to presentations 2 hopper presentations (large reinforcer) (small areinforcer).nd the right terminalIn other link sessionslead to 2 (large hopper right; presentation bottom panel),s (small the reinforcer). left terminal In linkother lead sessions to the (large small right;reinforcer bottom paneland), the the left right terminal terminal link link lead lead to tothe large small reinforcer. reinforcer These and thesessions right alternatedterminal link unpredictably lead to large via reinforcer.a pseudo-random These sessions binary alternated sequence. unpredictably The colors (red via and a pseudo green)- randomassociated binary with sequence.the left and The right colors (red terminaland green) links associated were counterbalanced with the left and across right pigeons. terminal In linksthe figure were redcounterbalanced and green lights across that pigeons.were In thedisplayed figure red during and greensession lights are represented that were displayed by diagonal during lines session and diamonds, are represented respectively. by diagonal See text lines and diamonds,for additional respectively. details. See text for additional details.

98 William Fetzner value indicates a bias for the right key). left and right keys, respectively. For all pi- Due to the nature of the PRBS, the pigeons geons, allocation of responses to each key had to determine the side that was associated was well controlled by the location of the with the larger reinforcer as the session pro- side that produced the larger reinforcer, in- gressed. Because each side produced access dicating that the pigeons were able to track to its terminal link an equal number of times it fairly consistently. Sensitivity and bias within each set of 6 cycles, each session values for each pigeon are presented within can be divided into 12 sections, or blocks. each graph. Sensitivity to reinforcement Acquisition of preference was determined by amount during baseline ranged from 0.47 to plotting log response ratios (L/R) across ses- 1.67 across pigeons. The performances of sion 12ths and was illustrated by an increas- Pigeons 17560 and 27938 were particularly ing preference for the side with the larger re- noteworthy; these pigeons showed high sen- inforcer across the session. sitivity and low bias throughout the last 30 Overall initial link response rates for each baseline sessions. session were analyzed to determine morphine’s effects on overall response output Morphine Administration (these would be considered “non-specific” drug effects). Overall initial-link response Figure 3 shows the effects of different rates were calculated by dividing the sum of doses of morphine (0.3 – 1.7 mg/kg) on the left and right responses during the initial the log ratio of responses for all pigeons. link by the total time spent in the initial link. For each plot, the unfilled circles and filled Separate one-way, repeated-measures squares show means from sessions when the ANOVAs were performed to analyze ef- larger reinforcer was on the left and right, fects of morphine on sensitivity to amount, respectively. The unconnected points above bias, and overall initial-link response rates. C and S show data for control (sessions im- Inferential statistical analyses for sensitivity mediately preceding injections) and saline and bias included saline and doses of mor- administration, respectively. The remaining phine that did not decrease overall initial-link points show effects of morphine. In this fig- response rates to below 80% of control rates. ure, only doses that did not affect overall re- Analyses for overall initial-link response sponse rates are shown. A morphine-induced rates included saline and the doses that were reduction in preference is depicted whenever tested in each pigeon. When an ANOVA re- the curves come together. Although there was sult was significant, Tukey’s post-hoc analy- some variability within subjects, this appears ses were conducted. All statistical tests were to occur, at least to some degree, in 5 of the performed using Vassarstats.net (alpha was 6 pigeons (except 4295), at one or two of the .05). doses (usually 1.0 and 1.7 mg/kg). Thus, in 5 of 6 cases, it appears that morphine tended Results to shift response ratios towards indifference. Effects of morphine on both sensitivity Baseline Performance to reinforcement amount and overall initial- link response rates for each pigeon are pre- Figure 2 shows log ratios for reinforce- sented in Figure 4. The sensitivity values ment amount (unfilled circles) and for re- are represented by the filled triangles, and sponses (filled squares) over the 30 sessions response rates are represented by the un- preceding the first saline injection. For re- filled diamonds; for each pigeon, sensitivity inforcement amount, positive (0.3, or 0.48 values are plotted only for doses that did not for Pigeons 17567 and 4295) and negative substantially alter response rates. For each (-0.3, or -0.48) values show sessions when pigeon, except 4295, effects of morphine on the larger reinforcer was associated with the sensitivity were selective. That is, at least

99 Explorations |Social Sciences Log Ratio (L/R)

Sessions

Figure 2. These graphs represent the daily log ratios (L/R) over the 30 days prior to morphine administrations. The unfilled circles represent the log ratio of the reinforcement amount. Positive log ratios for reinforcer amount (0.3 or 0.48) are from sessions when the larger reinforcer was associated with the left terminal link and negative log ratios (-0.3 or -0.48) are from sessions when the larger reinforcer was associated with the right terminal link. The filled squares represent log ratios of responses made on the left and right keys during the initial links. Positive values correspond to a greater number of responses made on the left side and negative values represent a greater number of responses on the right side. Sensitivity and bias values for each pigeon are inserted at the top right of each graph.

100 William Fetzner Log Ratio (L/R)

C S 0.3 0.56 1.0 1.7 C S 0.3 0.56 1.0 1.7 Morphine (mg/kg)

Figure 3. These graphs provide dose-effect functions for choice for individual pigeons by showing the log ratio of responses (L/R) for control (C), saline (S), and doses of morphine (0.3 mg/kg-1.7 mg/kg). Unfilled circles show data from sessions when the larger reinforcer was on the left key, and filled squares represent data from sessions when the larger reinforcer was on the right key. Data points are means. Error bars for saline and drug data show ranges; because the sample size for the control sessions was substantially larger, the error bars for these points show interquartile ranges. Data points without error bars indicate only one administration. If a subject did not finish a session due to a substantial decrease in overall initial-link response rates from the drug, then the data from these sessions were not included.

101 Explorations |Social Sciences Resp/Min Sensitivity

Morphine (mg/kg)

Figure 4. These graphs show morphine dose-effect functions for sensitivity (left y-axes) and overall initial-link response rates (right y-axes). . For points of sensitivity Only doses at which the pigeon finished the session are shown. Sensitivity values were calculated by plotting the log ratio of responses versus the log ratio of amount and determining the slope of the corresponding regression line. Note the individualized y-axis scales. Filled triangles with the solid black line denote the calculated sensitivity values. Unfilled diamonds with dashed line represent the response rates at the given dose. In order to represent the contrast of the significant decrease in responding, response rate points were plotted for the highest dose that each pigeon received. Doses higher than 1.0 mg/kg were not tested in Pigeon 409 because of issues with emesis (see Footnote 2).

102 William D. Fetzner one (and sometimes more than one) dose de- their responses across the sessions. That is, creased sensitivity to reinforcement amount as the session progressed, the log ratio of re- without substantially affecting response sponses increasingly favored the side with rates. This effect typically occurred at the 1.0 the larger reinforcer. For all pigeons, except mg/kg and/or 1.7 mg/kg. Selective effects 4295, morphine administration tended to were most prominent for Pigeons 17567 and reduce the acquisition of preference for the 17560. For Pigeon 409, the decrease in sen- larger reinforcer within each session (shown sitivity at 1.0 mg/kg was accompanied by a by the functions with open symbols). That is, slight decrease in responses rates. For Pigeon as the session progressed, the data for mor- 4299, 1.7 mg/kg produced a modest decrease phine are closer to indifference than the data in sensitivity without affecting response rate. for saline. This effect is shown most dramati- Figure 5 shows effects of morphine on cally in Pigeons 4299, 17560, and 27938. bias. Under baseline, one pigeon (409) had a For these pigeons, most of the data points relatively large bias for the right key and one following morphine administration fell out- pigeon (27938) had a relatively large bias for side the error bars for saline, and closer to the left key. A couple of other pigeons (4295 indifference, indicating a reliable reduction and 17567) had a slight bias for the right key. in the acquisition of preference based upon Morphine occasionally changed bias (409, reinforcement amount. 17567, 4295, & 27938), but the direction of the change was not systematic. More impor- Discussion tantly, the systematic changes in sensitivity shown in Figure 4 were not necessarily ac- Under baseline, the behavior of the six companied by a change in bias. pigeons was well controlled by reinforce- Group data for sensitivity, overall response ment amount, which was expected given rates, and bias are shown in Figure 6. One- past literature using similar procedures (e.g., way ANOVAs revealed a significant effect of Grace et al., 2003; Kyonka & Grace, 2008; morphine on sensitivity to amount [F(3,12) = Maguire et al., 2007, 2009). Sensitivity to 5.11; p=0.017]1, but not on bias [F(3, 15) = reinforcement amount in the present study 1.67, p = 0.216]. Post-hoc analysis revealed a was higher than has typically been reported. significant decrease in sensitivity at 1.0 mg/ For example, Maguire et al. (2007) reported kg. Morphine also produced a significant that whole-session values for sensitivity to decrease in response rates [F(4,20)=6.52, reinforcement ranged from 0.1-0.24. Indeed, p=0.0016]2. Importantly, post-hoc analyses the values obtained in the present study were revealed that only 1.7 mg/kg significantly de- similar to the highest reported sensitivity val- creased rates. Thus, for the group, morphine ues in the literature for reinforcement amount produced selective effects on sensitivity at (McLean and Blampied, 2001). 1.0 mg/kg. That is, this dose decreased sensi- One potential reason for the relatively high tivity without affecting bias or overall initial- degree of sensitivity to amount in the pres- link response rates. ent study was the use of a concurrent-chains Figure 7 shows effects of morphine on the procedure with a favorable ratio of initial- acquisition of preference within each session. link (VI 10 s) to terminal-link (FI 5 s) dura- These graphs show log ratios of responses tions (see also Kyonka & Grace, 2008). This across 12ths of the session. Lines with error would be expected to produce relatively high bars show data from saline sessions (positive levels of sensitivity to reinforcement param- and negative values are from sessions where eters (see Fantino, 1977; Grace, 1994), com- the larger reinforcer was on the left and right, pared to the standard concurrent-schedule respectively). These data illustrate that each arrangement used by Maguire et al. (2007, pigeon acquired a preference for the larger 2009). reinforcer within each session by adjusting

103 Explorations |Social Sciences Bias

Morphine (mg/kg)

Figure 5. These graphs show dose-effect functions for bias for each pigeons. Bias was calculated by finding the y-intercept of the same equation used to calculate sensitivity. All other features of these graphs are as described in Figures 3 and 4.

104 William Fetzner Resp/Min Sensitivity Bias

Morphine (mg/kg)

Figure 6. Group dose-effect functions for sensitivity and overall initial-link response rates (top), and for bias (middle). Each of the data points on the graphs are the group averages. Error bars indicate SEM. Data for doses producing statistically significant effects compared to saline are indicated by asterisks. All other features of these graphs are as described in Figures 4 and 5.

105 Explorations | Social Sciences Log Ratio (L/R)

Session 12ths

Figure 7. These graphs illustrate within-session acquisition of preference by showing log response ratios across twelves of the session, and depict effects of selected morphine doses on acquisition. The specific dose selected for each pigeon (indicated at the top of each graph) was one that did not substantially affect overall initial-link response rates. The lines without symbols on these graphs represent data that w obtained from sessions in which saline was administered. Data points show means and error bars show standard deviations. Effects of morphine are shown by the two lines of data with unfilled symbols. The circles and squares show data from sessions in which the larger reinforcer was associated with the left and right terminal links, respectively.

106 William Fetzner

Effects of Morphine issues noted above, there was considerable evidence that morphine selectively decreased Morphine decreased sensitivity to rein- sensitivity to reinforcement amount overall forcement amount in 5 of the 6 pigeons. (the group analyses presented in Figure 6), This suggests that given a choice between and in 4, and possibly 5, of the individual pi- a larger reinforcer and a smaller reinforcer, geons (the individual-subject data shown in these pigeons would not distinguish between Figures 4 and 5). the two as effectively under the influence of Some issues with the present data do sug- morphine. gest that any conclusions regarding selective Morphine produced a statistically signifi- effects should be made with caution. For cant decrease in sensitivity to reinforcement example, in a couple of cases, the effects of amount at 1.0 mg/kg. Importantly, this dose morphine on sensitivity were not directly did not significantly affect bias or overall dose-related (e.g., Pigeon 17560’s function initial-link response rates. Thus, on aver- was slightly U-shaped and Pigeon 27938’s age, the effects of 1.0 mg/kg on sensitivity function was slightly inverted U-shaped; see to amount were selective. That is, this dose Figure 4). Also, Figure 3 indicates that the ef- changed preference controlled by reinforce- fects of morphine on choice were somewhat ment amount without changing other aspects variable for some of the pigeons. Finally, for of behavior. Examining the individual-sub- Pigeon 4295, morphine did not alter sensitiv- ject data (Figures 4 and 5), relatively clear ity at any dose that did not decrease overall selective effects on sensitivity to amount response rates. Interestingly, response rates were obtained in 4 of the 6 pigeons, usually for this pigeon, and for Pigeon 409 (whose at more than one dose (17567 at 0.56, 1.0 effects on sensitivity to amount were not and 1.7 mg/kg; 4299 at 1.0 and 1.7 mg/kg, clearly selective), were decreased at lower 17560 at 0.3, 0.56, and 1.0 mg/kg; and 27938 doses than for most of the other pigeons. It at 0.3 and 0.56 mg/kg). Furthermore, hints is possible that the dose window between ef- of selective effects occurred at other doses fects on sensitivity to amount and effects on (e.g., 27938 at 1.0 mg/kg, although this dose overall response rates for Pigeons 4295 and slightly decreased overall response rates) and 409 simply was too narrow to reveal selec- in one of the other pigeons (409 at 0.56 mg/ tive effects. kg, although these effects were only slightly below saline, which was considerably below Possible Behavioral Mechanism of control). Morphine There are at least two reasons to why it is important to find a dose that selectively af- Despite the above considerations, the pres- fects sensitivity to reinforcement amount. ent data suggest that a decrease in sensitivity Such a finding allows us to conclude more to reinforcement amount is a potential behav- confidently that the effect on sensitivity was ioral mechanism of morphine’s effects on im- not the result of a lack of motivation to re- pulsive choice. Based upon the present data, spond, or the result of impairment in motor it could be predicted that under the influence ability. At higher doses, morphine and other of morphine (and perhaps other opiates), an opiates can significantly reduce appetite and individual would favor the larger reinforcer can impair motor functioning (see Maisto, less than usual, which would result in an in- Galizio, & Connors, 2011). We were par- crease in impulsive choice. This prediction ticularly interested in determining whether is consistent with the increases in impulsive morphine affects how subjects allocate re- choice previously reported (Harvey-Lewis sponses based upon reinforcement amount, et al., 2012; Kieres et al., 2004; Pattij et al., not simply whether or not it affects motor ca- 2009; Pitts & McKinney, 2005). pacity or willingness to respond. Despite the The present data are somewhat puzzling,

107 Explorations | Social Sciences however, when considered in the context of overpower the decreasing effects on sensitiv- Cummings’ (2010) data. Cummings found ity to amount, thus prompting a decrease in that morphine decreased sensitivity to rein- impulsive choice. A final possibility for the forcement delay. Thus, the results of these contradictory findings is that sensitivity data two studies make contradictory predictions obtained under rapid-acquisition concurrent- regarding impulsive choice. The decrease chains procedures may not relate to impul- in sensitivity to amount found in the pres- sive choice. That is, we may be attempting ent study predicts an increase in impulsive to generalize the data from these procedures choice, whereas the decrease in sensitivity to to circumstances in which they simply do not reinforcement delay reported by Cummings apply. suggests a decrease in impulsive choice. One possibility for these contradictory predic- Broader Implications tions is that there is some common variable across the two that is causing the sensitivities It has been shown that opiate abusers show to decrease for both delay and amount. It is more impulsive behaviors than do matched unclear, however, what that common variable controls. Although more studies clearly need might be, especially given that the effects on to be conducted (e.g., the direction of cau- sensitivity in both studies were selective. A sation has not been clearly established and second possibility is that the findings of one other variables likely are operating), the pres- (or both) of the studies simply are not reli- ent findings do suggest one potential reason able (e.g., they reflect a “Type I error”). Both why opiate abusers make more impulsive of these studies should be replicated to de- decisions than do non-abusers (e.g., Madden termine if the effects are reliable. A third et al., 1997). An opiate-abuser may behave possibility is that both sensitivity to amount more impulsively because of the direct ef- and sensitivity to delay actually are relevant fect of opiates on sensitivity to reinforcement behavioral mechanisms that contribute to amount. That is, under the influence of opi- morphine’s effects on impulsive choice. It is ates (either acutely or chronically), individu- possible that in most of the procedures pre- als are less sensitive to reinforcement amount viously used to examine effects of opiates, and, therefore, tend to make more impulsive the effects on sensitivity to reinforcement decisions. Based upon this interpretation, amount simply overpowered the effects on one avenue would be to study effects of sensitivity to reinforcement delay. This pos- chronically administered opiates on sensitiv- sibility is rendered more viable in light of ity to amount (and delay). previous findings that stimulant drugs, which The present research could be useful in un- typically decrease impulsive choice, also derstanding maladaptive effects of opiates, decrease sensitivity to both reinforcement both in those who abuse them and in those delay and reinforcement amount (Maguire who use them appropriately. If opiate effects et al., 2009; Pitts & Febbo, 2004; Ta et al. on impulsive behavior can be understood 2008). Thus, it may be that for opiates, the more completely, we might be in a better decreasing effects on sensitivity to amount position to develop procedures to help opi- overpower the decreasing effects on sensi- ate users make more adaptive choices. This tivity to delay, thus prompting an increase would be an important step in creating new in impulsive choice; whereas, for stimulants, methods to combat some of the maladaptive the decreasing effects on sensitivity to delay effects of opiates.

108 William Fetzner

Footnotes Acknowledgements

1 In order to be able to conduct the repeated- I would like to thank the lab gang and measures ANOVAs for sensitivity and bias up everyone who has been involved with the through the 1.0 mg/kg dose (which substan- gathering of my research throughout my tially decreased overall initial-link response numerous semesters in the lab. I would also rates for Pigeon 4295), the data for Pigeon like to thank my mentor, Dr. Raymond Pitts 4295 at 0.56 mg/kg were entered in for 1.0 for his considerable guidance throughout the mg/kg. That is, we adopted a conservative many semesters I have been working on this approach and assumed no effect of 1.0 mg/kg project. on sensitivity or bias for this pigeon. I would like to thank Dr. Christine Hughes for all of the editing help in creating this 2 Although Pigeon 409 usually responded at manuscript into the form it is in today. 1.0 mg/kg morphine, this dose sometimes I would also like to thank the reviewers and produced emesis in this pigeon. Therefore, editor of the Explorations Journal for taking higher doses were not tested in Pigeon 409. the time to review my manuscript and sup- In order to be able to include this pigeon’s porting the decision to publish it. data in the repeated-measures ANOVA for Lastly, I would like to thank The Center response rate, we also adopted a conserva- for Support of Undergraduate Research and tive approach here: the data obtained at 1.0 Fellowships (CSURF) for providing me with mg/kg for this pigeon were entered in at 1.7 $300 for the reimbursement of supplies that mg/kg. That is, no change was assumed, even were all desperately needed and well utilized though it seems probable that this pigeon’s for my thesis research. rate would have decreased at the 1.7 mg/kg dose.

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111 Explorations About the Student Authors Brinnae Bent core outcomes for children with Autism is currently a senior at North Carolina Spectrum Disorder. She aspires to continue State University studying Biomedical her education by pursuing a professional Engineering in the Joint UNC-NC STATE degree in occupational therapy, and hopes to BME Department with minors in Biological integrate her interest in sensory processing Sciences and Nanotechnology. Through and passion for individuals with autism caregiving roles in a nursing home for spectrum disorder into an impactful career. Alzheimer’s patients and with Cerebral Palsy of Mideast Wisconsin, she developed a sense William Fetzner of responsibility to use the skills she learned is currently an undergraduate student at the in her degree to solve the health care problems University of North Carolina Wilmington, that surround us, starting in local nursing where he is pursuing his two degrees in homes and working towards global solutions. Psychology and Biology with a concentration Outside of class, Brinnae is involved in being in Terrestrial Conservation. In addition to an Engineering Ambassador, K-12 STEM his studies, he is the secretary of the Beta outreach, and teaching engineering courses. Beta Beta Biological Honors Society club She is excited to continue pursuing medical at UNCW. In addition, he works with the research in a Neural Engineering graduate Student Recreation Center on campus as an program. intramural sports supervisor. He also works for Sea Coast Staffing Company, where he works as a server at different clubs and Sarah Burke centers in Wilmington, NC. He aspires to is currently an undergraduate student at East continue his education by pursuing a PhD in Carolina University, where she is pursuing the field research of animal behavior. With a her BS degree in Pubic History. In addition PhD in animal behavior he hopes to be able to her studies, she is currently working with to develop new, innovative methods for the the College of Education on a grant from the conservation of endangered species. He hopes Library of Congress that aims to integrate to integrate his experience of studying animal more primary sources into elementary-level behavior in both the fields of psychology and social studies curriculum. Post-graduation, biology to gain a unique perspective on his she plans to pursue a Master of Arts in behavioral field research. Teaching degree for elementary education. Courtney Hockett Kendyl Cole is currently an undergraduate student at the pursued her undergraduate degree at University of North Carolina at Pembroke, Meredith College where she received her where she is pursuing a bachelor’s degree B.A in Psychology and Spanish in May in Art Education with a minor in Creative 2015. In addition to her studies, she worked Writing. In addition to her studies, she is for many years with various individuals on active in the Pembroke Undergraduate the autism spectrum in clinical, therapeutic, Research Center (PURC) program on and recreational settings. She is currently a campus and has received over eight grants in Clinical Trials Assistant at the Duke Center the past two years. Courtney enjoys spending for Autism and Brain Development, where her weekends at the North Carolina Museum she is working on a study evaluating the of Natural Sciences where she works in the efficacy of umbilical cord blood to improve Discovery Room as a Weekend Captain; she

112 About the Student Authors also works within the Exhibits Department her graduate studies at Clemson University. in which she helps repair and build models There she hopes to obtain a Ph. D. in Biological inside the room. Her goal is to become a Sciences with a focus on physiological as Museum Educator, and achieve important well as pedagogical research. She hopes to creative and technical skills which will pursue a career in academia upon completion enable her to become a successful visual of her graduate studies. artist and writer. Sejiro Littleton Aleah Howell graduated from Davidson College in 2015 is a recent graduate of the University of North obtaining a BS in Biology, with a minor in Carolina at Chapel Hill, class of 2015, where Computer Science. He is currently working she earned a dual degree in studio art and as a Laboratory Technician in Dr. Sarafova’s journalism, graphic design. She is currently Lab researching the development of T cells a first-year graduate student at UNC working and the transcriptional regulation of the Cd4 towards her masters in Communications gene. His interests include bioinformatics, with a focus in visual communication and immunology, astrobiology, and the visual interactive multimedia. In addition to her arts. While working Sejiro is currently in studies, she is a research specialist in the the process of applying to PhD programs to UNC Library communications department further his education. where she specializes in information graphics and photo/video projects. Her research Dylan Ludwick interests include mental health awareness and recently graduated from Davidson College community journalism. in North Carolina with a BS in Biology. He is currently continuing his education in mathematics at Washington State University. Jack Koch He plans to further his understanding of is currently a graduate student at Oregon biology by pursuing graduate study in State University, where he is pursuing his biological physics. PhD degree in Integrative Biology in the laboratory of Virginia Weis. He recently graduated from the University of North Jack Twiddy Carolina Wilmington (UNCW) with his is a 2015 graduate of the University of North Honors BS degree in Marine Biology with Carolina at Chapel Hill where he graduated a minor in Chemistry. In addition to his with honors in Studio Art and a received studies at UNCW, he has taken field courses double major in Philosophy. His studio art in Marine Invertebrate Zoology and Ocean honors thesis, entitled “A Self-Fulfilling Acidification at University of Washington Prophecy” was motivated by ideas of radical life extension, longevity, mortality, and Friday Harbor Laboratories, and in Coral visual allegory. His other interests include Reef Ecology at the Bermuda Institute of the intersection between art and emerging Ocean Sciences. After obtaining his PhD, he science and technologies. He currently works hopes to integrate his interests in research for North Carolina State University, where and education as a professor at a university. he hopes to begin a Masters in Integrated Manufacturing Systems Engineering in Kaitlyn Leonard 2016. “A Self-Fulfilling Prophecy” and is a recent graduate from Wingate University its accompanying thesis statement can be where she obtained her Bachelors of Science viewed in the Carolina Digital Repository: Degree in Biology. She is currently pursuing https://cdr.lib.unc.edu/

113 Explorations About the Faculty Mentors

Acchia Albury, PhD Wade Dudley, PhD is currently serving as an Assistant Professor Wade G. Dudley, Ph.D.is a Teaching Professor at Wingate University in the Department of in East Carolina University’s Department Biological Sciences. Dr. Albury received of History, specializing in Naval and North her Bachelors of Science Degree in Biology Carolina History. Dr. Dudley holds an MA from Benedict College (Columbia, SC) and in Nautical Archaeology (ECU 1997) and her Ph. D. in Biology from the University a Ph.D. in History (University of Alabama of South Carolina (Columbia, SC) where at Tuscaloosa 1999).He is a member of the she focused on the underlying mechanisms university’s graduate faculty and advisor to of programmed cell death during flight the Lambda-Eta Chapter of Phi Alpha Theta muscle histolysis in the house cricket, History Honor Society. Professor Dudley’s Acheta domesticus. In addition, Dr. Albury publications include eight books and several completed a postdoctoral fellowship at the dozen chapters, articles, and short stories. University of Houston, where she focused on Among the books is Splintering the Wooden the molecular and functional consequences Wall: The British Blockade of the United of mutations of cardiac Troponin C and their States, 1812-1815, which received a John role in hypertrophic cardiomyopathy. Lyman Book Award from NASOH.

Cynthia Edwards, PhD Alper Bozkurt, PhD is a Professor of Psychology at Meredith serves as an Assistant Professor in the College. Edwards’ research focuses on the role Department of Electrical and Computer of peer social networks in the development of Engineering at North Carolina State girls and women. She has conducted a series University and as an associate faculty of studies on the social networks formed member in the Joint Department of by girls in dormitory and summer camp Biomedical Engineering-University of North settings, and her current project is a cross- Carolina Chapel Hill and North Carolina sequential study of cognitive-social networks State University. He graduated from and key life transitions among women. Bogazici University with a BS in Electrical Edwards enjoys working collaboratively and Electronics Engineering and earned with Meredith undergraduate students in an MS in Biomedical Engineering from the development of research projects, and Drexel University and a PhD in Electrical has mentored numerous students to national Engineering from Cornell University. His conference presentations in both psychology research interests include the development and interdisciplinary Honors. of microscale sensors, actuators and methodologies to unlock the mysteries of biological systems with an aim of engineering these systems directly or developing new Beth Grabowski, MFA engineering approaches by learning from is an artist and educator. She is a three-time these systems. recipient of support from the North Carolina Arts Council (two Artist Fellowships and a Project Grant.) Her work has been represented widely in national and international venues. Grabowski is the Zachary Taylor

114 About the Faculty Mentors

Smith Distinguished Professor of Art at the University of Canterbury in New Zealand. University of North Carolina-Chapel Hill, His current research focuses on the behavior where she has taught printmaking and book analysis and behavioral pharmacology of arts since 1985. Beth is co-author, with Bill choice and preference, with a focus on Fick, of Printmaking, A Complete Guide determining the behavioral mechanisms by to Materials and Processes, published by which drugs affect ‘impulsive’ and ‘risky’ Laurence King of London. The first edition choices. was released in 2009, and a second edition in the summer of 2015. She served as president of SGC International, the professional organization for print artists, educators, Brandon Sanderson, collectors, and enthusiasts from 2012-2014 MFA and currently serves on the SGCI Board of is a native of Kansas, who split his formative Directors. years between rural Kansas and Colorado Springs, Colorado. He holds a B.S. from Colorado State University-Pueblo in Printmaking and Computer Information Joseph R. Pawlik, PhD Systems and an M.F.A. in Printmaking from is a professor in the Department of Biology the University of South Dakota, where he and Marine Biology at the University of studied under Lloyd Menard. Sanderson has North Carolina Wilmington. He graduated been at the University of North Carolina- from the University of Minnesota, Twin Pembroke since 2008. He has participated in Cities with a BS in Biology and earned a PhD more than 350 venues since 2008, including in Marine Biology from Scripps Institution 70 international exhibitions. Since 2008, of Oceanography UC-San Diego, California. he has brought 36 visiting artists to UNCP His research group studies the ecology of and held 18 visiting artist workshops at 16 sponges on Caribbean coral reefs. universities nationally. He maintains a private printmaking and drawing studio in rural Red Springs, North Carolina. Raymond C. Pitts, PhD is a Professor of Psychology at the University of North Carolina Wilmington (UNCW). He Sophia Sarafova, PhD earned his Bachelor’s, Master’s, and PhD is an associate professor in the Biology degrees in Psychology from the University Department at Davidson College where of Florida, with specialties in Behavior she leads the Duke-Davidson Immunology Analysis and Behavioral Pharmacology. Partnership. She graduated from Swarthmore Prior to coming to UNCW, he completed College with a BA in biochemistry, continued a post-doctoral fellowship in Behavioral on to earn a PhD in immunology from Pharmacology in the Department of Columbia University and did postdoctoral Physiology and Pharmacology at the training at the National Cancer Institute. Medical School of Wake Forest University Her research interest is in the fields of and was a Research Assistant Professor at the gene regulation and its contribution to University of North Carolina Chapel Hill. In developmental decisions of immune system 2005 he was a Visiting Erskine Fellow at the cells.

115 Explorations Submission Process Who is Eligible? The primary author or authors must be undergraduates at a 2 or 4 year college or university in the state of North Carolina working on original research under the direction of a faculty mentor. Works may be co- authored. Students at North Carolina School of Science and Mathematics are also eligible.

What to Submit? We are seeking research papers, critical essays (literature/research reviews, articles written on a particular topic), or media submissions of performing/fine art endeavors. Text of papers should be no more than 6000 words. Explorations, the Journal of Undergraduate Research and Creative Activities for the State of North Carolina, provides opportunities for a variety of text and media submissions in the following categories:

Biological, Earth, and Physical Sciences Performing Arts Business and Legal Social Sciences Creative Writing and Discourse Technology and Engineering Humanities Visual Arts and Design Mathematics

How to Submit? Guidelines for publication: 1. Submit all articles (without images) in Word documents (.doc or .docx) only, and indicate where images, graphs, maps, or charts should appear. 2. Submit images, graphs, maps, and charts as separate files. For creating graphs and charts (in Excel, Illustrator, or Paint): make the image as LARGE as possible. This will ensure its visibility in the publication. In addition, also save figures as images (.jpg, see below). 3. Images need to be saved as .jpgs, at high resolution (at least 300dpi). 4. If images are not yours, please obtain permission in writing and cite the copyright owner. 5.Use grayscale (no colors) on all photos, charts, tables, and graphs as Explorations is not published in color. This is also true for visual arts submissions. 6. Submit everything in its original file. (Example: article as Word .doc, image as .jpg.) Do not convert files. Do not embed images into your article. Be sure you indicate image placement when you submit your final manuscript. 7. We ask that you have at least one faculty member other than the faculty mentor review the manuscript before you submit to Explorations. This reviewer should be well-versed in your discipline and able to provide comments to improve your paper. 8. Please attach your submission and all additional forms in an email addressed to: [email protected] .

116 Submission Process

Suggestions for Success 1. Make sure your name is on everything you submit. 2. Use your initials and submission title as the title of your paper and/or any other email attachments; this way everything can be easily identified. 3. Turn in your required student and faculty identification forms with your submission. Submissions sent without all required forms will not be accepted. Detailed information at www.uncw.edu/csurf/explorations/explorations.html. 4. Once your work has been approved by your faculty mentor and reviewed by another faculty member familiar with the research area, your faculty mentor may submit it on your behalf. If you are a single author, you will be the main contact. If you are one of multiple authors, decide who will be the main contact and have him/her submit on behalf of all. 5. Proofread, proofread, proofread.

Submission Deadline for Volume XI: June 1, 2016.

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