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Saltman | Quarterly

Undergraduate Biological Research Publication UCSD Division of Biological Sciences

Volume 4 Nos. 1, 2, & 3 sq.ucsd.edu

Saltman | Quarterly

UCSD Division of Biological Sciences Volume 4 Saltman Quarterly | Staff Editor-in-Chief│ Quynh-Anh Mai Research Editor│Amanda Chan Review Board Managers│Brannon Peralta and Gayatri Boddupalli Production Editors│Florencia Pierri and Frederick Lin Features Editor│Boris Arbit Technical Editor for Content│Daryush Tabatabai Technical Editor for Graphics│Jason Ly Webmaster│Jet Antonio Communications/Publicity Chair│Ellen Bailey Writers│Amanda Chan Christine Cho Alex Fortenko Sukhprit Mann Brenda Torres Billy Wang Christina Wang Interim Dean│Suresh Subramani Assistant Dean│Barbra Blake Staff Advisor│Patricia Walsh On the cover: The cover photo is of a red-eyed leaf frog (Agalychnis callidryas) encountered near the Refugio Eladios field station in the cloud forest reserve 14 km NE of Monteverde, Costa Rica. The frog, held by David Leong (UC Irvine), was photographed by Aria Jafari (UC San Diego), both students in the EAP Costa Rica- Tropical Biology and Conservation Program.

2006-2007

From the Editors: Dear Readers, Saltman Quarterly is now in its fourth year of existence, and this past year in particular has been one of learning, setting precedents, and establishing a stable and strong voice for the biological sciences undergraduate researchers here at UCSD. Not only is this journal now a showcase Dr. Paul D. Saltman for current undergraduate research, it has transformed into an educational tool that includes research reviews and informative features that reach out to the entire student population. The creators of this journal, Marika Orlov, Louis Nguyen, and Greg Emmanuel set out to honor the educational endeavors and success of Dr. Paul D. Saltman, former Provost of Revelle College and Vice Chancellor of Academic Affairs. Dr. Saltman created an enjoyable, scientific, and educational learning experience for all UCSD students, and we endeavor to continue with that tradition today. As Dr. Linda Strause, UCSD professor and former student of Dr. Saltman, once said, “I believe that Paul’s true passion was not in achieving the goal but in the process. We can always be a little bit better, learn something new each and every day. The process by which we travel this path is what he was so passionate about, the continued process of education and knowledge of teaching and learning.” We have bonded, learned, and grown as a team working towards the same goal: that all undergraduates will soon be inspired to seek out and take advantage of all the opportunities UCSD presents. We hope that this journal will provide you with encouragement, inspiration, and enough curiosity to expand your learning experiences outside of the classroom. Good luck in all of your endeavors, Quynh-Anh Mai Editor-in-Chief

Amanda Michele Chan Research Editor

sq.ucsd.edu

In This Issue│ Research Articles│Fall Preliminary Measurements of the Effect of Rosellinia Sp. on Coffea arabica Seedlings in Sun and Shade Conditions Elizabeth “Bones” Latham│p. 13

Intensity Discrimination in Patients with Auditory Neuropathy Brenda Lolli│p. 15

Research Articles│Winter Ring Species: A Living Laboratory for the Study of Speciation Linda Boettger│p. 19

Distribution and Abundance of Phototropic Meiofauna in Various Habitats of Moreton Bay Vince Si│p. 20

Evolution of the Bacterial Flagellum

Arezou Amidi, Tim Wong, Alexandra Dodds, Sara Siddiqi, Jing Wang, Tracy Yep, Dorjee G. Tamang, and Milton H. Saier, Jr.│p. 23

Research Articles│Spring Pathogenic Bacteria within the Dental Biofilms of a Human and a 130-pound Marine Predatory Fish Kjeld Aamodt│p. 26

Physical and Environmental Preferences of Mosquito Larvae (Culicidae) and Predation by Priapichthys panamensis in Cuajiniquil, Costa Rica Aria Jafari│p. 29

Noncompetitive NMDA Antagonist Dextromethorphan Reduces the Development of Acute Morphine Dependence Lusine Khachatryan│p. 31

Feature Articles│ Drugs Traditionally Used to Treat ADD and ADHD May Now Be the New “Academic Steroids” Amanda Chan and Sukhi Mann│p. 4

Wavefront and New Laser Technology Decreases Complications of LASIK Surgery Christine Cho│p. 6

UV Damage Caused by Tanning Alex Fortenko and Billy Wang│p. 8

Protein Treatment: An Effective Therapy for Parasitic Worm Infections Brenda Torres│p. 10

Thank you to all who submitted pictures for the photography contest. While we could only use one for the cover, above are some of our other favorites. Photographers from top: Will Parson, Shi Feng Xue, Alice Tsai, Aria Jafari, Alice Tsai, Jon Huntoon, Aria Jafari

Progress in Immunotherapy Leads to Advancements in Cancer Treatment Christina Wang│p. 11

Senior Honors Thesis Abstracts │p. 35 Acknowledgements│p. 37 Saltman Quarterly Staff │p. 38

Drugs Originally Used for Treatment of ADD and ADHD May Now Be the NEw

“Academic Steroid”

A Commentary by Amanda Chan and Sukhi Mann

Summary: For individuals with Attention Deficit Disorder (ADD) or Attention Deficit Hyperactivity Disorder (ADHD), there is no escape from unremitting distractions. ADD is a neurological disorder in which affected individuals are unable to concentrate on a specific task, are hyperactive, impulsive, or inattentive. Usually, doctors prescribe stimulants to people with symptoms of ADD, which allow their patients to concentrate. Current drugs are Ritalin, Concerta, and Adderall. Some students without these conditions found that use of these drugs greatly enhanced their ability to concentrate. The use of these drugs without a prescription is gaining popularity among many students and is potentially harmful.

Saltman Quarterly│UCSD Division of Biological Sciences│Volume 4│2006-2007

rugs used for ADD/ADHD treatment are amphetamines, or amphetamine-based substances that stimulate the central nervous system. They are believed to block the reuptake of dopamine and norepinephrine in presynaptic neurons, and instead, deliver these compounds to the extraneuronal space.3 As these processes are active in the regions of the brain responsible for concentration, a person taking these drugs experiences extended periods of increased ability to focus. Besides attentiveness, students can abuse these drugs for appetite suppression, alertness, and mood enhancement. ‘Study drugs’ are being misused annually by approximately 4% of late teens and young adults.6 Abuse of the drug or other similar substances may lead to severe psychological or physical dependence.7 This is not only dangerous but illegal. Ritalin is classified as a schedule II drug by law; possession without a prescription is as serious as being in possession of cocaine and may result in imprisonment. Adolescent children being prescribed Ritalin may encounter side affects, such as height and weight changes.1 Many students who take these drugs without a prescription are not being supervised by a physician. When taken in low doses, these drugs appear to have a small chance of addiction. Rarely, long-term use of these drugs can result in serious heart conditions such as irregular heart rhythm and high blood pressure.6

Past annual rates of non-medical use for Ritalin and related drugs ranged from zero to 25% at individual colleges.6 Many scientists claim that merely diagnosing ADD/ADHD can be problematic because there are no specific tests that indicate the presence of this disorder. Symptoms can

be easily researched by anyone and subsequently repeated to a physician. It is important to realize the danger therein. Pediatrician Dr. Lawrence Diller states: “The ADD-Ritalin issue reveals something about the kind of society we are becoming …. it throws a spotlight on some of our most sensitive issues: what kind of parents we are, what kind of schools we have, and what kind of health care is available to us … it raises fundamental philosophical questions about the nature of free will and responsibility.”2 One-hundred twenty-six biology students responded to an anonymous survey

Features │

given by the Saltman Quarterly Staff. The survey showed that thirty-six percent of those students would take ADD medication, such as Ritalin, if they saw that it had helped their friends on exams. Twenty percent said they have friends that have used these stimulants without a prescription. It is important to understand the psychological and physiological aspects of the drugs with which students may choose to self-medicate. As students, we need to be aware that what we consume has an impact on the way we think, act, and behave. References 1. Adesman, A.R. 2001. The Diagnosis and Management of Attention-Deficit/ Hyperactivity Disorder in Pediatric Patients Prim Care Companion J Clin Psychiatry, 3(2): 66–77. 2. Diller, 1998. Running on Ritalin: A Physician Reflects on Children, Society and Performance in a Pill. Bantam Books. 3. Markowitz, J.S. 2006. A Comprehensive In Vitro Screening of d-, l-, and dl-threo-Methylphenidate: An Exploratory Study J Child Adolesc Psychopharmacol, 16(6): 687-98. 4. McCabe, S.E., Teter, C.J., Boyd, C.J. 2006. Medical use, illicit use and diversion of prescription stimulant medication. J Psychoactive Drugs, 38(1): 43-56. 5. Miller, AR. 2001. Prescription of methylphenidate to children and youth. CMAJ 165(11): 1489–1494. 6. Sussman, S., Pentz, M.A., Spruijt-Metz, M., Miller, T., 2006. Misuse of ‘study drugs:’ prevalence, consequences, and implications for policy Subst Abuse Treat Prev Policy Volume, 1: 15. 7. Volkow, N.D., Wang G.J., Fowler J.S., Fischman M., Foltin R., Abumrad NN., Gatley S.J., Logan J., Wong C., Gifford A., Ding Y.S., Hitzemann R., Pappas N., 1999. Methylphenidate and cocaine have a similar in vivo potency to block dopamine transporters in the human brain. Life Science, 65(1):PL7-12.

New “Academic Steroid” | Chan & Mann

L A S I K Wavefront and New Laser Technology Decrease Complications of LASIK Surgery by Christine Cho

Saltman Quarterly│UCSD Division of Biological Sciences│Volume 4│2006-2007

Features │

Summary: Laser-Assisted In Situ Keratomileusis (LASIK), a form of refractive laser eye surgery, is used to treat patients with myopia, hyperopia, and astigmatism. As studies have continued in this field of ophthalmology, new techniques such as Wavefront technology and IntraLase have enabled scientists to make significant advances in treating severe vision conditions.

“I

t was incredible, because I almost forgot what it felt like waking up in the morning and seeing everything clearly.” After eighteen years of wearing glasses and contact lenses with a prescription that labeled him “legally blind,” 25-year old Mack Dawson was finally able to get 20/20 vision on January 2007. With such a rare prescription, Dawson would not have been able to get LASIK surgery if it had been five years ago. An eye surgeon at UCSD’s Shiley Eye Center, Dr. David J. Schanzlin, says, “Technology is changing every year, every month, every day. The complications are no longer 1 out of every 300 patients, but none out of 4000 patients.” Dr. Schanzlin, a Professor of Ophthalmology and Director of Refractive Surgery at UC San Diego, has played a key role in directing clinical trials using new technology. He focused on improving the process and outcome of refractive surgery. A recent tool that Dr. Schanzlin has been using at UCSD’s Shiley Eye Center is a Wavefront technology called iris registration. This new technique tracks the rotational movements of the eye, and provides valuable information about vision errors. The iris registration first sends waves of light into the eye and creates a customized 3-D map of the eye’s unique wave patterns. Whether the eye moves from left to right or up and down, the laser patterns detect the movements and stay accurately on the same spot on the eye. Dr. Schanzlin states that, “With this technology, we are not only trying to treat patients with contacts or glasses, but even patients with serious diseases.” Along with the Wavefront technology, Dr. Schanzlin has also been using a new technology called IntraLase, a laser that creates a flap in the cornea during LASIK surgery. Before, LASIK

was a surgical process that required a knife called a microkeratome to cut a flap in the cornea, the outer clear covering of the eye, in order to permanently alter its shape. The flap was then folded back, and the underlying cornea tissues were vaporized and reshaped with a laser. After the cornea was reshaped, the flap of the cornea was put back in place without any stitches. IntraLase creates a more precise flap, which reduces the risk of complications. It also shaves off the corneal tissue more precisely, leaving the eye with sharper vision. Even night vision improves. With IntraLase, laser surgery is an option even for patients with thin corneas.

“Technology is changing Every year, every month,

currently manufacturing this laser that is revolutionizing refractive and cornesurgery. The accuracy of this computercontrolled laser has shown to improve the overall outcome of LASIK surgery. According to TLC Laser Eye Centers, IntraLase has increased the number of successful surgical processes to 99 percent. Patients with nearsightedness, farsightedness, and astigmatism can have their vision restored. This new technology is considered safer and more effective than the use of a microkeratome. Dr. Schanzlin reports that, “Out of 1 million IntraLase surgeries, there have only been 5 surgical errors dealing with flap problems.” With the help of Wavefront technology, eye surgeons and researchers hope to cure patients with serious eye diseases. Although most of the eye diseases cannot be cured yet, Dr. Schanzlin stays hopeful and says, “I think it will be another 3 or 4 years until we can finally cure patients with serious illnesses.”

every day. The complications are no longer 1 out of every 300 patients, but none

out of

4000 Patients.” During this process, short-acting laser pulses pass through the cornea to create layers of microscopic bubbles beneath the corneal surface. A computer controls the laser for maximum precision, which is not possible with a microkeratome. After the bubbles are made, the flap in the cornea is opened, and another laser reshapes the inner corneal layer by removing unnecessary tissue. IntraLase Corporation is

References

1. Dawson, Mack. Telephone interview. 19 February 2007. 2. LASIK Eye Surgery. 12 July 2006. U.S. Food and Drug Administration. 06 February 2007. http://www. fda.gov/cdrh/LASIK/default.htm 3. Schanzlin, David J. Telephone interview. 19 February 2007. 4. Segre, Liz. “The LASIK Procedure: A Complete Guide.” All About Vision. September 2006. 02 March 2007. http://www.allaboutvision.com/visionsurgery/ lasik.htm

New Laser Technology | Cho

UV Damage Caused by Tanning

by Alex Fortenko and Billy Wang Summary: Melanoma and other skin cancers may develop as a consequence to overexposure to UV light. Unfortunately, there are plenty of ways in which to encounter this overexposure, and less ways to detect and protect the skin from UV damage. This article explores how damage to skin is caused by UV light, the cultural phenomenon of tanning, and how to avoid overexposure to UV rays.

or many, a typical Southern California summer day involves a flawlessly clear blue sky, and a trip to one of the many famous beaches that line the California coast from Los Angeles, down to San Diego. To the many of these beach enthusiasts it appears that summer in California is heaven on earth, and the comforting heat of the sun allows many to work on their tan, while enjoying a brilliant view of the warm ocean waters. But as summer turns into fall, and fall into winter, this heaven on earth goes into hiatus and takes with it those sunny afternoons that could be spent at the beach. Suddenly, beachgoers are left with simply their summer memories and the unwanted nippy sixty-degree weather. However, as days and weeks go by, the perfect summer tan begins to fade, leaving skin whiter and whiter the longer it is veiled from the sun. In the winter, the earth’s atmosphere is at an angle that facilitates the deflection of UV rays and this leads to drops in temperature. Without a sufficient amount of natural UV rays, Californians flock to the one destination that promises to renew their color for a nominal fee: the tanning salon and its UV rays. Make no mistake, there is nothing simulated about the so-called ‘simulated’ UV rays emitted by tanning beds. It has been deduced that “although the evidence is sparser and less clear, UV rays produced from indoor tanning beds also has been linked to melanoma risk.”1 The same UVA and UVB rays that are emitted by the sun are released by tanning lights. To many this may be somewhat of a deterrent. It has been a known fact that an increase in UV radiation may lead to adverse effects on the skin, ranging from harmless moles and freckles, to malignant melanoma, and other skin cancers. However, for many, including

Leanne B. of Highland, CA, the known risks are simply not a large enough factor in preventing her from tanning several times a week. Leanne is one of the unlucky victims in the battle of the tanning bed epidemic. She has tanned four times per week since the age of eighteen and now at twenty-nine, has experienced some of the more unpleasant effects UV radiation. She was twentyfive when a routine physical first unveiled a skin lesion of great concern. Since then, she has undergone endless procedures to remove over a dozen moles, 25% of which showed cancerous behavior. Recently, a particularly large and deeply rooted mole was removed from her stomach. A biopsy of the removed mole and regions surrounding it revealed it to be severely displastic; in other words, this mole and surrounding tissue could be melanoma infected. To ensure that all cancerous tissue was removed, a large amount of tissue going

Saltman Quarterly│UCSD Division of Biological Sciences│Volume 4│2006-2007

deep into the dermal layer had to be removed. The procedure left Leanne with twenty stitches and frightened of what further tanning may bring. But what is it that causes large amounts of UV rays to penetrate the skin, and through rather natural forms of radiation destroy living cells? And why, with these known effects, do so many Americans continue to place themselves in tanning beds? Ultraviolet radiation outputted by the sun is the primary form of radiation that causes sunburn and other forms of skin damage. Ultraviolet light is a form of electromagnetic radiation, as are xrays, visible lights, and radio waves. The wavelength of UV rays generally ranges from 200 to 400 nanometers (nm). The shorter the wavelength, the more energy the light photon carries but the less depth it can penetrate. Within this given range, there are three categories of ultraviolet radiation:

UVA, UVB, and UVC. UVC is mostly absorbed by the ozone layer and only minute amounts ever reach the earth’s surface. Exposure to these UVC rays will cause sunburn in as little as a minute, and cause plants to wither rapidly upon contact. Fortunately for life on earth, in most regions around the world, the ozone layer is thick enough to block a majority of these rays. However, UVA and UVB rays do penetrate the ozone layer and provide a major concern to

Features │ she was back tanning, though now down to about once per week. When asked why, she simply stated that she wanted to look her best and thought that the reduction in the amount of time tanning is sufficient to prevent her from contracting anything malignant. This appears to be an all too common occurrence, and with recent studies reporting that 85% of all tanning salon employees tell their customers that the process is safe, and of those, only 32% admit

a bow or bulge in a normally flush helical cylinder. Normally certain proteins can repair this damage. However, UVB (and sometimes UVA) radiation has the ability to damage and destroy the very proteins that contribute to the repair. When this occurs the cells have both a damaged blueprint and no means to fix it. This is one of the ways that cells may become cancerous—DNA alteration could affect the cellular clock, instructions for angiogenesis (cell sui-

“And why, with these known effects, do so many Americans continue to place themselves in tanning beds?” the health of the world population. UVA and UVB rays begin to impact the body by first stimulating skin cells in the uppermost layer of skin, called the epidermis. UVB radiation primarily affects the epidermis because there is no blood supply to any of the five sub-layers in the epidermis, so the cells in these layers are dead, dying, or will eventually die. However, the lowest layer of the epidermis, called the stratum basale, is able to receive nutrients by diffusion from the dermis layer.. The UV radiation stimulates melanocytes in the epidermis to produce melanin, a skin pigment that colorizes the skin as it proliferates with UV exposure. The increase of melanin is the body’s first mechanism to further protect the actively dividing cells of the dermis layer below. Melanin production, however, is limited in that its production can become saturated; the saturation point varies from person to person and is determined by skin type/ genetics. When pigmentation is not enough to protect the delicate dermis layer below the body compensates by increasing the thickness of the epidermis. Skin thickening is usually temporary, only lasting about a week after exposure is minimized. But with continued exposure, the thickening can become almost permanent, giving skin a leathery feel and appearance. UV radiation also damages the DNA. Primarily, the photons of energy cause bonds between each strand of DNA’s double helix to break. When the portions of the strand separate, each strand will tend to fold over on itself, causing

cide), and DNA checking mechanisms, and cause a rapid spread of these cells throughout the body. Fortunately, most UVB rays usually do not penetrate into the dermis, the live skin layer below the epidermis. However, extended exposure times will eventually allow UVB rays to affect the live cells of this layer. Symptoms such as severe sunburn are a sure sign that the integrity of the dermis has been compromised by UVB rays. The dermis is subdivided into the papillary region and the reticular region and is affected primarily by the more deeply penetrating UVA radiation. The reticular region holds various sensory nerves (pressure, pain, etc), small blood vessels called arterioles and venuoles, hair follicles, sebaceous (oil) glands, and sweat glands. UVA damages the dermis region by destroying or weakening collagen fibers. The body attempts to repair the collagen but is not always successful. Often times the repaired fibers will be abnormally shaped or disorganized in their attachment. The constant breakdown-repair process eventually causes the accumulation of these abnormal collagen fibers and gives way to wrinkles and loose skin. These symptoms usually won’t present themselves until years after damage is done. The simple risk of getting cancer or wrinkled, loose skin in the future does not outweigh the benefit of looking fabulous in the present. Leanne still presents a similar mindset, even after her melanoma scare. Less than six months after her frightening procedure,

to the risk of obtaining cancer,1 leads many more to continue on with this detrimental habit. Even with the enumerated dangers of active tanning, many still find excuses and reasons for why they tan. Some tanning beds advertise their supposed safety by claiming their lights only emit UVA rays so people believe they are safer to use because UVA rays generally do not cause sunburn. Others believe that tanning is healthy because UV rays are needed to produce sufficient amounts of vitamin-D. It is true that humans need a quantity of UV radiation to produce D-vitamins necessary for proper food absorption and ion regulation. However, only a few minutes of sunlight per day is needed to produce adequate amounts of D-vitamins for the individual. Essentially, simply walking to and from class, or having lunch out on the patio daily is all the sunlight one needs.

References

1. Culley, C.A., Mayer, J.A., Eckhardt, L., April J.B., Eichenfield, L.F., Sallis, J.F., Quintana, P.J.E., Woodruff, S. I. 2001. Compliance with federal and state legislation by indoor tanning facilities in San Diego. Journal of the American Academy of Dermatology, 44: 53-60. 2. Diffey, B.L. 1991. Solar ultraviolet radiation effects on biological systems. Physics in Medicine and Biology, 36: 299-328. 3. Foster, J. “Sunburn.” Mod. 24 Oct. 2005. http://www. emedicine.com/emerg/topic 798.htm (28 May 2007). 4. Meadows, M. 2003. Don’t be in the dark about tanning. FDA Consumer, 37: Nov-Dec. 5. Rados, C. 2005. Teen Tanning Hazards. FDA Consumer, 39: Mar-Apr. 6. Whitworth, A. 2006. Legislators combat melanoma, restrict teen tanning. Journal of the National Cancer Institute, 98: 1594-1596.

UV Damage from Tanning | Fortenko & Wang

Protein Treatment an Effective Therapy for Parasitic Worm Infections by Brenda Torres Summary: A certain bacterial protein, which is known in many agricultural circles as a pesticide in crop plants, is now under investigation as a possible drug treatment for hookworm. Scientists at UC San Diego and at Yale University have done extensive research on bacterial proteins that may help control strains. Such discoveries may help people suffering from anemia and malnutrition related to parasitic attachment.

hildren who do not receive necessary nutrients experience stunted growth and poor development. Unfortunately, the food that developed countries are able to provide cannot singularly alleviate their problems. Malnutrition does not just come from lack of food but may be a result of parasitic worms. For people in developed countries, it is unusual to be concerned about parasitic worm infections. Wider known illnesses, such as diabetes, AIDS, and Alzheimer’s take the forefront. However, diseases from parasitic worms are far-reaching, affecting two billion children and adults worldwide. It is a major concern in underdeveloped countries. Worms and their eggs, which sometimes cannot be seen by the naked eye, can infect anyone who comes in contact with them. Hookworm, whipworm (Trichocephalus trichiuris), and roundworm (Ascaris lumbricoides) are parasitic worms found in soil. Worm infections occur through penetration of the skin,

Dr. Raffi Aroian at UC San Diego.

produced by bacteria so that the plants themselves are able to survive organically without chemical pesticides. Studies on the protein’s effect on hookworms use infected hamsters. Protein treatment has proven comparable to current therapies. The proteins quell hookworm activity and development, therefore, the infected hamsters’ health improves. The findings are significant due to recent documented cas-

Worm causing gut hemorrhage. *

or from consumption of oral-fecal contaminated water and food. Upon infection, parasites colonize inside the intestinal tract. Larger worms can block the intestinal tract. All of these worms are able to eat the food that their hosts consume or feed on host blood. Because they are located in the intestinal tract, they usually are the first to take the consumed nutrients from the host. Symptoms include malnutrition that can result in abdominal pain, fever and anemia. Infection increases susceptibility to other diseases such as malaria and tuberculosis. Immunity cannot be acquired against these worms, and it is common to be infected multiple times. There are currently several treatments, including de-worming and chemotherapy. Dr. Raffi Aroian at UCSD and Dr. Michael Cappello at Yale are tackling the problem. Their idea is to use proteins produced by Bacillus thuringiensis to treat worm infections. Bacillus thuringiensis is a bacterium that produces proteins toxic to invertebrates. Transgenic plants were engineered to express the proteins originally

Saltman Quarterly│UCSD Division of Biological Sciences│Volume 4│2006-2007

Worm SAG at medium power. *

es of worms resistant to chemotherapy in Mali and Australia. The Aroian Lab’s involvement with the study includes purification of the proteins which are then used in treatment studies. Dr. Aroian says that it has been a dream to work with such a cornerstone study. The World Health Organization plans to treat 400 million school-aged children by 2010. Hopefully, this new treatment could be among those remedies used to help eradicate this disease.

Features â&#x201D;&#x201A;11 Aroian Lab From left: Cheng-Yuan Kao, PhD Audrey Bellier, PhD Yan Hu, PhD Chang-Shi Chen, PhD,

*Photos courtesy of Michael Cappello, MD Yale School of Medicine

References 1. Capello, M., et al. 2006. A purified Bacillus thuringiensis crystal protein with therapeutic activity against the hookworm parasite Ancylostoma ceylancium. PNAS., 103(41):15154-9. 2. Cromptom, D.W.T. 1999. How much human helminthiasis is there in the world? J. Parasitol., 85(3): 397403. 3. Bethony, J., et al. 2006. Soil-transmitted helminth infections: ascariasis, trichuriasis, and hookworm. Lancet, 367(9521): 1521-32. Review.

Progress in Immunotherapy Lead to Advancements in Cancer Treatment

by Christina Wang Summary: Scientists at UC San Diego are working on new ways to fight cancer. The innovative work done at the Moores Cancer Research Center has shown promising results using immunotherapy. Advances in Immunotherapy | Wang

urrent statistics state one in three people will be affected by cancer at some point in their lives. Cancer researchers today have turned to their understanding of vaccines in order to create new and viable treatments for this pervasive disease. Leaders in cancer research describe immunotherapy as “ushering in a new era of scientific progress” by mobilizing the body’s immune response against cancer cells. Immunotherapy prevents illnesses by stimulating the immune system’s natural defense mechanisms. Comparable to a battlefield, the immune system initiates a protective response combating harmful invaders, such as viral and bacterial pathogens. It also maintains memory of immunity to be used against future invasions. Killer B and T-cells, special lymphocytes that attack pathogens, travel to the infected area and release antibodies and cytotoxins. These special substances act like foot soldiers to help recognize and destroy harmful invaders. Vaccines harness disease-fighting capabilities of the immune system and provide a strong shield, defending the body from many diseases like smallpox, measles, and the common flu. In the same way, cancer immunotherapy primes the immune system and utilizes defense shields to help restore the body from cancer by suppressing the growth of dangerous cancer cells. Cancer occurs when a cell mutates to stimulate oncogenes (cancer-causing genes) and inactivates tumor-suppressing genes. As it is no longer a normal cell, it expresses genetic abnormalities and is unable to function. Killer B and T-cells transferred in vitro infiltrate the tumors, and prevent cancer cells from

“. . . the potential impacts of cancer vaccines lie in their ability to work in combination with other cancer therapies.” spreading and replicating in the body. The link between cancer and the body’s immune system was first discovered in the early 1890s by Dr. William B. Coley, a New York physician who is now known as the “Father of Immunotherapy.” When Dr. Coley treated patients who had cancer and streptococcal infections, he observed a reduction in malignant tumors. Intrigued by this finding, he conducted an experiment injecting live streptococci bacteria into a cancer patient to see whether it was responsible for shrinking the tumor. The doctor hypothesized that the immune system triggers a protective response in patients, targeting both in-

strengthen the body’s response to other treatments. In this way, cancer immunotherapy offers a more complete defense against the infiltration of cancer cells in the body.” Recent clinical studies on several vaccines reinforce the notion that combining cancer therapy offers greater protection from malignant cancer growths. Dr. Minev and his research team at Moores have developed a vaccine, called Melacine, to treat melanoma. In clinical trials, his patients were found to live twice as long and the size of their tumors was greatly reduced. This was done with fewer side effects than with traditional treatments. Many

fection and abnormal cancer cells. He observed full regression of the tumor realizing the experimental vaccine as a viable form of cancer treatment. The UCSD Moores Cancer Research Center is at the forefront of the field of cancer immunotherapy. According to Dr. Boris Minev, a research scientist at Moores, the potential impacts of cancer vaccines lie in their ability to work in combination with other cancer therapies. “Integrating vaccines into treatment will

vaccines treating breast cancer, prostate cancer, and other types of cancer are similarly promising. Such results illustrate how research advancements in immunotherapy are paving the way to many cancer treatments.

Saltman Quarterly│UCSD Division of Biological Sciences│Volume 4│2006-2007

References

1. Cancer and the Immune System: The Vital Connection. 2003. Cancer Research Institute and Cancer Immunology. May 28, 2007. http:// www.cancerresearch. org/immunology/immuneindex.html. 2. Gardasil. 1995-2007. Merck & Co., Inc. May 28, 2007. http:// www.gardasil.com. 3. Hayden M., Schroter S., Rehan N., Ma W., and Minev B. Trends in Cancer Vaccine Research. Nova Science Publishers, Inc. 1-32. 4. Immunotherapy. Wikipedia Online Encyclopedia. May 28, 2006. http:// www.wikipedia.org 5. National Cancer Institute. May 28, 2007. http://www. cancer.gov.

Research │13

Preliminary Measurements of the Effect of Rosellinia sp. on Coffea arabica Seedlings in Sun and Shade Conditions Elizabeth “Bones” Latham, Senior, Muir College, Ecology, Behavior, & Evolution major Education Abroad Program / Division of Biological Sciences, University of California, San Diego

osellinia, or white root rot, is becoming an increasing problem on Costa Rican coffee farms and secondary forest fragments. However, little is known about the natural history or infection process including associated growth conditions of this parasitic soil fungus. In my experiment, I quantified and described the effect of Rosellinia on Coffea arabica plants. Seedlings were inoculated with the fungus and positioned in either a shady or sunny environment to replicate the potential effects of a canopy. Measurements of leaf condition or quality, height, number of leaves, and fungal growth were taken at regular intervals to assess the level of and change in the infection. These experiments have shown, however, that over time these measurements are not affected by either treatment: sun vs. shade or infected vs. non-infected. Plants did decrease in overall leaf quality isolated from their inoculation. Because no signs of fungal infection were detected, it may be that effects would manifest themselves with more time or different inoculation techniques to better demonstrate the infection process and growth conditions of Rosellinia. Conversely, it could also be that this particular variety or strain of Coffea arabica, Caturra, is resistant to Rosellinia.

The American chestnut provides a profound example of the vast destructive potential of fungi. In its former glory, this tree was a powerful organism; its breadth extended throughout the northern and southeastern United States.11 Yet, before the turn of the 19th century, the fungal root pathogen Chestnut blight, Endothia parasitica, had killed 3.5 billion of these trees in less than 50 years, dooming them to extinction.11 Rosellinia sp., or white root rot, is another such root pathogen that is known to kill a wide range of plant species and has a capacity for causing economic and ecological catastrophe.1 Because Rosellinia disease is typically associated with monoculturally grown tropical crops, documenting it in secondary forest fragments on farms of the Monteverde Corridor was quite uncharacteristic, possibly because it is a strong generalist.5 Rosellinia is characterized by a loose mat of tubular filament known as hypha. The hypha is characterized by two sexual phases, the anamorph asexual stage and the stromata sexual phase, during which the fruiting bodies usually form.1 This implies that Rosellinia, of the family Xylariaceae, is a pleomorphic fungus, i.e., capable of existing in more than one form. However, the stromata phase of sporulation has never been observed or sampled upon infected tree roots; therefore, the only visual means of identification is through the hyphae, which appear as a gray to white wool surrounding the infected roots.1 Host death is caused by a toxin, which progresses from digesting finer roots first to larger ones later, eventually causing leaves to whither, turn yellow, and fall off. The host Coffea arabica accounts for 20% of all agricultural production in Costa Rica; consequently, millions of dollars and much time have been devoted to fighting fungal diseases such as ojo de gallo and roya do café.7 However, little attention has been given to Rosellinia despite the fact that it is negatively affecting many farms and conservation areas. D e s p i t e R o s e l l i n i a ’s prevalent damage in the area, particularly in the forest and coffee plants of Table 1: Differences in temperature.

the Leo Arguedas’ farm, the community does not know what actually happens to a plant exposed to Rosellinia. Questions that need attention include: how fast does infection take place, what happens to the leaves, how are infected leaves detected, and how do all of these regimes change under low and high intensity of light. This information would help farmers implement early detection of the infection, which may result in earlier control measures and preventing the spread of the infection. Furthermore, because it is unknown if many fungi, such as coffee berry borer, increase in shaded areas due to differences in light intensity or microclimate,7 the information presented here will help determine the efficacy of canopy tree use in growing coffee. Perhaps information gleaned will sway farmers towards planting a canopy, thus helping to reforest areas and restore habitat. Although the purpose of this study is not aimed at prevention methods against Rosellinia, the experiment may also be a step in that direction by furthering the understanding of the fungus and how it affects its host. With this in mind I have attempted to compile a list of methods used for control in agricultural environments. The most common method of control is solarization, which has worked for a wide range of annual crops.10 A hot water treatment is also used, though research indicates this method has only been effective on tubers.2 On the cutting edge of research is the use of lactic acid bacteria and its fermentation products, which were found to completely stop the growth of Rosellinia.3 Using a biocontrol such as Tricoderma (another fungus) is possibly the most environmentally friendly way of controlling Rosellinia if Tricoderma is found naturally in the surrounding soil and will not interfere with other ecological processes.13 To attempt to answer the question of the effect of solar flux on severity of Rosellinia infection, I placed coffee seedlings of the strain Caturra in shaded areas, which had a lower soil temperature, and also in direct sun. In both treatments, half the plants were inoculated with Rosellinia in an attempt to infect the plant, while the other half remained noninfected. Although the change in temperature between the two condiRosellinia and Coffea arabica

Figure 2: Change in average leaf condition as determined by leaf ranking for the sun infected, sun non-infected, shade infected, and shade non-infected treatments over the course of three weeks. Each observation period represents one week.

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INTRODUCTION │

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Figure 1: Layout of treatment groups. Four boxes with 10 plants each, half of which were infected.

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The Effect of Rosellinia sp. on Coffea arabica│Latham

Figure 3: Change in average plant height for the sun infected, sun non-infected, shade infected, and shade non-infected treatments over the course of the experiment. Each observation period represents approximately 11 days.

Figure 4: Change in average leaf number for the sun infected, sun non-infected, shade infected, and shade non-infected treatments over the course of the experiment. Each observation period represents approximately 11 days.

tions would be small, I hypothesized that it would be sufficient to change Rosellinia’s infection capacity based on the sensitivity of other soil fungi to heat change.9 With that in mind I also hypothesized that a sunnier environment would yield a more intense fungal infection because of the appearance of Rosellinia on coffee farms lacking shade and in fragments that consistently have higher temperatures.6 I also hypothesized that it would take less than three weeks for infection to be seen in the leaves and roots based on the rate for the tree Dama.13 I predicted wilting yellow leaves and woolly gray hyphal in plants that were inoculated with Rosellinia.

tocol provided by Stinson.14 It was hoped that this protocol would eliminate any biases when ranking the leaves and prevent any carelessness by consistently carrying out precautions against contamination. Special precautions were taken to help deter the spread of Rosellinia outside of the prevention ditches found inside the infected secondary forest. Basins filled with iodine as well as sponges were placed outside the ditch to disinfect shoes and to sterilize any equipment that touched the infected area or coffee seedlings. Special care was taken also in the disposal of the contaminated samples; all plants and soil were bleached. The seedlings themselves were kept in Ziploc bags throughout the experiment, and any water that collected in these bags was basted out and put in a bleach solution.

METHODS AND MATERIALS│

Forty seedlings were acquired from Café Monteverde on 16 May 2005; special attention was given to choosing healthy seedlings that showed no signs of disease or physical distress. The experiment took place in the secondary forest fragment and coffee plantation of the farm of Leo Arguedas in Los Llanos, which had previously been established as infected.5 Twenty seedlings were placed in direct sun, half of which were inoculated with Rosellinia. The other 20 were placed in a shady environment, half of which were also infected with the fungus, while the other half acted as a control. As a consequence, there were two treatments with four different set-ups: sun infected, sun non-infected, shade infected, and shade non-infected. The temperature of the soil inside the seedling bags and on the forest floor was measured. Randomly chosen leaves from each arrangement were tagged and ranked on a scale from 1 to 10, with 10 being in perfect condition. Rank was lowered depending on herbivory, physical damage, or fungal infection. Every measurement was part of the process of looking for signs of infection, which is characterized by rapidly wilting yellow leaves, beginning at the tips, which eventually fall off.1 See table 2 for specifics on plant location and treatment. Infection may, however, manifest itself in other ways, such as visible fungal growth on root tissue, decrease in growth rate, or increase in number of leaves lost. In an attempt to document the changes that Rosellinia may have, height was measured below the terminal bud of the main stem. In addition, the number of leaves on every seedling was counted at the beginning and end of the experiment. Special care was taken to ensure that every box of five plants had a broad range of plant sizes, as determined by the number of leaves, to avoid the possibility that the distribution of hearty or conversely weaker plants would affect the infection. All of these measurements were carried out approximately every week to continue the documentation of the effect or lack of effect of Rosellinia. A teaspoon of soil, with or without Rosellinia, was mixed with each plant for 10 minutes, taking care to touch at least some of the fine roots. To prevent bias, a third party kept track of what soil had been given to which plants, and I was not informed until the end of the experiment. The infected soil was taken from dying or dead plants that had previously been established to be afflicted with the disease, consistent with the pro-

RESULTS│

Over a period of 23 days, exposed coffee seedlings were compared to non-infected seedlings in the same physical conditions, but under two light systems. The difference in canopy density and light penetration of the two systems caused a minor, yet significant difference in soil temperature (Student’s t-test, p<0.05) (see table 1). The condition of the leaves for the four treatments (figure 1) showed a steady decline over the course of the experiment. Figure 2 illustrates the change in leaf condition as described by ranking 1-10 over a threeweek period; each line represents a combination of treatments. A Student’s t-test showed no significant difference in the height of sun-infected (p=0.729), sun non-infected (p=0.869), shade infected (p=0.861), or shade non-infected treatments (p=0.826) over the course of the experiment (figure 3). Similarly, there was no significant change in the numbers of leaves for sun infected (p=0.917), sun non-infected (p=0.720), shade infected (p=0.867), or shade non-infected (p=0.786), using a Student’s t-test (figure 4), indicating that there was no significant change in either height or leaf number, regardless of light conditions or inoculation. The descriptive aspect of the study shared a similar trend; i.e., there was no significant difference in notation or description of the leaves over time. For example, in Box 1, Plant 22 leaf A4 (table 2) was described three times as having a slight white dusting and one yellow dot for all three times. No results were obtained for the amount of time it takes for infection to take hold since leaf condition remained unextraordinary. P-values comparing the beginning and end times for the average height and number of leaves for each treatment were obtained, indicating there were no significant differences between the two times. There is an overlap in standard deviations for every treatment, indicating that neither height nor leaf number differed significantly from the first to the second observational period for any treatment. No evidence of fungal growth was observed after uprooting any of the 40 seedlings.

DISCUSSION│

Despite the fact that a significant temperature difference was observed in the two treatments, my hypothesis that increased light and tem-

Saltman Quarterly│UCSD Division of Biological Sciences│Volume 4│2006-2007

perature would cause an increased Table 2: Plant descriptions growth of Rosellinia, and therefore Box 1 Sun Infected Plant 22 5 leaves marked and described a greater decrease in plant and leaf 12 5 leaves marked with metal condition, was incorrect. Figure 2 Plant Plant 27 All leaves noted but not marked clearly shows a consistent slope for Plant 29 Leaves not marked 38 Leaves not marked plants both in sun and in shade. In Plant Box 2 Sun Infected the graph, all four setups of light Plant 21 All leaves noted but not marked Plant 17 5 leaves marked and described and fungal regimen decreased in avPlant 6 5 leaves marked with metal erage leaf ranking at approximately Plant 16 Leaves not marked the same rate, leading me to believe Plant 37 Leaves not marked Box 3 Sun Non-Infected that this change can be accounted for Plant 36 5 leaves marked and described Plant 13 All leaves noted but not marked by factors other than the Rosellinia. Plant 15 Leaves not marked Perhaps all the movements associ- Plant 19 Leaves not marked ated with being transplanted and Plant 26 5 leaves marked with metal 4 Shade Infected nearly daily measurement of some Box Plant 33 Leaves not marked kind were too stressful on the seed- Plant 8 Leaves not marked 25 All leaves noted but not marked lings. Also, herbivory continued in- Plant Plant 20 5 leaves marked and described side of the forest despite the fact that 5 leaves marked with metal 34 there are very few animals that feed Plant Box 5 Shade Non-Infected on coffee.7 However, this would also Plant 7 Leaves not marked 1 5 leaves marked and described account for change across all sce- Plant Plant 32 Leaves not marked narios. Plants that were infected had Plant 9 All leaves noted but not marked Plant 39 5 leaves marked with metal a consistently higher average leaf Box 6 Sun Non-Infected condition; however, this is a random Plant 2 5 leaves marked and described Plant 3 All leaves noted but not marked phenomenon and is not a byproduct Plant 23 Leaves not marked of the Rosellinia. Plant 14 5 leaves marked with metal Because groups that were inocu- Plant 30 Leaves not marked 7 Shade Non-Infected lated with the fungus and those that Box Plant 11 Leaves not marked remained non-infected have an insig- Plant 10 5 leaves marked with metal 40 5 leaves marked and described nificant change in average leaf con- Plant Plant 4 Leaves not marked dition, height, and number of leaves Plant 18 All leaves noted but not marked as well as no change in the amount Box 8 Shade Infected Plant 31 5 leaves marked with metal of fungal growth, it can be said Plant 24 5 leaves marked and described that experimentally measured signs Plant 35 Leaves not marked Plant 28 All leaves noted but not marked of infection were not seen and pos- Plant 5 Leaves not marked sibly that infection of Rosellinia did not take hold. One reason why infection may not have manifested itself is because the coffee seedlings did not receive enough water to induce fungal growth. Perhaps the plastic bag prevented too much water from penetrating the seedlings and the amount of water that I gave them was sufficient for their own growth, but not for the rapid visible growth of Rosellinia. Although this is a possible explanation, it is not very likely seeing that Rosellinia thrives in the temperate zone, where at least 75% less water is received, and past experiments involving infection of plants were done during the dry season with about half of the seedlings dying from the fungus.13 Another explanation for the lack of visible signs of infection could be that the experiment was just not given enough time. In previous studies, more than half of the seedlings studied were infected after only three weeks; however, nothing was said about the inoculation methods, so per-

Research │15

haps this number is inflated.13 For signs of infection (wilted and yellow leaves) to occur it would seem that Rosellinia must have to digest and destroy a large number of fine roots, eventually preventing the plant from absorbing water. Because coffee has waxy leaves that slow down transpiration and because these seedlings were bred commercially to be as strong as possible, it may be that it will take more than three weeks for infection to be visible and much longer for the plant to actually die. One of the most straightforward explanations would be that the inoculation technique was incorrect; however, past literature reported that infection resulted by mixing infected roots and soil into the soil of the study plant.4 It is possible that there were no living spores of Rosellinia in the soil used; however, this seems highly unlikely because past experiments proved that the fungus was present in the area sampled.14 It is also possible that the spores were dormant at that particular time or that transplanting the fungus from one plant to another caused a high level of stress and killed it. In future research different inoculation techniques could be performed to ensure the taking of the infection. The most encouraging possible explanation for the negative results is that this particular strain of Coffea arabica, Caturra, may be resistant or even immune to the fungus Rosellinia. This would also explain why coffee plants have grown adjacent to infected forest without showing any signs of infection. This is the most important direction in which future studies could go in the hope of finding ways to alleviate many agricultural pressures farmers are faced with on a regular basis.

REFERENCES│

1. Alexopoulos, C.J., Mims, C.W., and Blackwell, M. 1996. Rosellinia necatrix. In Introductory mycology. Canada: John Wiley & Sons, pp. 352-356. 2. Garcia-Jimenez, J. 2003. Control of Dematophora necatrix on Cyperus esculentus tubers by hot-water treatment. Journal of Phytopathology. 3. Kuwaki, S. 2004. Effects of the fermentation product of herbs by lactic acid bacteria against phytopathogenic filamentous fungi and on the growth of host plants. Journal of Bioscience and Bioengineering. 4. Lee, S.B. 2000. Resistance of selected malus germplasm to Rosellinia necatrix. Journal of the American Pomological Society, 54(4): 219-228. 5. Lopez, L. INTA. 2004. Official report published February 2004. 6. Lovejoy, T.E. 1986. The biological dynamics of tropical rainforest fragments: A prospective comparison of fragments and continuous forest. Bioscience. 7. Janzen, D. 1983. Costa Rican natural history. Chicago: The University of Chicago Press, pp. 86-88. 8. Kimani, M. 2002. Introduction to coffee management through discovery learning. Nairobi, Kenya, Africa Regional Centre. 9. Mata, M. 1999. Costa Rica mushrooms. Costa Rica, Instituto Nacional de Biodiversidad, pp. 11-14. 10. Pinkerton, J. 2002. The use of soil solarization for the management of soil borne plant pathogens in strawberry and red raspberry production. Plant Disease. 11. Rosenzweig, M. 1995. Species diversity in space and time. Cambridge: Cambridge University Press. 12. Rossman, A. 1998. Protocols for an all taxa biodiversity inventory of fungi in a Costa Rican conservation area. North Carolina: Parkway Publishers, Inc. 13. Smith, J. 2005. Forest regeneration in Rosellinia infected areas of Los Llanos, Monteverde and possible biocontrol agents (CIEE). 14. Stinson, S. 2004. Fungal distribution of Rosellinia species in Monteverde, Costa Rica. Analysis of horses as possible vectors in secondary forest fragments (EAP).

Intensity Discrimination in Patients with Auditory Neuropathy Brenda Lolli, Senior, Warren College, Biochemistry & Cell Biology major Division of Biological Sciences, University of California, San Diego

uditory neuropathy (AN) is a type of hearing impairment in which there is a loss of auditory nerve cells and/or a loss of firing synchrony in the auditory nerve, even though outer hair cells are intact.2 Psychoacoustic studies in AN patients show that intensity discrimination at low frequencies is often impaired, but at high frequencies is normal.16 This study explores the electrophysiology underlying the psychoacoustic findings. The negative peak at 100 ms after stimulus presentation (N100) of an event-related potential (ERP) is an electrical change recorded in the brain in response to a stimulus and reflects an early stage of sensory processing.13 Electrophysiological and psychoacoustic measures were performed at three different sound frequencies (250, 1000, and 4000 Hz). ERPs were recorded in response to random increases in baseline intensity by 2, 4, 6, or 8 dB in ongoing pure tones. We predicted that abrupt changes in intensity would elicit an N100 peak that was related to, and predictive of, perception. We found that the N100 peak amplitude was lower or not present in AN subjects compared to normal-hearing subjects at 250 Hz, but resembled normal amplitude and latency at 1000 Hz for all dB increases. However, at 4000 Hz the N100 peak amplitude and latency of the AN subject were less like normal than expected for high frequency. Overall, the characterization of intensity discrimination in AN subjects may allow for better means of diagnosing the disorder and possibly lead to new treatments. Presently, neither hearing aids nor cochlear implants significantly improve the hearing of patients with AN.11

Intensity Discrimination and Auditory Neuropathy│Lolli

INTRODUCTION │

Auditory neuropathy (AN) is a disorder that affects both infants and adults. The etiologies of AN are unclear at the present time, however some evidence points to genetics or illness.16 Hearing loss commonly associated with old age is due to loss of function of the outer hair cells of the cochlea, whereas AN has been narrowed down to problems with the inner hair cells of the cochlea, the hair cell-nerve junction, or the auditory nerve itself.11 Problems with the auditory nerve, such as loss of synchrony of the nerve fibers, lead to inadequate signaling of the brain stem and higher brain sound processing.2 When a sound enters the ear it travels through the auditory canal, causing vibrations of the tympanic membrane.4 The bones of the inner ear transmit the sound to the cochlea, in which the outer and inner hair cells pass the sound along to the auditory nerve.4 From there the sound information is sent to the brain stem and then to other cortical centers (figure 1). In patients with AN, the sound transmission to the brain stem is interrupted in the inner ear.2 Consequently, sound is not processed coherently. It is also particularly difficult for AN subjects to process sound amid a background of noise because AN distorts the frequencies.11 AN is diagnosed by testing for otoacoustic emissions (OAEs) and an auditory brain stem response (ABR).2 OAEs are sounds produced by the inner ear that can be measured with a microphone, and are a test of cochlear function.12 The presence of OAEs shows that the outer hair cells are intact and working.2 The ABR is a measure of auditory brain functioning commonly used on newborns to detect hearing loss because they are incapable of the concentration required for an audiogram.1 The absence of an ABR means that there is a problem with the inner hair cells or the auditory nerve, because the sound is not being passed along to the brain stem.2 Having OAEs but no ABR is characteristic of AN.2 Subjects with AN also show mild to moderate pure tone hearing loss on an audiogram, but have great difficulty understanding speech at similar decibel levels.11 A pure tone is a sound that only has one frequency, and the decibel is a measure of the loudness of sound.3,14 Therefore pure tone audiograms have little predictive value of speech perception in AN. This creates a need for a more reliable means of predicting speech perception. Electrophysiology can be used to examine the brain processes underlying psychoacoustic measures. Event-related potentials represent brain responses to thought or perception due to a stimulus and are measured by the electrical activity of the scalp.7 The ERP is one particular tool used to objectively examine the brain responses related to auditory stimuli. This is particularly helpful in populations in which psychoacoustic measures are often unreliable, such as the mentally handicapped. Early cortical potentials are electrical potentials recorded from the scalp, occurring milliseconds after stimulus presentation.13 The N100 is a negative peak occurring around 100 ms after stimulus presentation and is an early cortical potential thought to reflect basic sensory processing of stimuli.13 It has not been well characterized for intensity discrimination at low and high frequency tones in AN sub1.4 s jects. In AN subjects, most intensity discrimination studies conducted thus far have focused on psychoacoustic measures of a pure tone as a function of sensation level 100 m s (dB SL).16 Intensity Figure 2: A simulated part of the stimulus sequence with discrimination is an increase in intensity from the baseline pure tone (250, the ability to de1000, or 4000 Hz). Baseline intensities were 50 and 80 dB termine that there SPL, and intensity increases were 0, 2, 4, 6, or 8 dB. has been a change

semi circular canals elliptical circular

incus

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Figure 1: Anatomy of the ear. Areas of interest include the cochlea and auditory nerve. Image from http://www.wikipedia.org/wiki/ear.

in the intensity of a sound. Sensation level is the decibel level above the individual subject’s threshold of hearing for a given sound frequency. The threshold of hearing differs from one person to the next and represents the lowest decibel level at which a person can hear a sound at a certain frequency.9 These thresholds are tested for several frequencies with an audiogram.9 Sound frequency is measured in Hertz (Hz), which has units of s-1 and means cycles per second.10 It has been shown that AN subjects have elevated thresholds for detecting changes in intensity level of a 1000 Hz pure tone at 20 dB SL (sound level), whereas above 50 dB SL their intensity discrimination is very similar to that of normal-hearing subjects.16 In this study, we measured the N100 amplitude and latency of normal subjects and one AN subject when presented with varying intensities of low, middle, and high frequency tones (250, 1000, and 4000 Hz, respectively). Based on previous studies, AN subjects have a more difficult time Figure 3: Diagram of a 64-electrode neuroscan cap used for recordings, shown from the top down. Electrodes shown in front represent the left eye channels (VEOU, VEOL) and electrodes shown on either side in the front represent the temple channels (HEOL, HEOR). Major electrodes of interest to this study lie along the midline, particularly CZ, C1-C6, FCZ, and FC1-FC6. VEOL VEOU HEOL

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Saltman Quarterly│UCSD Division of Biological Sciences│Volume 4│2006-2007

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Research │17

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discriminating intensity increases in low frequency tones.16 Therefore we expect the N100 amplitude and latency to differ from normal at 250 Hz in AN subjects.

METHODS AND MATERIALS│

Subjects Seven healthy normal hearing adults (4 females, 3 males) participated in this study. All subjects had pure tone thresholds of 20 decibels or lower on their audiograms. One female AN subject was tested. Pure tone thresholds in the left ear were 75, 75, 45, 20, and 20 decibels for 250, 500, 1000, 2000, and 4000 Hz, respectively. The right ear had a cochlear implant that was turned off for the entire duration of the experiment. All subjects signed an informed consent following local Institutional Review Board (IRB) guidelines for testing human subjects. Each subject was paid for participating in the study. Stimuli All stimuli were generated digitally using Matlab. The stimuli consisted of a pure tone that contained shifts in baseline intensity lasting 100 ms. The frequencies used were 250, 1000, and 4000 Hz, and baseline intensities were 50 and 80 dB SPL (sound pressure level). SPL is the pressure deviation caused by a sound wave and is a physical quantity measured in decibels.15 Increases in intensity were presented every 1.4 seconds and randomly consisted of increases of 0, 2, 4, 6, or 8 dB. One hundred (100) repetitions of each transient were given, and each sequence was approximately 13 minutes long. (See figure 2.) Experimental Conditions Intensity stimuli were presented in two conditions for all subjects: 1. In the active condition, subjects listened to the stimulus and pressed a button as quickly as possible when the tone was perceived to get louder. Trials were arranged randomly for all subjects. Active conditions only included the 250 Hz pure tone because the low frequency was of most interest and also due to time constraints. 2. In the passive condition, subjects listened to the stimuli but did not press a button. Trials were arranged randomly for all subjects. Passive conditions included all three frequencies (250, 1000, and 4000 Hz). Normal subjects were tested at 50 and 80 dB SPL baseline intensity for both active and passive conditions. The AN subject was also tested in both active and passive conditions. The intensity for the 250 and 1000 Hz tones was increased to 95 dB SPL because the AN subject had hearing loss at these frequencies. This was done to make sure that the AN subject was hearing a similar sound intensity during the tests. Each subject was seated in a comfortable chair in a sound proof chamber. During trials subjects looked at a point straight ahead and were asked to move as little as possible. Passive conditions took approximately

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Figure 4: Grand mean ERP data, CZ electrode. Grand averaged potentials in the passive state are shown for the CZ electrode. The active state is not shown because there was only one trial done and was a means of confirming the psychoacoustic data. Measurements were made so that the CZ electrode on the neuroscan cap was placed on the top center of the scalp. This electrode usually gives the clearest and most pronounced electroencephalogram (EEG) readings on the computer. Therefore it is of major interest when recording ERPs. Graphs depict normal responses overlayed with the AN response as a function of frequency and intensity increase. From left to right, the graphs represent frequencies of 250, 1000, and 4000 Hz, respectively. From top to bottom are intensity increases of 8, 6, and 4 dB, respectively. Baseline intensity for normal subjects was 80 dB SPL, and for the AN subject it was 95 dB SPL. The stimulus was presented at time point zero.

13 minutes; active conditions were split in two so each trial lasted approximately 6 minutes. Recording and Analysis Scalp recordings were made with a 64-electrode neuroscan cap. The cap was elastic and fit to the head. Its 64 electrodes were filled with conductive gel down to the scalp. Electrodes were arranged according to the standard 10-20 system, in which the scalp is measured to ensure reliable results.6 Electrical impedance, which measures the resistance to current, was kept below 10 kΩ.5 (See figure 3.) Continuous files were corrected for eyeblink artifact, which is electrical activity on the EEG of non-cerebral origin.6 The EEG files were epoched in -200 to 500 ms windows surrounding each stimulus. N100 peak latency and amplitude were calculated for each intensity change in the active and passive conditions. Latencies were determined from the onset of the stimulus, and amplitudes were measured from the baseline.

RESULTS│

Grand Mean ERP Data The N100 component was evident in all conditions for normal averages (figure 4). As expected, the N100 was reduced and delayed for lesser intensity increases. For the AN subject the N100 is present but delayed in the 250 Hz 8 dB condition and disappears for the 4 and 6 dB increases. This was predicted based on the low frequency hearing loss characteristic of AN. At 1000 Hz, the AN responses for all intensity increases resemble that of the normal subject averages. At 4000 Hz, the AN response for 8 dB is reduced and delayed. For 6 dB the N100 is barely present, and for 4 dB cannot be distinguished. The results for 1000 and 4000 Hz are interesting because it was expected that the AN responses would most closely resemble normal responses at 4000 Hz and not as much at 1000 Hz. Overall N100 Latency and Amplitude Graphs of latency and amplitude are as expected for the normal averages (figure 5). Latencies are shown to decrease with increasing intensity, and amplitudes become more negative with increasing intensity. At 250 Hz, the AN latencies and amplitudes are outside the standard error bars for all conditions except the 8 dB increase. At 1000 Hz, the AN latencies are all within the standard error, and amplitudes also fall within the error except at 8 dB. At 4000 Hz, the AN latencies are outside the error bars for all but the 6 dB condition. All amplitudes at 4000 Hz are outside the error bars. These results are unexpected based on the prediction that latencies and amplitudes would be most similar to normal at 4000 Hz.

DISCUSSION│

Auditory neuropathy, a hearing disorder that affects the inner ear, has not been fully characterized. It is unknown whether a defect of the in-

Intensity Discrimination and Auditory Neuropathy│Lolli

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dB increase AN

Normal

Figure 5: Overall N100 latency and amplitude, CZ electrode. Latency and amplitude of the N100 peak at the CZ electrode in the passive state are shown. Graphs depict averaged normal responses overlayed with the AN response, as a function of frequency. From left to right, the graphs represent frequencies of 250, 1000, and 4000 Hz. Latencies are along the top row, amplitudes along the bottom. Error bars are shown for normal subjects, calculated using standard error: (Standard Deviation)/√N.

ner hair cells, the hair-cell nerve juncture, or the auditory nerve is responsible.11 AN subjects are usually most affected by signal distortion of low-frequency sound, and consequently signal amplification provided by hearing aids does little to improve hearing quality.2 Also, cochlear implants only help if the problem is within the cochlea and not the auditory nerve.11 At this point, not much can be done to help patients with AN unless the underlying etiologies are determined. Currently, in America it is only mandatory to screen newborns for hearing in 37 states.2 Hospitals use either OAEs or ABRs to test for hearing, but the diagnosis of AN requires both tests.2 Although it has been reported that 1 out of 20 infants with hearing loss has AN, many still go undetected.8 It is critical for infants to be tested for AN because other neuropathies may be present as well.8 This study focused on further characterizing intensity discrimination at low frequency sound in AN subjects. Based on previous psychoacoustic findings, it was predicted that intensity discrimination would be most impaired at a low frequency (250 Hz) and would most closely resemble normal-hearing subjects at a high frequency (4000 Hz). To encompass the entire spectrum, a middle frequency (1000 Hz) was also included in the trials. Specifically, we chose the amplitude and latency of the N100 early cortical potential as the focus of the EEG recordings. Our results showed that in fact the AN subject had the greatest difficulty discriminating the intensity increases at 250 Hz. Only the 8 dB increase from the baseline had an evident N100 peak, which itself had decreased amplitude from baseline compared to the normal averages. At 1000 Hz, the AN subject had near normal N100 peak amplitude and latency for all intensity increases (4, 6, and 8 dB). At 4000 Hz, the N100 peak was present but delayed and reduced in only the 6 and 8 dB increases from the baseline, and disappeared completely in the 4 dB condition. All normal subjects showed decreased latency and increased negative amplitude as the intensity increased. The AN subject followed this pattern most closely for the 1000 Hz condition and was outside standard error at the other frequencies. This result is surprising because it was expected that the 4000 Hz condition would be most similar to normal, whereas the results show that it was the 1000 Hz condition instead. However, these results are only preliminary because of the small sample size of both normal (n=7) and AN (n=1) subjects. Perhaps with more AN subjects the 4000 Hz condition would appear just as strong as at 1000 Hz. It is important to continue experimenting with AN in order to find a method to improve the hearing of patients with this disorder. AN subjects have trouble understanding speech and communicating in everyday life, and it is also difficult for infants and children with AN to develop communication skills.11 The disorder is not well understood; its most vi-

able options include sign language and lip reading, and there is no way to predict whether the hearing quality of AN subjects will improve or decline over time.11

ACKNOWLEDGEMENTS│

I would like to thank Dr. Arnold Starr for being my faculty mentor and giving me such a great opportunity to perform research in his lab as well as all members of the Starr lab for making me feel like part of their team: Andrew Dimitrijevic, Yuna Chun, Rie Irimajiri, Henry Michalewski, and Kevin Kawa. I also thank the University of California LEADS program for providing me with funding and support in conducting this research at the University of California, Irvine.

REFERENCES│

1. Anonymous. 31 Dec 2006. Auditory brain stem response. Wikipedia. http:// en.wikipedia.org/wiki/Auditory_brainstem_response. 22 Jan 2007. 2. Anonymous. 31 Dec 2006. Auditory neuropathy. Wikipedia. http://en.wikipedia.org/ wiki/Auditory_neuropathy. 22 Jan 2007. 3. Anonymous. 19 Jan 2007. Decibel. Wikipedia. http://en.wikipedia.org/wiki/Decibel. 22 Jan 2007. 4. Anonymous. 19 Jan 2007. Ear. Wikipedia. http://en.wikipedia.org/wiki/Ear. 22. January 2007. 5. Anonymous. 4 Jan 2007. Electrical impedance. Wikipedia. http://en.wikipedia. org/ wiki/Electrical_impedance. 22 Jan 2007. 6. Anonymous. 11 Jan 2007. Electroencephalography. Wikipedia. http://en.wikipedia. org/wiki/Electroencephalography. 22 Jan 2007. 7. Anonymous. 24 Nov 2006. Event-related potential. Wikipedia. http://en.wikipedia. org/wiki/Event-related_Potential. 22 Jan 2007. 8. Hain, T. 6 Sep 2006. Auditory neuropathy. Dizziness, imbalance, and hearing disorders. http://www.dizziness-and-balance.com/disorders/hearing/aud_neuropathy.html. 22 Jan 2007. 9. Anonymous. 7 Nov 2006. Hearing test. Wikipedia. http://en.wikipedia.org/wiki/ Hearing_test. 22 Jan 2007. 10. Anonymous. 17 Jan 2007. Hertz. Wikipedia. http://en.wikipedia.org/wiki/Hertz. 22. Jan 2007. 11. Hood, L. 31 Mar 2000. Auditory neuropathy: What is it and what can we do about it. LSU Health Sciences Center. http://www.medschool.lsuhsc.edu/Otorhinolaryngology/deafness_article1.asp. 22 Jan 2007. 12. Anonymous. 1 Jan 2007. Otoacoustic emission. Wikipedia. http://en.wikipedia.org/ wiki/Otoacoustic_emission. 22 Jan 2007. 13. Picton, T., Lins, O., and Scherg, M. 1995. The recording and analysis of eventrelated potentials. Handbook of Neuropsychology, p 10. 14. Anonymous. 13 Oct 2006. Pure tone. Wikipedia. http://en.wikipedia.org/wiki/Pure_ tone. 22 Jan 2007.

15. Anonymous. 16 Jan 2007. Sound pressure. Wikipedia. http://en.wikipedia.org/ wiki/Sound_pressure. 22 Jan 2007. 16. Zeng, F., Kong, Y., Michalewski, H. and Starr, A. 2004. Perceptual consequences of disrupted auditory nerve activity. Journal of Neurophysiology 93: 3050-3063.

Saltman Quarterly│UCSD Division of Biological Sciences│Volume 4│2006-2007

Ring Species: A Living Laboratory for the Study of Speciation

Research │19

Linda Boettger, Senior, Revelle College, General Biology major Division of Biological Sciences, University of California, San Diego

INTRODUCTION│

An important goal in evolutionary biology is to determine the ways in which new species are formed. Because the speciation process is presumed to occur through gradual change over an extended period of time, it has never been directly observed.1 One knows that two similar species must have diverged in the recent past, but it is not always easy to determine exactly how they diverged in the absence of intermediate forms. Another important question in the study of speciation is to determine if this process is feasible with gene flow still present between the two diverging groups. Both of these topics can be studied using a system called a ring species.2 (See figure 1.) Biologists differ somewhat in their exact definitions of what qualifies as a ring species. An ideal ring species is one that was founded by one ancestral population that gradually formed a ring around a geographic barrier, such as a desert. When the two terminal forms meet on the other side of the ring, they are reproductively isolated; however, gene flow around the remainder of the ring is extensive and uninterrupted between populations in close proximity.3 In this system, it is possible to see the steps toward divergence of the two terminal forms because the intermediate forms still exist. This paper will examine the specific example of a ring species, the Ensatina salamander. Additionally, it will discuss the complex nature of isolating barriers in ring species and how local adaptation increases divergence. The method of using ring species to study speciation with gene flow will also be examined. Finally it will analyze the utility of a broken ring.

ENSATINA SALAMANDER│

An interesting example of a ring species is the plethodontid salamander, Ensatina eschscholtzii. Seven subspecies of Ensatina exist in a ring surrounding the Central Valley in California, and the terminal forms live in sympatry in southern California with no apparent hybrid formation. This species is hypothesized to have migrated south from northwestern California and southwestern Oregon and to have subsequently differentiated, one group traveling along the coast and the other through inland mountains. The costal migrants gradually adapted a reddish-brown coloration, while the mountain migrants evolved a spotted color pattern.4 Although the color of the subspecies around the ring demonstrates a gradual change in phenotype between the two divergent forms, there appears to be restricted gene flow in several locations around the ring.5, 6 Additionally, there is a leak in the ring in the foothills of the Sierra Nevada where E. eschscholtzii xanthoptica (originally coastal) and E. eschscholtzii platensis exist in parapatry. These subspecies most likely differentiated in allopatry and regained contact in the Holocene. The hybrid zone has been examined for divergence levels using allozyme loci and mitochondrial DNA. The observed cline, or gradient, in allele frequency across a geographic range is narrow, signifying heavy selection against the hybrids.4 The story of the Ensatina salamanders is a complicated one, with inconsistent gene flow around the ring and a ring leak in the Californian central Sierra foothills. As such, the species is no longer considered an ideal ring; however, the system remains a useful tool in the study of speciation due to the varying degrees of divergence in its intermediate forms. Figure 1: This diagram illustrates an ideal ring species in which the ancestral form (blue) separated and differentiated, becoming fully speciated on the opposite side of the ring (pink/green). Picture courtesy of Wikipedia.

ISOLATING BARRIERS│

Although not mentioned in ring species literature, it appears that if a ring species were in existence for a long period of time, the present genetic incompatibilities are not necessarily those that originally caused its speciation. It is possible for each terminal form to develop new mutations that further separate them. In addition, because of the unique circumstance of gene flow between the terminal species (passing new mutations around the ring), it should theoretically be possible for these populations to overcome old isolating barriers. The two-locus model of Dobzhansky-Muller incompatibilities portrays a situation in which two different substitutions are made in the descendants of an ancestral form that are incompatible with each other when present in the same individual (hybrid). However, because ring species can pass mutations around the ring, it is possible that terminal species could re-substitute an ancestral allele from an intermediate form that does not have an incompatibility. Because of these complications, if one wishes to directly trace the original incompatibilities that lead to speciation, the ring species is a good indicator but still an imperfect model.

ADAPTATION│

Ecological differences between directly hybridizing populations around the ring perhaps aid the speciation process through local adaptation,9 but ecological differences on opposite sides of the ring do not seem to be an impediment.1 The greenish warbler (Phylloscopus trochiloides), a classic example of a ring species, demonstrates these principles. In this system there are reproductively isolated populations in sympatry with intermediate forms connecting them around the Tibetan plateau. Analysis has demonstrated continuous and gradually changing intermediate forms around the ring, both genetically and phenotypically. There is only one gap in the ring, which will be discussed later. The varying ecology around the ring is said to have resulted in rapid evolutionary adaptation, as there appears to be less gene flow in areas with a sharp ecological gradient.1 The likely explanation for this is that individuals of one population do not survive as well in their neighbor’s habitat.7 However, similar ecological changes on opposite sides of the ring do not seem to interfere with speciation. Although the ancestors of the terminal species were exposed to similar ecological pressures, they likely adapted in different ways. In the greenish warbler example, the birds evolved more complex songs while diverging around the ring, but the authors are quick to point out that there are numerous ways in which to evolve song complexity. This resulted in different mating songs and thus a reproductively isolated species.1

SPECIATION WITH GENE FLOW│

A ring species is an ideal example of speciation despite gene flow. When studying two reproductively isolated species, it is often difficult to prove that there was gene flow during the time of speciation. This problem is avoided with ring species because gene flow still exists.2 Again, the Ensatina eschscholtzii salamanders surrounding California’s Central Valley demonstrate this principle. Between the two terminal forms in the ring is “a chain of morphologically distinct subspecies, each integrating with its neighbor.”5 Through this chain, gene flow between the terminal forms still exists. It is possible that because these species are, and were, connected by gene flow, their speciation might take longer to occur than if no gene flow were present.8 Gene flow makes it more difficult for the terminal forms to acquire unique mutations that could result in incompatibilities. Gene flow causes sharing of the alleles, decreasing the possibility for speciation. However, the speciation process may not be drastically affected as long as gene flow is limited.2 The ring species of North Atlantic white-headed larid gulls displays a moderate level of gene flow between the intermediate forms (admittedly with potential gaps), and the reproductively iso-

Ring Species: A Living Laboratory for the Study of Speciation│Boettger

lated terminal forms display a striking genetic similarity. These data not only indicate that the gulls are recently diverged but also that a high level of divergence is not necessary to create separate biological species.9

INFORMATION IN BROKEN RINGS│

Although an ideal ring species has continuous gene flow around the ring with the exception of the two terminal forms, a recent break in the ring hardly reduces its scientific value. An ideal ring species is very difficult to find because a ring is difficult to maintain. Although there is nothing about the ring itself that directly promotes discontinuities,10 the likelihood of a change in geography or the extinction of a subspecies is significantly increased when examining such a large system. As long as the populations on either side of a gap in the ring demonstrate a close genetic and phenotypic relationship, and evidence suggests that gene flow was present around the ring in the recent past, it is still a good representation of a ring species.1 Such a case would still adequately demonstrate the speciation process with intermediate forms between the diverged species in addition to exemplifying divergence despite gene flow. The greenish warbler is a good example of this. Not only do the birds on either side of the gap possess little genetic differentiation but they also respond “strongly”1 to each other’s mating calls. In addition, there is evidence that this gap is caused by recent human deforestation.1 Therefore the greenish warbler should be considered a good example of a ring species.

FUTURE DIRECTIONS│

Although ring species are useful systems to study speciation, there are presently very few well-documented examples. The reason for this is most likely related to their rarity. However, superficial investigations of several other potential ring species have yielded promising results,11, 12, 13, 14 but have yet to be studied in depth. One such potential ring species is the deer mouse, Peromyscus maniculatus, found in areas of Texas and Oklahoma. In this system there are five main ranges of the species that form a ring around the Red River. The presumptive terminal forms have been shown to be more closely related to populations of supposed intermediate forms than to each other, despite their overlapping geographical range. Furthermore, there were no hybrids found in the area where the two supposed terminal forms are sympatric, suggesting reproductive isolation.11 Although this research indicates that the deer mouse is a potential ring species, further phenotypic and genotypic analyses must be done on each population of the deer mouse in this area to determine its closest relative. In addition, a geographic survey must be conducted to determine the frequency of migration across the Red River and to understand the geographic history of the area.

CONCLUSION│

Unlike allopatric speciation, where it might be difficult to determine conclusively the way in which two present day species arose from an ancestral species, ring species contain all intermediate forms in the present day. Although the original genetics of speciation might still be difficult to determine, rings demonstrate the stepping stones needed for speciation to occur. In addition, ring species are an excellent example of speciation occurring despite gene flow because reproductively isolated forms are shown to still have some level of gene flow through genes being passed around the ring.2 Even ring systems that have a gap should still be considered good examples of ring species as long as the break is shown to be recent.1 Unfortunately, there are very few well-documented instances of ring species, but initial studies of possible ring species have yielded promising results and will hopefully be investigated further in the future.

REFERENCES│

1. Irwin, D.E., Bensch, S., and Price, T.D. 2001. Speciation in a ring. Nature, 409: 333-337. 2. Irwin, D.E., Irwin, J.H., and Price, T.D. 2001. Ring species as bridges between microevolution and speciation. Genetica, 112-113: 223-243. 3. Coyne, J.A., and Orr, H.A. 2004. Speciation. Sunderland, Massachusetts: Sinauer Associates. 4. Wake, D.B., and Alexandrino, J. 2005. Strong selection against hybrids at hybrid zone in the ensatina ring species complex and its evolutionary implications. Evolution, 59: 1334-1347. 5. Wake, D.B., and Yanev, K.P. 1986. Geographic variation in allozymes in a ‘ring species’, the plethodontid salamander Ensatina eschscholtzii of western North America. Evolution, 40: 702-715. 6. Wake, D.B., and Schneider, C.J. 1998. Taxonomy of the Plethodontid salamander Genus ensatina. Herpetologica, 54: 279-298 7. Gavrilets, S. 2000. Waiting time to parapatric speciation. Proceedings of the Royal Society - Biological Sciences (B), 267: 2483-2492. 8. Dobzhansky, T. 1958. Species after Darwin. In: A century of Darwin, S.A. Barnett (Ed.). pp. 19–55. London: Heinemann. 9. Snell, R.R. 1991. Interspecific allozyme differentiation among North Atlantic white-headed larid gulls. Auk, 108, 319–328. 10. Irwin, D.E., Bensch, S., Irwin, J.H., and Price, T.D. 2005. Speciation by distance in a ring species. Science, 307: 414-416. 11. Caire, W., and Zimmerman, E.G. 1975. Chromosomal and morphological variation and circular overlap in the deer mouse, Peromyscus maniculatus, in Texas and Oklahoma. Syst. Zool., 24, 89–95. 12. Grant, P.R. 1986. Ecology and evolution of Darwin’ finches. Princeton, New Jersey: Princeton University Press. 13. Thiollay, J.M. 1994. Family Accipitridae (hawks and eagles). In: Handbook of the birds of the world. Vol 2. J. del Hoyo, A. Elliott, and J. Sargatal (Eds.). New World Vultures to Guineafowl, pp. 52–205. Barcelona: Lynx Edicions. 14. McKnight, M.L. 1995. Mitochondrial DNA phylogeography of Perognathus amplus and Perognathus longimembris (Rodentia: Heteromyidae): A possible mammalian ring species. Evolution, 49: 816-826.

Distribution and Abundance of Phototropic Meiofauna in Various Habitats of Moreton Bay Vince Si, Senior, Warren College, General Biology major Division of Biological Sciences, University of California, San Diego

eiofauna are microscopic organisms that live in the interstitial spaces of the sediment. Only recently have major studies besides taxonomic identification been performed that indicate the importance of these organisms; in particular, they constitute a major food source for macrofauna and fish, which in turn are important food sources of humans. Distribution and abundance of meiofauna were examined in environments that had varying nutrient quality, grain size, and amount of predation. Rocky shore habitats contained significantly more meiobenthic organisms and species than seagrass beds and sandy shores.

INTRODUCTION │

Relatively little research has been performed on the microscopic organisms found in the benthic region (oceanic floor) of intertidal zones, which are the areas on the shore that are submerged at high tide and exposed at low tide. These microscopic organisms, which range in size from 40 μm to 500 μm, live between the grains of the sediment in the intertidal zones. These organisms, known as meiofauna, provide important trophic

links in the food chain of the intertidal zones; they are a food source for a variety of macrofauna and commercially important fish.1 More information about these organisms will therefore give scientists and fishing companies a better understanding of the impacts of dredging and pollution at the lowest trophic levels and how this affects higher trophic levels, all the way up to humans. If the meiofauna are affected by pollution, for example, it could possibly have a ripple effect and reduce fish population,

Saltman Quarterly│UCSD Division of Biological Sciences│Volume 4│2006-2007

methods and materials│

Study Area The study took place along the western coast of North Stradbroke Island at the Moreton Bay Research Station in Dunwich (27’ 2˚ S and 153’ 3˚ E). Three different types of habitats were chosen: a sandy shore with large grains, a rocky shore with small, fine grains, and a seagrass bed with intermediate size grains. Sampling Three samples of meiofauna were obtained over a two-day period from the three habitats, giving a total of 18 samples. These samples were all collected near the Moreton Bay Research Station on North Stradbroke Island. At each of the different habitats, a tablespoon, with a volume of approximately 15 cm2, was used to gather the top layer of sediment, while avoiding the anoxic layer, a layer devoid of oxygen, at a depth of 20 cm. Each sample was obtained from an area of approximately 30 cm2 and was deposited into a plastic vial, which was then filled with 20 mL of sea water. At each of the habitats, the three samples were obtained 1 meter apart. Over the two-day period, the samples were collected at roughly the same time and locations. The vials were then taken to a dark room where a light gradient was established using a cold light source in order to attract the phototropic meiofauna. As the meiobenthic organisms swam towards the light, they were taken out via pipette and placed into Petri dishes and concave glass slides for identification and rough quantification under compound and dissecting microscopes. The Petri dishes were examined under 4x magnification, and the concave glass slides were examined under 10x magnification. Once the quantity and diversity of organisms were obtained, the density of the organisms in each population in a one square meter area was extrapolated using the average number of organisms in each habitat per 30 cm2. Statistical Analyses Due to the fact that there are multiple variables, such as rocky shore, sandy shore, and seagrass beds, an Analysis of Variance, ANOVA, was performed. This test is a statistical test that determines how significantly different two variables are from each other, in this case, the abundance in each habitat and the species diversity in each habitat. A post-hoc test was also performed to determine if a significant difference existed. Two ANOVA tests were performed with a p value of 0.05, one for the abundance variable and one for the diversity variable. Post-hoc tests were performed to ensure the reliability of the data.

Research │21

RESULTS│

The rocky shore habitat contained the greatest number of meiofauna and the sandy shore habitat the least, with the seagrass habitat in the middle (figure 1). In the sandy shore habitat, the average number of meiobenthic organisms was 2.92 in an area of 20 cm2. The seagrass habitat contained approximately 8 times as many, with an average of 23.5 organisms in a 20 cm2 area. The rocky shore habitat contained almost 4 times as many as the seagrass and 30 times as many as the sandy shore habitat with an average of 97.5 organisms in a 20 cm2 area. However, according to the ANOVA and post-hoc tests, figure 1 and table 1, respectively, the abundance of organisms in the sandy shore habitat compared to that in the seagrass was not significantly different from each other. The ANOVA and post-hoc tests did show a significant difference between the number of organisms in the rocky shore habitat and the number found in both the sandy shore and seagrass habitats. Once the average number of organisms was obtained per 30 cm2, the number of meiofauna per m2 was calculated. In the sand, 972.22 organisms m-2 were extrapolated; in the seagrass, 7833.33 organisms m-2 were calculated; and in the rocky shore habitat, 32,500 organisms m-2 were obtained. In regard to the diversity of the meiobenthic organisms, the rocky shore had greater species richness than the other two habitats. The sandy shore habitat contained an average of two species, the seagrass had an average of four species, and the rocky habitat had an average of 8.5 species (figure 2). The sandy shore habitat had an abundance of copepods, particularly Calanus, and amphipods, of the genus Parathemisto. The seagrass beds had organisms from the genus Favella and Nanomia in addition to the organisms observed in the sandy shore. The rocky shore had all the aforementioned organisms in addition to organisms from the genus Lucifer, Tomopteris, Salpa, and Oithona. With the ANOVA and post-hoc tests, a significant difference in species diversity was shown between all three habitats. The sandy shore habitat had the least amount of species diversity, and the seagrass had slightly more, whereas the rocky shore habitat contained more than four times as many species as the sand and twice as many species as the seagrass.

DISCUSSION│

In the study, diversity and abundance were directly related; as the number of meiofauna increased so did the number of taxa. This correlation can be attributed to the factors that affect both the diversity and abundance of the organisms. One major factor that contributes to the distribution pattern observed in the study is the size of the grain particles.6 Since meiofauna live in the interstitial spaces of the sediment, the size of the particles is important. As the grain size increases, the interstitial space also increases, which allows more flow of water. The increase in Vertical bars denote 0.95 confidence intervals

No. of Organisms

which would ultimately harm humans, especially in developing countries that rely heavily on fishing. In comparison to macrofauna, meiofauna are only 3% in biomass when compared to that of macrobenthos, but provide 15% of the food that is consumed in a sublitteral community.2 Meiofauna, therefore, are responsible for about five times the total benthic metabolism of the macrofauna. In addition to being a common food source for higher organisms, meiofauna are responsible for 51% of total primary production, which is the production of energy by photosynthetic organisms.3 Moreover, meiobenthic communities contribute significantly to the recycling of nutrients in the sediment4 and can serve as signals for environmental disturbances.5 Because they are important food for macrofauna and fish, changes in meiofaunal populations will likely impact higher trophic levels. A small amount of information is available concerning these organisms due to their relatively recent discovery. Moreover, it was viewed in the past that meiofauna were relatively unimportant. However, as previous studies have cited, meiofauna are an important ecological facet of the marine world; studies of full ecosystems with multiple trophic levels and the biodiversity of animals and microbial organisms are essential. This study sets out to examine the biodiversity and abundance of phototrophic meiobenthic organisms in different environments on North Stradbroke Island in Southeast Queensland, Australia. Located on the Island is the Moreton Bay Research Station. This study will also attempt to identify the various factors that may contribute to the differences found in the abundance and diversity of the different environments.

Sandy Shore

Seagrass

Rocky Shore

SUBSTRATE Figure 1: Average number of organisms in each habitat. The average number of meiofauna was highest in the rocky shore habitat. The seagrass habitat had the next highest amount of organisms and the sandy shore, the least. With the 0.95 confidence bars, the sandy shore and the seagrass do not differ significantly, but the rocky shore does differ significantly.

Distribution and Abundance of Phototropic Meiofauna of Moreton Bay│Si

Sandy Shore

Seagrass

Rocky Shore

0.373074

0.002338

Sandy Shore

0.005178

Seagrass

0.00017

Rocky Shore

0.00018

Seagrass

0.373074

Rocky Shore

0.002338

0.005178

Table 1: Post-hoc test of number of organisms in each habitat. The rocky shore habitat is significantly different from the other two habitats, indicated by the low numbers. However, the sandy shore and seagrass habitats are not significantly different from each other, indicated by higher numbers.

Habitat:

Average Extrapolated Density (meiofauna/m2 )

Sandy Shore

972.22

Seagrass 7833.33 Rocky Shore 32500.00 Table 2: Extrapolated density of meiofauna per m2. The predicted amount of meiobenthos in each of the habitats. The density increases from sandy shore to seagrass to rocky shore.

space makes the habitat unsuitable for meiofauna because the meiofauna become too small. The increase in water flow, when coupled with a larger space, can flush out the meiofauna. The grain size of the different habitats in this study decreased from the sandy shore to the seagrass to the rocky shore. The sandy shore had much larger sand particles with clear water; the seagrass had smaller particles and was slightly more turbid; the rocky shore’s grains were muddy and much more turbid, indicating small grain size and more nutrients. The findings of this study supports the finding of Coull and Bell’s 1979 study7 that the abundance of meiofauna is greater in mud than in equivalent sand substrates. However, grain size cannot be confidently related to the number of species present in the substrates. In the study, samples from each habitat varied in number, which provides a confounding variable. In order to be able to confidently state that grain size is related to the number of species, subsamples with the same number of meiofauna as the sandy shore need to be taken from the seagrass beds and the rocky shore and compared. The increase in nutrients is also related to the number of meiofauna present.8 As the amount of nutrients decreases, only certain types of species are able to live in these areas. In rocky shore habitats, nutrients were more abundant because the water was murky, indicating the presence of dissolved organic matter and salts. In contrast, in the sandy shore habitats, the water was very clear, indicating it was oligotrophic,

No. of Species

Vertical bars denote 0.95 confidence intervals

Sandy Shore

Seagrass SUBSTRATE

Rocky Shore

Figure 2: Average number of species in each habitat. The rocky shore contained the most diverse amount of meiofauna and the sandy shore the least. The vertical bars of 0.95 confidence indicate that the diversity of meiofauna is significantly different in all three habitats.

Sandy Shore

Seagrass

Rocky Shore

0.00017

0.00018 0.00017

0.00017

Table 3: Post-hoc test for number of species in each habitat. The low numbers indicate that the diversity of meiofauna is significantly different in all three habitats.

or lacking any dissolved nutrient salts necessary for supporting life. The seagrass beds’ water was intermediate, with murkier water than the sandy shore but less murky water than the rocky shore. Consequently, in the rocky shore habitat, where nutrients are abundant, more types of meiofauna can thrive, whereas the seagrass has fewer nutrients and only a few species can survive, and in the sandy shore, nutrients are the lowest and very few species can survive. Another factor that could have contributed to the observed distribution of the meiobenthic organisms is predation. During the collection process, at low tide during the day, large groups of soldier crabs were seen along intertidal regions of the sandy shore. This large number of soldier crabs could contribute to the low numbers of organisms found in the sandy shore habitat. Moreover, in the seagrass habitat, juvenile fish were often seen, and meiobenthos are a required diet for these fish. In the rocky shore habitat, many predators were found, yet the number of meiofauna was highest in this habitat. This confounding observation could be attributed to the high volume of nutrients found in the rocky habitat, which may outweigh the predation factor. This study had some limitations that may have affected the results. The samples were taken approximately at low tide during the day over a two-day period; but the low-tide period shifts daily, which could bias the results. However, the procedures used in the experiment attempted to account for this bias by taking samples at all habitats at the same time. Essentially, this tidal shift variable was held constant. Other studies have shown that meiofauna aggregate in clumps, and the distribution of the organisms is non-uniform but rather patchy.9 This patchy distribution may have skewed the results because one sample may have randomly been collected at an aggregate of meiofauna whereas another sample may have been collected at a patch with few or no meiofauna. However, an increase in the number of samples will reduce this factor. Another issue that may have affected the results is the estimation of the amount of meiofauna in the samples. Due to their small size, the meiobenthos were difficult to accurately quantify, and a rough estimation had to be used. Consequently, the accuracy of the results could be very low. Because the samples were only taken over a two-day period, there were a limited number of samples, which lowers the confidence of the results. If more samples had been taken over a longer period of time, more definitive results and trends would have been obtained. Despite these limitations, the method of collection used in this study provides a good way to get a general idea of the distribution and variety of meiofauna in a small area. This study’s examination of meiofauna has important implications, such as how habitat may influence the distribution, abundance, and diversity of meiofauna. Their distribution may, in turn, help to explain the distribution, abundance and diversity of larger organisms, such as fish, crabs, and other meiobenthic predators. As stated previously, humans can also be indirectly impacted by meiofauna, and vice versa. There are important future studies that need to be performed, especially those dealing with how pollution affects meiofauna. If human pollution on intertidal shores is affecting meiofauna adversely, it will ripple through the food chain and impact humans as well. This study’s purpose was to obtain a general idea of how meiobenthos are distributed among different habitats and to provide the groundwork for studies on the effects of pollution on these organisms. Future research could therefore shed light on the interactions between habitat, predation, and pollution.

Saltman Quarterly│UCSD Division of Biological Sciences│Volume 4│2006-2007

Research │23

REFERENCES │

1. Hann, B.J., and Kowalchuk, S. 2006. Zoobenthos and Meiobenthos communities in Lake Winnipeg. (Department of Zoology, University of Manitoba) 2. Gerlach, S.A. 1971. On the importance of marine meiofauna for benthos communities. Oecologia (Berl.), 6: 176-190. 3. Stead, T.K., Schmid-Araya, J.M., and Hildrew, A.G. 2005. Secondary production of a stream metazoan community: Does the meiofauna make a difference? Limnology and Oceanography, 50: 398-403. 4. Nalepa, T.F., and Quigley, M.A. 1983. Abundance and biomass of the meiobenthos in nearshore Lake Michigan with comparisons to the macrobenthos. J. Great Lakes Res, 9, 4:530-547. 5. Pequegnat, W.D. 1975. Meiobenthos ecosystems as indicators of the effects

6. 7. 8. 9.

of dredging. Cronin, L. E. ed., Estuarine research. Geology and engineering. (New York, Academic Press) 2: 573-583. Mclntyre, A.D. 1969. Ecology of marine meiobenthos. Biological Reviews, 44: 245-290. Coull, B.C., and Bell, S.S. 1979. Perspectives of marine meiofaunal ecology. Livingston, R. J. ed., Ecological processes in coastal and marine ecosystems. New York, Plenum Press. pp. 189-216. Thorsen, G. 1966. Some factors influencing the recruitment and establishment of marine benthic communities. Neth. J. Sea Res., 8: 267-293. Coull, B.C., and Wells, J.B.J. 1981. Density of mud-dwelling meiobenthos from three sites in the wellington region. New Zealand Journal of Marine and Freshwater Research, 15: 411-415.

Evolution of the Bacterial Flagellum Arezou Amidi, Senior, Earl Warren College, Molecular Biology major; Tim Wong, Thurgood Marshall College Alumnus, Molecular Biology major; Alexandra Dodds, Senior, Revelle College, General Biology major; Sara Siddiqi, Revelle College Alumna, Molecular Biology major; Jing Wang, Senior, Warren College, General Biology major; Tracy Yep, Senior, Revelle College, Molecular Biology major; Dorjee G. Tamang; and Milton H. Saier, Jr., Professor, Molecular Biology Division of Biological Sciences, University of California, San Diego

reationists (‘intelligent designers’) have considered bacterial flagella to be ‘irreducibly complex,’ leading them to profess that these structures could not have appeared via an evolutionary process. Here we present evidence favoring the notion that they are modular structures consisting of (1) a basal body, (2) a filamentous propeller, (3) an interconnecting hook complex, (4) a rotary motor, (5) a secretion/assembly system, (6) a secretion energizing ATPase, and (7) various ancillary proteins. We show that each module could have evolved independently of the others from a primordial system of related subfunction having nothing to do with cell motility. Complexity arose by domain and protein recruitment as well as by intragenic and extragenic duplication events. The net result has been the co-evolution of many structurally and functionally distinct flagella, present in various bacteria, sharing only about half of their protein constituents.

INTRODUCTION │

Proponents of intelligent design (ID) claim that the bacterial flagellum (BF) is ‘irreducibly complex,’ so complicated that it could not have arisen through an evolutionary process involving natural selection and gradual change. Its construction, like that of a clock, had to be designed by an intelligent entity.11 One reason for this claim is the supposition that each component of the flagellum serves only the purpose of flagellar construction. Further, each protein is assumed to have appeared independently of other proteins. Finally, in accordance with creationist thinking, they are presumed to have been created for the sole purpose of motility.18 If these assumptions are valid, then concluding that the BF could not have evolved through natural selection to its present state of complexity with a reasonable probability might be justified. In this brief article, we summarize published evidence from many laboratories as well as the results of our own studies showing that none of these assumptions is justified. The BF apparently evolved as a modular system with many of its components having been derived from various simpler protein complexes. These modules will be discussed sequentially in this article following presentation of various evolutionary principles.

THE MODULAR CONSTRUCTION OF FLAGELLA│ FliD FlgK

Identifiable flagellar modules include: (1) the basal body and its set of rings that anchor the flagellum to the bacterial cell

FliC

FlgE FlgL FlgH

FlgG

FlgI

FlgB , FlgC, FliE , FlgF FlhB

FlhA

FliF FliG, FliM, FliN FliO, FliP, FliQ, FliR FliH

FliI

Figure 1: Physical structure of the E. coli flagellum showing the basal region embedded in the bacterial cell envelope with its envelope-associated rings (bottom), the hook (middle), and the filament (top). The approximate positions of various flagellar proteins in the overall structure are shown.

envelope, (2) the hook complex that connects the basal body to the filament, (3) the filament that serves as the propeller for motility, (4) the motor for flagellar rotation, (5) the secretion system that exports flagellar subunits, and (6) the ATPase complex that energizes secretion. It also requires a number of ancillary proteins for its proper synthesis and assembly.14 How these modules appeared during evolutionary history is becoming increasingly clear as will be shown in this article. Several functionally dissimilar proteins of the BF arose from a single common ancestral protein via ancient gene duplication events. Some additional constituents were constructed by fusing previously recognized protein domains. Others increased in size and complexity due to intragenic duplication events. Several modular BF proteins have homologues that serve functions unrelated to motility. The physical structure of the Escherichia coli flagellum is shown in figure 1, which reveals where the different structural proteins can be found within the flagellum. A complete tabulation of flagellar proteins in E. coli can be found in the excellent, more detailed review by Pallen and Matzke.13

VARIATION IN FLAGELLAR STRUCTURES│

BF are not uniform in construction. They include a range of related structures that differ from organism to organism with constituents that are essential for optimal motility in that particular organism. For example, while Gram-negative bacteria have two membranes, Gram-positive bacteria have just one membrane, and consequently the flagella of these two bacterial types differ in ways that reflect these two envelope types. While Gram-negative flagella have basal bodies that include an M-ring that interacts with the inner membrane, a P-ring that interacts with the peptidoglycan cell wall, and an L-ring that interacts with the lipopolysaccharide-containing outer membrane, Gram-positive bacteria, lacking the outer membrane, lack the L-ring and its constituents. They also lack the typical Gram-negative bacterial P-ring. Because many other constituents of the two flagella, including the proteins of the M-ring, are demonstrably homologous, they were evidently derived from a common ancestral system that underwent modifications to allow optimal embedment in the various cell envelopes. Interestingly, of the approximately 40 constitu-

Evolution of the Bacterial Flagellum│Amidi et al.

ents of the enteric bacterial flagellum present in E. coli, only about 20 of them are common to all bacteria.14 What is essential for flagellar function in one bacterium may not even be present in another.1

SIMILARITIES BETWEEN BACTERIAL FLAGELLA AND TYPE III PROTEIN SECRETION SYSTEMS│

Bacterial flagella and virulence related type III protein secretion systems (T3SSs) are clearly derived from a common ancestral system sharing at least nine homologous constituents.1,12 But which was the precursor of the other? Some have argued that T3SSs preceded the BF basal construct while others have argued the opposite.3,15 We would like to suggest that both systems arose from a simpler primordial secretion system. In fact, Gram-negative bacteria have evolved many distinct mechanisms for exporting proteins across the two membranes of their cell envelopes, and some of these systems share protein constituents.16,17 T3SSs secrete substrate proteins directly from the Gram-negative bacterial cytoplasm across the inner and outer membranes of the bacterial envelope either to the external medium or directly into the cytoplasm of a host plant or animal cell with which the bacterium forms a symbiotic or pathogenic relationship.7 T3SSs consist of thin, rigid, hypodermic needle-like protein complexes anchored to the envelope by basal structures resembling flagellar basal bodies (figure 2). Because in some instances both types of systems can secrete the same proteins across the two-membrane envelope, T3SSs and flagellar secretory systems overlap structurally and functionally. T3SSs are often encoded on mobile plasmids and ‘pathogenicity islands,’ both of which can be transferred laterally between Gram-negative bacteria.5 By contrast, flagellar systems are almost always chromosomally encoded and appear to have been transmitted to progeny bacteria largely by vertical descent (ref. 12, and unpublished results). Phylogenetic analyses have established that the proteins of flagellar basal bodies and those of T3SSs form distinct branches on the phylogenetic tree when any of the homologous protein constituents of these systems are examined. This implies that the divergence of proteins comprising T3SSs and flagella occurred during the early evolutionary history of these systems.

RELATED FLAGELLAR STRUCTURAL PROTEINS│

Sequence similarity is observed between certain protein constituents of the flagellum although not all of these share the same domains. Thus, the FlgB, FlgC, FlgF and FlgG ‘transmission shaft’ rod proteins all exhibit regions of sequence similarity with the FlgE ‘universal joint’ hook protein (figure 3). For example, FlgG has 3 domains (N, C1, and C2) that are homologous to these domains in FlgE, but the central domain of FlgE (M) is absent in FlgG (figure 3). The simplest explanation is that the evolutionary precursor of these proteins resembled the smaller ones (FlgG and FlgF) and that a novel domain was inserted into FlgE to generate the larger one. The differing subflagellar locations and functions of these homologous constituents presumably resulted in part from domain insertion and in part from sequence divergence during evolution. The Flg rod proteins, the FlgE hook protein and the FlgK hook associ-

Figure 2: Comparative schematic depiction of the E. coli flagellum (left) and Gram-negative bacterial type III secretion systems (T3SS, right). Homologous structures in the basal regions of both structures are shown with the same shading. Non-homologous structures are shown as distinct structures. IM, inner (cytoplasm) membrane; PG, peptidoglycan cell wall layer; OM outer (lipopolysaccharide-containing) membrane. The multilayered MSring interacts with the IM, the P-ring anchors the structure to the PG, and the L-ring interacts with the OM. ATP hydrolysis via the FliI ATPases provides the energy required for protein export and assembly.

ated protein-1 (HAP1) all share a common C terminal domain (C2), the DUF1078 domain in figure 3, and most have similar N terminal targeting sequences, the Flg-bb-rod domain in figure 3. The latter directs the newly formed cytoplasmic proteins to the general secretory apparatus. The function of the common C2 domain is unknown, but it could be involved in assembly. It seems clear that the complexity of the flagellarrod/hook/filament complex arose in part by gene duplication events followed by sequence and domain divergence. Such mechanisms suggest a possible basis for the evolution of dissimilar protein-protein interactions since homo-multimeric proteins always have self-associative properties.

THE HOOK AND FLAGELLAR FILAMENT│

Subunits of bacterial flagellar filaments, called flagellins, vary tremendously in amino acid sequence as well as in quaternary structures of the assembled filament. These filaments can be curly or straight, right-handed or left-handed helices, and either flexible or rigid. Some are modified by methylation or glycosylation. In different strains of E. coli, there are nearly 50 sequence divergent flagellins, a surprising observation since each E. coli strain usually has just one. This observation suggests that the genes encoding these proteins have been transferred between bacteria with high frequency. Moreover, an Arctic sulfate-reducing bacterium, Desulfotalea psychrophila, has dozens of flagellins encoded within its genome. In this case, we do not know what their functions are. However, we do know that while some flagellar filaments consist of a single flagellin, others contain many. Surface exposed residues in flagellins, which are strongly antigenic, are Figure 3: Results of a search of the National Center for Biotechnology Information (NCBI) Conserved Domain Database (CDD) much more variable than the buried using the Escherichia coli FlgG flagellar basal-body rod protein as the query. The accession number for FlgG is AAN79818. Bars indicate regions of homology for different flagellar proteins: FlgF, FlgE, FlgC, FlgK and FlgB. The common N-terminal residues, which are poorly antigenic. domain is labeled Flg_bb_rod while the common C-terminal domain is labeled DUF1078. Amino acyl residue position in FliG is This surface variability provides a mechanism for immune evasion by provided at the top. The web address for the NCBI CDD is http://www.ncbi.nih.gov/structure/cdd/cdd.shtml. pathogenic bacteria and is easily explained by natural selection (see ref. 13 for a more detailed review). In spite of the tremendous sequence variation in flagellins, most if not all of them probably share a common ancestry. In fact, flagellins are also homologous to the hook-associated protein-3 (HAP3 or FlgL). Moreover, they share domains with the hook-associated protein-1 (HAP1; FlgK), which in turn shares

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Figure 4: Generalized structure of F-type ATPases of prokaryotes, mitochondria and chloroplasts. The heterohexameric a3b3 ATPase complex in F-ATPases is homologous to the homohexameric (FliI)6 of the flagellum. The dimeric FliH protein of the flagellum, which is known to interact with (FliI)6, is equivalent of the fused b (N-terminal) and d (C-terminal) subunits of the F-ATPase which interact with and regulate the a3b3 ATPase. The figure shows the membrane lipid bilayer as well as the movement of a proton from outside to inside the bacterium accompanied by ATP synthesis.

domains with other flagellar proteins as noted in the previous section. Finally, flagellins may be partially homologous to the secreted needle complex proteins in T3SSs. This last observation might be expected since the BF and T3SSs share a common origin.

EVIDENCE FOR INTERNAL REPEAT UNITS IN FLAGELLAR PROTEINS│

One of the most common evolutionary mechanisms for increasing biological complexity and generating diversity involves genetic duplication followed by sequence divergence. Our bioinformatic studies revealed that FlgK of E. coli, a hook-filament junction protein, shows significant sequence similarity throughout much of its length with internal regions of bacterial autotransporter-2 proteins, adhesins that contribute to bacterial virulence. The autotransporter-2 proteins are known to contain multiple repeat units of 7 to 60 amino acyl residues each.10 We could confirm the presence of tandem repeats in FlgK homologues, and other flagellar proteins exhibit repeated sequences as well. Several structural constituents of the BF evidently grew in size and complexity by multiplication of repeat units.

THE MOTOR│

The BF motor consists of two proteins (MotA and MotB) that are homologous to (1) the motor for a dissimilar form of motility called ‘gliding’ that otherwise does not depend on constituents of the BF and (2) the energizers of outer membrane receptors that concentrate large molecules in the periplasm of the Gram-negative bacterial cell, between the inner and outer envelope membranes.19 These proteins comprise transmembrane proton channels.6 MotAB probably evolved as a simple channel complex to allow gated proton flow across the bacterial membrane. It may have thus allowed dissipation of acid (H+) for cytoplasmic pH stabilization. Once these channels existed, they could have been recruited for their various currently known functions of motility and outer membrane transport energization.9 In contrast to the better characterized flagella of enteric bacteria that rotate either clockwise or counterclockwise, other proteobacteria possess flagella that rotate in only one direction, but with two modes, rotating and stalling, or three modes: fast, slow, and stop. Different bacteria evidently use different motor mechanisms to achieve directed motility.

THE FLAGELLAR ASSEMBLY ATPASE│

Flagellar assembly ATPases, the FliI proteins, are homologous to (1) T3SS ATPases and (2) F-type ATPase a- and b-subunits of prokaryotes and eukaryotic organelles such as mitochondria and chloroplasts.8,12 ATPases are known to energize numerous biological processes, so FliI could easily have evolved independently of flagellar function, having later been recruited to energize flagellar assembly. In addition to FliI, other constituents of the flagellar assembly system may have been derived from a primordial F-type ATPase. Like the BF, F-type ATPases use a rotary motor driven by the flow of protons (H+) down their electrochemical gradient.2 Similarities in sequence and

structure are observed for the dimeric FliH protein (which interacts with and regulates the hexameric FliI ATPase), and two subunits of the F-type ATPase, the dimeric b- and d-subunits (which interact with the homologous hexameric a/b-subunit F-ATPase complex). Moreover, our colleagues Jiwon Youm and Seul-a Shin have noticed that in Mycobacterial species, the b- and d-subunits of F-type ATPases are fused together as in FliH. These observations indicate that both FliI and FliH have coevolved from F-type ATPase-like subunits for the purpose of energizing and regulating flagellar assembly.

FLAGELLAR-SPECIFIC AUXILIARY PROTEINS DERIVED FROM NON-FLAGELLAR SOURCES│

The P-ring assembly chaperone protein, FlgA, is homologous to other assembly proteins in bacteria, such as pilus chaperone proteins. These proteins share the ‘b-clip fold’ domain with many enzymes and accessory proteins. A common origin correlates with their shared fold and subfunction. Another example of a flagellar auxiliary protein sharing a common origin with non-flagellar proteins is FlgJ. It has a C-terminal amidase domain that locally hydrolyzes the bacterial cell wall in preparation for flagellar construction. This domain is homologous to many type IV amidases serving a variety of functions unrelated to motility. Finally, expression of flagellar genes involves transcription using a flagellum-specific sigma factor to initiate mRNA synthesis by RNA polymerase. This sigma factor (sF or FliA) is homologous to many other sigma factors in bacteria.4 sF could have evolved from a primordial vegetative s.

CONCLUSIONS│

As a result of research conducted in hundreds of laboratories over the past several decades, we now recognize that the modular bacterial flagellum may have evolved from several different sources unrelated to an organelle of motility. For example, (1) the flagellar subunit secretion apparatus and T3SSs were derived from an ancestral secretion system that used ATP and an ancient ATPase to drive protein export. (2) This ATPase and its regulatory protein share a common ancestry with subunits of rotary F-type ATPases. (3) The filament and parts of its connecting ‘hook complex’ may have arisen from a bacterial adhesin. (4) The motor for flagellar rotation evidently derived from a proton-conducting channel complex that also evolved into motors for gliding motility and molecular uptake. (5) Increased complexity from relatively simple homopolymeric structures resulted from both intragenic and extragenic duplication events, giving rise to multiply interacting protein constituents. (6) Sequence divergence and domain insertion resulted in functional specialization that rendered each protein irreplaceable. (7) Finally, flagellumspecific accessory apparati were recruited to facilitate flagellar assembly. Natural selection can thus account for the development of flagellumdriven bacterial motility. Scientific postulates base their conclusions on that which is known, recognizing that much has yet to be understood. The realm of religion and mythology are left to deal with the unknown. Oscar Wilde once stated, “Science is the record of dead religions.” Regardless of whether science proves a religious postulate correct or false, that postulate then enters the arena of science and exits the realm of religion. The BF has entered that arena.

REFERENCES│

1. Aizawa, S.I. 2001. Bacterial flagella and type III secretion systems. FEMS Microbiol. Lett., 202: 157-164. 2. Dimroth, P., von Ballmoos, C., Meier, T., and Kaim, G. 2003. Electrical power fuels rotary ATP synthase. Structure, 11: 1469-1473. 3. Gophna, U., Ron, E.Z., and Graur, D. 2003. Bacterial type III secretion systems are ancient and evolved by multiple horizontal-transfer events. Gene, 312: 151-163. 4. Gourse, R.L., Ross, W., and Rutherford, S.T. 2006. General pathway for turning on promoters transcribed by RNA polymerases containing alternative sigma factors. J. Bacteriol., 188: 4589-4591. 5. Groisman, E.A., and Ochman, H. 1996. Pathogenicity islands: bacterial evolution in quantum leaps. Cell, 87: 791-794. 6. Hosking, E.R., Vogt, C., Bakker, E.P., and Manson, M.D. 2006. The Escherichia coli MotAB proton channel unplugged. J. Mol. Biol., 364: 921-937. 7. Hueck, C.J. 1998. Type III protein secretion systems in bacterial pathogens of

Evolution of the Bacterial Flagellum│Amidi et al.

animals and plants. Microbiol. Mol. Biol. Rev., 62: 379-433. 8. Iino, R., Rondelez, Y., Yoshida, M., and Noji, H. 2005. Chemomechanical coupling in single-molecule F-type ATP synthase. J. Bioenerg. Biomembr., 37: 451-454. 9. Ito, M., Terahara, N., Fujinami, S., and Krulwich, T.A. 2005. Properties of motility in Bacillus subtilis powered by the H+-coupled MotAB flagellar stator, Na+-coupled MotPS or hybrid stators MotAS or MotPB. J. Mol. Biol., 352: 396408. 10. Kim, S.H., Chao, Y., and Saier, M.H., Jr. 2006. Protein-translocating trimeric autotransporters of Gram-negative bacteria. J. Bacteriol., 188: 5655-5667. 11. Musgrave, I. 2004. Why intelligent design fails: a scientific critique of the new creationism. Young, M. and Edis, T., Eds. Rutgers University Press, Piscataway, N.J., USA. p. 72-84. 12. Nguyen, L., Paulsen, I.T., Tchieu, J., Hueck, C.J., and Saier, M.H., Jr. 2000. Phylogenetic analyses of the constituents of type III protein secretion

systems. J. Mol. Microbiol. Biotechnol., 2: 125-144. 13. Pallen, M.J., and Matzke, N.J. 2006. From The origin of species to the origin of bacterial flagella. Nature Rev., 4: 784-790. 14. Pallen, M.J., Penn, C.W., and Chaudhuri, R.R. 2005. Bacterial flagellar diversity in the post-genomic era. Trends Microbiol., 13: 143-149. 15. Saier, M.H., Jr. 2004. Evolution of bacterial type III protein secretion systems. Trends Microbiol., 12: 113-115. 16. Saier, M.H., Jr. 2006. Protein secretion systems in Gram-negative bacteria. Microbe, 1: 414-419. 17. Saier, M.H., Jr. 2007. Protein secretion and membrane insertion systems in Gram-negative bacteria. J. Membr. Biol., in press. 18. Wilkins, A.S. 2006. ‘Intelligent design’ as both problem and symptom. Bioessays, 28: 327-329. 19. Zhai, Y.-F., Heijne, W., and Saier, M.H., Jr. 2003. Molecular modeling of the bacterial outer membrane receptor energizer, ExbBD/TonB, based on homology with the flagellar motor, MotAB. Biochem. Biophys. Acta, 1614: 201-210.

Pathogenic Bacteria within the Dental Biofilms of a Human and a 130-pound Marine Predatory Fish Kjeld Aamodt, Senior, Muir College, General Biology major Division of Biological Sciences, University of California, San Diego

he dental plaque biofilm of a captive subtropical marine gulf grouper, Mycteroperca jordani, was cultured for the first time. Isolates reflect the presence of the pathogenic dental bacterium, Vibrio carchariae, which has been implicated in causing an antibiotic-resistant infection in humans bitten by the great white shark, Carcharodon carcharias. In addition, the presence of Gram-positive Streptococci in the grouper’s dental biofilm is unusual for the marine environment, warranting future investigation. It is possible that this bacteria is similar to that found to infect farmed fish (Streptococcus iniae), leading to increased rates of morbidity in fish stocks. The bacteria isolated from the human subject served to verify the presence of typical human oral bacteria (Streptococcus mutans, Lactobacillus casei) and to corroborate the efficacy of the selective culturing methods used in this experiment.

INTRODUCTION │

Only a handful of research papers have investigated the oral microbiota of marine vertebrates,3 therefore little is known about the composition of dental biofilms on marine organisms such as “Harvey,” a captive 29year-old, 130-pound gulf grouper, Mycteroperca jordani. As a top predator of the near-shore subtropical marine ecosystem, gulf groupers feed on large, bony fish, lobsters, and even juvenile hammerhead sharks. The presence of pathogenic bacteria in a gulf grouper’s mouth could reflect the presence of an infected population of fish lower down the food chain, or could be indicative of a symbiotic relationship involving bacterial degradation of leftover food particles, aiding oral hygiene. Moreover, it is even possible that a potentially pathogenic bacterium could serve as a bacterial “poison” to aid the capture of bitten prey similar to the strategy employed by the formidable Komodo dragon.15 Gulf groupers may reach up to 6 1/2 feet long and 200 pounds within their near-shore natural habitat range from La Jolla, California to Baja California, Mexico. Based on diet, gulf groupers occupy a similar niche to comparably sized young great white sharks before the sharks switch to a diet consisting of mammals. Shark attacks on humans are generally non-fatal, with about three-fourths of bite-victims surviving.1 In a high percentage of attacks, a significant health risk is due to infection from the antibiotic-resistant bacterium V. carchariae transmitted from the teeth of the sharks. Bacteria, rather than loss of tissue from the bite itself, remain a silent threat.3 Harvey is one of the longest-term resident fish of Scripps Institution of Oceanography. Adding to his allure, Harvey is extremely sociable with humans, possibly arising out of the lack of symbiotic grooming organisms within its habitat tank and its long exposure to humans since being captured as a juvenile. Achieving popularity for his endearing doglike behavior with the aquarists and aquarium divers, he presently inhabits the Magdalena Bay tank at Birch Aquarium, Scripps Institution of Oceanography, University of California, San Diego, where he can be witnessed to follow divers in the water while opening his mouth, seeming to enjoy his body being massaged and his teeth brushed. It is this unusual behavior for a fish that inspired this research and enabled the bacteria

samples to be collected without sedation or invasive methods. Harvey even lifted his mouth out of the water for sampling, which decreased the potential that the ambient seawater contaminated the samples. Although the dental biofilm of gulf groupers had not been investigated previously, there has been significant research into the composition of human dental biofilms. Human dental plaque forms primarily from Streptococcus mutans, Lactobacillus casei, and filamentous Actinomyces naslundii. However, it is estimated that human plaque contains up to 400 distinct bacterial species.6,11 Working cooperatively through the use of intercellular signaling mechanisms, the oral bacteria construct an organized three dimensional architecture called a “biofilm.” The pathogenic bacteria considered in this experiment are all known to contribute to disease in humans, either septicemia, gastroenteritis,2,14 or the formation of tooth decay. The mechanism of tooth decay begins with the infection of facultatively anaerobic, lactose-fermenting bacteria. The lactic acid from anaerobic sugar fermentation destroys tooth enamel at the site of the plaque by lowering the pH at the tooth surface, resulting in demineralization of the tooth structure. Both human and grouper teeth are composed of distinct layers of tissue, including an extremely durable outer layer called enamel, which is the hardest material in the human body and the most important layer for the resistance to tooth decay. Its mineral structure consists primarily of a crystalline calcium phosphate known as hydroxyapatite. This high mineral content (96%) not only contributes to its strength but also to its brittleness and susceptibility to demineralization.4,5 Marine fish teeth are composed of minerals similar to those of human teeth, although they differ in morphology and number. Marine toothed organisms may be protected from inherent demineralization from the high pH of seawater, which is usually slightly basic, with a pH range between 7.5 and 8.4. Such a high ambient pH may preclude the formation of the acidic environment in a dental biofilm that leads to tooth demineralization even if anaerobic acid producing bacteria exist on the teeth. The objective of this study is to investigate the microbial constituents of dental plaque biofilms. Particularly, the purpose of this study is to look for the presence of various known pathogenic bacteria in the plaque of a captive large marine osteichthyes fish and a human. The hypothesis

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being tested is that the typical pathogenic oral bacteria of humans will only be found in the human subject and the pathogenic oral bacteria of the grouper will be Vibrio sp.

METHODS AND MATERIALS│

Samples were obtained from the teeth of the fish while his head was raised out of the water during grooming to avoid contamination from ambient seawater (figure 1c). Samples of grouper and human plaque were plated on various selective media to test whether certain types of possibly pathogenic bacteria were present. Six types were targeted: 1. Streptococci sp. using Mitis-Salivarius (MS) agar (Difco)8 2. Streptococcus mutans using Mitis-Salivarius with added Bacitracin antibiotic (MSB)8 3. Lactobacilli sp. using MRS medium (Difco)13 4. Lactobacilli casei using MRS with added Vancomycin antibiotic (MRSV)13 5. Actinomyces sp. using Actinomyces Enrichment medium (protocol of Dr. Paul Jensen, Scripps Institution of Oceanography, UC San Diego) 6. Vibrio sp. using TCBS medium (Difco)10 Isolated colonies were obtained, Gram-stained, and if found to be Gram-negative, were investigated using BIOLOG database identification. Descriptions of Specialized Selective Media Mitis-Salivarius Agar (MS) -- is readily available from Difco and was modified in this experiment to contain a greater concentration of sucrose to serve as a broad inhibitor of several non-cariogenic Streptococcus bacteria8 because S. mutans has been shown to be resistant to high concentrations of sucrose.9 To 1000 mL of de-ionized water, 90g of dehydrated mitis-salivarius agar powder was combined with 150g of sucrose. The mixture was heated and stirred to dissolve the components and then autoclaved at 121°C for 15 minutes. The medium was then cooled to 45°C and poured into Petri dishes and dried overnight. Mitis-Salivarius Agar with Bacitracin (MSB) -- MS agar was prepared and altered as above, then additionally modified to contain 0-2 units of Bacitracin per mL. MSB has been shown to be selective and non-inhibitory for the bacterium Streptococcus mutans.8 MRS (Lactobacilli media) -- Rehydrated MRS broth (Oxoid) was prepared according to the manufacturer’s instructions. It was used in this experiment to select for Lactobacilli bacteria.13 MRS-V (Lactobacilli media with Vancomycin) -- was prepared by the methods of previously described researchers. 2 mL of 0.05 g vancomycin (Sigma Chemical Co., Castle Hill, Australia)/100 mL solution was added to 1 L of MRS broth to obtain 1 mg/L vancomycin as a final concentration. Agar powder was added to each broth and the media were autoclaved at 121°C for 15 min. It has been shown that MRSV is effective and non-inhibitory for Lactobacilli naslundii.13 Actinomyces Enrichment Medium -- was prepared Figure 1: A) Harvey in Magdalena Bay, B) K. Aamodt sampling Harvey, C) Close up of the outof-water swab-sampling. Photos taken by A. Lennard and K. Aamodt.

Figure 2: 1000x magnification of coccishaped bacteria isolated from MS media at 37°C.

Figure 3: 1000x magnification of Lactobacilli isolated from MRS media at 20°C.

following the protocol of Dr. Paul Jensen, Scripps Institution of Oceanography. TCBS media (Thiosulfate Citrate Bile Sucrose Agar) -- is used for the isolation and selective cultivation of enteropathogenic Vibrio sp. and closely related species.10 It was prepared in this experiment as specified by its distributor, Gallade Chemical. Sampling and Culture Methods Bacteria samples were taken from the dental plaque of both Harvey and the human specimen using long cotton swabs, then re-suspended from the swabs in 1 mL of sterile marine broth or phosphate buffered saline solution for each specimen, respectively. Two hundred (200) microliters of this suspension were plated on each type of media: TCBS, Actinomycete Agar, MRS, MRS-V, and MSB Agar. The media plates were incubated at two temperatures, 20°C and 37°C, to simulate the ambient temperatures in the marine and human oral environments, respectively. From the primary streak plates, a new set of secondary plates was streaked from each isolated colony to obtain genetically homogenous strains of each bacterium of interest. As a control for the efficacy of each sample culture, an extra plate was streaked on each medium using a sterile sample. The controls were incubated along with the other plates at both temperatures and checked for unintended growth. For the human samples, phosphate-buffered saline solution was used as the control sample, whereas sterile seawater was used for the marine samples. Characterization and Identification of Bacterial Colonies From the secondary streak plates, each pure strain was observed and photographed at 1000x magnification for the deduction of bacterial shape and structure. The strains were also Gram-stained to corroborate the identification of bacterial species from the selective media. The Gramnegative colonies were incubated at their respective culture temperature in BIOLOG identification plates, and the results were entered into the online database for potential matches. Based on selective media, Gramstaining, microscopy, and BIOLOG plates, the identity of each colony was deduced. Further tests to confirm the exact identity of the bacteria could include 16S rRNA analysis and genomic sequencing; however, due to time and budget constraints, they were not utilized.

RESULTS AND DISCUSSION│

Three distinct colony phenotypes were isolated from the grouper sample on TCBS plates. Although one colonial phenotype was unable to be identified using the BIOLOG database, the other two were identified as Vibrio carchariae and Aeromonas sp. With similarity index match (SIM) of 0.617 for V. carchariae, the SIM value was quite good, as a SIM value > 0.500 is required for the positive identification under standard BIOLOG protocol for incubation periods of 24 hours. For the other colony with the genus Aeromonas, the SIM value for the closest species match was Aeromonas caviae, SIM=0.4 (table 1). V. carchariae, synonymously known as a subspecies of Vibrio harveyi,7 is the same virulent bacterium that has been found in the dental biofilm of the great white shark (Carcharodon carcharias). It has been implicated to cause the antibiotic-resistant infection in shark-bitten humans.3 Finding these bacteria also present on Harvey’s teeth could suggest a greater re-

Pathogenic Oral Bacteria│Aamodt

Marine Medium

Actin..

TCBS

MRS

MRS-V

MSB

(Actino.)

(Vibrio)

(Lactobac.)

(L.casei)

(Strep.)

(S.mutans)

Harvey 200ul Sample

~200 CFU; multicolored colonies

About 16 CFU; creamy, 2mm, umbonate circular.

Human 200ul Sample

~ 500 CFU; very small colonies

~500 CFU; small colonies

Light green, Dark green, Yellow irrecular umbonate colonies -

~200 CFU; 5mm white matte colonies, small (.5mm) white colonies

~200 CFU; 5mm white matte colonies

Table 1: Growth of bacterial colonies on selective media incubated at 20°C. Incubated @ 37 C

Marine Medium

Actin.

TCBS

MRS

MRS-V

MSB

(Actino.)

(Vibrio)

(Lactobacilli)

(L. casei)

(Strep.)

(S.mutans)

10 irregular dark umbonate colonies (3mm) >500 CFU; dark small raised colonies (<0.5mm)

Harvey 200ul Sample

~200 CFU multicolored colonies

10 CFU; creamy umbonate colony

Human 200ul Sample

~ 40 small colonies

6 large (6mm) white pulvinate dull circular colonies, 12 whiteish irregular (2mm) raised colonies, myriad small (<0.5mm) white colonies.

18 small white (2mm) irregular pulvinate colonies

Table 2: Growth of bacterial colonies on selective media incubated at 37°C. Legend for Tables 1 & 2: No Growth is indicated by a (-) sign. CFU = Colony Forming Units. Umbonate = having a concave profile. Pulvinate = having a convex profile. For Media Type Abbreviations, see Methods and Materials section.

lationship between the oral microbiota of phylogenetically distant macroscopic marine predators. It could also suggest the bacterium is present on shared prey species between C. carcharias and M. jordani, or that the bacterium may possess a symbiotic role in the oral microbiota of large marine predators. Additionally, this finding could potentially provide a new model organism for studying marine oral pathogens. Since groupers are significantly more docile and easier to keep in captivity, they would prove a much better study organism than the fastidious white sharks, which to date have only been successfully held in captivity at the Monterey Bay Aquarium for limited periods of time. It is interesting to note that Harvey shares his tank with several large nurse sharks (Ginglymostoma cirratum). Although the sharks and the ambient tank-water were not tested, the presence of V. carchariae may have originated from Harvey’s proximity to these large sharks. Future studies should investigate this possibility. Additionally, the grouper sample incubated at 37°C on MS agar yielded several flat dark colonies (figure 2), and through Gram-staining, they were found to be Gram-positive. Because MS is selective for Streptococci, digital microscopy revealed that the bacteria are cocci-shaped and that the colony is Gram-positive, it is safe to assume that these colonies are indeed of the genus Streptococcus. This finding challenges the hypothesis that Streptococci would not be found in the marine samples. Further investigation should include genomic analysis to confirm the exact identity of this bacterium. Streptococci, a Gram-positive group of bacteria not typically associated with marine environments, have been implicated as a culprit for invasive infections that plague fish farms.12 Potentially, the species isolated from Harvey’s teeth could be Streptococcus iniae. The presence of Streptococci could indicate that either Harvey or other fish in the shared water of the aquarium are suffering from an invasive Streptococci infection. Further study could help draw connections between the communication of disease within a marine aquarium and within fish farms. Conversely, the presence of these two potentially pathogenic types of

~400 CFU; dark small raised colonies (<0.5mm)

bacteria in Harvey’s mouth could be completely natural, possibly aiding oral hygiene or the capture and/or digestion of prey. Moreover, further investigation of the dental biofilms of marine macropredators such as sharks and large groupers could show that the presence of V. carchariae is ubiquitous in this group’s niche. The human dental biofilm samples were incubated on the same media and at the same temperature as the marine samples. Sample cultures grew as anticipated, supporting the hypothesis that Vibrio sp. would not be present in the human sample. Streptococcus mutans were isolated along with other Streptococci, as were various Lactobacilli including the desired species, casei. The identities were deduced from the specific selectivity of the media employed. These data serve to verify the viability of the methods used to isolate the target species and to verify the presence of the target species in the human biofilm sample.

ACKNOWLEDGEMENTS│

I gratefully acknowledge Angie Lennard, Mark Ball, and Vincent Levesque, all aquarists of the Birch Aquarium at Scripps Institution of Oceanography, for their support and encouragement and the guidance of Douglas Bartlett, Vera Tai, and Bianca Brahamsha. I also thank Lolly Almazan for her help in obtaining many of the specialized media and antibiotics used in this experiment.

REFERENCES│

1. Baldridge, H.D. 1974. Shark attack. A program of data reduction and analysis. Contributions from the Mote Marine Laboratory, vol. 1, no. 2. (Sarasota, Fla, Mote Marine Laboratory). 2. Blake, P.A., Weaver, R.E., and Hollis, D.G. 1980. Diseases of humans (other than cholera) caused by vibrios. Annual Review of Microbiology, 34, 341-367. 3. Buck, J.D., Spotte, S., and Gadbaw, J.J., Jr. 1984. Bacteriology of the teeth from a great white shark: Potential medical implications for shark bite victims. J Clin Microbiol., November; 20(5): 849–851. 4. Currey, J.D. 1999. The design of mineralised hard tissues for their mechanical functions. J Exp Biol., Dec., 202(Pt 23): 3285-94. 5. Ferrari, M., Goracci, C., Sadek, F., Eduardo, P., and Cardoso, C. 2002. Microtensile bond strength tests: Scanning electron microscopy evaluation of sample integrity before testing. Eur J Oral Sci., Oct; 110(5): 385-91. 6. Foster, J.S., Palmer, R.J., Jr. and Kolenbrander, P.E. Human oral cavity as a model for the study of genome-genome interactions. Biol Bull. 2003, Apr;204(2):200-4. 7. Gauger, E.J., and Gomez, C.M. 2002. 16S ribosomal DNA sequencing confirms the synonymy of Vibrio harveyi and V. carchariae. Diseases of Aquatic Organisms, 52(1), 39-46. 8. Gold, O., Jordan, H.V., and van Houte, J. 1973. A selective medium for Streptococcus mutans. Arch Oral Microbiol., 18: 1357-64. 9. Ikeda, T., and Sandham, H.J. 1972. A high-sucrose medium for the identification of Streptococcus mutans. Arch Oral Biol., Apr; 17(4):781-3. 10. Lotz, M.J., Tamplin, M.L., and Rodrick, G.E. 1983. Thiosulfate-citrate-bile salts-sucrose agar and its selectivity for clinical and marine vibrio organisms. Ann Clin Lab Sci., 13: 45-48. 11. Palmer, R.J., Kasmerzak, K., Hansen, M.C., and Kolenbrander, P.E. 2001. Mutualism versus independence: Strategies of mixed-species oral biofilms in vitro using saliva as the sole nutrient source. Infection and Immunity, Sept., p. 5794–5804 Vol. 69, No. 9. 12. Roy, P.E., and Francis-Floyd, Y&R. 2002. “Streptococcal Infections of Fish.” Circular 57, Institute of Food and Agricultural Sciences, University of Florida. 13. Tharmaraj, N., and Shah, N.P. 2003. Selective enumeration of Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Bifidobacteria, Lactobacillus casei, Lactobacillus rhamnosus, and Propionibacteria, J. Dairy Sci., 86: 2288-2296. 14. Yeung, P.S., and Boor, K.J. 2004. Epidemiology, pathogenesis, and prevention of foodborne Vibrio parahaemolyticus infections. Foodborne Pathog Dis., Summer;1(2):74-88. 15. Montgomery, J.M., Gillespie, D., Sastrawan, P., Fredeking, T.M., and Stewart, G.L. 2002. Aerobic salivary bacteria in wild and captive Komodo dragons. J Wildl Dis., Jul; 38 (3): 545-51.

Saltman Quarterly│UCSD Division of Biological Sciences│Volume 4│2006-2007

Research │29

Physical and environmental preferences of mosquito larvae (Culicidae) and Predation by Priapichthys panamensis in Cuajiniquil, Costa Rica Aria Jafari, Junior, Revelle College, Animal Physiology & Neuroscience major Education Abroad Program / Division of Biological Sciences, University of California, San Diego

he interface between nature and human beings is strikingly apparent in the case of mosquitoes (Culicidae). These insects, which belong to the order Diptera, develop in standing water and utilize the blood of vertebrates to complete their reproductive life cycle. For this reason, many serve as vectors of very serious illness prevalent in the tropics, such as malaria and dengue. This study considers the environmental factors that foster larval development and explores the predator–prey interaction of Priapichthys panamensis (Poecilidae) fish on mosquito larvae of different sizes in the local context of Cuajiniquil, Guanacaste. I sampled a total of 33 pools of standing water and found that the incidence of encountering larvae was highest for street puddles (84.62%), followed by household items (54.55%), and then by river sites (11.11%). I also found that a pH level above 7.35 is an important indicator of larval presence in aquatic environments. Since neutral waters harbor many predators, the ability of an ovipositing mosquito to distinguish more basic water may be an anti-predatory behavior. Although a relationship between pH and larval presence or absence was observed, environmental factors were found to have no correlation with number of individuals present. Lastly, I found that Priapichthys panamensis demonstrates no significant difference in the rate of consumption of large or small larvae.

INTRODUCTION │

# of individuals

Adult female mosquitoes feed on the blood of vertebrates to obtain nourishment for ovarian and egg development.3 This ecological interaction is of great economic and medical importance, as many prevalent tropical medical afflictions such as malaria and dengue fever utilize Culicidae as arboviral vectors. These illnesses not only have serious and sometimes fatal implications for people but also adversely affect livestock, pets, and wildlife.3 Although not all mosquitoes are disease vectors, their bites may also cause secondary infections, allergic reactions, pain, irritation, redness, and itching.3 Thanks to intensive vector control programs and the use of antimalarial drugs, the incidences of malaria in Costa Rica are infrequent. In contrast, dengue fever remains an important and rapidly growing health concern in the region because of a lack of vaccines, prophylactic drugs, or specialized treatment.5 Mosquitoes require bodies of standing water to develop from egg to the larval and pupal stages.7 In Cuajiniquil, Costa Rica, a coastal fishing community of about 2,000 people, there is an abundance of transient street pools and household items such as empty cans or tires that collect rainwater and host development of potentially disease-carrying mosquitoes. In 2005, 121 cases of dengue fever were reported, prompting the local government to initiate chemical fumigation and education programs (Briceño, personal communication). However, chemical methods of mosquito control alone have not proven effective for a variety of reasons, primarily due to cost, lack of cultural acceptance, and insufficiency in controlling the rapidly reproducing larvae. A much more affordable, safe, and environmentally friendly means of control harnesses the natural predatory behavior of small fish within the Poecilidae family. These small fish inhabit puddles or slow-moving shallow river waters and present a notable preference for the consumption of mosquito larvae.2 Priapichthys panamensis (Poecilidae), which is very common in Río Cuajiniquil, was used in this study. In 2004, an inter-institutional study conducted in El Salvador showed that Poecilids are very effective in consuming mosquito larvae, with a rate between 100 to 450 larvae per fish per day.1 Although these figures reveal the high effectiveness of the fish as predators, they also present a notably 100 90 80 70 60 50 40 30 20 10 0

Aedes Anopheles

broad range of consumption rates. Authors of the paper speculate that this range is due to a difference in consumption rates when preying on small versus large larvae. This study involved two phases of investigation. The first phase addressed the question whether or not Culicidae genera exhibit habitat preference and whether or not environmental factors affect the presence or absence of larvae and the number of individuals present. The second phase addressed the relationship between larval size and the effectiveness of predation by Priapichthys panamensis.

METHODS AND MATERIALS│

Study Site This study was conducted from 13 November 2006 to 26 November 2006 in Cuajiniquil, Guanacaste, NW Costa Rica. Priapichthys panamensis fish were collected from Río Cuajiniquil, a slow-flowing freshwater river that originates in Santa Rosa, Guanacaste and flows northwest to Cuajiniquil, where it empties into the Pacific Ocean. Phase I: Water and Larvae Field Work Larvae and water samples were collected from 33 different sites characterized by standing water. Of these sites, 13 were categorized as street puddles, 9 as river pools, and 11 as household items. All sites were easily accessible and contained standing freshwater. At each site, water temperature using a standard thermometer and analyzed water samples for pH, alkalinity, carbon dioxide, and turbidity levels were determined using a LaMotte chemical kit. A 4.0 cm, fine mesh household strainer was used to test for Culicidae larvae. A strainer was run through the surface, middle, and bottom levels of the water. If larvae were found, 10 additional minutes were spent for collection. Following collection, the captured larvae were extracted using a soft tweezer and/or a 1 mL dropper. The collected larvae were placed in a numbered 2 mL vial and preserved in 80% alcohol for further analysis. Observations such as the presence or absence of fish, sunlight exposure, and location were recorded at each site. The samples were taken to the laboratory for observation using a stereoscope at 4.0x magnification. Larvae were counted individually and identified to genus level using a dichotomous key.4 Larvae were returned to the vials for preservation.

Wyeomia Limatus Culex Trichoprosopon

Street puddle

River

Household

Habitat

Figure 1: The composition of genera within each habitat. Household items have the highest number of individuals (n= 139), followed by street puddles (n=46) and river (n = 8). N =193.

Phase II: Fish Laboratory Trials Priapichthys panamensis (Poecilidae) fish were collected using a mosquito net from Río Cuajiniquil. Upon field collection, live fish were brought to the laboratory where they were placed into a large, oxygenated aquarium with minimal organic matter to sustain the fish until they were ready for trial. Each fish was measured from head to caudal peduncle using a caliper. A 4.0 cm fish was selected from the larger aquarium and placed into a smaller aquarium for closer observation. Rocks, plants, and leaves were

Habitat, Environment and Fish Predation of Mosquito Larvae│Jafari

Genera Aedes Wyeomyia Anopheles Culex Limatus Trichoproposon Total

No. of Individuals 126 49 15 1 1 1 193

Table 1: Distribution of Culicidae larvae by genera. Data represent samples taken from 33 sites. The number of individuals is reported to the right of the genera.

used to simulate the natural habitat of the fish. Live Culicidae larvae were placed on ice to slow down movement and then measured with a microscopic ruler. Two groups were established based on larval size, 0.3-0.4 mm being ‘small’ and 0.4-0.5 mm being ‘large.’ Ten individuals were selected from each group per trial, for a total of four trials per group. Using a stopwatch, the time it took for the fish to consume all of the larvae was monitored. Statistical analyses JMPin 4.0.4 was used to determine the statistical significance of the data. For phase I of this study, a χ² test was conducted to see if Culicidae exhibited habitat preferences. Furthermore, to test for the effect of environmental factors on presence or absence of larvae, a logistic regression analysis was performed. Two factors were considered in this analysis: water temperature and pH. Water temperature was selected due to its very low p-value during the χ² analysis for environmental affect on the number of individuals, and pH was selected based on trends observed in the raw data. A multiple linear regression analysis was used to observe the environmental effect on the number of individuals present. In phase II of this study, two distinct ‘rounds’ of predation by the fish were noted. For each trial, the rate of consumption of large and small larvae was determined by dividing the number of larvae consumed by time in seconds for its respective round. In order to test whether or not larvae of different sizes have an effect on the rate of predation by fish, three t-tests were conducted, one for each round of predation and a final t-test for overall rate of predation.

RESULTS│

Phase I: Water and Larvae Field Work Figure 1 shows the general distribution of 193 larvae collected for this study: 139 were found in household items, 46 in street puddles, and 8 in river pools. Of this total, 126 were identified as Aedes, 49 Wyeomyia, 15 Anopheles, 1 Limatus, 1 Culex, and 1 Trichoprosopon, for a total of 6 genera (table 1). Field observations revealed that 18 of 33 sites (54.55%) contained Culicidae larvae. The incidence of encountering larvae was greatest for street puddles (84.62%), followed by household items (54.55%), and least for river sites (11.11%). The range of the data for temperature was 24ºC to 37.5ºC ( x = 28.56 ± 3.46), for pH was 6.2 to 9.5 (x = 7.49 ± 0.78), for alkalinity was 30 ppm to 500 ppm (x = 208.30 ± 132.25), for CO2 was 0 ppm to 85 ppm (x = 5.94 ± 16.27), for turbidity was 0 jtu to 300 jtu (x = 17.65 ± 16.27). Culicine genera and habitat preference Figure 1 demonstrates a tendency for certain genera to occur in particular habitats (χ²=100.82, d.f.= 12, p <.0001). Aedes showed a primary preference for household items and secondary preference for the street puddle habitat. Wyeomyia showed a strong preference for household items. Anopheles showed a preference for the river and street puddle habitats (figure 1). Habitat preferences of Culex, Limatus, and Trichoprosopon were not able to be determined due to low sample numbers (table 1). Environmental affect on presence or absence of larvae Figure 2 shows that temperature was not related to presence or absence of larvae, while pH shows a strong relationship (R²= .2451, d.f. = 1, p<.008). The pH level at which larvae are most likely to be present

Figure 2: Logistic regression plot. When a horizontal line is drawn through the graph at a probability of .50, it intersects the graph at a pH level of 7.35. This value corresponds to the lowest pH value at which larvae are most likely to be present and infers that basic water is a strong predictor of larval presence. N= 33.

appears to be above 7.35, indicating a preference for basic waters (figure 2). Environmental affect on the number of individuals Table 2 reveals the results for a multiple regression analysis. These data show that temperature, pH, alkalinity, CO2 concentration, and turbidity levels do not play a significant role in determining the number of individuals present in standing water. Phase II: Fish Laboratory Trials Larval size and fish predation rate As shown in table 3, fish were observed to consume larvae in two distinct rounds. During the first round, the average rate of consumption was high for both small (x = .34 larvae/sec. ± .10) and large (x = .29 larvae/sec. ± .01) larvae. However, the second round showed a much lower average consumption rate for small (x = .01 larvae/sec. ± .00) and large (x = .02 larvae/sec. ± .02) larvae (figure 3). T-tests conducted per round, and the final t-test taking the overall average consumption rate of fish did not reveal any statistically significant differences between rate of predation and size of larvae.

DISCUSSION│

For the purpose of this study, it is assumed that greater numbers of larvae occur where they are most suited for survival and development. This suitability is equated with preference, given the study sites, when comparing the occurrence of genera in different habitats. Although, on average, 54.55% of all site samples contained mosquito larvae, there was an exceptionally low incidence of encountering larvae in river pools (11.11%). This low incidence can be attributed to the presence of Priapichthys panamensis fish near the sample sites. Since the height of the river fluctuates daily due to the tide, at certain times of the day the river provides access for the predatory fish thus decreasing the chances of encountering larvae at these sites. For all isolated river habitats, predatory fish were noted in the nearby river. River sites are also more susceptible to salty or brackish waters due to the daily tidal cycle. This is especially important in Cuajiniquil because the study site was less than 1600 m from where the river empties into the Pacific Ocean. Interestingly, Anopheles was the only genus that was present in the river habitat. This may be due to the fact that Anopheles is one of the few culicine genera that have species (A. atropos and A. bradleyi) specially adapted to survive in salty or brackish water. Furthermore, the low number of Anopheles individuals in the street puddle sites and absence from household items was expected because larvae adapted to cope with salty waters do not compete well with freshwater species.3 Further investigation involving the salinity of the river study sites would be helpful in fully determining the factors responsible for this observation. Furthermore, the ability of Anopheles mosquitoes to survive in unfavorable conditions may also explain their preference for the street puddle habitat. Such habitats are harsh for larval development, since they are generally shallow, low in organic matter, and have broad environmental fluctuations due to exposure to sunlight.5 However, Anopheles mosquitoes and larvae are able to survive under these conditions by using a diapause

Saltman Quarterly│UCSD Division of Biological Sciences│Volume 4│2006-2007

Research │31

Rate of consumption

Since basic waters (above pH 7.35) seem to be more likely to be inhabited by larvae, it may be that adult female mosqui B-level p-level toes choose to oviposit in basic water to avoid larval predators Temperature .306861 .171404 R2 = .12904630 such as Poecilid fish in neutral waters such as rivers. FurpH -.197903 .416446 n = 33 thermore, these conditions may be more favorable due to an CO2 level -.345221 .180893 f(5,27) = .80010 increased food supply. Incidentally, the single river site that Turbidity .081599 .668220 p< .55938 tested positive for larvae was basic (pH 8.2). This observation Alkalinity .375460 .158945 has great implications for vector control, but further studies Table 2: Number of individuals showing no significant relationship to five variables: temperature, are needed to identify what, specifically, is responsible for the pH, CO2 level, turbidity, and alkalinity. Multiple regression analysis was conducted to test signifi- presence of larvae in these conditions. cance. N= 193. Observations in phase II showed that the rate of predation is not significantly different for large or small larvae. This can t p be attributed to the fact that the time spent by the fish differentiating size Round 1 of predation -.982 .3638 would slow down the rate of predation, thus impeding fitness. However, Round 2 of predation .754 .4792 df = 6 the size difference between the two categories may not have been large Total predation .604 .5682 N=8 enough to merit a different predatory response by the fish. Nonetheless, this observation challenges previous literature which attributes large Table 3: T-test values showing no significant relationship between size of larvae and ranges of consumption rates to larval size. rate of predation d.f.= 6, N=8. In summary, it appears that mosquito larvae are able to survive in a wide mechanism.3 During diapause, an organism can survive harsh conditions variety of conditions. However, certain genera appear to exhibit habiby minimizing metabolic processes until conditions improve. Addition- tat preference. Additionally, the pH of the water seems to be important ally, in contrast to other mosquitoes, Anopheles larvae lack a respiratory when considering the likelihood of larval presence. The significance of the phases of predation observed in phase II is unclear. Further research siphon and, for this reason, position themselves parallel to the surface of on this peculiar feeding behavior may be valuable to more completely unthe water, making depth of water less of a requirement for survival. Household items were the second most likely habitat to be inhabited derstanding the predator–prey relationship between mosquito larvae and by larvae. Wyeomyia preferred this habitat, characterized by completely Poecilid fish. Understanding habitats and environmental factors, specifistagnant, relatively clean, above-ground water. Since, naturally, Wyeo- cally pH, that influence the presence and composition of mosquitoes is myia mosquitoes are phytotelmata ovipositors, it may be that the above- necessary to effectively control these potentially harmful organisms. ground water present in household items artificially replicates this enviACKNOWLEDGEMENTS│ ronment (Chavarría, personal communication). First and foremost, I would like to thank Ramsa Chaves for her patience and willingness to help through every step of this project. I would also 0.50 like to thank Frank Joyce for all of his valuable advice and support. Spe0.45 cial thanks to Marielle Decker, David Leong, and Katerina Morici for helping me in the field. 0.40 0.35 0.30

Round 1

0.25

Round 2 Total

0.20 0.15 0.10 0.05 0.00 1

2 Size of Culicidae larvae

Figure 3: Average rate of predation for round 1 and round 2 and total average rate of predation. The left side shows fish predation rate for small larvae (0.3-0.4 mm). The right side shows fish predation rate for large larvae (0.4-0.5 mm).

REFERENCES│

1. Academia para el desarrollo educativo (AED). 2004. Ensayo para evaluar la utilidad del genero Poecilia sp. como biocontrolador de los estadíos acuáticos del Aedes aegypti. 2. Bussing, W.A. 1997. Peces. Universidad De Costa Rica, San José, Costa Rica. 3. Eldridge, B.F. 2003. Mosquitoes. In V. Resh and R. Cardé (Eds.). Encyclopedia of insects, Elsevier Science USA. pp. 743 -749. 4. INBio (Instituto Nacional de Biodiversidad). 2005. Clave fotografica para larvas de zancudo (Diptera: Culicidae) presented in Centroamerica y Panama. http://www.inbio.ac.cr/papers/Culicidae_larvas/. 5. National Center for Infectious Diseases, Division of Parasitic Diseases. 2004. Anopheles mosquitoes. http://www.cdc.gov/malaria/biology/mosquito/. 6. Vargas, M.V. 2003. Uso de peces larvívoros como controladores biologicos de larvas de Aedes Aegypti: una participación comunitaria. Rev. Col. De MQC De Costa Rica 9 (3): 1-5. 7. Zumbado, M. 1999. Diptera of Costa Rica. Instituto Nacional De Biodiversidad, Heredia, Costa Rica.

Noncompetitive NMDA Antagonist Dextromethorphan Reduces the Development of Acute Morphine Dependence Lusine Khachatryan, Senior, Eleanor Roosevelt College, Biochemistry & Cell Biology major Division of Biological Sciences, University of California, San Diego

oth humans and animals become dependent on morphine with as little as a single injection.11 The current study analyzed the effects of dextromethorphan, an NMDA receptor antagonist, on the development of acute morphine dependence. Previous results have shown that dextromethorphan and similar compounds that bind non-competitively to the NMDA receptors can prevent the development of tolerance to morphine.21 In the current study, acute morphine dependence was measured by withdrawal-induced elevations in brain stimulation reward thresholds. Rats treated 4 times at daily intervals with morphine (5.6 mg/kg) that received vehicle treatment prior to each morphine injection showed a great increase in brain reward thresholds from day 1 to day 4, while the rats that were injected with different doses of dextromethorphan prior to morphine showed a reduced development of this withdrawal response at the higher doses of dextromethorphan tested. Consequently, the results indicate that dextromethorphan pretreatment attenuates the development of acute morphine dependence as measured by the withdrawal effects of naloxone.

Noncompetitive NMDA Antagonist Dextromethorphan│Khachatryan

INTRODUCTION │

Acute opioid dependence, “a state in which abstinence can be demonstrated or precipitated after either a single dose or a short-term infusion of morphine,”3,14 is demonstrated by withdrawal-like symptoms brought about by opioid antagonists after the administration of a single dose of opioid agonists.14 This dependence is measured based on withdrawal effects induced by the morphine antagonist naloxone after the administration of morphine.11 Morphine acts as the agonist because it readily binds to its receptor protein and activates a cellular response. Four hours after morphine is injected into the animals, the withdrawal drug naloxone is administered, which binds to the receptor and prevents the agonist from binding, thus blocking the receptor and inhibiting cellular response. As a result, naloxone acts as the antagonist. Although this particular ligand is recognized by the morphine receptor, it does not activate the receptor once it binds because of its low efficacy. Naloxone is a very potent drug, which is evident in its ability to bring about withdrawal effects when administered 20 hours after morphine injection.21 There is an increase in the severity of the naloxone-induced withdrawal signs with increased exposure to morphine. Even at low doses of the agonist morphine, antagonist naloxone is able to elicit opiate withdrawal signs when administered in high dose. Escape jumps, abundant salivation, and diarrhea are examples of withdrawal signs observed after the first exposure to morphine while teeth chattering, irritability and vocalization are brought about by naloxone after the second exposure to morphine.18 Evidently, due to its ability to produce strong “somatic signs of withdrawal” at low doses, naloxone is being widely used in drug addiction studies because depending on the strengths of withdrawal signs associated with different dosages of morphine, conclusions can be drawn on how effective certain medications are, e.g., dextromethorphan, that aim to treat drug addiction.18 This is accomplished by measuring elevations in brain stimulation reward thresholds elicited by naloxone. Studies on human subjects have revealed that withdrawal from acute opioid dependence may produce somatic, physiological and subjective symptoms similar to withdrawal from chronic opioid dependence.2 In addition, studies performed on animal models have also shown that acute opioid dependence is similar to chronic opioid dependence in terms of somatic and affective withdrawal symptoms such as “anxiety, restlessness, and depression/dysphoria.”11 The results of both studies indicate that withdrawal from acute opioid pretreatment increases with the administration of acute doses of the agonist morphine. In other words, continued treatments with morphine produces more severe withdrawal symptoms upon the administration of the antagonist naloxone.1,2,11,18,19,20 Therefore, with repeated exposure to morphine, acute opioid dependence may progress to chronic opioid dependence since the state of acute opioid dependence appears to indicate the preliminary responses to the agonist morphine.20 If acute opioid dependence does indeed lead to chronic opioid dependence, then this would suggest that the initiation of acute opioid dependence would be inhibited by treatments that have been shown to repress the initiation of chronic opioid dependence when co-administered with the opioid. Substantial evidence indicates that the development of neuroadaptive responses to addictive drugs requires the activation of NMDA (N-methyl-D-aspartate) receptors.21 NMDA receptors have a non-competitive site, otherwise referred to as an allosteric site. Dextromethorphan has a high affinity for this site and by bindFigure 1: Stereotax is a small apparatus that holds small animals securely in place for the electrode implantation. The horizontal bars are called ear bars, which are used to hold the animal’s head in a tight and stationary position, while the vertical bar holds the electrode in place. Picture from: http://wwwkopfinstruments.com/Stereotaxic/900.htm.4

ing to it blocks the NMDA receptor preventing activation.21 Furthermore, NMDA receptor antagonists, such as dextromethorphan, have been shown to hinder the development of sensitization and physical dependence on morphine.21 Consequently, as demonstrated by Trujillo, the administration of NMDA receptor antagonist dextromethorphan with morphine inhibits the development of tolerance, sensitization or physical dependence on the drug.21 Furthermore, several other studies that have primarily focused on the side effects of NMDA antagonist drugs such as dextromethorphan suggest that this drug may be clinically very useful in inhibiting tolerance to morphine because it does not display any apparent side effects when co-administered with morphine.12 The purpose of the current study was to ascertain whether the initiation of acute opioid dependence might be blocked or attenuated by the high-affinity noncompetitive NMDA receptor antagonist dextromethorphan as measured by withdrawal-associated elevations in brain reward thresholds.

methods and materials│

Animals The laboratory subjects tested were male Wistar rats (n=80, Harlan Labs, Indianapolis, IN) that weighed 380-470 g at the time of testing. The rats were pair-housed in standard rodent cages in a humidity and temperature controlled room. The room also had 12-hour light and 12hour dark cycles (lights on at 6:00 am). The subjects had unlimited access to food and water. The training and testing were done between 10:00 am and 4:00 pm Monday through Friday. All experimental procedures were carried out according to the “Guide for the Care and Use of Laboratory Animals” and were approved by the Subcommittee on Animal Studies of the VA San Diego Healthcare System. Drugs Morphine sulfate (donation from the National Institute on Drug Abuse - NIDA), dextromethorphan, and naloxone hydrochloride (purchased from Sigma) were used in this experiment and were prepared for injection in sterile physiological saline with concentration of 0.9%. The subjects received subcutaneous injections of 0.1 mg per 0.1 kg body weight for morphine and naloxone and introperitoneal (IP) injections for dextromethorphan. Brain stimulation reward procedure To implant brain stimulation reward electrodes, rats were anesthetized with isoflurane and secured in a Kopf stereotaxic instrument (figure 1). A stainless-steel polar electrode (Plastic Product, Roanoke, VA) was implanted in the lateral hypothalamus of the rats. Half the rats received implants on the right side of their brain while the other half received implants on the left side of the brain in order to offset any possible functional brain asymmetries. The stereotaxic coordinates for implantation, based upon the altas of Pelligrino and Cushman, were as follows: AP-0.5 mm from bregma, L ±1.7 mm, -8.3 mm ventral from the durma, incisor bar +5.0 mm above interaural line. After surgery, the rats were allowed one week to recover. Complete descriptions of the surgery, procedure, and apparatus are available elsewhere.11,13 Constant current stimulators delivered electrical stimulation in the form of 60 Hz sinusoidal waves, with a train duration of 250 ms. A discrete-trial current-intensity threshold procedure was utilized.8 To start a trial, a rat received a non-contingent electrical stimulus. A correct response, which involved rotating a wheel at least ¼ turn within 7.5 seconds of the non-contingent stimulus, resulted in a second, contingent stimulation that was in every way identical to the first. An intertrial interval (ITI) averaging 10 seconds followed every correct response. If there was no response recorded within 7.5 seconds of the non-contingent stimulus, then the trial would end followed by an ITI. If response was detected during the ITI, the start of the next trial would be delayed by 10 seconds. Reward thresholds were determined by varying the intensities of the stimuli in alternating ascending and descending series (two of each) with a step size of 5 µA between each intensity. Three trials were presented at each stimulus intensity, and at least two correct responses were required at

Saltman Quarterly│UCSD Division of Biological Sciences│Volume 4│2006-2007

Research │33 Habituation Phase (Mon-Fri) Baseline Week

Day 4 of Morphine Brain Reward Threshold

Percent of Baseline Threshold

170

Post-S1

160 150

Post-S2

140 130 120 110 100 Vehicle

0.1

(Mon) Test Week

Morphine Day 4 (Friday) Test Week

IP: SQ:

Veh Veh

Veh or Dex* Mor 5.6

SQ:

Veh

Nal 1.0

Table 1: Details of experimental design and treatment conditions for habituation phase and test day 4. *Separate groups received Vehicle 0.10, 1.0, 10.0 mg/kg DexM, with each group receiving same IP drug prior to morphine on each Morphine Day. Abbreviations: Veh = vehicle; Dex = dextromethorphan; Mor =morphine; Nal = naloxone.

Dose of dextromethorphan (mg/kg)

Figure 2: Effects of acute and repeated doses of dextromethorphan, morphine, and naloxone on brain reward thresholds. The graph shows the gradual decrease in brain reward threshold brought about by dextromethorphan as compared to vehicle. Dextromethorphan administered at the high doses of 10 mg/kg showed the most significant reduction in the brain threshold compared to the vehicle treatment. *Significant difference is evident between dextromethorphan 10.0mg/kg and vehicle

a given stimulus intensity for it to be considered above threshold. In order to determine the estimated threshold for each session, the mean of the four series thresholds was taken. The midpoint was set between the current intensity level at which the correct responses were fewer than two and the level at which a minimum of two correct responses was recorded being defined as the threshold of each series. The data obtained from the experiment were then analyzed to look for changes in the pattern of wheel-turning and changes in brain reward thresholds when saline alone was administered during the baseline period and when naloxone was injected post morphine and dextromethorphan treatment during the testing period. Acute dependence and withdrawal testing regimen Rats received two test sessions per day for four days. Session 2 was separated from session 1 by approximately 4 hours. Once responding to the task was stabilized (less than 15% variation in threshold from the average of 5 consecutive days), rats were habituated to subcutaneous vehicle injections for 6 days. Vehicle is injection of saline solution (0.9% concentration), and its purpose is to familiarize the subjects with the needle in an attempt to avoid the interference of the effects of the needle on their behavior during the testing period. Otherwise, saline has no effect on the behavior of the subjects. Furthermore, the six thresholds obtained during habituation were averaged to provide the baseline against which all changes induced by drug treatment were assessed. The rats in the control group were treated with intraperitoneal (IP) injections of vehicle 15 minutes prior to subcutaneous injection of morphine (5.6 mg/kg). Four hours after the first such treatment in the control group, naloxone (1.0 mg/kg) was injected subcutaneously and thresholds were determined. These rats then received an additional 3 days of vehicle/ morphine treatment, with naloxone again injected after the fourth morphine treatment, and thresholds were again measured at that time. Test groups received one of several doses of dextromethorphan (0.1, 1.0, 10.0 mg/kg), but otherwise were treated the same as the control group. The regimen for the habituation phase and test day 4 are depicted in table 1.

RESULTS│

Naloxone (1.0 mg/kg) induced a significant increase in reward threshold in the vehicle group on day 1 of morphine treatment (mean ± SEM = 127.8 ± 7.3), with further significant increase in threshold on day 4 of morphine treatment (mean ± SEM = 154.1 ± 7.2) as revealed by a onefactor repeated measure ANOVA. Groups treated with varying doses of dextromethorphan showed increased thresholds similar to the vehicle control group on day 1 of morphine treatment, but higher doses of dextromethorphan appeared to attenuate the increase in threshold produced by repeated morphine treatment. Figure 2 clearly illustrates the decrease in brain reward thresholds as a result of administration of dextromethorphan at different doses. One-factor ANOVA revealed that this dose-de-

pendent reduction in threshold on morphine day 4 was statistically significant, and comparison of each dextromethorphan dose group to vehicle controls on day 4 revealed a statistically significant reduction in threshold produced by the highest dose of dextromethorphan, 10.0 mg/kg (figure 2). The figure shows a gradual decrease in threshold after each treatment of dextromethorphan at different doses. However, it is evident that compared to the vehicle, the highest dose of dextromethorphan (10.0 mg/kg) has produced the greatest and the most statistically significant decrease in brain threshold.

DISCUSSION│

The results of the experiment indicate that dextromethorphan blocks the potentiation of morphine dependence produced by repeated administration over 4 days, as measured by a reduced withdrawal effect brought about by antagonist naloxone. When only vehicle was administered prior to morphine treatment each day, the brain reward thresholds increased substantially across days of treatment. This explains the significant withdrawal effects produced after a single exposure to morphine when naloxone was administered 4 hours later.11 Consequently, repeated administration of morphine resulted in progressive elevation in the effectiveness of naloxone. According to Schulteis et al., the “development of dependence on opiates begins with [as little as] a single dosing.”18 However, upon the injection of different doses of dextromethorphan, the reward thresholds were significantly lower after four days of treatment at the highest dose of dextromethorphan tested. There was a gradual decrease in threshold compared to vehicle when the subjects were treated with lower doses of dextromethorphan (figure 2). However, the results depicted in figure 2 confirm that the administration of dextromethorphan at the highest dose (10.0 mg/kg) resulted in the most significant reduction in brain threshold compared to vehicle (saline 0.9%). Several other studies, including Manning et al.,12 have demonstrated that dextromethorphan inhibits the development of dependence and tolerance produced by chronic treatment with morphine along with reducing withdrawal symptoms brought about by morphine antagonist naloxone. Compared to rats that are treated with morphine only, subcutaneous co-administration of dextromethorphan with morphine reduces naloxone-induced hyperalgesia (increased sensitivity to pain).12 Our current findings support the notion that the initial development of acute opioid dependence is dependent upon NMDA receptor activation in the same way that chronic opioid tolerance and dependence are, providing further evidence that acute opioid dependence reflects the initial stages of neuroadaptation responsible for the progressive development of opioid dependence. However, dextromethorphan is able to successfully prevent the activation of NMDA receptors, which leads to the reduction of withdrawal-like symptoms when the morphine antagonist naloxone is administered, portraying the decrease of dependence on morphine. Consequently, it is apparent that dextromethorphan has potential clinical applications including the capacity to prevent subjects from developing tolerance to analgesia produced by morphine.21 In fact, the National Institute on Drug Abuse suggests that dextromethorphan and other similar NMDA antagonists may potentially treat drug addiction5,6 by reducing dependence on drugs. Further research, however, is necessary in order to determine what behaviors are associated with pain and addiction

Noncompetitive NMDA Antagonist Dextromethorphan│Khachatryan

and discover drugs with high-affinity, non-competitive antagonist characteristics that will reduce or eliminate these behaviors without producing “psychotomimetic side-effects” or carrying the potential for abuse.7, 9, 10, 15, 16, 17, 21, 22 Additionally, since studies conducted on animal subjects produced consistent results with the data obtained from human studies regarding the elevation in the effectiveness of naloxone after increased exposure to morphine, this animal model “proves useful in identifying the neural substrates mediating acute dependence.”18

ACKNOWLEDGEMENTs│

I would like to extend my deepest appreciation to Dr. Gerhard Schulteis for his guidance throughout the experiment and assistance in furnishing this manuscript. He is a very accomplished individual who was a great mentor to me, and the skills that I learned in his laboratory will guide me in future research endeavors. In addition, I have learned so much as a result of my experience at the Schulteis lab, and I am truly grateful to everyone who contributed to my educational development.

REFERENCES│

1. Adams, J.U., and Holtzman, S,G. 1990. Pharmacologic characterization of the sensitization to the rate-decreasing effects of naltrexone induced by acute opioid pretreatment in rats. J Pharmacol Exp Ther, 253;483-9. 2. Azorlosa, J.L., Stitzer, M.L., and Greenwald, M.K. 1994. Opioid physical dependence development: effect of single versus repeated morphine pretreatments and of subjects’ opioid exposure history. Psychopharmacology, 114:71-80. 3. Bickel, W.K., Stitzer, M.L., Liebson, I.A., and Bigelow, G.E. 1988. Acute physical dependence in man: effects of naloxone after brief morphine exposure. J Pharmacol Exp Ther, 244:126-32. 4. David Kopf Instruments. 2007. Model 900 Small Animal Stereotaxic. 21 May. http://www.kopfinstruments.com/Stereotaxic/900.htm. 5. Herman, B.H., and O’Brien, C.P. 1997. Clinical medications development for opiate addiction: focus on non-opioids and opioid antagonists for the amelioration of opiate withdrawal symptoms and relapse prevention. Semin Neurosci, 9:158–172. 6. Herman, B.H., Vocci, F., and Bridge, P. 1995. The effects of NMDA receptor antagonists and nitric oxide synthase inhibitors on opioid tolerance and withdrawal. Medication development issues for opiate addiction. Neuropsychopharmacology, 13:269–293. 7. Kemp, J.A., and Leeson, P.D. 1993. The glycine site of the NMDA receptor – five years on. Trends Pharmacol Sci, 14:20–25.

8. Kornetsky, C., and Esposito, R.U. 1979. Euphorigenic drugs; effects on reward pathways of the brain. Fed Proc, 38:2473-6. 9. Leeson, P.D., and Iversen, L.L. 1994. The glycine site on the NMDA receptor: structure-activity relationships and therapeutic potential. J Med Chem, 37:4053–4065. 10. Lipton, S.A. 1993. Prospects for clinically tolerated NMDA antagonists: open channel blockers and alternative redox states of nitric oxide. Trends Neurosci, 16:527–532. 11. Liu J., and Schulteis, G. 2004. Brain reward deficits accompany naloxoneprecipitated withdrawal from acute opioid dependence. Pharmacology, Biochemistry and Behavior, 103:104. 12. Manning, B.H., Mao, J., Frenk, H., Price, D.D., and Mayer, D.J. 1996. Continuous co-administration of dextromethorphan or MK-801 with morphine: attenuation of morphine dependence and naloxone-reversible attenuation of morphine tolerance. Pain, 67:79–88. 13. Markou, A., and Koob, G.F. 1993. Intracranial self-stimulation thresholds as a measure of reward. In: Sahgal A, Ed. Behavioral neuroscience, a practical approach. Oxford: pp. 93-115. 14. Martin, W.R., and Eades, C.G. 1964. A comparison between acute and chronic physical dependence in the chronic spinal dog. J Pharmacol Exp Ther, 146:385-94. 15. Parsons, C.G., Danysz, W., and Quack, G. 1999. Memantine is a clinically well-tolerated N-methyl-D-aspartate (NMDA) receptor antagonists – a review of preclinical data. Neuropharmacology, 38:735–767. 16. Rogawski, M.A. 1993. Therapeutic potential of excitatory amino acid antagonists: channel blockers and 2,3-benzodiazepines. Trends Pharmacol Sci, 14:325–331. 17. Rogawski, M.A., and Porter, R.J. 1990. Antiepileptic drugs: pharmacological mechanisms and clinical efficacy with consideration of promising developmental stage compounds. Pharmacol Rev, 42:223–286. 18. Schulteis, G., Yackey, M., Risbrough, V., and Koob, G.F. 1997. Opiate withdrawal signs precipitated by naloxone following a single exposure to morphine: potentiation with a second morphine exposure. Psychopharmacology, 129:56-65. 19. Schulteis, G., Heyser, C.J., and Koob, G.F. 1999. Differential expression of response-disruptive and somatic indices of opiate withdrawal during the initiation and development of opiate dependence. Behav Pharmacol, 10:235-42. 20. Schulteis, G., Morse, A.C., and Liu, J. 2003. Repeated experience with naloxone facilitates acute morphine withdrawal: potential role for conditioning processes in acute opioid dependence. Pharmacol Biochem Behav, 76:493-503. 21.Trujillo, K.A. 2000. Are NMDA receptors involved in opiate-induced neural and behavioral plasticity? Psychopharmacology (Berl), 151:121-41. 22. Willetts, J., Balster, R.L. and Leander, J.D. 1990. The behavioral pharmacology of NMDA receptor antagonists. Trends Pharmacol Sci, 11:423–428.

Saltman Quarterly│UCSD Division of Biological Sciences│Volume 4│2006-2007

Senior Honors Thesis Research UCSD Division of Biological Sciences The Division of Biological Sciences Senior Honors Thesis Program (BISP 196) is open to undergraduate biology majors who have an overall, and major, GPA of 3.7 or higher, have senior standing, and commit to three consecutive quarters of research during their senior year. The goals of the program are to increase

one-to-one interaction between students and faculty and to encourage more biology majors to pursue research. The Biological Sciences Student Affairs Office oversees the program, which includes a poster session in spring quarter to enable these students to discuss their research with faculty and other students.

Tobacco-mediated Induction of ILK via EGFR Family Proteins, EGFR/ERBB-1 and ERBB-2/HER2/NEU in Carcinogenesis Coney Bae, Revelle College, Biochemistry & Cell Biology major, Zana Ahmad, Jay Patrick Lopez, Aleksander Ciric, Weg M. Ongkeko* *(All authors contributed equally to this project.)

Principal Investigator: Dr. Martin Haas, Adjunct Professor, UCSD Division of Biological Sciences, & UCSD Cancer Center While it has been demonstrated that EGF ligand induces ILK (integrin-linked kinase) via EGFR family, the role of tobacco in ILK induction remains to be unveiled. Using the siRNA knock-down of EGFR/ErbB-1 and ErbB-2/Her2/neu in head and neck carcinoma and lung cancer cell lines, 10B and A549 respectively, we investigate the potential role of cigarette smoke condensate in the induction of ILK via the EGFR proteins. Our preliminary data indicate that the knock-down of EGFR and ErbB-2 shows a significant decrease in ILK expression upon the introduction of tobacco. Also, EGFR and ErbB-2 manifest a synergistic relationship in ILK induction. As an architectural component and an upstream stimulator of a potent oncogene, Akt/PKB, ILK seems to be a promising drug target for chemotherapy. Furthermore, this project suggests one of the possible mechanisms of tobacco - mediated carcinogenesis and metastasis via EGFR as various cancer types are known to up-regulate their EGFR expressions.

Characterization of the Ca2+ Release-Activated Calcium Channel (CRAC-C) Family Madeleine G. Matias, Marshall College, Biochemistry & Cell Biology major Principal Investigator: Dr. Milton H. Saier, Jr., UCSD Division of Biological Sciences The Ca2+ release-activated Ca2+ (CRAC) channel consists of the CRAC channel protein, Orai1, complexed with the STIM1 (Stromal Interaction Molecule) protein. This complex is involved in Store-Operated Ca2+ entry that is release of calcium from intracellular stores. Intracellular free Ca2+ is essential for sustained nuclear translocation of the transcription factor NFAT whose activation is critical for a productive immune response. Patients with severe combined immune deficiency (SCID) syndrome contain a genetic mutation in the Orai1 protein. Seventeen homologues have been identified showing similarity throughout each hydrophobic domain. These proteins are derived exclusively from animals. Orai1 consists of four well conserved hydrophobic domains that are likely to comprise the CRAC channel. It is activated by the endoplasmic reticulum Ca2+ sensor, STIM1, upon Ca2+ store depletion. Phylogenetic analyses reveal that both the Orai1 and STIM1 proteins cluster according to organism type. Domain analysis reveals several conserved regions that are likely to be of functional significance. This report provides detailed bioinformatic analysis of the Ca2+ Release-Activated Calcium Channel (CRAC-C) family.

P-type ATPase Homologues in Proteobacteria Steve R. Stanford, Marshall College, Biochemistry & Cell Biology major Principal Investigator: Dr. Milton H. Saier, Jr., UCSD Division of Biological Sciences P-type ATPases comprise a huge superfamily of ATP hydrolyzing enzyme transporters that catalyze the transport of a variety of ions. Each of the nine families within the superfamily is specific for a different set of ions. The various members of the family have been the subject of many studies in recent years, but desPrincipal Investigatorte attempts to unravel functional and structural properties, important aspects of their mechanism still remain a mystery. We have analyzed the P-type ATPases from 56 proteobacteria with fully sequenced genomes. These include the five groups of proteobacteria: alpha, beta, gamma, delta, and epsilon. The proteins were retrieved from the Transport Classification Database (TCDB), and for each of the five groups of proteobacteria, redundancies, small sequences, and non-homologous sequences were eliminated before generating a multiple alignment. The multiple alignments revealed conserved residues and have allowed us to identify the nine recognized conserved motifs (which characterize P-type ATPases). Representative, well-characterized members of the super family (also taken from TCDB) were then included to the alignments for each of the five groups. The alignments were used to generate phylogenetic trees for each group. Clustering on the phylogenetic tree is usually in accordance with specificity for the transported ion(s). The phylogenetic trees revealed clustering patterns of our proteins with the well-known members of the super family that allowed us to identify which families (within the super family) the many identified proteins belonged to. The results of the study provide details regarding the distribution of each P-type ATPase family member within each of these bacteria and each of the proteobacterial groups. Interestingly, it was found that in each tree, there were proteins present that did not fit into any of the established families. From these findings, we have identified eight new P-type ATPase families of unknown specificity. We have defined their phylogenetic relationships to each other, their topological features, and the sequences of their conserved motifs. These unknown proteins are of great interest because they may represent novel functional types.

Senior Honors Thesis Abstracts | 35â&#x20AC;&#x192;

Affects of a Non-histone Protein on DNA Binding by an RNA Polymerase III Transcription Factor Ryan McHenry Muir College, Molecular Biology Major, and George A. Kassavetis, Ph.D., Adjunct Professor, UCSD Division of Biological Sciences, & Center for Molecular Genetics Principal Investigator: Dr. E. P. Geiduschek, UCSD Division of Biological Sciences In the transcription of RNA polymerase III class genes, RNA polymerase III (polIII) is greatly dependent on multiple DNA binding transcription initiation factors. The transcription of tRNA genes in particular requires transcription factor III C (TFIIIC), the first protein to bind these genes, to recognize and bind downstream promoter elements known as boxA and boxB. The successful binding of TFIIIC at these sites is the first step in recruiting the remaining proteins to assemble the functional polIII transcription apparatus. The overall process of gene regulation and transcription has been shown to be affected by the presence of HMG class non-histone DNA binding proteins. These proteins interact with and bend DNA. In this study we investigate the affects of Nhp6, an S. cerevisiae HMG B class protein, on the DNA binding of S. cerevisiae TFIIIC to tRNA and U6 snRNA genes. We do this using plasmid constructs of SUP4-tRNA genes with increasingly spaced boxA and boxB binding sites and boxB knockouts. Purified transcription proteins and Nhp6 were used to observe the overall change in levels of transcription. We show that in the presence of Nhp6, transcription is greatly increased, there is essentially no limit to the distance between the TFIIIC binding sites boxA and boxB in vitro, and Nhp6 increases the affinity of a TFIIIC domain for its binding site. We also investigate the placement of another transcription factor (TFIIIB), which depends on the location of the TFIIIC binding site boxA, by TFIIIC in the presence of Nhp6 by using SUP4 constructs with adjusted TFIIIB binding sites. The nature of Nhp6’s interactions with DNA and TFIIIC to cause these affects is discussed, as well as the relevance of these findings to other RNA polymerases and eukaryotes.

Evaluating a Novel Link Between Metabolic Stress and p53 Regulation Justin De la Cruz, Muir College, Molecular Biology major Principal Investigator: Dr. Geoffrey Wahl, Adjunct Professor, UCSD Division of Biological Sciences & Salk Institute for Biological Studies In response to nutrient and environmental stress, mammalian cells can restore bioenergetic homeostasis through compensatory changes in the regulation of metabolism. Energetically demanding, cellular proliferation occurs only if regulatory pathways allow for nutrient uptake and if sufficient resources, such as ATP, are available. p53, an evolutionarily conserved tumor suppressor protein, is implicated in the response to metabolic stress, although the mechanisms by which this occurs are not clear. Many tumor cells rely on glycolysis for ATP production and therefore may be particularly sensitive to treatment with agents that mimic metabolic stress. In this report, we evaluate whether glucose deprivation or treatment with 2-deoxyglucose (stalls glycolysis), AICAR (activates AMP-activated protein kinase, AMPK) and phenformin (reduces glucose production) activates the p53 pathway. Strikingly, we found that these conditions lead to downregulation of Hdmx, a critical negative regulator of p53. Intriguingly, Hdmx possesses a phosphorylation motif for AMPK, a sensor of metabolic stress. Further studies are in progress to determine the functional significance of these observations.

Using C. elegans to Study the Role of a Mutated Protein in Microcephaly Hayley Pemble, Sixth College, General Biology major Principal Investigator: Dr. Amy Pasquinelli, UCSD Division of Biological Sciences Centrioles perform two important functions: (1) they recruit pericentriolar material (PCM) to form centrosomes, which are required to set up a proper mitotic spindle, and (2) they serve as basal bodies to direct the formation of cilia. Cilia are cell surface projections that serve various motile and sensory functions. Due to their structural and functional diversity, cilia have been implicated in a wide variety of disorders. Recently, mutations in SAS-4, a core centriolar protein, have been linked to the neurological disorder microcephaly. Despite normal brain architecture, patients with microcephaly exhibit reduced size of the cerebral cortex. Here we used C. elegans as a model system to determine whether mutations in a centriolar protein that lead to a human neurological disorder are a consequence of abnormal centrosome function during cell division, or the inability of centrioles to act as basal bodies during ciliogenesis.

Effect of Pollen Availability and a Colony’s Pollen Demand on Pollen Foraging in Bombus impatiens Traci Kitaoka, Marshall College, Biochemistry & Cell Biology major Principal Investigator: Dr. James Nieh, UCSD Division of Biological Sciences Bumble bees play an important ecological role because they pollinate a wide variety of native plants. An important aspect of their role as pollinators is their ability to communicate information about a nectar source via pheromones and excitatory movements inside the nest. In our first experiment, we investigated whether a bumblebee forager, from the species Bombus impatiens, could also directly communicate about the availability of a pollen source. By measuring the colony’s recruitment response, we were able to conclude that successful pollen foragers can activate nestmates to forage for pollen. There is a significant increase in the number of bees going in and out of the nest when there is pollen available versus when the pollen is absent. This result led us to the next experiment, which examined whether the bees are able to use information obtained from monitoring the pollen pots to initiate a recruitment response. We hypothesized that nestmates monitor pollen levels to determine the need for pollen foraging. We therefore manipulated the pollen stores by directly adding a small amount of pollen into the colony’s pollen pots. There was no effect on recruitment response within 30 minutes after adding pollen. Thus there was no short-term effect. However, 24 hours after the pollen was added to empty pots, the overall recruitment response was higher than on days when pollen was added to full pots. This suggests that the bees were monitoring the pollen pots and initiating a recruitment response that was reflective of the colony’s demand for pollen. Moreover, this monitoring affects colony behavior on a relatively long timescale (24 hours). Since pollen is being constantly used and brought in during the day, a longer timescale may be advantageous because it allows the colony to more accurately integrate information about pollen availability.

Saltman Quarterly│UCSD Division of Biological Sciences│Volume 4│2006-2007

Acknowledgements│ Review Board│2006-2007

Dean Suresh Subramani│

Allison Ashley Aka

We gratefully acknowledge the support provided by Interim Dean Suresh Subramani not only by committing the financial and staff resources needed but also through your strong belief in the value of SQ as an educational tool for the Division’s biology majors and as a means of showcasing undergraduate research in the biological sciences.

Sophomore, Earl Warren College, Biochemistry and Cell Biology major and Music minor

Lisa Michelle Casey Junior, Roosevelt College, General Biology major

Cathy Ao Chang Senior, Revelle College, Biochemistry & Cell Biology major

Vicki Pei-Chun Cheng Senior, Revelle College, Biochemistry & Cell Biology major

Matthew Charles Croskey Freshman, Warren College, General Biology major

Fernanda Delgado Senior, Muir College, Molecular Biology major

Alex Fortenko Junior, Roosevelt College, Microbiology major

Nick James Griff Senior, Muir College, Human Biology major

Patricia Elizabeth Griffiths Freshman, Marshall College, Human Biology major

Peggy B.K. Leung Junior, Warren College, Biochemistry & Cell Biology major

Frederick H. Lin Senior, Marshall College, Biochemistry & Cell Biology major

Tiffany Liu Senior, Earl Warren College, Biochemistry & Cell Biology major

Faculty Advisors│

From Left: Dr. Lisa Boulanger, Dr. Robert J. Schmidt

We express our sincere gratitude fo the time, effort, and dedication From Left: provided by Saltman Brody, Quarterly’s Faculty Adviso- Dr.Dr.Stuart Christopher ry Committee throughout J. Wills the entire year. You gave us such valuable insights into crafting well-written research articles. Thank you for taking the time to sit down with each student author to provide productive comments, suggestions, and support. Also, thank you for conducting the annual manuscript review workshops for the student reviewers.

Jason Ly

Staff Advisor Patricia Walsh│

Senior, Sixth College, Human Biology major

Lauren Ashley Miller

This year was certainly a learning experience for all of us. Thank you, Pat, for your patience in guiding us carefully, for being open to our ideas and being open to learning with us, and for really believing and working for our vision for SQ. We couldn’t have done it without you.

Junior, Revelle College, Molecular Biology major

Ralitza Parina Freshman, Revelle College, Biochemistry & Cell Biology major

Halley Park Junior, Revelle College, Biochemistry & Cell Biology major

Hayley Pemble Senior, Sixth College, General Biology major

Matt Russell Junior, Muir College, Biochemistry & Cell Biology major

Patrick Shih

Assistant Dean Barbra Blake │ Thank you for your crucial role in establishing SQ and for your continuing interest and support.

Junior, Warren College, Microbiology major

Brenda Torres Senior, Eleanor Roosevelt College, Microbiology major

Olivia Truong Freshman, Revelle College, Human Biology major

Billy Chen Wang Freshman, Warren College, Human Biology major

Tania Yaser Junior, Muir College, General Biology major

the

Faculty of the Division of Biological Sciences│

Thank you for your guidance and support in the classroom and in the laboratory, and for your commitment to nurturing the next generation of scientists.

Thank you!

Staff│

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Division of Biological Sciences University of California, San Diego 9500 Gilman Drive La Jolla, CA 92093-0376 (858) 534-3112 http://sq.ucsd.edu sq@biomail.ucsd.edu

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