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Signal transduction during nociception in the hornworm, Manduca sexta

By: Fred E. Arreola and Tyler Deniston

Cellular & Molecular Biology

Faculty Advisor: Dr. Megumi Fuse, Dr. Christopher Moffatt, and Dr. Kimberly Tanner

Nociception describes the process of encoding noxious stimuli by the central nervous system. Nociceptive sensitivity occurs when noxious stimuli such as inflammation or mechanical damage alter the nociceptive response, making animals more sensitive to normal touch. This provides a defensive advantage to animals but can also result in medical conditions including hyperalgesia and allodynia. The biochemical basis of how nociceptive sensitivity is mediated is still not fully understood. Different research models suggest roles for the second messengers, cAMP and cGMP, and results remain variable. The hornworm, Manduca sexta, has a defensive strike in response to touch, which has been shown to be sensitized in response to a noxious pinch. Using the vertebrate SUDO and Up-down methods to assess nociception, we tested the responses to (i) noxious stimuli, (ii) pharmacological agents of the cGMP signaling pathway, (iii) inhibition of protein synthesis. We found that both a pinch (mechanical stress) and cold (thermal stress) reduced the threshold of sensitivity significantly. Moreover, injections of 8-Bromo-cGMP revealed an attenuation in nociceptive sensitivity, while inhibiting the production of cGMP, using methylene blue, increased nociceptive sensitivity. Finally, inhibition of protein synthesis using cycloheximide blocked the onset of long-term sensitivity. Our data suggest that nociceptors in M. sexta respond to multiple stimuli. Sensitization requires protein synthesis, and is inhibited via cGMP, possibly to aid in analgesic effects. Further pharmacological agents will verify this and determine whether NO is involved in this signaling.

35 GL

Phylogeny of Deep Rooted Thaumarchaeota DS1 Energy Metabolism

By: Gilberto H. Gonzalez

Cellular & Molecular Biology

Faculty Advisor: Dr. José R. de la Torre

Thaumarchaeota are a very diverse phylum of archaea that have recently been appreciated for their influence of ammonia oxidation metabolism in ecological environments. Ammonia monooxygenase (AMO) has been largely theorized to be necessary in the ammonia oxidation pathway. Thaumarchaeota DS1's genome was extracted from Yellowstone National Park, Dragon Spring as part of a metageonomic study. Dragon Spring has a high sulfur low pH environment, which are factors known to inhibit ammonia oxidation metablism. A phylogenetic analysis using 40 universally ribosomal proteins categorized this organism as a Thaumarchaeota; DS1 was consistently found to be more deeply rooted than any other previously found species. I will attempt to analyze how DS1 diverged from ammonia oxidizing Thaumarchaeota by doing a comparative phylogenetic study on its energy metabolism, primarily oxidative phosphorylation and its utilization of sulfur.

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Lasers, Arrays, and Informatic Approaches for Studying the Human Placenta

By: Oliver Oliverio

Cellular & Molecular Biology

Faculty Advisor: Dr. Carmen Domingo and Dr. Susan Fisher

Cytotrophoblasts (CTBs) are the major cell types of the placenta, an organ that mediates nutrient transfer and gas exchange between the mother and fetus. Malformation and misregulation between CTBs leads to pregnancy complications like preeclampsia and preterm birth. To understand possible molecular mechanisms between CTB populations, we performed laser microdissection (LMD) on the placenta, isolating these compartments: syncytial, column, and invasive CTBs. We subsequently performed microarray analysis and employed a non-linear principal components analysis (PCA) approach for determining differentially expressed genes between CTB types. Our PCA results were biologically verified using immunohistochemistry, western-blotting, and confocal microscopy. Using this methodology, we've discovered two potential biomarkers for placental development, cannabinoid receptor-1 and neurotensin. These proteins are endogenously expressed in the brain and intestine. However, it's functions have yet to be explored in the placenta.

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Studying the effects of streptomycin on the gut microbiome of Manduca sexta

By: Ryan Marder, Brenda Cisneros, and Raphael Feliciano

Microbiology

Faculty Advisors: Dr. Lily Chen and Dr. Megumi Fuse

Streptomycin is a broad-spectrum antibiotic that prevents the growth of both Gram-positive and Gram-negative bacteria by inhibiting protein synthesis through irreversibly binding to the host’s 30S-subunit proteins and 16S rRNA. It is used to treat bacterial infections as well as inhibit fungal growth on plant-based foods. The use of streptomycin can also alter a host’s normal gut bacterial flora that has been shown to play a significant role in bacterial pathogenesis. The focus of our study is to observe the microbial and physiological effects of streptomycin by using the model organism Manduca sexta, a tobacco hornworm. M. sexta hatchlings have five developmental stages (L1 – L5), and they were divided into two groups. An experimental group of larvae (N=8) were fed a gypsy moth wheat germ diet without streptomycin and the control group (N=8) was fed with streptomycin added to their diet. Stool samples were collected from each developmental stage and inoculated for bacterial culture. Streak plates were performed to characterize bacterial colonies present at each stage, and each colony was Gram stained to determine bacterial morphology. Daily weights and developmental timing were also recorded for all larvae at each developmental stage. / Our results show distinct differences in bacterial morphology between streptomycin positive and streptomycin negative of each larva. Larvae fed with streptomycin mostly contained Gram-positive cocci-shaped bacteria, while larvae fed without streptomycin either had an equal ratio of Gram-positive and Gram-negative bacilli-shaped bacteria, or a majority of Gram-negative bacilli-shaped bacteria. Daily weights of M. sexta raised without streptomycin were higher than animals that had streptomycin in their diet. Developmental timing at each larval stage was shorter for animals raised on a diet without streptomycin. Contrary to previously published studies using streptomycin and kanamycin in M. sexta diets, we found that the developmental timing and weights were not comparable; however, the gut microbiota were similar. Further analysis is in progress to identify the phylogeny of the different bacterial species. The data generated from this project will illustrate the possible mechanism of bacterial pathogenesis related to the gut microbiome.

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Evaluating the dnd Gene Cluster of Phosphorothioate Salmonella

By: Beatriz Kowalski and Steven Flammer

Microbiology

Faculty Advisors: Dr. Lily Chen and Dr. Joseph Chen

Salmonella is the second most common cause of foodborne illness in the US; infecting more than one million people annually. It is known modifications to a bacterium’s genome may confer antibiotic resistance, toxin production, or phenotypic alteration. For an example, phosphorothioation is a post-replicative modification that replaces a non-bridging oxygen in the DNA backbone with a sulfur molecule, which was found to protect bacteria from foreign DNA and offer resistance to hydrogen peroxide. The phosphorothioation modification has been found in Salmonella which is closely associated with the presence of the dnd gene cluster (dndBCDE). Although it is hypothesized that the dnd gene cluster protects bacterial genome from foreign genetic material as well as from environmental damage, little is known about its true biological function(s) and the effect it has on microbial pathogenesis or the role it has on the invasion process. This study aims to examine the functional roles of the dnd gene cluster in Salmonella. Two Salmonella enterica serotypes are used in this study; S. serotype Saintpaul (PT+) and S. Brandenburg (PT-). The DNA damaging effects of H2O2 and ultraviolet (UV) light against PT+ were first examined by observing cell growth in a nutrient rich environment with varying levels of H2O2 and determining percent survivability when exposed to UV light, respectively. Contrary to previous reports, it was found that non-modified Salmonella (PT-) grew just as well in the presence of H2O2 when compared to PT+ Salmonella. It was also found that ≥10 seconds of UV exposure resulted in no significant difference in the survivability of PT+/- Salmonella. No apparent differences in cell morphology were observed under an electron microscopy study. We are in the process of generating knock-out mutants for the genes in the PT gene cluster using the λ-red recombination system in order to more comprehensively study the role this novel DNA modification plays in the Salmonella lifecycle and infection. The λ-red recombination system utilizes the λ-phage recombinase to create double stranded, site specific recombination event to knock out regions of interest by using DNA constructs homologous to loci of interest. Other ongoing experiments in PCR and qRT-PCR are underway to assess levels of dnd gene expression change during infection of host cells¬¬¬¬. Together, these experiments will provide important insights to whether dnd genes affect physiologic states of Salmonella that may confer a selective pathogenic advantage in PT+ Salmonella.

39 GL

Influence of host phylogeny and environment on the microbiome of terrestrial salamanders /

By: Alicia Bird

Physiology & Behavioral Biology

Faculty Advisors: Dr. Andrew Zink and Dr. Vance Vredenburg

Amphibians are major ecological drivers, serving as secondary consumers, and thus influencing nutrient and carbon cycling. Unfortunately, many amphibian species are in jeopardy of extinction or population collapses, with disease being a major factor. Disease may be avoided by some species due to their skin microbiome, the community of microorganisms living on an amphibian host, which often plays an important role in host health and function. However, a lack of knowledge about the ecology and host acquisition of microbiomes limits our understanding of microbiome-disease dynamics. Understanding the modes of microbe acquisition could facilitate implementation of microbial therapies that could increase amphibian survival against disease. This study aims to describe the association between an amphibian host and its skin microbiome by considering the effects of three factors: host traits (i.e. host phylogeny), environmental variables, and disease. Using Next Generation sequencing, I will characterize the skin microbiome across the phylogeny of 11 subspecies of the terrestrial salamander Ensatina eschscholtzii, and assess the extent to which different disease statuses, host traits and environmental variables contribute to microbiome composition. Assuming the skin microbial community composition is primarily influenced by host intrinsic traits, I expect differences in community composition to correlate to evolutionary distances of hosts. I expect that environmental variables and disease prevalence will likely play secondary roles in microbial community composition. This research will create a foundation for future studies on terrestrial amphibian microbiomes and their interaction with pathogens. Further understanding of skin microbiome-pathogen dynamics may provide important insight for developing therapies to mitigate disease in amphibians.

40 GL

Manduca sexta as an Insect Model for Modulation of Nociception

By: Dennis Tabuena

Physiology & Behavioral Biology

Faculty Advisor: Dr. Megumi Fuse

Current preclinical models of nociception in animals such as mice or rats require multiple levels of protocol and logistical regulation. This presents an economic burden to research in the field. Here we characterize a model for hypersensitization of nociception in the insect Manduca sexta. This terrestrial caterpillar can bridge the gap between aquatic invertebrate and traditional, mammal preclinical models. This work was based on a previous behavioral assay where a hypersensitive state was induced with a noxious stimulus. In this study, we identified the afferent nerve pathway from the body wall to the ventral nerve cord using mechanical stimulation with calibrated von Frey filaments and simultaneous extracellular nerve recordings, by systematically eliminated nerve projections. We then assayed both hypersensitive and non-treated animals in this manner, using a pinch as the noxious stimulus. We found that force required to induce a half maximal response in the ventral nerve cord was significantly reduced (1.64mN vs. 0.571mN respectively, P = 0.02) after the pinch. However, the response in afferent nerves was unchanged (2.83mN vs1.70mN respectively, P = 0.22). The lack of significant change in afferent nerve pressure response or basal firing rate suggests that the hypersensitivity to nociception is driven by signaling factors at or after synapses in the ventral nerve cord ganglion. This system provides a novel in vitro model for studying modulation of nociception and “pain” signaling in a cheaper and less strictly regulated animal.

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Investigating Alumni and Faculty Perspectives on UC Berkeley’s Mo’orea Course

By: Brianna McCoy

Ecology & Conservation Biology

Faculty Advisor: Dr. Kimberly Tanner

Many biology research opportunities exist for undergraduates, all with the goal of providing a practical context to use their knowledge. A residential, field-based course is one such opportunity that also provides informal opportunities to interact with faculty and to gain autonomous research experience. UC Berkeley offers a course titled the Biology and Geomorphology of Tropical Islands, hereinto referred to as the Mo’orea course. This course has been conducted for 20 years with only short-term, course-content-based-student evaluations given, but no one has investigated further to see what, if any, impact this course has on the lives of its alumni. This study looks to identify the alumni and faculty perceptions of the Mo’orea course and identify what, if any, influence the course has on the participants’ lives personally, professionally, or in their understanding of the nature of science. Another component of the study will compare alumni perspectives to faculty goals in order to see how, if at all, the two align. To address the questions, the study will interview a subset of both the alumni and faculty populations involved in the course between 1994-2013. After completing 26 alumni interviews and 7 faculty interviews the data have been coded and emergent themes identified in order to establish what the perceptions indicate was impactful on the study populations. Themes will be compared within the population of alumni, using individuals prior research experperience, involvment in different careers and different years and again between the alumni and faculty population. I predict that alumni involved in science-related careers will express how the course influenced their choice or understanding of science. Overall, this study will provide insight into the long-term impact of a residential, field-based course and how that impact changes over the course of 20 years.

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Exploring Student Talk in Active Learning Biology Classrooms

By: Ellen Young

Ecology & Conservation Biology

Faculty Advisor: Dr. Kimberly Tanner

Picture a university science lecture course, and answer the following questions: who is talking? Is the instructor lecturing? Are students occasionally raising hands to ask questions? Are students talking about science in small groups? Do the same students speak repeatedly in the same class session? Talking about course content has been shown to benefit student learning (e.g. Smith 2009). While verbal participation has been extensively investigated in K-12 education, less research exists on student verbal participation in university science classes. For example, Think-Pair-Share (an activity where students first think and talk with a partner before sharing an answer) is a strategy shown to increase participation in elementary school settings (Teobald, 2011). However, the impact of Think-Pair-Share on student verbal participation has not been investigated in university science classes. / / Through community-engaged research, I aim to investigate the patterns of in-class verbal participation and the impact of Think-Pair-Share in four biology lecture courses at San Francisco State University. Specifically I hope to answer the following four research questions: What proportion and population of students verbally participate during class? Of the proportion of students who verbally participate, in what types of verbal participation are they engaged? What proportion and population of students verbally participate after Think-Pair-Share activities compared to other classroom verbal participation events? What are students’ perceptions of their own and their colleagues’ in-class verbal participation? I will investigate these questions through in-class observations and an online student-perception survey. The results of this study will provide a uniquely detailed description of student verbal participation university science classrooms and insights into how students perceive talking as part of class activities. These findings may provide insights for educators and researchers about how to craft a more equitable classroom talk environment.

43 GL

Social Behavior and Batrachochytrium dendrobatidis (Bd): A dangerous dynamic for Batrachoseps salamanders

By: Kendra Ritchie

Ecology & Conservation Biology

Faculty Advisor: Dr. Andrew Zink and Dr. Vance Vredenburg

Many of the world’s amphibian populations are currently suffering rapid declines due to the fungal pathogen Batrachochytrium dendrobatidis (Bd). While amphibians typically encounter the zoospores of this pathogen in their aquatic habitats, transmission can likely take place in terrestrial species via skin-to-skin contact. Little is known about how Bd transmission rates may differ in amphibians that exhibit social behaviors, such as the terrestrial salamander Batrachoseps attenuatus. I propose that the aggregation behavior of this species, which includes frequent body contact with conspecifics, may be increasing their individual risk of becoming infected. I intend to determine whether the size of social aggregations affects Bd transmission through groups. I will also investigate how infection status influences these salamanders’ inclination to aggregate. Through extensive behavioral monitoring and frequent skin swabbing (to assess infection status and intensity), I will be able to quantify the relationship between disease and behavior in these salamanders. I expect to see that an individual’s risk of Bd infection is magnified in larger groups, suggesting that social amphibian species experience a higher risk of this disease. I also expect to see that infection status and intensity can cause overall behavioral changes, perhaps decreasing an individuals tendency to aggregate socially. This research will help to clarify disease dynamics in social amphibians and provide key information for predicting the impacts that Bd could have on wild amphibian populations.

44 GL

Differential Susceptibility to Chytridiomycosis in two Co-occurring Terrestrial Salamanders

By: Mae Cowgill

Ecology & Conservation Biology

Faculty Advisors: Dr. Andrew Zink and Dr. Vance Vredenburg

The fungal pathogen Batrachochytrium dendrobatidis (Bd), which is credited with the decline of many amphibian species worldwide, has already been identified in several California amphibians. However, many species have yet to be tested for susceptibility to Bd. This fungus infects amphibian epithelial cells, preventing effective electrolyte transport and ultimately causing death in many cases. In order to understand the dynamics of this disease in the terrestrial amphibian community, we will (1) document the current prevalence of Bd in two co occurring terrestrial salamanders, Aneides lugubris (Arboreal Salamander) and Batrachoseps luciae (Santa Lucia Mountains Slender Salamander), (2) investigate both species' susceptibility to Bd in lab infection trials, and (3) determine historic prevalence of Bd in both species by analyzing museum specimens for infection. Preliminary results suggest that A. lugubris may not exhibit serious disease symptoms, while B. luciae does suffer typical symptoms in the lab. By comparing lab susceptibility and wild prevalence in both species with presence and density of the opposite species, we can speculate on the role of each species in the community: whether typically symptomatic or asymptomatic disease carrier.

45 GL

Investigating Student Conceptions of Antibiotic Resistance and Use of Intuitive Thinking

By: Melissa Richard

Ecology & Conservation Biology

Faculty Advisor: Dr. Kimberly Tanner

Bacterial resistance to antibiotic medications is a biological concept with growing relevance in society; however it is also a process still frequently misunderstood. Past studies have shed light on common misconceptions and how pedagogical changes can help to clarify these concepts. Little research, however, has focused on the degree to which misconceptions may be based in intuitive, deep-seated cognitive patterns, rather than in the complexity of biological concepts themselves. In cognitive psychology these intuitive patterns are referred to as cognitive construals, and are informal assumptions or predispositions developed at an early age to make sense of the world. In this study I investigate undergraduate students’ application of three construals— teleological, essentialist, and anthropocentric thinking— to a series of challenge statements on antibiotic resistance. While cognitive construals are formed because they are often useful in reasoning about everyday scenarios, they frequently lead to inaccurate conclusions when applied in scientific reasoning. Study of intuitive thinking has historically been limited to young children, and the persistence of cognitive construals into undergraduate students has significant implications for understanding how biology misconceptions persist and how an awareness of intuitive thinking can inform changes in biology education. /

46 GL

Genetic variation across trophic levels in a diverse lizard community

By: Erica Rutherford

Ecology & Conservation Biology

Faculty Advisor: Dr. Eric Routman

Under a standard neutral model of population genetics theory, the size of a population is expected to have a large effect on its genetic diversity. However, very little empirical research has focused on this topic in natural populations of vertebrates. We used a group of sympatric, closely related lizard species from a single locality in order to collect a dataset which minimized potential confounding environmental variables. In this study, we used trophic level (herbivory vs. carnivory) and habitat distribution as a proxy for long-term effective population size. Tissue samples from four species of lizards, two herbivorous and two carnivorous, were collected in the Mojave Desert. One mitochondrial and three autosomal genes were sequenced and used to compare intraspecific genetic diversity. Data analysis, using standard methods for estimating genetic diversity, is ongoing. Results from these four species will be compared with those from other species previously sampled from the same locality.

47 GL

Disease and the skin microbiome of an endangered frog, Rana sierrae

By: Silas Ellison

Ecology & Conservation Biology

Faculty Advisor: Dr. Vance Vredenburg