SQ Volume 21 (2023-2024)

Front Cover

Manta Ray(Mobula birostris) feeding off the coast of Kona, Hawaii.

Photo by: Darren Wong

Inside Cover

White Tiger(Panthera tigris) swimming at the Singapore Zoo.

Photo by: Dominic Tse

Back Cover

SALTMAN FAMILY AND

The views expressed in this publication are solely those of Saltman Quarterly, its principal members, and the authors of the content of this publication. While the publisher of this publication is a registered student organization at UC San Diego, the content, opinions, statements, and views expressed in this or any other publication published and/or distributed by Saltman Quarterly are not endorsed by and do not represent the views, opinions, policies, or positions of the ASUCSD, GSAUCSD, UC San Diego, the University of California and the Regents or their offices, employees, or agents. The publisher of this publication bears and assumes the full responsibility and liability for the content of this publication.

In an effort to engage the UC San Diego Community, Saltman Quarterly holds an annual photo contest. The winners of this contest have their images featured on the cover and interior pages of the journal.

Green Sea Turtle(Chelonia mydas) on a night dive in the Florida Keys.

Photo by: Darren Wong

DEAR READER, LETTER from the EDITORS

photo by: Harnoor Sidhu

Thank you for picking up this year’s edition of SaltmanQuarterly and for celebrating biology’s newest discoveries with us, ranging from advancements in personalized medicine to the anthropological relationship between humans and elephants. We are proud to share the work of our writers, editors, illustrators, photographers, and production designers who have spent the last year carefully crafting these pages while also juggling the demands of their rigorous academic lives and never-ending social commitments.

Our staff’s enthusiasm for science communication is born from a desire to share the stories being created on our campus that we believe you should know. In an era seemingly saturated with disheartening information—whether on the order of world events or everyday school pressures—it can be difficult to appreciate the innovations slowly propelling society forward. However, as our Features writers met with scientists unraveling the mechanisms behind disease and drivers of climate change, we found pockets of hope worth sharing.

For example, one of our stories on page 22 is about how Alzheimer’s disease, once a creeping death sentence, may soon be clinically treatable in ways that could restore cognitive function and improve an individual’s quality of life. Another article on page 6 focuses on how scientists are analyzing protein misfolding to battle diseases like cystic fibrosis or retinal degeneration. These novel approaches to solving human health crises are powered by people on our campus dedicating their lives to making our world a safer place.

Looking beyond the systems of the human body, this year’s manuscripts are a testament to how our undergraduate researchers are also improving the world by examining shifting environmental patterns and indicators of ecological health. Three manuscripts are the result of students traveling around the world to assess the impact of climate change on the abundance of species like salamanders, am-

phibians, and avians. In the future, these findings can support policymaking that protects our ecosystems and in turn protects us. Despite the variety of topics, the common thread between these articles is the compassion behind the research. As science communicators, we believe in our responsibility to contextualize the findings we present and to explain why this work is more than just data. We’ve included an opinion editorial found on page 26 for the first time this year, which describes new molecular approaches to fighting malaria, but also insists that we shift our perspectives of malaria to include more culturally conscious research when finding treatments. The author highlights UC San Diego’s efforts to decrease this gap, proving once again that the pursuit of science must be intertwined with humanity.

Though our magazine can only fit a few ways in which our community advances the field of biology, we encourage you to seek out these stories on your own as well. As we sat down with our staff every week, sifting through the articles and interviews we chose to share with you, we felt a strong sense of pride in our scientific community. We hope that as you read through this year’s edition, you feel similarly.

With heartfelt gratitude, we are proud to present Volume 21 of Saltman Quarterly.

Warmly,

Amoolya Chandrabhatta and Emily White Editors-in-Chief, Saltman

Quarterly 2023-2024

Saltman Quarterly thanks the Saltman Family for their generosity and support. Their contributions have allowed SQ to continue to spread Dr. Paul Saltman’s ideals of science, communication, and education.

FEATURES

Protecting the Proteome by Kalisa Kang

New Weapons in the Battle against Cancer by Sahana Kashyap

Compassionate Conservation by Esha Bhattacharya

How to Taste with Your Arms byAmy Li

Bouncing Back by Emma Svendsen

Open-Ended: Malaria Moves Back West by Kaz Nuckowski

RESEARCH, BREVIAS, & REVIEWS

Comparison of Bird Communities in Forested and Urban Habitats on Pulau Tioman, Malaysia by Sally Guan

Spatiotemporal Expression of srcr142 throughout the Development of the Painted Sea Urchin Lytechinus pictus by Gloria D.Renaudin &Amro Hamdoun

Effect of Abiotic Microhabitat Conditions on Coastal Giant Salamander Larvae Across and Within Streams byAshAbel,Sanchita Biswas,Cleo Lu,SierraVasquez

Anuran Distribution and Richness in a Tropical Montane Forest in Monteverde, Costa Rica – An updated survey byTaryn Cornell,Frank Joyce,Federico Chinchilla

SENIOR HONORS THESIS

Undergraduates in the Biology Honors program are required to complete a written thesis detailing their scientific research. The Senior Honors Theses section, which presents the abstracts of their individual theses, highlights the achievements of accomplished student researchers.

49 58 SALTMAN DEDICATION

STAFF

Meet the members of the 2023-2024 Saltman Quarterly staff who worked throughout the year to bring you this issue, as well as our online content, quarterly insiders, and community outreach initiatives.

DR. PAUL SALTMAN

Dr. Paul Saltman, the namesake of our beloved student-run organization, continues to live vicariously through the lasting legacy he has left behind as both a renowned biologist and professor at UC San Diego. His love of learning and enthusiasm for science laid the groundwork for our organization’s mission to present scientific research in an informative way that is accessible to all. Today, nearly twenty-five years after his passing, Dr. Saltman is still regarded as a revolutionary researcher in nutritional science, dedicated professor, and mentor who went above and beyond to share his passion for biology and guide students to their future endeavors.

Dr. Saltman received his B.S. in Chemistry in 1949 and his Ph.D. in Biochemistry in 1953 from the California Institute of Technology. Upon completing his degrees, Dr. Saltman did postgraduate studies in biochemistry at the College de France in Paris and later became a professor at the University of Copenhagen and at Murdoch University in Australia. He then made his way back to the United States, where he joined the biochemistry department as a teaching professor at the University of Southern California

in Los Angeles. Fourteen years later, Dr. Saltman joined the UC San Diego community as the provost of Revelle College in 1967 and then as the Vice Chancellor of Academic Affairs in 1972. After serving as Vice Chancellor, Dr. Saltman returned to his true passions: teaching and research.

Dr. Saltman’s influential research in the field of nutritional science centered around understanding the impact of trace metals such as zinc, iron, copper, and manganese on human physiology. His studies had a wide range of applications, resulting in enhanced dietary and supplemental strategies to ensure proper nutrition for growth and development. Additionally, clinical applications of his research included preventing anemia, reducing free radical heart damage, and improving skeletal metabolism. An expert in all things nutrition, Dr. Saltman also authored TheUniversityof California Nutrition Book, which aims to educate the general public on nutritional health and food, as well as The New Nutrition, which provides an in-depth compilation of nutrition-related readings from medical and scientific literature.

Paul Saltman’s profound legacy was not only rooted in academia and research, but also in his dedication to mento-

ring his students as they embarked on their journeys as future scientists. Dr. Saltman was known to be compassionate and dedicated, going great lengths to build relationships with his students as a role model. When asked what made a great teacher, Dr. Saltman underscored the importance of upholding the ‘teacher-pupil relationship,’ an interactive process of giving and sharing. He found it imperative to understand the process of human learning and comprehension to inspire students and fuel their eagerness to learn. Dr. Saltman left a lasting mark on each of his students, many of whom credit him for their successes as scientists and leaders.

Former University of California President Richard Atkinson noted that "Paul Saltman played a key role in defining the nature of UCSD. He held to the highest academic standards, was

a brilliant teacher, and showed excellent judgment in guiding the activities of the University.”

Throughout his career as a researcher and teaching professor, Dr. Saltman garnered recognition for his outstanding contributions. In 1994, he received the Career Teaching Award from UC San Diego, setting the stage for numerous accolades from Marshall, Muir, Revelle, and Warren colleges. The establishment of the Paul D. Saltman Chair in Science Education at UC San Diego further stands as a poignant tribute to his impact within the fields of science and education.

Beyond his fervor for academic excellence, nutritional science, and human health, Dr. Saltman exuded a myriad of passions. He actively engaged in shaping scientific discourse on both national and international le-

vels, serving on editorial boards for prestigious scientific journals and offering his expertise as a consultant to the National Institutes of Health. Dr. Saltman further served on the National Science Foundation Committee on Science Education, a testament to his dedicated pursuit of effective science communication.

Dr. Saltman's commitment to fostering a broader understanding of science manifested in his appearances on numerous radio and television programs, where he sought to bridge the gap between scientific knowledge and public education. Notably, his National Educational half-hour series Patterns of Life and television program for the Public Broadcasting Service strived to enhance science communication for the general public. Additionally, Dr. Saltman’s presence extended to the documentary film, WhyManCreates, where he engaged in discussions about the nature of creativity. Parallel to his endeavors in science communication, Dr. Saltman also lent his voice as a contributor to the National Endowment for the Humanities newspaper, contributing to the discourse on “America and the Future of Man.”

In this multifaceted way, Dr. Paul Saltman left an enduring mark as a pioneer in nutritional science and science communication. His groundbreaking research not only steered modern preventative medicine against various health concerns and conditions but further showcased his steadfast dedication to academic excellence, education, and public engagement. It is Dr. Saltman’s commitment that serves as the driving force behind Saltman Quarterly’s unwavering dedication to foster learning within the UC San Diego community and beyond.

In the enduring legacy of Dr. Paul Saltman, Saltman Quarterly commits to honoring his memory through effective science communication. Recognizing the profound impact he had on individuals both within and beyond the scientific community, whether as a researcher, teaching professor, vice chancellor, author, editor, broadcaster, father, or friend, the Quarterly remains dedicated to celebrating the unwavering kindness and selfless contributions of Dr. Paul Saltman to people, science, and beyond.

written by: Samantha Kawai & Ava Selami

UC San Diego is at the forefront of scientific discovery and exploration as a hub of biological research. The Features section highlights some of the groundbreaking work accomplished by researchers affiliated with the UC San Diego campus.

photo by: Ella McLaren Colors and textures of a San Diego tree

THE CHAPERONE CHRONICLES OF DFM 1

WEAPONS IN THE BATTLE AGAINST CANCER

written by SAHANA KASHYAP illustrated by MAIA LAZOR

NOVEL APPROACHES TO IMMUNOTHERAPY AND PRECISION MEDICINE

Precision Medicine as a Potential Weapon

The ability to be truly oneself arises from the freedom to exude individuality, expressing one’s experiences, thoughts, ideas, and background in any way they choose. It is what makes us diverse, and this diversity filters into the body—its appearance and genetic variation. While the development of healthcare in its initial stages sought to cater to the masses, and help as many people as possible with each breakthrough, more recent research has delved into precision medicine. Precision medicine aims to cater to every individual based on their characteristics, and is a more complex and ambitious outlook to medicine. Variabilities in response to treatment methods include the individual’s lifestyle, such as diet and regular medications, phenotype such as weight, ethnicity, as well as genotype of the individual, which could have which are variations in a DNA sequences in regions of interest.1 Dr Hannah Carter at the UC San Diego School of Medicine is one such researcher who is interested in precision medicine, and some of her exciting work is in the field of cancer immunotherapy.

An Introduction to the Enemy Cancer, or ‘The Emperor of All Maladies,’ as Siddhartha Mukherjee coined it, has been an ubiquitous health concern since 2500 B.C.2 It is characterized by an uncontrolled growth of cells in the body due

to failure of cell cycle regulation.3 This disease overrides healthy functioning of the immune system, especially its ability to impede cell proliferation. In an attempt to better tackle the disease, the scientific community seeks to understand tumor growth, tumor interactions with cells in the body, and the genes of the immune system. With more clarity on the genetics of the immune system in relation to cancers and tumor growth, immunotherapy techniques can be modified for better, more personalized patient care.

Building

the Weapon:

Tumors, TIME and eQTLs

While the field of immunotherapy has expanded over the years, one drawback has been low response rates to current immunotherapies, with no definitive cause.4 The Carter Lab believes that the potential of immunotherapy could be recognized in part by delving into the tumor immune microenvironment (TIME).4 This microenvironment consists of both inhibitors as well as supporters of tumor growth, and learning from each body’s response could help develop therapies to attack the TIME.5 One family of components in TIME is immune infiltrates, which are immune cells that enter the tumor from the bloodstream.6,7 In Dr. Carter’s research, TIME was described through a collection of immune phenotype (IP) components that included immune infiltrating cells, measured from gene expression levels.4

Figure 1: Simplified progression of cancer in the body. With and without Immunotherapy: Healthy Cancer cells (in blue), due to genetic (as well as environmental factors), can develop into cancer cells. They can progressively multiply to become groups of cancer cells and eventually tumors. However, with immunotherapy efforts, the body’s defense system is strengthened and immune responses can impede cancer cell growth.

To further understand TIME and the genetics involved in immunotherapy, an understanding of eQTLs is needed. Many phenotypic traits in the body are quantitative in nature, meaning that there are multiple genes contributing to an expression of one specific phenotype.8 While it is difficult to isolate and determine the contribution of each of these specific genetic loci towards a complex trait, these loci all contribute to gene expression, and are called quantitative trait loci (eQTLs).8 Dr. Carter’s work identifies and utilizes TIME eQTLs to delve into potential immunotherapy targets.4 Here, the loci contribute to IPs in the tumor microenvironment, and could provide insight into a better understanding of TIME.4 The ease of data collection and quality of modern technology has allowed the growth of precision medicine, and cancers can be analyzed to impute associations, paving way to treatment methods.

Summary: The Research Approach

On understanding the key terms in Dr. Carter’s research, a more thorough look reveals the group’s aims—elucidating cancer risk, survival, as well as response to a method of immunotherapy known as Immune Checkpoint Blockade (ICB).4 They study changes in TIME and its evolution, as well as response to immunotherapy in different genetic backgrounds to identify potential target areas and breakthroughs.4 In this process of providing potential targets for immunotherapy, Dr. Carter tests her computational models using different datasets, and further uses in-vivo mouse models to test other targets.4 As a broad framework, their research first identifies genes and IPs of interest before narrowing in on eQTLs of interest.4 The research specifically identifies TIME eQTLs and analyzes them downstream for correlations across different cancers and tumor types.4 Further, the research delves into predicting cancer risk using inferred associations, along with survival rates.4 To address the response to current ICB immunotherapies as well, analysis was done across different studies to check for strong association of eQTLs identified with ICB response.4 In addition to identifying eQTLs, their correlation with risk, survival and therapy response, the lab built a model to assess the potential for immune genetics to affect the three main areas of focus and yield potential targets, and tested this model on different cohorts and cancer types.4

Figure 2: An overview of the research approach. Dr. Carter’s research uses databases, such as TCGA, to identify genes of interest, which on analysis provide eQTLs of interest. Further computational analysis and polygenic models are used to impute key markers—one such marker, gene CTSS, was tested in mice, and showed powerful correlations with tumor growth, cancer risk and survival.

Connecting TIME eQTLs to Cancer Risk, Survival and Immunotherapy Response

To determine contributions of TIME to tumor development and possible immunotherapy approaches, initial analysis included identification of heritable characteristics (ones that could contribute to the phenotype) in TIME that could affect tumor immune responses.4 On normalization and analysis, 733 total IP components of interest were derived from an atlas of data of over 11,000 patients' tumor and matched normal samples, identified across 30 different cancer types known as The Cancer Genome Atlas.4 32% of these could potentially be SNP-heritable, meaning that 32% of these components could significantly influence the phenotype (expressed as the TIME).4 However, since a majority of these IPs did not pass a threshold for such heritability, researchers looked at a subset of 157 heritable immune genes that were SNP-heritability associated.4 A genome wide association study (GWAS) was conducted on these remaining genes, which identified frequent variations found in diseased samples when compared to healthy samples.4 From this GWAS study, 890 TIME eQTLs were identified.4 On analysis of the genes of interest identified, genes of macrophages (immune system components that which engulf foreign cells) and lymphocytes (produce antibodies) were found in the highest quantity.4 Additionally, a large correlation was seen in genes coding for MHC complexes, which are responsible for antigen presentation.4 The majority of the eQTLs are not cancer-type-specific, and of the 890 TIME eQTLs identified, only one (rs146336885) was specific to tumor type.4 Dr. Carter’s group used analysis methods in human cohort datasets such as TCGA and other databases such as UK Biobank to assess that eQTLs were relevant to existing cancer data, and not just simply gene expression.4 A high variation was found with both predictions of cancer risk, as well as survival using TIME eQTLs identified, and 525 of those eQTLs were of significance for ICB response as well.4 Overall, Dr. Carter’s approach looks at various tumor stages to identify significant heritable modifiers of the immune system, which could yield potential drug targets in the body.4

Using Machine learning models and Polygenic Scores

“THE CARTER LAB’S RESEARCH SIGNALS THAT A PATIENT’S GENETIC BACKGROUND IN RADIATION THERAPY PROVIDES HOPE FOR GROWTH OF PRECISION CANCER THERAPY. ”

To assess cancer risk with TIME eQTLs, Dr. Carter’s Lab applied a machine learning model known as XGBoost to calculate polygenic risk scores, which calculates the potential risk of developing a given disease based on the genetic background of the individual.4 This approach is unique since it is not a linear risk calculation, simply based on adding the effects of different variations.4 Rather, it determines the risk and progression of a cancer in an individual, based on a comprehensive study, learning and analysis of their specific variants’—thus increasing the degree of personalization.4 Three main areas of analysis included risk, survival and immunotherapy response. The former two showed that the body’s immune system makes efforts to suppress the cancer throughout tumor development.4 A novel approach in Dr. Carter’s research was the attempt to study whether immunotherapy would be beneficial to the patient, or have life-threatening side effects based on the immunotherapy response aspect of the ML approaches.4 This model showed, for the first time, that hereditary immune genetic traits can provide insight into responders to immunotherapy compared to those expected to suffer detrimental outcomes.4

This research looked at two different cancer types—melanoma and prostate cancers; melanoma is a cancer type with tumors containing high amounts of immune infiltrates and good response to immunotherapy, while prostate cancer has low amounts of immune infiltrates and poor immunotherapy response4,9,10 Risk scores for developing both cancers were significantly different in the top 10% and bottom 10% of population.4 Further, eQTLs related to a gene of interest, CTSS, and MHC genes were considered most important in both cancer types.4 Similarly promising results were seen for patient survival when tested using Polygenic survival scores.4

“IMMUNOTHERAPY TECHNIQUES CAN BE MODIFIED FOR BETTER, MORE PERSONALIZED PATIENT CARE. ”

To assess response to an immunotherapy known as Immune Checkpoint Blockade, the researchers constructed a similar model: immunotherapy response-specific polygenic risk score using melanoma cohorts with ICB.4 The researchers imputed 15 genes that were expressed differently in responders to ICB as compared non-responders, and one such gene was CTSS. CTSS is a gene that is known to help with antigen presentation in the MHC protein complex and in cancer, it is associated with increased tumor progression.4,12 Identifying CTSS as a potential inhibiting genes with negative ICB response, the Carter lab tested the effect of inhibiting CTSS in-vivo.4 They found that mice with this inhibition showed slower tumor growth and better survival, and also provided more insight into macrophages as key infiltrates, where M1 macrophages showed increased infiltration and M2 showed the opposite.4 Through these experiments, the Carter Lab aims to provide a procedure for determining putative immunotherapy targets in various cancer types through analysis of TIME and its eQTLs.4

Dr.Carter’s Other Projects: More Applications for Precision Medicine

Immunotherapy is one of many types of treatments for cancer and Dr. Carter’s work looks at other forms of precision cancer therapies. One such widely used approach that contributes to almost 40% of cancer treatments is radiation therapy. In collaboration with Dr. Ida Deichaite and Dr. Vitali Moiseenko, the Carter Lab worked to identify the biomarkers correlated with post-radiation toxicity, an array of side effects caused by penetrating radiation therapy.11 Head and neck squamous cell carcinoma patients at the Moores Cancer Center at UC San Diego were studied with somatic tumor sample sequencing and transcriptome RNA-seq amongst other methods.11 The patients underwent radiation therapy and were grouped based on whether they exhibited symptoms or signs of radiation toxicity.11 They were then studied for associations with specific germline variants, or mutations that were inherited but not developed during the individual’s lifetime, and their clinical toxicity outcomes.11 The study concluded that germline genetic biomarkers can indeed predict outcomes of radiation therapy, including the post-radiation toxicity.11 Similar to the genetic study of immunotherapy, The Carter Lab’s research signals that a patient’s genetic background in radiation therapy provides hope for growth of precision cancer therapy.

Implications for future battles

While this research is a promising step in the path to precision medicine, identifying potential avenues for personalized medicine is just the beginning. Clinical trials are necessary to validate the discussed targets and approaches. As a future direction, partnering with clinicians could help understand whether, and how patients would benefit from such therapeutic methods.13 In addition, the

analysis suggests that one therapy that seems better suited to a patient based on genetic factors could be considered while considering treatment options.13 The Carter Lab plans to further utilize polygenic scores to also look at somatic characteristics, and plans on exploring pathology-based predictions among others.13 The over-representation of people with European ancestry in most data sets also limits generalizability.13 Investigations require greater diversity to determine whether the same genetic loci are significant across populations and whether epigenetics plays a role in immunotherapy.13

Conclusion

As the potential impact of precision medicine for cancer is slowly yet surely being recognized, the work of Dr. Carter and her lab is truly inspiring to passionate researchers. While there is a lot of ground to cover in this battle against cancer, the addition of precision immunotherapy to the arsenal is sure to pave the way for more such meaningful and impactful discoveries in oncology.

References

[1] Akhoon N. Precision Medicine: A New Paradigm in Therapeutics. Int J Prev Med. 2021;12:12. doi:10.4103/ijpvm.IJPVM_375_19

[2] Cancer: A historic perspective. Cancer.gov. [accessed 2023 Nov 27]. https://training.seer.cancer.gov/disease/history/

[3] What is cancer? National Cancer Institute. 2007 Sep 17 [accessed 2023 Nov 27]. https://www.cancer.gov/about-cancer/ understanding/what-is-cancer

[4] Pagadala, M., Sears, T.J., Wu, V.H. et al. Germline modifiers of the tumor immune microenvironment implicate drivers of cancer risk and immunotherapy response. Nat Commun 14, 2744 (2023). https://doi.org/10.1038/s41467-023-38271-5

[5] Ino, Y., Yamazaki-Itoh, R., Shimada, K. et al. Immune cell infiltration as an indicator of the immune microenvironment of pancreatic cancer. Br J Cancer 108, 914–923 (2013). https://doi. org/10.1038/bjc.2013.32

[6] Baghban R, Roshangar L, Jahanban-Esfahlan R, Seidi K, Ebrahimi-Kalan A, Jaymand M, Kolahian S, Javaheri T, Zare P. Tumor microenvironment complexity and therapeutic implications at a glance. Cell Commun Signa. 2020;18(1). https://doi.org/10.1186/ s12964-020-0530-4

[7] Zou R, Gu R, Yu X, Hu Y, Yu J, Xue X, Zhu X. Characteristics of infiltrating immune cells and a predictive immune model for cervical cancer. J Cancer. 2021 https://doi.org/10.1186/s12964020-0530-4

[8] Nica AC, Dermitzakis ET. Expression quantitative trait loci: present and future. Philosophical Transactions of the Royal Society B. 2013;368(1620):20120362. https://doi.org/10.1098/ rstb.2012.0362

[9] Jochéms C, Schlom J. Tumor-infiltrating immune cells and prognosis: the potential link between conventional cancer therapy and immunity. Experimental Biology and Medicine. 2011;236(5):567–579. https://doi.org/10.1258/ebm.2011.011007

[10] Mills CD. M1 and M2 Macrophages: Oracles of Health and Disease. Critical Reviews in Immunology. 2012;32(6):463–488. https://doi.org/10.1615/critrevimmunol.v32.i6.10

[11] Deichaite I, Hopper A, Krockenberger L, Sears TJ, Sutton L, Ray X, Sharabi AB, Navon A, Sanghvi P, Carter H, et al. Germline genetic biomarkers to stratify patients for personalized radiation treatment. J of Transl Med. 2022;20(1). https://doi.org/10.1186/s12967-02203561-x

[12] McDowell SH, Gallaher SA, Burden RE, Scott CJ. Leading the invasion: The role of Cathepsin S in the tumor microenvironment. Biochim. Biophys. Acta doi:10.1016/j.bbamcr.2020.118781

COMPASSIONATE CONSERVATION

written by ESHA BHATTACHARYA

illustrated by CHRISTINA ZHA0

AN INCLUSIVE APPROACH TO SUSTAINABLE DEVELOPMENT

Your food reserves are running low; you are beginning to doubt you will be able to feed your family in the upcoming week. Resources have been scarce for a while, but it became especially difficult to find anything after the new neighbors moved in. They have been demolishing and rebuilding upon much of the land you use to gather food, and have even started approaching your home. You are starting to feel threatened, with this intruder only exacerbating your previous problems. If they do not leave soon, you might have to do something drastic to drive them away.

As human beings, our innate reaction may be to empathize with the person we infer to be experiencing these feelings and concerns. We are generally inclined to believe that only other humans encounter anxieties around feeding themselves and keeping their families safe. However, the effort for survival is a universal experience —transcending boundaries of species and resonating with all living beings. Recent explosions in human development, and resulting encroachment into previously undisturbed animal habitats, have led to increased conflict between human and animal needs. Elephant communities have been particularly affected with regards to land-use and resource distribution. However, within these instances of conflict, human and elephant groups are both equally negatively impacted, and often for the same reasons. As such, it is possible to reshape destructive human practices by placing the needs of both human and animal communities at the forefront of sustainable development.

EXPLORING THE ROOTS: WHAT ARE

THE CAUSES OF CONFLICT

To understand what sustainable practices should aim for, it is necessary to first characterize the current systemic threats to elephant communities. The most prominent is habitat fragmentation. Rapid human population growth and resulting needs for infrastructure, housing, agriculture, and mining have led to human expansion into historic elephant domains. This has fragmented long-ranging elephant habitats and prevented access to essential migratory pathways.1 Elephant habitats now increasingly overlap with human settlements, often in areas where human activity dominates and actively separates previously large elephant communities. The following genetic isolation has led to

a loss of gene flow between populations and an increase in inbreeding. The cascading effects have resulted in a reduction of genetic diversity and a reduced adaptive potential. Isolated elephant populations, with limited phenotypes to “pick from,” have less adaptive capacity for external threats, both human and non-human, and a greater vulnerability to extinction (see Figure 1).2

A reduced range also means that elephants can no longer sustain themselves solely on the resources within their habitats. These animals often turn to seasonal migration to ensure consistent resource access.3 However, habitat fragmentation impedes migratory behaviors, which are especially vital for elephant communities facing resource scarcity. When elephants do attempt to migrate, they encroach on human settlements and cause damage to subsistence crops and livestock. This may result in retaliatory human behavior, which usually ends in endangerment or loss of life for both populations.3 Excessive human-elephant conflict can become detrimental to both groups, and conservation practices must take this into account.

Historic approaches to conservation have considered this problem, yet they have sometimes taken it to the extreme. Dubbed “elephantocentric” conservation, this approach prioritizes the reconstruction and maintenance of theoretically ideal elephant communities to be released into sanctuaries and protected habitats.4 On the other side of the spectrum, it accounts solely for the ecological needs of elephants without wider consideration of human landuse needs or local ecosystems. Constructing an ideal elephant community to reside in a “pristine” natural world, completely untouched by human influence, is unattainable. Elephantocentric approaches are also not feasible given the current trajectory of human growth in areas where elephant conservation is most needed. In addition, this outlook does not consider the history of human-elephant coexistence in many parts of the world. Elephants play major social and cultural roles in local communities, including reciprocal relationships developed through cen-

turies of elephant husbandry practices.5 While human needs shouldn’t be considered over elephant needs, ignoring the local history of human-elephant relationships and supplanting these philosophies with a largely Western perspective is also counterproductive to elephant conservation. Current research conducted by the de Silva Human-Nature Lab at UC San Diego, which considers a more interdisciplinary and intersectional approach to human-elephant dynamics, addresses this very gap in traditional conservation strategies.

FINDING

THE KERNELS: WHAT ARE ELEPHANT NEEDS?

There have been increasing global critiques of modern land usage, primarily concerning agricultural expansion on a scale often unsustainable for local ecosystems. The pesticide and nitrogenous fertilizer usage, strenuous land use, and enormous scale of ultra-efficient commercial farming practices may increase crop yields, but they simultaneously wreak havoc on local ecosystems. Modern agriculture must turn towards new methods which can boost crop production without stripping the land of its resources.6 As a result, implementing sustainable land use practices, which balance the needs of both animal and human inhabitants, is key to preventing further escalation of these issues. Elephants, as adaptable, wide-ranging animals, become bolder the more their habitats are threatened and force human communities to confront their impacts on the wider ecosystem.3 Utilizing this reasoning, the de Silva Lab focuses their research on Asian elephants as a valuable case study to aid in the development of their intersectional approach.

“ PREVIOUS AREAS OF SUSTAINABLE COEXISTENCE INDICATE THAT HUMAN PRESENCE ITSELF IS NOT AN INHERENT DETRIMENT TO ELEPHANT POPULATIONS. RATHER, IT IS HUMAN PRACTICES THAT ARE HARMFUL. ”

The de Silva Lab and collaborators developed an ecological niche model to map and predict the distributions of Asian elephants through their habitats within South and Southeast Asia. With this model, researchers hoped to better understand the core ecological needs of elephants, as well as how those needs overlap with human ones. The process of developing this model can be divided into three main stages: formulating inputs, running the inputs through machine learning algorithm MAXENT to develop the outputs, then refining outputs.7

The primary inputs of the ecological niche model were elephant occurrence data and ecological covariates. Ecological

covariates refer to specific variables that indicate a region’s suitability to elephant needs, such as wood harvest rate, human densities, forest cover, and climate. For this model, the researchers drew the covariates from historical datasets, concerning patterns in land use, and finer resolution datasets, which set a “benchmark” for environmental variables. Once the ecological covariates and elephant occurrence data were fed into MAXENT, two different map model outputs were constructed from the associations between covariates and the elephant occurrence. The outputs were initially continuous models, but they were binarized for analysis by establishing a cutoff which determined locations

as either suitable or unsuitable habitats. From this binarized state, the two models were combined into a single map and a corresponding mathematical model for the covariate-elephant associations. This model can now project elephant habitat suitability back into years where there are no records, like predicting an “x” and “y” for a linear model given its equation (see Figure 2).7

Land-use models are beneficial in allowing researchers to reconstruct a past more distant than existing records. By analyzing historic correlations between human and elephant groups, this model demonstrates that suitable elephant habitats and populations have existed in areas with recorded human presence. Previous areas of sustainable coexistence indicate that human presence itself is not an inherent detriment to elephant populations. Rather, it is human practices that are harmful. This can prompt a shift in conservation practices to prioritize sustainable human presence instead of trying to reconstruct a “pristine” ecosystem that never existed.

GROWING THE GA RD EN: WHAT ARE HUMAN NEEDS?

The growing importance of sustainability has also prompted interest in incorporating Indigenous land-use philosophies into modern agricultural practices. Indigenous communities, such as those in South Asia and elsewhere, generally embed the health of the surrounding ecosystem into their cultural values, which then positively influences their land management.8 However, Indigenous practices are often tailored to the particular ecosystems they have been developed in. Similar to “elephantocentric” conservation strategies that ignore local elephant histories, applying the practices of Indigenous peoples outside of Asia to Asian land-use practices, without holistic consideration of greater impacts, may cause more harm than good.

One example of difficulty transferring research findings across different contexts is the potential implementation of “bee-fences” to protect crops from elephant damage. Previous research showed that African elephants had adverse reactions to African honey bees, and researchers hypothesized that Asian elephants would react similarly to Asian honey bees. The bee-fence initially caught the attention of researchers as it would provide a more ecologically conscious alternative to protecting human crops. A bee-fence would harness an elephant’s innate reaction to honey bee sounds, instead of using damaging man-made materials and constructing physical barriers. However, studies conducted in Asia observed mixed

Figure 1: Habitat fragmentation. There are a multitude of problems plaguing modern elephant populations, including loss of migratory pathways and resources, as well as decreased gene flow and genetic diversity between populations.1,2 Majority of these problems have a common source: widespread habitat fragmentation due to increased human activity in elephant habitat ranges.

HOW TO TASTE WITH YOUR ARMS

written by AMY LI

illustrated by

VICKIE NGUYEN

CHEMOTACTILE RECEPTORS FROM BIOPHYSICS TO EVOLUTION

Octopuses and squids have differing predation strategies due to their distinct sensory capabilities, mediated by sensory receptor specializations. This high-impact study from the Hibbs Lab at UC San Diego features two keystone values of science: curiosity and collaboration.

Ashrimp pounding against the wall of its tank, as if it knows what is to come. In the opposite corner: an octopus, its arms probing the surfaces of their enclosure. It meanders, seemingly aimless but for its searching arms. Water, rippling. Suddenly the octopus lunges, as if it has caught the shrimp's scent. You already know what happens next. Except—how did the octopus 'smell' the doomed shrimp? Well, by 'tasting' it.

A biologist looks on, curiosity—that lifeblood of science—sparked.

Wading IN : Cephalopods and Structural Biology

If fish were gamblers, coleoid cephalopods would win the evolutionary jackpot (note that cephalopods are not technically fishes).1

Boasting the largest nervous systems observed in invertebrates, coleoid cephalopods—a phylogenetic subclass that comprises octopus, squid, and cuttlefish—are the ocean's darlings. With sophisticated behaviors arising from their complex nervous systems, cephalopods have long fascinated scientists. However, their evolution remains poorly understood, and particularly murky is the molecular basis for the substantial behavioral diversity across the 750-some species of cephalopods.1,2

Bellono Lab to collaborate on characterizing the newly discovered CRs.

The Hibbs Lab, previously located at the UT Southwestern Medical Center, moved to UC San Diego in 2023. Headed by UC San Diego alumnus Dr. Ryan Hibbs, the lab focuses on the biophysics and pharmacology of nicotinic and GABAA receptors as well as their involvement in certain autoimmune disorders. Using cryogenic electron microscopy (cryo-EM), the lab constructs high-resolution images of ion channels to understand how they work, putting into practice the oft-repeated biological maxim “structure determines function."

“ O CTOPUS, SQUID, AND CUTTLEFISH ARE THE OCEAN'S DARLINGS. ”

One interesting behavioral difference between octopus and squid lineages is found in their feeding strategies. The eight-armed octopus feels along the seafloor to find its food, while the squid conceals itself before springing into action to capture prey with hook-like suckers on its eight arms and two longer tentacles.3 What adaptations underpin this difference?

The answer lies in chemotactile receptors (CRs), a cephalopod-specific family of proteins that the Bellono Lab at Harvard University first reported in 2020.4 These CRs are expressed in sensory cells on the outer lining of cephalopod arms and tentacle suckers (at a greater level in octopi than in squid), and were found to play a role in prey detection.4 CRs share sequence similarities with nicotinic acetylcholine receptors, a crucial ligand-gated ion channel found at the neuromuscular junction.3 Nicotinic receptors are found in many animals, including humans, and activate muscle contraction when they bind to acetylcholine, a neurotransmitter released by neurons. Well-known for their work on the structural biology of nicotinic receptors, the Hibbs Lab at UC San Diego was approached by the

CRs are in the ballpark of 160 angstroms long—just 1/62,500th of a millimeter!5 How exactly does cryo-EM enable researchers to visualize such tiny proteins? A technique for the elucidation of biological macromolecular structures, cryo-EM serves a purpose similar to the more traditional X-ray crystallography. An issue with X-ray crystallography is that membrane proteins like CRs are difficult to crystallize due to solubility issues, since they have both hydrophobic and hydrophilic regions; cryo-EM bypasses this obstacle.6 In this study, solutions of the CRs were flash frozen, suspending randomly oriented CRs in vitreous ice just nanometers thick. Unlike typical ice, vitreous ("glass-like") ice has no crystalline lattice, which prevents it from distorting the CRs' structures. A beam of electrons is then aimed at the sample, passing straight through the ice but not the CRs to produce projection images—like casting shadows. Millions of these two-dimensional projection images in different orientations were averaged and mapped onto a simplified three-dimensional model of what the CR was expected to look like—in this case, a sim plified nicotinic receptor. This method generated images with a resolution of 3.13 angstroms, al lowing the lab to visualize CR structures down to the atomic level.3

The Plunge: Active Site Structures

Using the cryo-EM images, the lab con firmed that octopus and squid CRs, like nicotinic receptors, are ligand-gated cation channels. When a ligand binds to the receptor, its channel opens, and positively charged ions like sodium and potassium move across the mem brane. This leads to depolarization, generating an action potential—the basis of fast electrical signaling. CRs and nicotinic receptors bind ligands at the same site, located in the extracellular do-

“ IN THE CONTEXT OF A SENSORY RECEPTOR LIKE A CR, CHANGING THE MOLECULES THAT CAN BIND TO IT MEANS CHANGING THE STIMULI THAT THE ORGANISM CAN DETECT AND RESPOND TO. ”

main. However, the CR site does not bind to acetylcholine as the nicotinic receptor site does. A few key alterations render CRs insensitive to acetylcholine: one loop of residues (amino acids that are part of a protein chain) is shorter due to missing tyrosine residues and a missing disulfide bond.3,7

Additional biochemical differences between squid and octopus CRs reveal how they sense different molecules, which in turn can explain the contrast in their behaviors. In the context of a sensory receptor like a CR, changing the molecules that can bind to it means changing the stimuli that the organism can detect and respond to.

The family of CRs found in squid are known as chemotactile receptors for bitterants (CRBs), so named because they bind to bitter compounds. CRB1, which

was imaged by the Hibbs Lab, was found to interact with denatonium—a hydrophilic bitterant containing an amide group. The amide group has a partially positively charged nitrogen that is attracted to the negative charge character of two key structural features in CRB1's active site, allowing CRB1 to bind denatonium. First is an aromatic cage, which has several aromatic ring-containing residues. Aromatic rings, of which the archetypal example is benzene, are hydrophobic and thus would typically repel hydrophilic compounds like denatonium. However, aromatic rings are resonance-stabilized; in other words, their electrons are delocalized between the ring's carbons. Since these carbons are “holding onto” their electrons more loosely than usual, the electrons are more available to quench positive charges. The stabilizing effect

of the cage's characteristic delocalized electrons on the positively charged nitrogen in denatonium's amide group contributes to CRB1's binding of denatonium. Secondly, a favorable electrostatic interaction between the positive nitrogen and the negatively charged glutamate-39 (by convention, residues are identified by their numerical position in a protein's amino acid chain) positioned above the aromatic cage further coordinates denatonium within the ligand-binding site (see Figure 1).3

Being hydrophilic, bitterants can diffuse readily in seawater. As a result, squid can detect their prey from a distance in a manner analogous to terrestrial predators’ sense of smell. This sensory capability equips squid for their observed ambush predation strategy.3

Thinking back to the ill-fated shrimp, we recall that octopuses have a different approach to catching prey; this is reflected in their divergent CRs. In octopuses, the CRs bind to terpenes (an extensive class of large, insoluble, hydrophobic molecules naturally secreted by marine plants, animals, and fungi), and as such, they are called chemotactile receptors for terpenes (CRTs). In CRT1, the tyrosine-166 of one protein strand protruded out between two other strands in a sheet, disrupting the hydrogen bond network that held the cage structure together. This produced a flat, hydrophobic pocket at the ligand-binding site instead of the aromatic cage found in nicotinic receptors and CRB1. The 'open' binding site is

Figure 1. Simplified active site structures of squid CRB1 and octopus CRT1. In CRB1, the positively-charged bitterant denatonium is ensnared by a cage of partially negative aromatic rings, and pinned in place by an electrostatic interaction with the overt negative charge on glutamate-39. An 'open' hydrophobic pocket in CRT1 allows the larger terpenoid diosgenin to slot in.

more accessible to large terpenes; since “like attracts like,” hydrophobic terpenes have affinity for the hydrophobicity of the pocket. To demonstrate this, the Hibbs Lab imaged CRT1 with diosgenin, a terpenoid compound, bound to it (see Figure 1).3,7

In direct contrast to the squid’s sense of “smell” for soluble bitterants, octopuses require physical contact to detect the poorly soluble terpenes found on the surface of prey animals and left in their tracks.3,8 “Taste-by-touch” sensation explains why octopuses use their arms to search their environments while hunting.3

The Unexplored Depths

Only recently discovered, CRs still present numerous avenues for future research. One potential direction is to investigate marine natural products. The CRB1 ligand denatonium does not occur naturally in squid's prey—it is a commercially available bitter compound (also used in Nintendo Switches to prevent children from eating the game cards).9 The Hibbs Lab is potentially acquiring marine natural products from the Scripps Institute of Oceanography, which would enable investigations of truer-to-life CR interactions with its ligands.

For another, the Hibbs and Bellono Labs do not know if CRB1 and CRT1 are the predominant CRs found in squid and octopus. Since ion channels are embedded in the cell membrane, scientists have yet to extract them without destroying them in the process. Instead, for both CRB1 and CRT1, the Hibbs Lab chose one gene encoding a CR subunit and expressed it in vitro; homopentameric channels then self-assembled from five identical subunits. These are the only CRs that have been imaged thus far, but octopuses also express heteropentameric CRTs—five-subunit receptors that have two or more different subunits—and there is a possibility of heteropentameric CRBs in squids as well. Based on the researchers' understanding of nicotinic receptors, the most biochemically interesting and complex interactions tend to occur at the interfaces of different subunit types in heteropentamers (see Figure 2). If it turns out that heteropentameric CRs are more important than the thus-far documented CRB1 and CRT1, it would mean that research has only skimmed the surface of the molecular basis for cephalopod sensation.10

The Hibbs Lab's biophysical analysis helps unravel how CRs diverged from the ancestral nicotinic receptor, pivoting from neurotransmitter receptor to sensory receptor; and how sensory specialization affects behavior, which in turn drives octopuses and squids to fill different ecological niches.3 While CRs are one factor among many, it is remarkable that the evolution of these two cephalopod lineages—a grand process with a time frame

Figure 2. Homopentam ers vs. heteropentamers Both CRB1 and CRT1 are homopentamers (left) composed of five identical subunits. Heteropentameric (right) CRs are a promising direction for future research; more subunit types means more complexity, particularly where two different subunits meet.

“ BOTH LABS DESCRIBE THIS WORK AS 'CURIOSITY-DRIVEN' [...] AND THERE IS VALUE IN THAT—BEAUTY, EVEN—IN SCIENCE FOR ITS OWN SAKE: NOT MOTIVATED BY A PROBLEM, BUT RATHER A QUESTION. ”

of hundreds of millions of years—can be traced down to specific amino acid modifications in one receptor family. Thanks to the collaborative nature of this study, researchers from UC San Diego and Harvard were able to synthesize their different areas of expertise into a multilevel understanding of octopus and squid: from the molecular to the behavioral and evolutionary. We might expect that researchers investigated CRs with the goal of improving our understanding of the nicotinic receptor, to gain insight into human health and disease. However, both labs describe this work as "curiosity-driven"—they were enchanted by octopus and squid behavior, and wanted to find out how and why 10,11 And there is value in that—beauty, even— in science for its own sake: not motivated by a problem, but rather a question.

REFERENCES

[1] Hanlon RT, Messenger JB. 2018. Cephalopod behaviour. New York. Cambridge University Press.

[2] Albertin CB, Medina-Ruiz S, Mitros T, Schmidbaur H, Sanchez G, Wang ZY, Grimwood J, Rosenthal JJC, Ragsdale CW, Simakov O, and others. 2022. Genome and transcriptome mechanisms driving cephalopod evolution. Nat Commun. (13)2427. https://doi.org/10.1038/ s41467-022-29748-w

[3] Kang G, Allard CAH, Valencia-Montoya WA, Giesen LV, Kim JJ, Kilian PB, Bai X, Bellono NW, Hibbs RE. 2023. Sensory specializations drive

octopus and squid behavior. Nature. (616):378383. https://doi.org/10.1038/s41586-02305808-z

[4] Giesen LV, Kilian PB, Allard CAH, Bellono NW. 2020. Molecular basis of chemotactile sensation in octopus. Cell. (183)3:594–604.e14. https://doi.org/10.1016/j.cell.2020.09.008

[5] Unwin N. 2013. Nicotinic acetylcholine receptor and the structural basis of neuromuscular transmission: insights from Torpedo postsynaptic membranes. Q Rev Biophys. 46(4):283–322. https://doi.org/10.1017/S0033583513000061

[6] Callaway E. 2020. Revolutionary cryo-EM is taking over structural biology. Nature. [accessed 2023 Nov 27]; https://www.nature.com/articles/d41586-020-00341-9.

[7] Allard CAH, Kang G, Kim JJ, Valencia-Montoya WA, Hibbs RE, Bellono NW. 2023. Structural basis of sensory receptor evolution in octopus. Nature. (616):373–377. https://doi.org/10.1038/ s41586-023-05822-1

[8] Mollo E, Fontana A, Roussis V, Polese G, Amodeo P, Ghiselin MT. 2014. Sensing marine biomolecules: smell, taste, and the evolutionary transition from aquatic to terrestrial life. Front Chem. (2). https://doi.org/10.3389/ fchem.2014.00092

[9] HAL 90210. 2017. Nintendo admits it has made Switch cartridges taste unbearably bitter. The Guardian. [accessed 2023 Nov 27]; https:// www.theguardian.com/technology/2017/ mar/02/nintendo-switch-game-cartridges-taste-bitter.

[10] Li A, Hibbs RE. 2023. Interview for Saltman Quarterly.

[11] Research. Bellono Lab. 2023 [accessed 2024 Jan 8]. https://www.bellonolab.com/

BOUNCING BACK

written

Emma Svendsen

by

illustrated by Kristiana Wong

SYNAPTIC RESILIENCE AS A POTENTIAL

THERAPEUTIC AVENUE FOR ALZHEIMER’S

Introduction

When one is sick, the comforting mentality tends to be: “When I get better…” The focus shifts to improvement—the cough will abate, the fever will break, and eventually, the illness will become a thing of the past. But what if there was no hope of this relief? For those with conditions like Alzheimer’s disease, or AD, there isn’t. AD is a neurodegenerative disease that leads to loss of cognitive function, including loss of memory and reasoning, behavioral changes, and eventually, loss of brain volume. AD affects more than 6 million individuals in the United States and is the leading cause of dementia. For those with AD, current treatments cannot offer hope of improvement—any cognition lost will remain lost.1 The best a patient or their family can hope for is a slower rate of cognitive decline: abilities will remain slightly longer and their loved one will lose cognition at a slower rate. But what about AD makes it so difficult to restore cognition?

AD is characterized by an accumulation of beta-amyloid protein plaques and neurofibrillary tau tangles in the brain.2 Beta-amyloid plaques and tau tangles are dense aggregates of malformed proteins. The build-up of these insoluble protein aggregates has devastating effects on neurons. Neurons are the cells in the brain responsible for sending electrical and chemical signals that carry information that is essential for cognitive processes like memory. In addition to forming these insoluble plaques, beta-amyloid proteins also take a soluble form in the brain. These soluble proteins become toxic when they aggregate in excess, as in AD brains. Beta-amyloid protein aggregation occurs at synapses, or the gaps between neurons where inter-neuron signaling takes place. This aggregation then disrupts neuron signaling. The combination of neuron death and synaptic damage leads to the loss of cognitive function, eventual loss of brain volume, and death, characteristic of AD. However, it is worth noting that not all instances of beta-amyloid presence are known to cause Alzheimer’s, and that beta-amyloid accumulation has been found in brains that did not demonstrate a cognitive deficit.3

One common therapeutic target in the treatment of Alzheimer’s is the accumulation of these beta-amyloid plaques. However, this approach can only slow the progression of AD, as plaque removal does not restore cognition or synaptic health. At UC San Diego, the Dore Lab has an approach that can address the most devastating aspect of Alzheimer’s disease: the seemingly unstoppable loss of cognitive function.

“ FOR THOSE WITH AD, CURRENT TREATMENTS CANNOT OFFER HOPE OF IMPROVEMENT—ANY COGNITION LOST WILL REMAIN LOST. ”

A Promising Start: The

Dore Lab and PSD-95

Rather than focusing on beta-amyloid plaques, Dr. Kim Dore and her lab focus on the synapse. More specifically, the proteins around the synapse, like scaffolding proteins (proteins responsible for maintaining molecular spatial arrangements). The Dore Lab specifically investigates a scaffolding protein known as postsynaptic density protein 95 (PSD-95), which is a key element responsible for synaptic transmission. By altering PSD-95 expression in AD model mice, the Dore Lab demonstrated that levels of PSD-95 are directly correlated with synapse health and cognitive ability and even have the potential to undo the damage caused by AD: a previously unattainable feat. Levels of PSD-95 were measured through cognitive tests in mice and through post-mortem electrophysiological imaging of brain tissue samples. These results suggest that increasing levels of PSD-95 may provide a novel path towards increased cognition in patients.

Scaffolding Proteins and PSD-95

Scaffolding proteins like PSD-95 are a family of proteins responsible for coordinating the activity of signaling molecules. These proteins bind to these molecules and associate them with their intended receptor, enabling the specificity of a signaling pathway.4 PSD95 is a scaffolding protein that acts at the N-methyl D-aspartate (NMDA) receptor. NMDA is a key receptor that helps mitigate the impact of beta-amyloid on the synapse by blocking the impact of beta-amyloid on the spatial arrangement of the NMDA receptor. It is the conformational change induced by beta-amyloid on NMDA that correlates with loss of synapse function.5 The essential neurotransmitter glutamate acts at the NMDA receptor, and exposes the neuron to calcium in the process.6 In brains affected by AD, excess glutamate exposes the brain to damagingly-high levels of calcium, causing cognitive decline (Figure 1).

Maintaining receptor and synaptic health for as long as possible can slow decline. However, the presence of PSD-95 alone is not sufficient to maintain synaptic health: only palmitoylated PSD-95 remains at synapses. Palmitoylation is the process of adding fatty acids, like palmitic acid, to proteins. Not all forms of PSD-95 interact with the NMDA receptor in a way that promotes synaptic health, so it is essential to evaluate levels of expressed PSD-95 as well as levels of palmitoylation (Figure 2).

PSD-95 and the AD

Brain

To evaluate the effect of PSD-95 on AD brains, the Dore Lab induced the expression of PSD-95 in advanced AD-model mice, which are animals that have developed AD over a period of months and exhibit a marked decrease in cognitive function. To monitor and compare changes in cognitive function, baseline cognitive ability was established through performance in a memory test. A platform was submerged beneath the surface of a large pool of opaque liquid. AD mice were then placed in the pool and monitored as they attempted to find the platform. This test was

Figure 1: PSD-95 and the N-methyl D-aspartate (NMDA) Receptor. Glutamate (pink) interacts with this receptor (teal), which is responsible for regulating calcium (white).

PSD-95 (chain of blocks and spheres) can regulate NMDA receptor activity even in the presence of glutamate to maintain appropriate calcium levels—and synaptic health by extension.

then repeated with some of the mice acting as a control group (no change in PSD-95 expression) and the rest behaving as a treatment group (genetically-increased expression of PSD-95). Mice with increased PSD-95 levels expressed a greater ability to remember and successfully locate the platform compared to control mice.7 In human patients, cognition is tested via multi-part recall tests and task assessments, such as a patient's ability to draw a clock.8

These cognitive tests are not the only way to evaluate the impact of PSD-95 on an AD brain.

Post-mortem imaging of brain tissue samples from PSD-95-increased mice showed increased dendritic spine health compared to control mice.7

Dendritic spines are extensions of neurons that act as markers for synaptic health: increased amounts of spines correlate to more functional synapses.9

The larger presence of dendritic spines in mice with increased PSD-95 indicate its restorative effect on the AD phenotype, and validate the improved cognitive ability of PSD-95 treated mice.

Factors Affecting PSD-95 Activity

Several factors beyond expression impact PSD-95 activity at the synapse. For example, the upregula-

tion of protein kinase C alpha (PKCα) reduces the functionality of PSD-95.10 While the exact mechanism behind this interaction is not fully understood, the overall result is an increase in synaptic depression (lowered levels of neurotransmitter release) and more cognitive decline.11 However, there are mutations known to lead to PKCα upregulation, but they are not therapeutically viable targets because genetic editing is not currently a clinically viable solution. While these kinds of genetic modifications may be a possibility in the future, The Dore Lab has found an approach that is accessible today: addressing the process of palmitoylation, or protecting levels of functional PSD-95.

Just as there are known classes of enzymes responsible for palmitoylation, the inverse is also true. Depalmitoylase enzymes are responsible for removing the fatty-acid chains essential for interaction between PSD-95 and NMDA receptors. The Dore Lab targets these enzymes, seeking to degrade them and thus increase levels of active, palmitoylated PSD-95. They hope that these enzymes provide a clinically viable, druggable target that could lead to increased cognition in AD models and eventually offer reprieve to AD patients and their families.

Membrane

Calcium shuttles the NMDAR receptor

Increased binding of glutamate leads to increased Ca2+ flux

Moving Forwards Thinking Clinically

As the relationship between PSD-95 and cognition becomes increasingly clear, the focus now turns to maximizing its application. This research not only provides a path to create potential therapeutics for patients suffering from AD, but may also provide insight into the biological mechanisms behind the range of impacts AD has on different patients. Dr. Dore and her team discovered that only female mice were impacted by increased levels of PSD95. The reason for this difference remains unexplained; however, this discrepancy aligns with another observed disparity AD. Women are more likely to get Alzheimer’s disease than men, making PSD-95 an incredibly effective treatment for a more at-risk population.12 Reasons behind this disparity are currently being explored. Additionally, levels of PSD-95 in brain tissue could serve as a biomarker for Alzheimer’s and provide insight as to why some parts of the brain react differently to amyloid plaques. The presence, or lack thereof, of PSD-95 may also clarify why some individuals have AD but maintain their cognitive function. Dr. Dore intends to investigate this further through post-mortem analysis of human AD brains, where levels of PSD-95 and dendritic spine health can be compared against the reported disease phenotype and severity to investigate why some advanced AD patients maintain cognition where others do not. This investigation into post-mortem brain tissue will be beneficial in addressing the limitations in the translatability of the mouse model into human physiology. In the meantime, exploration on the effect of degradation of depalmitoylation enzymes provides one future path to address these discrepan-

cies and offer some relief to impacted patients and families.

Final Takeaways

For too long, AD treatment and research approaches have focused only on slowing disease progression with little consideration of restoring ability. The work of the Dore Lab with PSD-95 provides an approach that restored cognitive function to AD-model mice within five days. This novel approach of addressing the mechanism behind the most devastating symptom of Alzheimer’s (cognitive decline), rather than its fundamental cause (plaques), may be the answer to improve quality of life in those living with this devastating disease. While genetic editing to alter human PSD-95 levels is not currently feasible, depalmitoylation enzymes might be a clinically viable target. For patients who have never before had the comfort of recovery, the work done by the Dore Lab provides hope that those suffering from Alzheimer’s can indeed bounce back.

References

[1] How Is Alzheimer’s Disease Treated? National Institute on Aging. [accessed 2024 Jan 8]. https://www.nia.nih.gov/health/alzheimers-treatment/how-alzheimers-disease-treated#:~:text=Cholinesterase%20inhibitors%20 prevent%20the%20breakdown.

[2] National Institute on Aging. 2021 Jul 8. What Is Alzheimer’s Disease? National Institute on Aging. https://www.nia.nih.gov/health/alzheimers-and-dementia/what-alzheimers-disease. [accessed 2024 Jan. 23]

[3] Baker-Nigh A, Vahedi S, Davis EG, Weintraub S, Bigio EH, Klein WL, Geula C. 2015. Neuronal amyloid-β accumulation within cholinergic basal forebrain in ageing and Alzheimer’s disease. Brain. 138(6):1722–1737. doi:https:// doi.org/10.1093/. [accessed 2024 Feb 18]. https://academic.oup.com/brain/article/138/6/1722/2847057.

[4] Hu L, Zou F, Grandis JR, Johnson DE. 2019 Jan 1. Chapter 4 - The JNK Pathway in Drug Resistance. Johnson DE, editor. ScienceDirect. 3:87–100. [accessed 2024 Jan 8]. https://doi. org/10.1016/B978-0-12-813753-6.00004-4.

[5] Doré K, Carrico Z, Alfonso S, Marino M, Koymans K, Kessels HW, Malinow R. 2021. PSD95 protects synapses from β-amyloid. Cell Rep. 35(9):109194. doi:https://doi.org/10.1016/j. celrep.2021.109194.

[6] Jewett BE, Thapa B. 2023. Physiology, NMDA Receptor. PubMed. https://www.ncbi.nlm.nih. gov/books/NBK519495/

[7] Svendsen E, Doré K, 2023. Interview for Saltman Quarterly

[8] Cognitive Screening and Assessment. 2024. Alzheimer’s Disease and Dementia. [accessed 2024 Jan 8]. https://www.alz.org/professionals/ health-systems-medical-professionals/cognitive-assessment#:~:text=Patient%20assessment%20tools&text=The%20Mini%2DCog%20 is%20a.

[9] Hering H, Sheng M. 2001. Dendritic spines: structure, dynamics and regulation. Nat Rev Neurosci. 2(12):880–888. https://doi. org/10.1038/35104061. https://www.nature. com/articles/35104061.

[10] Lordén G, Wozniak JM, Doré K, Dozier LE, Cates-Gatto C, Patrick GN, Gonzalez DJ, Roberts AJ, Tanzi RE, Newton AC. 2022. Enhanced activity of Alzheimer disease-associated variant of protein kinase Cα drives cognitive decline in a mouse model. Nature Commun. 13(1). https:// doi.org/10.1038/s41467-022-34679-7.

[11] Salmasi M, Loebel A, Glasauer S, Stemmler M. 2019. Short-term synaptic depression can increase the rate of information transfer at a release site. Hennig MH, editor. PLoS Comput Biol. 15(1):e1006666. https://doi.org/10.1371/ journal.pcbi.1006666.

[12] Beam, Christopher R et al. “Differences Between Women and Men in Incidence Rates of Dementia and Alzheimer's Disease.” J Alzheimer’s Dis. vol. 64,4 (2018): 1077-1083. doi:10.3233/JAD-180141

Figure 2: The Effect of Depalmitoylase Enzymes on PSD-95. Depalmitoylases (red) are a class of enzymes that remove fatty acids from proteins like PSD95 (orange and yellow). Only PSD-95 with an intact fatty acid tail is able to interact with NMDA, so PSD-95 must be available and palmitoylated to protect synaptic health.

Translocase enzyme degradation allows fatty acid tail to bind.

Translocase enzyme removes fatty acid tail, inhibiting binding.

MALARIA MOVES BACK WEST

written by: Kaz Nuckowski

Climate Change and Colonization in Biotechnologies for “Tropical” Disease

Transmitted by female biting anopheles mosquitoes carrying the plasmodium parasite,1 malaria thrives in the heat. It kills over 600,000 people annually, and more than half of all malaria deaths occur in just four sub-Saharan African countries.2 With climate models predicting a lengthening of the malaria season3 and insecticide resistance becoming more widespread,4 new solutions are required. Preventing malaria in Africa remains challenging because of sparse public health infrastructure. Biologists at UC San Diego have joined the research trend reimagining malaria prevention using novel genetic biotechnology.

Gene drives make use of CRISPR to create “selfish genes” which conserve themselves in offspring.5 One gene drive model, the “suppression drive,” forces deleterious traits into a population—akin to genetic insecticide.5 Dr. Andrea Smidler’s team followed a suppression approach to create ifegenia: inherited female elimination by genetically encoded nuclei to interrupt alleles. Named for the sacrificed daughter Iphigenia of Greek mythos, this technology knocks out the femaleless gene—necessary for female mosquito development—to cause female death.6

Importantly and interestingly, ifegenia is not a gene drive. In contrast to gene drives’ 100% heritability, ifegenia follows classical Mendelian inheritance patterns (Figure 1).7 This renders ifegenia resource-intensive, as multiple releases are required for suppression.7 However, this weakness can also be a strength. “You can’t control where a mosquito goes, so you can’t control where a gene drive goes,” Dr. Smidler notes. This makes gene drives “unconfinable,” in contrast with ifegenia. 7 “It doesn’t cross borders, which is a big point of contention among African nations.”7 When considering antimalarial biotechnologies, we must weigh the politics of their implementation.

Disease control programs that don’t involve local stakeholders are usually ineffective. The failure of the Global Malaria Eradication Program (GMEP) serves as one example. The program was created in 1955 by the WHO, spurned by the elimination of malaria from the US a few years prior.8 The GMEP’s aggressive implementation of only insecticide spraying contributed to its discontinuation just 14 years later. The sub-Saharan African countries home to the majority of malaria cases did not have adequate public health infrastructures to support spraying. Perhaps more problematically, they were not consulted on the program’s feasibility.8 In the context of disease control, it’s important

to consider colonial histories of paternalism and othering in tropical medicine.

Malaria is typically described as a “tropical” disease. But malaria was present in the US until the early 1950s.8 Tropicality embroils discussions of malaria in environmental determinism, where non-temperate climates and their inhabitants are painted as inherently unhealthy or extreme harbors of disease.8 As a field, tropical medicine was developed as a colonial tool to create dependence in African and Asian subjects of empire.8 Accordingly, it is not climate which makes the global South vulnerable to malaria. Robust public health systems enabled American elimination in the 1950s — and colonialism impeded the development of infrastructures to do the same in sub-Saharan Africa.9

Popular discussions of gene drive technology can reinforce the concept of “tropical” disease. NPR articles on malaria10 and antimalarial gene drive technology,11 for instance, discuss the wide-ranging impacts of malaria in Africa and Asia in the same stroke as a few US cases. Such parallelism risks implying these problems are of equal severity. The potential that climate change renders malaria once again locally transmitted in the global North is important. But malaria in the global South deserves attention as a health crisis in its own right. As such, Smidler et al. only mention the deadliness of malaria in sub-Saharan Africa as the significance of their research.6 “As we discuss these technologies, we really need to remind ourselves we are not the ones benefitting,” said Dr. Smidler.7

Involving community partners helps ensure that malaria prevention interventions garner community support. Gaining informed consent is complex, however, when colonial impacts restrict the development of local scientific expertise. One effort to increase research capacity is the bilateral exchange program between UC San Diego (UCSD) and Universidade Eduardo Mondlane (UEM) in Mozambique,12 one of the countries where malaria deaths are highest.2 Given that there is no “silver bullet” for malaria,7 African scientists with insight into their own communities can help develop technologies with diverse local contexts in mind.

Smidler et al.’s research on mosquito biotechnology holds promise for malaria prevention, but we must reframe our discussions of such solutions. Focusing on malaria as an ongoing issue in Africa, poised to worsen with climate change, requires that we unpack malaria’s status as a looming “tropical” threat to the global North. Ultimately, Dr. Smidler’s research aims to protect African health, and implementation of such a technology requires cross-cultural collaboration. UCSD facilitation of research capacity at UEM will help foster new generations of biologists to determine their communities’ futures.

References

[1] Talapko J, Škrlec I, Alebić T, Jukić M, Včev A. 2019. Malaria: The past and the present. Microorganisms. 7(6).

[2] WHO. 2023 Dec 4. Malaria. World Health Organization. [accessed 2024 Feb 5]. https://www.who.int/ news-room/fact-sheets/detail/malaria.

[3] Colón-González FJ, Sewe MO, Tompkins AM, Sjödin H, Casallas A, Rocklöv J, Caminade C, Lowe R. 2021. Projecting the risk of mosquito-borne diseases in a warmer and more populated world: A multi-model, multi-scenario intercomparison modelling study. The Lancet Planetary Health.

[4] Ranson H, Lissenden N. 2016. Insecticide resistance in african anopheles mosquitoes: A worsening situation that needs urgent action to maintain malaria control. Trends in Parasitology. 32(3):187-196.

[5] Bier E. 2022. Gene drives gaining speed. Nature Reviews Genetics. 23(1):5-22.5. Dye-Braumuller KC, Kanyangarara M. 2021. Malaria in the USA: How vulnerable are we to future outbreaks? Curr Trop Med Rep. 8(1):43-51.5

[6] Smidler AL, Pai JJ, Apte RA, Sánchez C HM, Corder RM, Jeffrey Gutiérrez E, Thakre N, Antoshechkin I, Marshall JM, Akbari OS. A confinable female-lethal population suppression system in the malaria vector, anopheles gambiae. Science Advances. 9(27):eade8903.

[7] Nuckowski KR, Smidler AL. 2024. Interview for Saltman Quarterly.

[8] Nájera JA, González-Silva M, Alonso PL. 2011. Some lessons for the future from the global malaria eradication programme (1955-1969). PLoS Med. 8(1):e1000412.

[9] Shahvisi A. 2019. Tropicality and abjection: What do we really mean by “neglected tropical diseases”? Developing World Bioethics. 19(4):224-234.

[10] Huang P. 2023 Dec 15. The U.S. is unprepared for the growing threat of mosquito- and tick-borne viruses. NPR. [accessed 2024 Feb 5]. https://www.npr.org/sections/health-shots/2023/12/15/1219478835/arboviruses-mosquito-tick-borne-viruses-tropical-disease.

[11] Allen G. 2024 Jan 26. New gene-editing tools may help wipe out mosquito-borne diseases. NPR. [accessed 2024 Feb 5]. https://www.npr. org/2024/01/26/1226110915/gene-editing-bioengineering-mosquito-disease-dengue-malaria-oxitec.

[12] UC San Diego School of Medicine. Mozambique. Division of Infectious Diseases & Global Public Health. [accessed 2024 Feb 5]. https://medschool.ucsd.edu/ som/medicine/divisions/idgph/research/international/ Pages/Mozambique.aspx

Figure 1 illustrated by: Julia Dingman

RESEARCH

Biology students at UC San Diego often choose to enrich their educational experience by joining labs and conducting their own research. This section showcases original research manuscripts and review papers produced and written entirely by undergraduate students.

photo by: Tommy Murphy Chicken (Gallus gallus domesticus) wandering the streets of the Arroyo Grande Village with its flock.

COMPARISON OF BIRD COMMUNITIES IN FORESTED AND URBAN HABITATS ON PULAU TIOMAN, MALAYSIA

Sally Guan

UC San Diego in collaboration with the National University of Singapore, Faculty of Science, Dr. Chan Kin Onn, Movin Nyanasengeran

ABSTRACT

Lying just east of the Malaysian peninsula, Pulau Tioman is known for its incredible biodiversity that attracts many tourists and researchers to the island. With various topographies ranging from the tropical rainforest to the rocky and sandy beaches, Tioman Island is home to many wildlife that is governed by the state.18 Despite state governess, increasing human disturbances are causing the island to experience great changes. While Pulau Tioman relies heavily on tourism for its economic growth, the increasing influx of tourists is exerting significant pressure on its ecosystem.17 For instance, the natural coastal and coral ecosystem is heavily disturbed by tourist activities and fisheries, making conservation efforts of corals notably important.18 In a recent development, the Berjaya Group, a company deeply invested in tourism expansion, has proposed the construction of an international airport on the island.4 Local residents and non-governmental organizations alike have raised concerns about the proposed land development, asserting that the project poses a significant threat to Pulau Tioman’s coral reefs, seagrass beds, and other natural treasures.4 Given the existing disturbances, the addition of an international airport has the potential to further exacerbate the current conditions that are impacting the island’s various ecosystems. With the lack of research on bird communities in Tioman Island, we aim to establish the impact of ongoing anthropogenic activities on the island’s avian population. A comparison study was conducted to investigate the bird communities in forested and urban habitats in Pulau Tioman, Malaysia. We hypothesized that there would be a difference between species diversity in forested and urban habitats on the island. To investigate, we went on various transects in the forested and urban habitats and observed the abundance of birds. A data analysis was conducted to examine the differences in species diversity and richness in the two habitats, as well as to compare the functional traits of the bird species. The results of the species diversity and richness indicated that there was no significant difference. However, our functional traits analysis showed notable differences in bird species composition. These findings emphasize the importance of considering functional trait differences in the formulation of conservation strategies and for guiding future studies on Tioman Island.

INTRODUCTION

Located in Southeast Asia, the island of Pulau Tioman lies just east of coastal Malaysia (Figure 1).10 While the climate on the island tends to be consistently warm and humid10, the overall landscape is divided into four distinct categories: coastal vegetation, mangrove, lowland forest, and hill dipterocarp forest.7 The various landscapes and biodiversity on Tioman may be vulnerable to human impact.7 In an effort to protect the marine fauna and floras from fishing and other anthropogenic activities, the waters surrounding Tioman are protected by the government.18 In addition, numerous bird species inhabit the island; however, humans have exploited bird nests in caves for commercial gain.18 Previous studies have highlighted the amazing biodiversity of Tioman and further urged researchers to conduct studies in efforts to preserve its natural habitats.10 This is extremely important as anthropogenic disturbances continue to impact the island’s various ecosystems. The proposed introduction of an airport on the island is anticipated to escalate air pollutant levels, result in biodiversity and landscape losses, and cause significant disturbances to habitats and organisms on a large scale.4 In an article that discusses the air pollutants that birds suffer from, birds tend to be more vulnerable to airborne particles than humans.23 In addition, on Tioman Island, there are an abundance of birds that serve as seed dispersers, pollinators, scavengers, and contributors to nutrient cycling.5,13 These species play an integral role in the ecosystem, serving as bioindicators that assess the overall health of the environment.11 Birds are often used as bioindicators due to their well-understood ecology, strong connections within their ecological niche, adaptability to various environmental conditions, and easy detectability.11 With continuous anthropogenic disturbances, studies

have shown that there is an ongoing trend in which urban habitats tend to have lower bird species compared to forested habitats.9 The comparison revealed that the lower bird species may be due to the different vegetation that urban habitats harbor relative to forested habitats.9 Previous studies have suggested that vegetation heterogeneity and overall landscape can have the potential to have a direct impact on species richness and diversity.8 Thus, past studies have shown the Shannon-Wiener index for bird species to be higher in forested habitats as opposed to urban environments.8 These considerations prompted us to pose the question: Is there a difference in bird communities between forested and urban habitats in Pulau Tioman, Malaysia? To measure this distinction, we examined the abundance of birds in each habitat, assessed their classification as generalists or specialists, and studied the diverse feeding guilds of the birds. We predict that there would be a difference between forested and urban habitats and bird community assemblages. We expect to see a notable contrast in species diversity between forested and urban habitats, with a prevalence of generalist species over specialists in urban habitats. Urban habitats tend to have a wider range of environmental conditions, which are key features to allow generalists species to thrive in. A null hypothesis testing would therefore signify an absence of observable, quantifiable differences between the two habitats and bird community assemblages. These findings could offer valuable insights into how urbanization has affected the species richness and diversity of birds in Pulau Tioman. As the prospect of an international airport looms over the island, our study aims to furnish evidence suggesting a potentially heightened impact on the loss of diversity and species richness.

MATERIALS AND METHODS

Study area and sampling

Birds were surveyed on the island across the span of four days from July 7th, 2023, to July 10th, 2023 between the hours of 7:00 AM to 11:00 AM. In consideration of the peak in bird activity during the early morning hours, we deliberately selected this time frame for

Fig 1. Location of Pulau Tioman island relative to Malaysia. The areas that are shaded in yellow indicate where the settlements are, or areas that are open for urbanization.17 And the red line that runs horizontally across the island shows the area in which there is a single inland road.17

our surveys, aiming to enhance the likelihood of encountering an optimized range of bird species. We split our team into two groups of four observers. In each habitat, we had a transect of 500 meters, where we stopped at every 100 meters to observe (Figure 2). We utilized the "Runkeeper" app in order to measure the length of our transect.16 Upon taking our first step, we clicked the start button on the app to measure the distance traveled. Our study is an observational study that considered both visible birds and bird calls. To observe the birds, we used binoculars at 10x magnification. Once a bird was seen, we tried to describe the birds based on bodily features, upon which The Birds Southeast Asia was used as reference for clear identification.12

Data Analysis

number of species.19 We also used a t-test to compare the ShannonWiener Index between forested and urban habitats. A t-test was performed using R studio a space that uses R programming language to perform data analyses.15 We also categorized the birds based on their functional traits, which entail the morphological, physiological, and phenological traits indirectly impacting the fitness of the species.20 Our functional trait analysis considered whether the birds are generalists or specialists, as well as their feeding guilds. By comparing whether the birds are generalists or specialists, we can look at how well-suited the birds are to a specific environment.21 The identification of feeding guilds also provides insight into the dietary preferences and foraging habits of the observed birds. We then calculated and compared the percentage of functional traits to look at how bird composition varies between the two habitats. In addition to looking at the percentage breakdown, a Pearson’s Chi-squared test was used to test the comparison of functional traits—generalists and specialists.

RESULTS

Most abundant bird species and species richness and evenness

During our four-day observational period, we observed a total of 22 forested bird species and 22 urban bird species. The top three bird species in the forested habitat included the hill myna, green imperial pigeon, and pacific swallow. The top three bird species in the urban habitat included the hill myna, house crow, and pacific swallow, with the hill myna and pacific swallow identified as the overlapping top three birds in each habitat.

Shannon-Wiener Index and p-value

Upon calculating the Shannon-Wiener Index (Table 1), we compared the 5 points on a box plot to visualize the results (Figure 3). The

the locations of

and urban habitats. The forested habitat

green. The urban habitat transects are colored in black.

are

We used the Shannon-Wiener Index to calculate the diversity index for both species richness and species evenness. A Shannon-Wiener Index is often used as a diversity index, under the assumption that all species are represented in the sample.19 While acknowledging the Shannon-Wiener Index’s sensitivity to both sample size and the randomness of sampling procedures, we leveraged these factors to derive a value closely approximating the community’s diversity index. For each point along our transec, we calculated the Shannon-Wiener Index using the following formula: In a Shannon-Wiener Index equation, p is the proportion of individuals found in a particular species, divided by the total number of individuals found, while s accounts for the total

mean Shannon-Wiener Index for the forested habitat is 1.31, while the mean Shannon-Wiener Index for the urban habitat is 1.55. Subsequently, a 95% confidence interval of [-1.19, 0.71] was conducted. Based on the Shannon-Wiener Index, there is a higher species richness and evenness in the urban habitat than the forested habitat. To assess the statistical significance between the two habitats, a t-test was performed, yielding a p-value of 0.5787. The results of the statistical analysis therefore show that there is no significant distinction between the forested and urban habitats. Consequently, we fail to reject the null hypothesis.

Functional traits findings

When examining generalists and specialists in forested and urban habitats, we found forested habitats tend to host a higher percentage of specialists at 72.7% in comparison to generalists at 27.3% (Figure 4). Contrastingly, urban habitats contain a lower percentage of specialists at 18.2% and a higher percentage of generalists at 81.8% (Figure 4). After performing a Pearson’s Chi-squared test, the result with a Chi-squared value of 2.00 and critical value of 0.157. The Chi-

Fig 2. Annotated map with

our transects in the forested

transects

colored in

Table 1 Shannon-Wiener index forest and urban transects. A table that contains the Shannon-Wiener index for each of the points on our transect.

squared value is greater than the critical value that was extrapolated, indicating that there is insignificant variation between generalists and specialists in the urban habitat and forested habitat. And thus, rejecting the null hypothesis. Examining another functional trait, feeding guilds revealed a diverse bird community (Figure 5). In forested habitats, insectivores are more common–constituting 51.9% of the observed bird species (Figure 5). Conversely, omnivores were exclusive to urban habitats, constituting 11.5% of observed birds (Figure 5). We further noticed that carnivores are more common in urban habitats, comprising 15.4% of the birds observed, compared to only 3.7% in forested habitats (Figure 5). Monitoring these functional traits is highly efficient in understanding the overall species composition, diversity, and habitat composition.14

DISCUSSION

Our findings contradicting past studies

Our findings did not reveal significant differences in the Shannon-Wiener Index between forested and urban habitats, thereby contradicting previous studies.8,9 Such discrepancy may be attributed to the forest being heavily disturbed by human activity. Throughout our survey days, we noticed the abundance of all-terrain vehicles [ATVs] entering the forest in groups of five or more typically after 9:00 AM, coinciding with our survey times and the majority of our forest point count locations. In addition, our transect is located along the Mother Willow Trail, which is a popular hiking route. The abundance of hikers contributed to increased forest disturbance levels.2 Hikers may elevate noise pollution levels,6 widen trails,11 and potentially lead to fauna mortality.6 Our data shows that anthropogenic disturbances, particularly from forest disturbances, may contribute to a decrease in species diversity in forested habitats. This likely accounts for the statistically insignificant difference between species diversity in forested and urban habitats.

Limitations

We recognize that our sampling constraints may have led to an underestimate of forest bird species diversity. In thinking about bird detectability, we recognize that in our forest transects, there is generally dense vegetation surrounding the trail. The forest is almost continuously covered by a canopy that impacts our visibility in observing birds. In the urban transects, there is sparse vegetation, making it relatively easy for visual detection over a greater distance. Unlike the forest, the absence of a canopy cover in the urban transects enabled us to easily spot flyovers. The variation in habitat composition may have led to sampling errors, influencing the obtained results. Prior studies have shown that urban habitats have more generalist species and fewer specialist species.14 Our data supports past studies and showed further evidence that urban transects harbored more generalist species. An example of this would be the Pacific Swallow, ranking among the top 3 species found in urban transects. Generalists tend to occupy a rather wider range of ecological niches.21 They are more adaptable to habitats and have a broader tolerance. Specialists, on the other hand, occupy very specific niches to thrive,21 rendering them less adaptable with lower urban tolerance. We also examined the feeding guilds. Past studies have suggested that the number of insectivores would decline with

Fig 3. The box plot to visualize our results – the Shannon-Wiener Index.

photograph by Christabel Yow Qian Yang

trail near Paya Resort exhibited more pronounced disturbances compared to the forest in Tektek. Recording all the birds in sight proved challenging, with a potential bias toward bird species that are detectable by their colors, size, and calls. This could lead to bias favoring more noticeable birds, risking doublecounting. In a study that is purely observational relying on sight and hearing, efforts were made to count within our designated points, yet we acknowledge the possibility of double-counting, especially when birds were in flight. For future studies, we recommend sampling across multiple days for a broader picture and more statistically significant. In addition, we suggest conducting forest transects off trails to minimize disturbances from crowds and potentially reach areas with less canopy cover. Lastly, we suggest using sound level meters to quantify any noise pollution to strengthen future studies.

CONCLUSION

To reiterate some key findings, our study suggested that there is no significant difference in species diversity between forest and urban habitats. We have failed to reject the null hypothesis. Our functional trait analysis has shown some interesting and notable trends that could be taken into consideration in future research studies. Our data have shown that there are more generalists and fewer specialists in urban habitats. On the contrary, there are more specialists than generalists in forested habitats. With the continued urbanization of Tioman Island and the impending development of the Tioman Island airport, the likelihood of continuous increases in noise pollution and human disturbance is apparent. As such, we suggest that future research studies and conservation efforts should be primarily focused on specialists, given their reduced abundance in urban settings. Since specialists are less adaptable to environmental changes, we urge future research studies and conservation biologists to formulate plans to address this concern prior to the installation of the Tioman airport—as an airport is expected to draw major environmental changes. It is also equally important for future studies to consider having studies that could possibly work alongside with policymakers to have greater intervention. Prior studies on Tioman Island have heavily focused on the abundance of biodiversity on the island, and while that is great, it does not pose any concern on the greater issue of environmental concern and habitat loss from anthropogenic impacts. We urge future studies to take greater incentive in trying to conserve the biodiversity of Tioman Island—even with what is left.

urbanization while predatory birds and scavengers would increase.3 Our data further supports this assertion as we saw fewer insectivores in urban areas. This decline could be attributed to elevated air pollution levels as caused by vehicles or the burning of waste, directly affecting the invertebrate populations. Additionally, our observations indicated a rise in carnivores and omnivores in urban habitats compared to forested habitats. This could be due to the increased foraging opportunities in urban environments. Our study is not a perfect induction on the difference in bird communities in forested and urban habitats on Pulau Tioman Island. Rather, we recognize our limitations and encourage future studies to consider our imposed considerations. The forest in Tektek posed challenges for data collection, with an obstructed path riddled with rocks and roots, which made it quite difficult to traverse. Consequently, we adapted our approach and most of our data from the forest closer to Paya Resort. However, this proximity to the resort attracts a higher number of tourists, leading to increased disturbances. Whether it is hiking along the trail that coincides with our transect or the ATV presence that may have impacted the birds we observed, the hiking

ACKNOWLEDGEMENTS

I would like to thank the National University of Singapore for their support in conducting research in Pulau Tioman. I would also like to thank Dr. Chan Kin Onn and Movin Nyanasengeran for their guidance and expertise in biodiversity research, field work, and overall wisdom and knowledge of the world. They helped me a lot with the study and supported me throughout the process of publication. Lastly, thank you to my team members: Valerie Cheung, Jia Le Ng, Manroop Kaur Sandhu, Qian Ying Christabel Yaw, and Iris Yee for their incredible hard work during our trip to Pulau Tioman, and allowing me to publish our research findings. In particular, thank you Christabel for her pictures that were used throughout the paper.

Fig 4. The bar graphs to visualize the differences in abundance of generalists and specialists in forested and urban habitats.

Fig 5. The bar graphs to visualize the proportions of feedings guilds in the forested and urban habitats.

REFERENCES

[1] Bonier F, Martin PR, Wingfield JC. Urban birds have broader environmental tolerance. Biol Lett [Internet]. 2007;3(6):670–3. Available from: http://dx.doi.org/10.1098/rsbl.2007.0349

[2] Bötsch Y, Tablado Z, Scherl D, Kéry M, Graf RF, Jenni L. Effect of recreational trails on forest birds: Human presence matters. Front Ecol Evol [Internet]. 2018;6. Available from: http://dx.doi. org/10.3389/fevo.2018.00175

[3] Chamberlain D, Kibuule M, Skeen RQ, Pomeroy D. Urban bird trends in a rapidly growing tropical city. Ostrich [Internet]. 2018;89(3):275–80. Available from: http://dx.doi.org/10.2989/003065 25.2018.1489908

[4] Ejolt. New Airport on Tioman Island, Malaysia: Ejatlas [Internet]. 2021. Available from: https:// ejatlas.org/conflict/new-airport-on-tioman-island

[5] Gaston KJ. Birds and ecosystem services. Current Biology [Internet]. 2022;32(20). Available from: http://dx.doi.org/10.1016/j. cub.2022.07.053

[6] Karasin L. All terrain vehicles in the Adirondacks - issues and options [Internet]. 2010. Available from: https://library.wcs.org/doi/ctl/ view/mid/33065/pubid/DMX548400000.aspx

by Christabel Yow Qian Yang

[7] Latiff A, Faridah Hanum I, Zainudin Ibrahim A, Goh M.W.K., Tan H.T.W. On the vegetation and flora of Pulau Tioman, Peninsular Malaysia. The Raffles Bulletin of Zoology. 1999;6:11-14.

[8] Lee AT, Ottosson U, Jackson C, Shema S, Reynolds C. Urban areas have lower species richness, but maintain functional diversity: Insights from the African Bird Atlas Project. Ostrich. 2021;92(1):1–15. doi:10.2989/0030652 5.2021.1902876

[9] Naithani A, Bhatt D. Bird community structure in natural and urbanized habitats along an altitudinal gradient in Pauri district (Garhwal Himalaya) of Uttarakhand State, India. Biologia. 2012;67(4):800–8. doi:10.2478/ s11756-012-0068-z

[10] Ng PKL, Yong HS, Sodhi NS, Biodiversity Research on Pulau Tioman Peninsular Malaysia: A Historical Perspective. The Raffles Bulletin of Zoology. 1999;6:5-10.

[11] Padoa-Schioppa E, Baietto M, Massa R, Bottoni L. Bird communities as bioindicators: The focal species concept in agricultural landscapes. Ecological Indicators. 2006;6(1):83–93. doi:10.1016/j.ecolind.2005.08.006

[12] Robson C. Birds of Southeast Asia: Thailand, Peninsular Malaysia, Singapore, Vietnam, Cambodia, Laos, Myanmar. Princeton, NJ: Princeton University Press; 2005.

[13] Sodhi NS, Briffett C, Lee BPY-H, Subarak R, An Annotated Checklist of the Birds of Pulau Tioman, Peninsular Malaysia. The Raffles Bulletin of Zoology. 1999;6:125-130.

[14] Zakaria M, Rajpar MN. Bird species composition and feeding guilds based on point count and mist netting methods at the Paya Indah Wetland Reserve, peninsular Malaysia [Internet]. U.S. National Library of Medicine; 2010 [cited 2023 Jul 25]. Available from: https://www.ncbi.nlm.nih.gov/ pmc/articles/PMC3819076/

[15] RStudio Team. RStudio: Integrated Development Environment for R [Internet]. Boston, MA; 2015. Available from: http://www.rstudio.com/ [16] The ASICS Runkeeper App [Internet]. 2023. Available from: https:// runkeeper.com/cms/

[17] Lechner AM, Verbrugge LNH, Chelliah A, Ang ML, Raymond CM. Rethinking tourism conflict potential within and between groups using participatory mapping. Landscape and Urban Planning. 2020;203:103902. doi:10.1016/j.landurbplan.2020.103902

[18] Abdul J. An Introduction to Pulau Tioman. 1999;(Supplement6: 3-4). Available from: https://lkcnhm.nus.edu.sg/wp-content/uploads/sites/10/ app/uploads/2017/04/s6rbz003-004.pdf

[19] Ortiz-Burgos S. Shannon-weaver diversity index. In: Encyclopedia of Estuaries. Dordrecht: Springer Netherlands; 2016. p. 572–3.

[20] Violle C, Navas M-L, Vile D, Kazakou E, Fortunel C, Hummel I, et al. Let the concept of trait be functional! Oikos [Internet]. 2007;116(5):882–92. Available from: http://dx.doi.org/10.1111/j.0030-1299.2007.15559.x

[21] National Geographic Society. Generalist and Specialist Species [Internet]. education. nationalgeographic.org. 2023. Available from: https:// education.nationalgeographic.org/resource/generalistand-specialist-species/

[22] Riley K, Cook R, Carr E, Manning B. A systematic review of the impact of commercial aircraft activity on air quality near airports. City and Environment Interactions. 2021 Aug;11:100066.

[23] 1.Qin K. Birds suffer from air pollution, just like we do [Internet]. Audubon California. 2016. Available from: https://ca.audubon.org/news/birds-suffer-airpollution-just-we-do

photograph

photograph by Christabel Yow Qian Yang

EFFECT OF ABIOTIC MICROHABITAT CONDITIONS ON DICAMPTODON TENEBROSUS ACROSS

AND WITHIN STREAMS

Ash Abel1, Sanchita Biswas2, Cleo Lu3, Sierra Vasquez4

1UC Santa Cruz, Santa Cruz, CA, USA 2UC Davis, Davis, CA, USA 3UC Berkeley, Berkeley, CA, USA

4UC San Diego, Muir College, Ecology, Behavior, and Evolution, 2025

ABSTRACT

Changes in the abiotic characteristics of a habitat can impact the feeding behavior of top predators, triggering top-down trophic cascades. Coastal giant salamanders (Dicamptodon tenebrosus) are of particular interest due to their importance in California riparian habitats and their sensitivity to abiotic changes. Our research studies how different creek systems and the presence of D. tenebrosus larvae affect the growth and density of other surrounding larvae. We predicted that D. tenebrosus population density and body size would be highest in creek pools with the greatest rock cover density and lowest in pools with the most silt. Furthermore, we predicted that larger creeks would have more cover rocks, which would correspond to higher population densities and body sizes, while smaller creeks would have more silt, corresponding to lower densities and body sizes. We surveyed D. tenebrosus across three northern California (USA) streams with varying substrates and flow rates to examine the effect of abiotic factors on larval population density and body size. We found that larval density increased with rock density, while larval size was found to be smaller in smaller creeks and in the presence of silt. We also found a positive correlation between salamander population density and the size of salamander larvae. Conservation efforts of D. tenebrosus must thus incorporate maintenance of microhabitat conditions, such as substrate composition and water flow.

INTRODUCTION

Changes in the population of top predators can have far-reaching effects on lower trophic levels via a trophic cascade.1 These changes are caused by factors including relative body size or abundance, influencing the feeding behavior of top predators in a top-down ecosystem. In this ecological framework, consumers exert control over the behaviors and densities of species lower in the food web.2 For example, overfishing has reduced the density of cod fish (Gadus morhua), a top predator, leading to larger populations of smaller predators occupying lower trophic levels (such as herrings, Clupea harengus, and macroinvertebrates). Another study found that anthropogenic habitat destruction has reduced predatory bird populations, which in turn triggered a trophic cascade that increased florivorous invertebrate populations, intensifying damage to plant populations.2 Studies on trophic cascades often focus on changes in the population density of key species, such as top predators, in response to the destruction or alteration of their habitat, but less understood are the effects of factors that impact predator function, such as size, which may in turn trigger trophic cascades.

An example of body size demographic changes affecting topdown control is seen in the common whitetail dragonfly (Plathemis lydia).3 The increased diversity of size classes in populations of the whitetail drove changes in top-down trophic control by promoting cannibalism of smaller individuals. This, in turn, promotes antipredator behavior in these smaller individuals. In this way, the largest dragonflies control the behavior and abundance of the lower trophic level, consisting of the smallest whitetails. Furthermore, conspecific predation reduced predation pressure on other insect

species, indicating larger trophic effects as a result of populationlevel changes in body size. Although whitetails undergo seasonal changes in body size demographics, habitat changes may also affect predator body sizes, rather than predator population density alone.

The effects of habitat changes on body size have been studied in organisms of intermediate trophic levels, such as reef fishes in San Diego, CA.4 The mean body sizes of these fishes were highly dependent on microhabitat conditions, such as substrate composition, with ecotone and sandy substrate correlating with larger body lengths. In this case, smaller fishes preferred rocky substrate due to cover necessity, less frequently occupying the more exposed ecotone and sandy region. Furthermore, numerical fish density was highest in rocky regions due to favorable feeding conditions. Thus, microhabitat conditions strongly affect both the distribution and body size of aquatic organisms. These factors may alter trophic cascades by changing the function of top predators in top-down regulated ecosystems.

At the core of our study is the coastal giant salamander, Dicamptodon tenebrosus, which is a predator species that is affected by microhabitat composition. They inhabit Pacific Northwest coastal riparian areas where creeks are relatively shallow. Due to the limited space and resources in these conditions that are unsuitable for larger predators, D. tenebrosus is the top of the aquatic food chain for this region. As the top predator, D. tenebrosus consists of over 90% of predator biomass in many creeks.5 Known for their voracious diet, salamander larvae consume a variety of freshwater macroinvertebrates and fish hatchlings, and stabilize the creek ecosystem through a trophic top-down control. Therefore, any changes in D. tenebrosus larvae populations can drastically alter the populations of other stream organisms. Additionally, like the whitetail dragonfly, D. tenebrosus is cannibalistic. Larger salamander larvae can incorporate larger prey into their diet as they grow, such as the smaller larval D. tenebrosus. Thus, both the population density and the size of D. tenebrosus directly affect the population of their prey and could even affect the distribution of smaller D. tenebrosus larvae as they avoid predation by larger conspecifics.

The abiotic factors that affect D. tenebrosus have been predominantly studied in the context of effects on population density. Larval density is considered to be best predicted by microhabitat creek conditions, such as substrate composition.6 Parker (1991) showed a strong positive correlation between loose rock availability and larvae abundance in Northern California, indicating that the density of rock cover was a limiting factor of D. tenebrosus abundance. Further, they demonstrated that D. tenebrosus larvae may favor small, slow-moving streams.6 However, this pattern may not be consistent between streams, as slow-moving streams have higher silt deposition, a factor that is known to reduce salamander larval density.7 Specifically, the deposition of fine sediment may create unsuitable habitats for amphibians, as it fills interstitial spaces between rock crevices and reduces overall habitat availability.7 Furthermore, fine sediment also promotes the growth of microflora and mold that are harmful to salamander gill health, reducing population density.8 Thus, it may be that wider, fastermoving streams are preferred by D. tenebrosus larvae. Although studies have determined some effects of abiotic

factors on the density of D. tenebrosus larvae, none have examined the effect of abiotic factors on larval body size.

Based on creek conditions and population dynamics, D.tenebrosus body size may be affected by substrate composition or intraspecies interactions. For instance, increased rock cover provides surface area for more macroinvertebrates.9 Since salamander growth is indeterminate and growth rates depend on food availability, an increase in rock cover could indirectly provide D. tenebrosus larvae with more food and increase their body size.10 Moreover, the potential for cannibalistic interactions may drive smaller individuals to avoid cohabitating with larger individuals, leading to their reduced preference in streams with larger larvae. This dynamic could decrease both the average body size and overall abundance of larvae, as any remaining smaller larvae are likely to be preyed upon by larger counterparts. Considering these factors, we were interested in exploring how differing microhabitat characteristics affect salamander body size and density within the same larger ecological system.

To understand how creek size variation results in distinct microhabitats and influences D. tenebrosus larval population densities and size distributions, we conducted a study of three creeks within the Angelo Coast Range Reserve in Mendocino County, California, USA. While the creeks are situated in the same old-growth forest, they each have varying drainage levels, which potentially leads to distinct microhabitats caused differed by water flow and substrate composition of the creek bed.11 We hypothesize that in all creeks, an increase in the density of cover rocks will correlate with an increase in D. tenebrosus larval density. Conversely, we predict a rise in the percentage of silt within a pool will lead to a decrease in larval density. Due to observable differences in creek substrate composition, we anticipate that larger creeks will have a higher density of cover rocks, lower silt levels, support higher salamander larval densities, and produce larger salamander larvae. In contrast, smaller creeks will have a lower density of cover rocks, higher amounts of silt, lower salamander larval density, and smaller salamander larvae. Additionally, the presence of a large D. tenebrosus larvae within a pool will have a positive effect on the mean body size of other D. tenebrosus larvae.

MATERIALS AND METHODS

Natural History

The coastal giant salamander (Dicamptodon tenebrosus) is native to the coastal regions of British Columbia, Oregon, Washington, and Northern California.12 Here, they primarily feed on invertebrates and rarely on small snakes, shrews, young rodents, and even their own kind. These amphibians prefer temperate regions of fresh flowing water within forested communities.

To survey D. tenebrosus, freshwater pools were studied within the UC Angelo Coast Range Reserve, CA, USA from August 1st to August 5th, 2023. Angelo Reserve is situated in an expansive old-growth forest in Mendocino County. The region harbors a multitude of terrestrial and aquatic habitats, including redwood groves, mixed conifer-deciduous forests, salmon-bearing mainstream rivers, and tributary streams.13 Creeks of Angelo Reserve include Fox Creek, McKinley Creek, and Elder Creek, all of which are tributaries of the South Fork Eel River.

During periods of low flow (April through November), the creek channels form wide, shallow pools connected by short riffles.14 These conditions allow for a wide range of aquatic larval organism–such as mayflies, stoneflies, dobsonfly larvae, caddisfly larvae, and damselflies–which constitute the diet of D. tenebrosus

From observation, Fox Creek is characterized by medium flow rates, a drainage area, and a greater abundance of gravel. Conversely, McKinley Creek with its slow-moving water and greater levels of silt contrasts with Elder Creek, which is characterized by fast-moving water and levels of rock and gravel similar to Fox Creek. With drainage areas used as a proxy for water flow and thus size, McKinley Creek is a small creek with a drainage area (DA) of 0.58 km2, Fox Creek is a medium-sized creek with a DA of 2.8 km2, and Elder Creek is a large creek with a DA of 13.5 km2 11, 15

Pool Selection and Characteristics

To survey this system, we examined 20 pools from all three creeks (Fox Creek, McKinley Creek, and Elder Creek). Pools were defined

Figure 1: Relationship between number of D. tenebrosus in a pool per m2 and number of rocks in a pool per m2. A linear regression test was used to analyze the relationship between number of D. tenebrosus in a pool per m2 and number of rocks in a pool per m2. The number of rocks over 7.5 centimeters was counted by removing and counting every unembedded rock in the pool that was 7.5 centimeters. The number of D. tenebrosus was also counted. Rocks per m2 and D. tenebrosus per m2 was calculated by dividing total number of rocks and D. tenebrosus by area of the pool. Data points represent salamander density (individuals/m2) and rock density (rocks/m2) in each of 60 pools. The trend line shows that as the number of rocks per m2 increases, the number of D. tenebrosus per m2 also increases (N=60, r2=0.34, p<0.0001).

as bodies of slow or stagnant water bound by exposed rocks. The length and width of surveyed pools were restricted to 0.5 to 2 meters, and the depth did not exceed 35 centimeters. This was to ensure the feasibility of our observations, as larger pools could potentially harbor inaccessible larvae hidden in rocks or sediment. To determine the surface area of each pool, the longest length and width were measured in meters. The formula for the area of an oval was used to determine the area of each pool. To understand pool-level sediment characteristics of the D. tenebrosus microhabitat, every team member conducted a percent visual estimate for rocks and silt in each pool, which was then averaged amongst the group. We collected data moving upstream to minimize the risk of interfering with other study pools via substrate dislodgement or salamander displacement.

Dicamptodon tenebrosus Survey Method

A total of 80 salamanders were surveyed from Fox, McKinley, and Elder Creek. The collection of D. tenebrosus larvae started with an initial survey of the pool to collect visible individuals with aquarium nets. Rocks were then removed and placed to the side of the pool before another larvae observation was conducted. Per Parker (1991), we counted all unembedded rocks over 7.5 centimeters to measure rock cover density. Rock density was calculated by dividing the number of rocks by the pool’s area. Before leaving the pool, a thorough examination was conducted by inserting hands beneath the pool’s rock, gravel, and silt to flush out any remaining larvae that may have been overlooked.  Observed D. tenebrosus were transferred to a dip net placed in adjacent creek sections to avoid double counting and to conduct individual measurements. In the dip net, snout-to-vent length (SVL) was measured to the nearest centimeter. After a maximum observation period of 25 minutes, we counted the total number of larvae collected and returned them, along with the rocks, back to their original positions. D. tenebrosus larval density was calculated by dividing the total number of larvae by the area of the pool.

Analysis

All statistical analyses were conducted using JMP statistical software v17 (SAS Institute Inc., Cary, NC, 1989–2023). Linear regression was used to test if rock density affected D. tenebrosus larval density and to determine if the quantity of silt in each pool affects the density of D. tenebrosus larvae. For analyzing differences in abiotic factors between creeks, an ANOVA was used to compare the visual percent cover of silt among all three creeks and to examine differences in salamander larval density and SVL between creeks.

RESULTS

As the number of rocks over 7.5 cm per m2 increased, the number of D. tenebrosus per m2 in a pool also increased (n=60, r2=0.34, p<0.0001; Fig 1).  However, no relationship was found between the number of D. tenebrosus per m2 and the percentage of silt cover in a pool (n=60, r2=0.0004, p=0.88).

The large creek had a marginally higher percent cover of rocks comparedtothemediumandsmallcreeks(nsmall=20,nmedium=20,nlarge=20, F=2.64, p = 0.08); the small creek had a higher percentage cover of silt compared to medium and large creeks (nsmall=20, nmedium=20, nlarge=20, F=18.93, p<0.0001). However, there was no difference in D. tenebrosus larval density between the three creeks (n=60, F=0.07, p=0.93).

Interestingly, D. tenebrosus larvae in the small creek had a significantly smaller SVL compared to those in the medium and large creeks (nsmall,=32, nmedium=30, nlarge=19, F=9.56, p=0.0002; Fig 2). The presence of a large D. tenebrosus in a pool had a marginally positive effect on the mean SVL of the remaining D. tenebrosus in the same pool (n=38, t=2.09, p=0.055; Fig 3).

DISCUSSION

We found evidence for our hypothesis that a higher density of cover rocks correlates with an increase of larval D. tenebrosus density. Contrary to our hypothesis, there was no discernible difference in rock density between the creeks of different flow rates, nor did we find a correlation between larval density and the visual percent silt. However, silt was more prevalent in the smaller creek, which also contained the smallest larvae. This finding suggests that stream size and flow rate might influence larval size for a variety of reasons. Our results differed from expectations, as no salamander size difference existed between large and medium creeks, implying that their water flow rates are not as distinct as initially predicted. Across all creeks, D. tenebrosus larvae had a higher density in pools with more unembedded rocks, which confirms previous findings that suggest larvae prefer areas with more objects that can provide cover.14 These observations correlate with studies demonstrating that greater rock coverage provides more surface area for aquatic macroinvertebrates, which is a large part of the D. tenebrosus diet.9 Therefore, not only do rocks provide possible protection, but they also likely provide habitats for the main food sources of salamander larvae. We did not find any difference in the rock density between separate creeks with differing water flows. This could be because rock cover does not vary with creek flow, whereas factors like silt cover are carried and washed away by increases in water flow.

An increase in silt is often characterized by creeks with slower creek flow rates and a reduced amount of water, making the size of streams an important factor in our study system. We found that the slowest-moving creek had the highest abundance of silt, as well as the smallest overall salamander size. However, we also found no significant correlation between larval salamander density and the presence of silt. This is contrary to our initial predictions, as previous research shows that silt has a negative impact on salamander health.8 Although D. tenebrosus larval population density did not decrease in the presence of silt, the presence of silt correlated with a decrease in larval body size. Salamander larval size and silt abundance have not been linked before, so future studies could examine this relationship further with different concentrations of silt. It has been suggested by recent experiments with similar salamander densities that the salamanders could be using the silt to burrow into for cover as an alternative to using rocks.16 This suggests a possible positive relationship between

silt cover and larval salamander density. Furthermore, it was more difficult to collect salamanders in pools containing silt because slight disturbances stirred up sediments and reduced visibility, which could be a desirable anti-predator microhabitat trait for larvae. Although larvae of D. tenebrosus did not show any differences in density between the pools containing silt, this could be explained by the ways both silt and rock can provide hiding opportunities.  Like silt, abundance of macroinvertebrates is also affected by creek size. Faster-moving water means more invertebrate movement between pools and more available invertebrate habitat space.9 Furthermore, in flood years, when water is most abundant, salamanders have twice the amount of food in their stomachs than in non-flood years.17 Since having more available food results in salamander larvae growing faster and metamorphosing sooner, increased water flow could contribute to higher larval body size.18 This could explain why the medium and large creeks have larger salamander larvae since they both have faster-moving water than the small creek. It follows that the larvae within the large and medium creek may be growing larger and metamorphosing sooner due to the food abundance brought on by faster-moving water.

Other factors could also be affecting D. tenebrosus larval size. Contrary to our results, past studies have found that as larval salamander density increases, salamander size decreases due to competition.17 The low water flow in the small creek results in pools being significantly farther apart by land rather than by wide areas of moving water, making it more challenging for larvae to move between pools. Although we found that larval salamander density was approximately equal across all three creeks, the physical characteristics of the low-flow creek may limit travel for both salamanders and the influx of food sources, condensing the larvae population into fewer pools. Therefore, it is possible that D. tenebrosus larvae compete much more in the low-flow creeks than in larger creeks. A study on artificially isolated crayfish found that high-density populations depleted resources and experienced reduced growth rates.19 Increased D. tenebrosus larval interaction

Figure 2: Comparison of Snout of Vent Length (SVL) in centimeters across three Creeks. We used ANOVA to determine differences in mean SVL between Fox (small), McKinley (medium), and Elder (large) Creek. Snout to vent length (SVL) was measured as the distance, in centimeters, from the tip of the snout to the most posterior opening of cloaca from the dorsal side of D. tenebrosus. The whiskers at the top and bottom of the plot represent the maximum and minimum values of SVL in that creek, respectively. The dots represent outliers. The 25% quantile, median, and 75% quantile are represented by the horizontal lines in each boxplot from bottom to top. There was a difference between the SVL of D. tenebrosus in Small Creek compared to the SVL of D. tenebrosus in Medium and Large Creek (NFox=30, NMcKinley,=32, NElder=19, F=9.56, p=0.0002).

Figure 3: The effect of presence of an individual over or at 5cm on the mean SVL of the rest of D. tenebrosus in the pool. A t-test was utilized to understand how the presence of D. tenebrosus individuals affected the mean SVL of the remaining individuals within that same pool. We analyzed D. tenebrosus that have a SVL at or over 5 cm as a large individual and categorized all the pools as “yes” or “no” based on the presence or absence of an individual at this size. Then, we took out the large individual and calculated the mean SVL for the rest of D. tenebrosus in the pool. There is a marginally significant difference between groups, and with the presence of a large individual, the mean SVL is likely higher for the rest of D. tenebrosus in the same pool (N = 38, t=2.09, p=0.055).

within pools of the slow-moving creek could account for the smaller overall size of larvae since both resources and space are restricted within this system. A future experiment that isolates pools from new resources and salamander pool-hopping could investigate further the effect of pool isolation on salamander size.

Our results show that across all the creeks, the presence of a large salamander may be an indicator of larger overall salamander size in the surrounding pool. This could be because large salamander larvae are cannibalistic towards smaller larvae.20 Large D. tenebrosus larvae may either eat all the smaller larvae in the immediate vicinity, or the smaller larvae could avoid them. This implies that the only larvae cohabitating are those that are too large to consume. In this way, salamander larval size has an effect on trophic dynamics, as the smaller salamanders being eaten are going down a trophic level.

CONCLUSION

We found that abiotic differences across creeks in the same region can affect larvae size but found no support for an effect on population density, and it is important to consider how we can use this information going forward. Understanding what substrate is preferred and, consequently, which creeks are more suitable for salamanders can help us understand how abiotic factors, such as microhabitat substrate composition, affect their physical appearance and health.

Changes in salamander population density and size demographics in turn impact the feeding behavior of salamander larvae. This changes macroinvertebrate and fish hatchlings’ abundance, therefore having a cascading effect on plant materials and algae that these macroinvertebrates and fishes feed on. Trophic relationships work in a delicate balance. Any changes in higher trophic levels will have cascading effects on lower trophic levels. For this reason, salamander larval populations are important to consider when making conservation decisions regarding species in lower trophic levels, as well as understanding how the conservation of salamander populations affects their prey and

beyond. Given the strong influence of microhabitat conditions on the function of this top predator in the creek ecosystem, habitat conservation must consider the maintenance of small-scale abiotic factors, such as the substrate composition of creek beds.

ACKNOWLEDGEMENTS

This work was done at the Angelo Natural Reserve DOI: 10.21973/ N3R94R. This reserve is part of the University of California Natural Reserve System. We would like to gratefully acknowledge the Cahto People, who have historically cared for this land. We are also deeply grateful to all those who played a role in the success of this project, especially Dr. Timothy Miller, Dr. Renske Kirchholtes, and PhD candidate Lydia Dean for their unwavering guidance and support. We would like to thank Peter Steel for his support in our research.

REFERENCES

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[6] Welsh H.H., Lind A.J. “Multiscale Habitat Relationships of Stream Amphibians in the Klamath-Siskiyou Region of California and Oregon.” The Journal of Wildlife Management. 2002;66(3):581–602. https://doi.org/10.2307/3803126

[7] Ashton D.T., Marks S.B., Welsh H.H. “Evidence of continued effects from timber harvesting on lotic amphibians in redwood forests of northwestern California.” Forest Ecology and Management. 2006 Jan;221(1–3):183–93. https://doi. org/10.1016/j.foreco.2005.09.015

[8] Lefcort H, Hancock K.A., Maur K.M., Rostal D.C. “The Effects of Used Motor Oil, Silt, and the Water Mold Saprolegnia parasitica on the Growth and Survival of Mole Salamanders (Genus Ambystoma).” Arch Environ Contam Toxicol. 1997 May 1;32(4):383–8. https://doi.org/10.1007/s002449900200

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[12] Stebbins R, McGinnis S. Field Guide to Amphibians and Reptiles of California. University of California Press; 2012. 85–88 p.

[13] Natural History – Angelo Coast Range Reserve [Internet]. [cited 2023 Aug 8]. Available from: https://angelo.berkeley.edu/about-angelo/natural-history/

[14] Parker M.S. “Relationship between Cover Availability and Larval Pacific Giant Salamander Density.” Journal of Herpetology. 1991;25(3):355–7. https:// doi.org/10.2307/1564597

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SPATIOTEMPORAL EXPRESSION OF SRCR142 THROUGHOUT THE DEVELOPMENT OF LYTECHINUS PICTUS

Gloria D. Renaudin & Amro Hamdoun

Center for Marine Biology and Biomedicine Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA, 92037, USA

ABSTRACT

Sea urchins develop in the water column as larvae, but then metamorphose and settle on the benthos as adults. This transition involves the innate immune system, particularly coelomocytes that transcribe scavenger receptor cysteinerich (SRCR) genes. In invertebrates, SRCR genes play a crucial role in regulating and developing the immune response. While sea urchins lack adaptive immunity, Strongylocentrotus purpuratus’s genome encodes over a thousand SRCR genes to combat the microbial-rich benthos post-metamorphosis. Srcr142 in S. purpuratus is expressed during the early pluteus stage, yet it is unknown whether this is the case in Lytechinus pictus. While S. purpuratus was the original sea urchin model organism, L. pictus has a shorter generation time, making it a better candidate for developmental immunology. To further understand the role of SRCRs in innate immunity, HCR-FISH was used to visualize the expression of srcr142 throughout the development of L. pictus. We anticipate that our findings will reveal an upregulation in the expression of srcr142 within pigment cells as they approach metamorphosis, to prepare for the higher microbial activity in the benthos.

INTRODUCTION

Scavenger receptors (SRs) are a large family of pattern recognition receptors (PRRs). SRs can recognize both damage-associated molecular patterns (DAMPs) such as oxidative stress and pathogenassociated molecular patterns (PAMPs) such as Gram-positive and Gram-negative bacteria. SRs are also innate immune recognition receptors as they can cooperate with Toll-like receptors (TLRs) to identify and mediate the phagocytosis of various bacterial pathogens. They are involved in autoimmune disorders, as they bind Amyloid-β peptides (Aβs), oxidized low-density lipoproteins (LDLs), and apoptotic cells. Aβs are neurotoxic proteins known to cause Alzeihmer’s disease but can be cleared through SRs yet mediate its inflammatory response which contributes to the disease. High levels of LDLs are known to cause a buildup of cholesterol in the arteries leading to atherosclerosis, an inflammatory disease connected to SRs. Apoptotic cells give rise to autoimmune diseases if not cleared, such as through SRs.1 Thus SRs are crucial receptors in the innate immune system. There are 12 classes of SRs.1 The scavenger receptor cysteinerich (SRCR) domain is present in both class A and class I SRs. This highly conserved domain plays a crucial role in the recognition and clearance of pathogens. These receptors, which may exist as either membrane-bound or soluble, are divided into two groups: group A and B. Group A has 6 cysteine residues encoded in two exons, while group B has 8 cysteine residues encoded in a single exon.7 Invertebrate SRCRs are highly diverse compared to mammals. However, some invertebrates exhibit a similar or lower diversity of SRCRs to those in mammals, while other invertebrates, such as sea urchins, show a great expansion of SRCRs.3 More specifically, humans have 81 SRCR genes, while the purple sea urchin, Strongylocentrotus purpuratus, has 1,095 SRCR genes.8 This is particularly interesting, given that SRCRs play a crucial role in the immune system, and sea urchins, despite their vast SRCR repertoire, lack adaptive immunity.

The sea urchin innate immune system consists of blastocoelar cells and pigment cells, which mature and become known as coelomocytes post-metamorphosis as they reside in the coelomic cavity–the fluid-filled cavity inside a sea urchin. In the blastocoel, the central cavity of the blastula, various types of blastocoelar cells exhibit distinct motility and phagocytic capabilities. These include filopodial cells, ovoid cells, globular cells, and amoeboid cells, which collaboratively identify and phagocytize foreign particles. Filopodial cells, characterized by small cell bodies and long projections, form connections within a larger syncytial network capable of expressing immune effector genes to elicit an immune response. After an immune response is triggered, ovoid cells will appear and aggregate at the site of infection to phagocytize microbes. Globular cells, while continuously moving within the blastocoel, actively seek out microbes and can interact with other blastocoelar cells and pigment cells by forming filopodial projections. Amoeboid cells, highly motile in nature, rapidly migrate to the site of infection and interact with other immune cells, including pigment cells. In contrast to blastocoelar cells, pigment cells move along the ectoderm and not within the blastocoel. They are characterized by a vivid red color derived from polyketide synthase (pks1), which synthesizes echinochrome A—a naphthoquinone used in pharmaceutical drugs. Thanks to their color, pigment cells are easily discernible under a microscope and can be seen migrating to the site of infection. As larvae approach metamorphosis, both pigment cells and blastocoelar cells collectively become known as coelomocytes, now present in the adult sea urchin’s coelomic cavity. Coelomocytes retain their motility, opsonizing then phagocytizing foreign particles.2

All these immune cells express SRCRs. More specifically, pigment cells are known to express srcr142 in the early pluteus stage, only 3 days post fertilization (dpf). In the early pluteus stage of S. purpuratus, the expression of srcr142 is co-localized with pks1 and undergoes significant upregulation after 42 hours post-fertilization (hpf).2 Nevertheless, the spatiotemporal expression of srcr142 is unknown throughout development and has not been studied in Lytechinus pictus Due to their larger, more transparent eggs and faster development time, L. pictus is a better model organism for developmental gene expression studies than S. purpuratus. Notably, L. pictus undergoes metamorphosis at 3 weeks post fertilization, in contrast to the more prolonged 1-3 months observed in S. purpuratus.4,6 This renders L. pictus a preferred choice for the study.

Fig. 1. Lateral-view of the expression of srcr142 and pks1 in L. pictus’ early blastula stage. Small specks of srcr142 expression are seen in early blastula embryos (n = 38/166).

Fig. 2. Lateral-view of the expression of srcr142 and pks1 in L. pictus’ late gastrula stage. Expression of srcr142 was expressed in 95/98 late gastrula embryos on a cellular level. Srcr142 is colocalized with pks1 throughout the embryo except at the apical plate where srcr142 expression is found without pks1 expression.

Fig. 3. Side-view of the expression of srcr142 and pks1 in L. pictus’ 1st larval stage (L1). The expression of srcr142 and pks1 are adjacent to one another throughout the body (n = 50/50) with more expression of srcr142 in the left coelomic pouch (n = 26/50). B) Close-up of srcr142 expression in the left coelomic pouch with no pks1 expression in proximity.

Preliminary data from Cody Hargadon and I on Lytechinus pictus shows that, during an immune challenge, srcr142 and pks1 remain colocalized. However, srcr142 undergoes upregulation in both coelomic pouches, while pks1 predominantly localizes to the gut. Notably, the expression of srcr142 varies slightly from pigment cells during an immune challenge, suggesting a potential divergence in srcr142 expression as sea urchins progress through development. Drawing support from prior findings in S. purpuratus, we hypothesized that srcr142 would be expressed in pigment cells throughout development. The expression of srcr142 should also be upregulated as larvae approach metamorphosis, in response to heightened microbial activity while settling in the benthos. Determining the location of srcr142 throughout development will help pinpoint its function in the sea urchin’s innate immune system.

MATERIALS AND METHODS

Culturing

The larvae were raised in seawater filtered through 1µm followed by 0.2µm filters to remove impurities. Their 4L beakers consisted of an air pick with a rotating paddle. The air pick ensures the water remains oxygenated, while the paddle keeps the larvae in the water column rather than at the bottom of the beaker. The paddle is crucial to the survival of large batches of larvae. The larvae were fed 5,000 cells/mL of red microalgae, Rhodomonas lens twice a day. Any excess Rhodomonas on the bottom was siphoned with a serological pipette. This prevents the larvae from sticking to the Rhodo dot, which would in turn catch other larvae and crash the beaker. To maintain a stable environment for larval development, water changes were not performed unless absolutely necessary. Once competent, metamorphosis was triggered within two hours by adding the competent larvae to a biofilm plate that was kept in running seawater for months. Metamorphosis was triggered to synchronize the development of the juveniles as they do not usually metamorphose all at once.

Fixations

The first fixation occurred at 6.5-7.5 hours post fertilization (hpf) during the early blastula stage. The second fixation occurred at 18hpf, capturing the late gastrula stage. Subsequently, larvae at six distinct developmental stages were systematically collected. The first larval stage (L1) was gathered at 3 days post-fertilization (dpf), followed by L2 at 4-5dpf, L3 at 7dpf, L4 at 8-10dpf, L5 at 10-12dpf, and L6 just before competency, precisely at 3 weeks post fertilization. The final two stages encompassed specimens at competency and those at 2-4 hours post metamorphosis, representing the juvenile stage.6

The fixation protocol employed in this study was adapted from Molecular Instruments, specifically designed for the purple sea urchin, Strongylocentrotus purpuratus’s embryos. The steps were modified to fix the white painted sea urchin, Lytechinus pictus’s larvae and juveniles. The larvae were observed under a microscope for the correct developmental stage. Upon confirmation, approximately 100 larvae were collected in a 2 mL tube and rinsed with 1 mL of filtered seawater (FSW). Next, 300 µL 4.45M NaCl and 100 ul of fix buffer were added to a previously aliquoted cryogenic tube containing 400 ul of 16% PFA. After thorough mixing, the 1mL larvae sample were transferred to the PFA and fix buffer tube. The sample was left on a rocker at 4°C for 24 hours. Subsequently, the tube was transferred onto ice, and 100 ul 1M glycine (1mM EDTA in calcium-magnesium-free seawater (CMFSW)) was added. Washes were conducted at 10-minute intervals. After removing as much solution as possible, the sample underwent two rinses with 1 mL 1xPBST (phosphate-buffered saline with Tween20) and 100 µL 1 M glycine combined. A single wash with 1 mL 1xPBST then followed. The final washes involved three rounds of 70% Ethanol (EtOH). These samples were stored at -20°C in 70% EtOH until imaging. In situ hybridization chain reaction (HCR)

After fixing all stages, HCRs were performed using probes designed based on the L. pictus transcriptome. This HCR protocol was optimized for L. pictus larvae utilizing the HCR RNA-FISH protocol from Molecular Instruments for S. purpuratus embryos. The larvae were rehydrated 3x with 200 ul of 5x SSCT and left to sit 10 minutes in between rehydrations with 5x SSCT. The majority of the 5x SSCT was then aspirated to add 50 µL of probe hybridization buffer to the tubes

Fig. 4. Side-view of the expression of srcr142 and pks1 in L. pictus’ 2nd larval stage (L2). The expression of srcr142 and pks1 are adjacent to one another throughout the larvae (n = 9/9). There is no higher expression of srcr142 in the left coelomic pouch as previously seen.

containing the larvae. The larvae were pre-hybridized for 30 minutes at 37°C. The following steps are all performed at 37℃. During this time, the probe solution was prepared by adding 0.4 pmol of each probe set to 50 µL of probe hybridization buffer. After the larvae are pre-hybridized, the probe solution was added to the tube containing the larvae to reach a total volume of 100 µL. The solution was then gently mixed with a pipette and left to incubate for 24 hours. The following day, 200 ul of probe wash buffer was added to each tube and incubated again for 5 minutes. To remove excess probes, the larvae were washed with 200 µL of probe wash buffer twice for 10 minutes and twice for 30 minutes. Finally, the larvae were then washed twice for 1 minute with 200 ul of 5x SSCT. This is the last step at 37℃.

For HCR’s amplification stage, 2 ul of 3 µM hairpin stock were snap cooled (90 seconds at 95℃) to prepare 6 pmol for each hairpin used. Once cooled, both hairpins were added to 100 µL of room temperature amplification buffer. Next, the 5x SSCT was aspirated from the tube containing the larvae. The hairpin solution was then added to the larvae tube and allowed to sit overnight in the dark at room temperature. The next day, 200 µL of 5x SSCT was added to the larvae and left to incubate for 5 minutes at room temperature. To remove excess hairpins, the larvae were washed with 200 µL of 5x SSCT, twice for 10 minutes and twice for 30 minutes. During the second 30 minute wash, the larvae were washed with 200 µL of 1 µL DAPI in 1000 ul 5x SSCT to a final concentration of 300 µM DAPI in 5x SSCT. The longer the larvae are incubated in DAPI, the better the larvae are stained so this wash can be an hour long instead of 30 minutes. Afterwards, the larvae were rinsed with 200 µl 5x SSCT twice for 10 minutes to remove the DAPI. This will help minimize any background noise during imaging. Finally, the samples were then stored at 4℃ in the dark.

The first HCR completed in this study used a different probe set that did not hybridize well to the srcr142 mRNA, as expression was not seen. Thus, a second HCR was performed with a different probe set, thereby yielding the expression patterns as illustrated in the figures. Imaging and image processing

Larvae samples were imaged using high-content imaging on a Molecular Devices ImageXpress HT.ai. For the early blastula through L1 larvae, a stepwise rinse (70/30 5xSSCT:100%Glycerol, 50/50, & 20/80) was utilized to image them while in 80% glycerol which prevents them from shifting during imaging. Once in 80% glycerol, approximately 25 of each stage were transferred to each well of a Cellvis glass bottom 384-well plate and imaged using a 10X objective. For all stages after L1, a single sea urchin was transferred to a well of a 96 well plate and imaged with a 20X objective. The juveniles were imaged on both sides after gently moving them with a 10 ul pipette tip. The z-stack images were subsequently edited using ImageJ to remove any background fluorescence by decreasing overall fluorescence until the background was visually black. The images were then compiled for analysis.

RESULTS

The following results are presented as figures, where srcr142 is represented in blue, pks1 in green, and DAPI in gray to show the animal cells. As expected, srcr142 expression was absent in the early blastula stage of L. pictus, since pigment cells are not yet present (Figure 1). The speckles of srcr142 expression observed did not coassociate with cells and were only seen in 38 out of 166 early blastula embryos. These were determined to be noise and not indeed srcr142 signal. On the other hand, the pks1 expression shown is merely background due to the presence of autoflouroscence resulting from the use of hairpins in the 647-channel.

However, the expression of genes become co-associated with cells starting in the late gastrula stage. As seen in Figure 2, the cyan color results from an overlap in the expressions of pks1 and srcr142 This overlapping expression is seen at the vegetal plate in 95 out of 98 embryos. There is also a distinct expression of srcr142 that is not in proximity to pks1 in the apical plate. This is present in all the embryos. This slight difference in expression is also seen in the first stage of larval development (L1). While all 50 L1 larvae’s srcr142 and pks1 expressions overlapped, 26 of these larvae had a higher expression of srcr142 in the left coelomic pouch (Figure 3). This is important

Fig. 5. Side-view of the expression of srcr142 and pks1 in L. pictus’ 3rd larval stage (L3). The expression of srcr142 and pks1 are adjacent to one another throughout the larval body (n = 11/11) with more srcr142 expression in the aboral hood (n = 10/11) and stronger srcr142 expression on both sides of the gut as lines and not on a cellular basis (n = 2/11).

Fig. 6. Side-view of the expression of srcr142 and pks1 in L. pictus’ 4th larval stage (L4). The gut is located with the yellow asterisk and the rudiment is shown with the pink asterisk. The expression of srcr142 and pks1 are adjacent to one another throughout the larval body (n = 11/12), with more srcr142 expression on the aboral hood (n = 10/12).

Fig. 7. Side-view of the expression of srcr142 and pks1 in L. pictus’ 5th larval stage (L5). The gut is located with the yellow asterisk and the rudiment is shown with the pink asterisk. B) A slice from the DAPI z-stack to better showcase the rudiment developing alongside the gut. The expression pks1 is present throughout the larval body, with 4/10 larvae showing a localization of pks1 expression to the gut. Meanwhile, the expression of srcr142 is heavily localized to the gut (n = 10/10).

Fig. 8. Bottom-view of the expression of srcr142 and pks1 in L. pictus’ 6th larval stage (L6). The gut is located with the yellow asterisk and the rudiment is shown with the pink asterisk. B) A slice from the DAPI z-stack to better showcase the rudiment alongside the gut. The expression of srcr142 and pks1 are in close proximity to one another in the arms. When srcr142 was expressed (n = 9/10), it was more localized to the gut.

Fig. 9. Side-view of the expression of srcr142 and pks1 in L. pictus’ competent larval stage. The dark region in the transmitted light image is the juvenile before metamorphosis. When srcr142 was expressed (n = 9/10), it was strongest surrounding the rudiment (n = 6/9). Meanwhile, pks1 is still expressed throughout the body but in a lower expression, yet with a higher expression around the rudiment as well.

to note as the left coelomic pouch gives rise to the rudiment, ultimately leading to the development of the adult sea urchin.

However, this distinct expression pattern of srcr142 is conspicuously absent in L2 larvae. All 9 L2 larvae had srcr142 and pks1 expressions in close proximity to one another throughout the body without any higher expression of srcr142 in either coelomic pouch (Figure 4).

The same spatial expression pattern of srcr142 and pks1 is seen throughout the body in L3 larvae, with 10 out of 11 larvae showing a higher expression of srcr142 in the aboral hood (Figure 5). Two of the larvae showed a strong srcr142 expression on both sides of the gut in a linWear fashion not co-associated with cells.

In L4, although the rudiment starts to develop, gene expressions remain consistent with those observed in L3. The expression of srcr142 and pks1 remain in close proximity throughout the larval body, with 10 out of 12 larvae showing a higher srcr142 expression in the aboral hood (Figure 6). As the rudiment continues to develop in L5, the expression of srcr142 changes drastically. All 10 of the L5 larvae exhibited a pronounced localization of srcr142 to the larval center, with increased expression around the gut. Yet, pks1 is still present throughout the larval body, with 4 larvae showing a localization of pks1 to the gut (Figure 7).

The same is seen in L6 larvae, as 9 out of the 10 larvae displayed a higher localization of srcr142 expression in the gut. Nonetheless, srcr142 and pks1 are still adjacent to one another in the arms and around the aboral hood (Figure 8). Finally, in the competent larvae, the rudiment is fully matured with tube feet and mineralized spines6 Srcr142 was expressed in 9

Fig. 10. Aboral-view of the expression of srcr142 and pks1 in young L. pictus juveniles. These juveniles were fixed 4-6 hours post metamorphosis (hpm). This is the aboral side of the juvenile in Figure 11. B) Representative image through a microscope of a juvenile 4-6 hpm. Srcr142 is expressed in a higher amount on the aboral side of the young juveniles, yet the expression is still very low (n = 7).

out of 10 competent larvae with 6 of the larvae showing a strong expression surrounding the rudiment. Pks1 also seems to be more highly expressed around the rudiment yet is still dimly expressed throughout the larval body (Figure 9).

Between 4-6 hours post metamorphosis, a noticeable shift occurs in gene expression patterns among the 7 young juveniles, as they no longer exhibit the robust srcr142 expression observed in the competent larvae. The expression of pks1 is also greatly diminished post-metamorphosis. The expression of srcr142 and pks1 in the young juveniles is more pronounced on the aboral side. However, contrary to previous stages, srcr142 and pks1 expressions are no longer in close proximity, with srcr142 exhibiting a lower expression compared to pks1 (Figure 10 &11).

Overall, the expression of srcr142 begins in the late gastrula stage and localizes to the center of the larva after L4. It is barely expressed in the young juveniles, with a slightly elevated expression on the aboral side (Fig. 12.). Srcr142 and pks1 are also largely in close proximity from the late gastrula stage to L6, with a gradual increase in distance from one another as the larvae approach metamorphosis. There is a higher expression of srcr142 in the apical plate of late gastrula embryos. This higher expression is then seen in the left coelomic pouch of L1 larvae, the precursor to the developing rudiment. However, this is not further seen in L2 larvae, instead showing an increased expression in the aboral hood of L3 and L4 larvae. Finally, at L5 and L6, the rudiment is further developed and srcr142 is localized

Fig. 11. Oral-view of the expression of srcr142 and pks1 in young L. pictus juveniles. These juveniles were fixed 4-6 hpm. This is the oral side of the juvenile in Figure 10. Srcr142 was expressed as small specks on the oral side with very low expression (n = 7).

Fig. 12. Expression of srcr142 throughout the development of Lytechinus pictus. A) Early L. pictus stages including early blastula to L1 were taken using a 20x objective. B) Stages L2-Juvenile were taken using a 10x objective.

to the gut. Once competent, srcr142 is heavily localized to the mature rudiment. Concurrently, pks1 localizes to the mature rudiment, albeit to a lesser degree, and exhibits significantly lower expression in young juveniles. While both pks1 and srcr142 are involved in the sea urchin innate immune system, their roles may differ such that srcr142 could be involved in protecting the developing adult sea urchin and its germline, while pks1 is involved in the larva’s general immunity.

DISCUSSION

Evolution of the expression of srcr142 and pks1 throughout development

The observed extensive proximity of srcr142 and pks1 expressions from late gastrula to L6 suggests a predominant presence in pigment cells. However, this assumption is challenged by the varying location of srcr142 to the gut as larvae approach metamorphosis, while pigment cells remain present throughout the body. The cessation of srcr142 expression in pigment cells after L4 may be attributed to the growth of the rudiment in L5. It is plausible that srcr142 relocates to the gut as a defense against bacteria, thereby safeguarding the rudiment. This can be seen in L1 larvae, as there is a higher expression of srcr142 in the left coelomic pouch with no pk1 expression around. This observed expression in L1 larvae may be indicative of a transient defense mechanism aimed at temporarily protecting the developing rudiment from any bacteria present in the water. This occurrence coincides with their first ingestion of microalgae from the water column. The significance of the rudiment to the reproductive success of sea urchins is underscored by its role as the emergence site for the adult. The larval body is merely a vessel for the growing juvenile. As pigment cells are mobile, they could have moved to the mature rudiment to pass these to the young juvenile. This is seen in competent larvae as the expression of pks1 and srcr142 are both localized in the mature rudiment. However, these genes are not well expressed on the oral and aboral side of young juveniles. We were unable to see the expression of pks1 and srcr142 inside the juveniles due to the thickness of the juvenile tissue. While there may be more expression of these genes in the coelomic cavity, where coelomocytes are located, such expression cannot be visualized through HCRs. Furthermore, there should be more pks1 expression on the aboral side of the young juveniles since they are covered in red pigment cells, as seen in Figure 10.B. The lack of gene expression is therefore likely due to the thickness of the juvenile tissue as hypothesized.

Conclusion

Sea urchin larvae grow up in a very microbial dense ocean from which they must protect themselves. Unlike humans, sea urchins lack an adaptive immune system, making their innate immune system crucial. A better understanding of their innate immune system would allow us to further comprehend the evolutionary origins of the adaptive immune system. This study on srcr142 brings to light several components of the innate immune system that warrant further research. Srcr142 is expressed by pigment cells but also by cells in the left coelomic pouch, which harbor primordial germ cells giving rise to gametes and the future adult. As the larvae further develop, the expression of srcr142 quickly localizes to the gut which may be to protect the growing rudiment from ingested bacteria and foreign particles. Knowing that srcr142 could be involved in protecting L. pictus’s germline, this could be the case in other organisms. Srcr142 can be linked to protecting the germline from mutations due to viral infections as well as other pathogenic threats. These germline mutations can cause a range of catastrophes, including cancer and autoimmune disorders.

Future Directions

To further ground these results, another round of HCRs would be required on biological replicates. Bacterial exposures should also be done on L4-6 larvae to verify that srcr142 is indeed targeting the gut and not the rudiment. To determine if the expression of srcr142 is in fact in the coelomic cavity of the young juveniles, one could try to cut the samples along the dorsal-ventral axis to visualize the inside or perform RNA-Seq.

FUNDING

This research was made possible through the URS Hiestand scholarship from UCSD given to Gloria Renaudin. As well as from NIH funding to Amro Hamdoun’s lab.

AVAILABILITY OF DATA AND MATERIALS, CONFLICTS OF INTEREST

Data can be provided upon request. The authors declare no competing interests.

ACKNOWLEDGEMENTS

The authors thank Alexis Cody Hargadon for designing the probes and corresponding hairpins.

REFERENCES

[1] Alquraini A, El Khoury J. Scavenger receptors. Current Biology. 2020 Jul;30(14):R790–5.

[2] Sarrias MR, Gronlund J, Padilla O, Madsen J, Holmskov U, Lozano F. The Scavenger Receptor Cysteine-Rich (SRCR) Domain: An Ancient and Highly Conserved Protein Module of the Innate Immune System. Critical Reviews in Immunology. 2004;24(1):1–38.

[3] Dierking K, Pita L. Receptors Mediating Host-Microbiota Communication in the Metaorganism: The Invertebrate Perspective. Frontiers in Immunology. 2020 Jun 16;11.

[4] Sodergren E, Weinstock GM, Davidson EH, Cameron RA, Gibbs RA, Angerer RC, et al. The Genome of the Sea Urchin Strongylocentrotus purpuratus. Science. 2006 Nov 10;314(5801):941–52.

[5] Chun E, Buckley KM, Schrankel CS, Schuh NW, Taku Hibino, Solek CM, et al. Perturbation of gut bacteria induces a coordinated cellular immune response in the purple sea urchin larva. Immunology & Cell Biology. 2016 Jul 5;94(9):861-74.

[6] Nesbit KT, Hamdoun A. Embryo, larval, and juvenile staging of Lytechinus pictus from fertilization through sexual maturation. Developmental dynamics : an official publication of the American Association of Anatomists [Internet]. 2020 Nov 1 [cited 2023 Mar 31];249(11):1334–46. Available from: https://www. ncbi.nlm.nih.gov/pmc/articles/PMC8153651/#:~:text=Juveniles%20reach%20 sexual%20maturity%20at

[7] Heyland A, Hodin J. A detailed staging scheme for late larval development in Strongylocentrotus purpuratus focused on readily-visible juvenile structures within the rudiment. BMC Developmental Biology. 2014;14(1):22.

[8] Nesbit KT, Hargadon AC, Darin E, Renaudin GD, Lee Y, Hamdoun A, Schrankel CS. Characterization of cellular and molecular immune components of the painted white sea urchin Lytechinus pictus in response to bacterial infection. (2024). Manuscript submitted for publication.

[9] HCR RNA-FISH protocol for whole-mount sea urchin embryos (Strongylocentro-tus purpuratus). Molecular Instruments. (2023, February 13). Retrieved April 13, 2023, from https://files.molecularinstruments.com/MI Protocol-RNAFISH-SeaUrchin-Rev8.pdf.

ANURAN DISTRIBUTION AND RICHNESS IN A TROPICAL MONTANE FOREST IN MONTEVERDE, COSTA RICA – AN UPDATED SURVEY

Taryn Cornell*, Frank Joyce§, Federico Chinchilla§

*UCSD, Revelle, General Biology, 2023;§Tropical Biology & Conservation Program, Education Abroad Program, University of California, Monteverde, Costa Rica

ABSTRACT

Montane ecosystems support high endemism and biodiversity. As Neotropical communities encounter climactic pressures, measuring species richness and abundance across an elevational gradient provides insight into their responses. Amphibians such as frogs and toads (Order: Anura) are uniquely sensitive to environmental change and are experiencing severe declines globally. In this study, Anuran surveys were conducted across four distinct Holdridge life zones in Monteverde, Costa Rica, to compare with a corresponding study in 2019. Holdridge life zones are characterized by three characteristics of a region: heat, precipitation, and moisture. Visual encounter surveys (VES) and audio encounter surveys (AES) methodologies were used at five non-continuous locations. Data from 134 individuals across 17 species was collected during May, 2023. Fewer species were encountered as elevation increased, as expected. An analysis of observed species’ zonal distributions revealed two species (C. bransfordii and C. stejnegerianus) outside of their expected Holdridge life zones based on recent species distribution guides. A high variability in precipitation was observed, consistent with decades of climate change data. Our results did not indicate a significant difference in Anuran community composition related to precipitation. This study demonstrates the necessity for sustained, long-term collection of amphibian data in biodiversity hotspots. In turn, a cohesive understanding of community responses can inform conservation efforts.

INTRODUCTION

Globally, about 40% of amphibian species are threatened and populations continue to decline at an alarming rate.1 A series of interconnected factors are extinguishing this unique taxon, including climate change, habitat destruction and fragmentation, contamination from pesticides, and disease—especially chytridiomycosis.2 As Neotropical ecosystems rapidly change in response to climate change and anthropogenic factors, communities deteriorate and populations migrate to more suitable regions. This leads to extinctions as organisms fail to adapt quickly enough to their new environments. The amphibians of Costa Rica are a resounding demonstration of this phenomenon, with three species confirmed extinct and 59 endangered.3 Tropical montane forests accommodate a variety of microclimates, high biodiversity, and endemism.4 Endemic species are those confined to a single geographic area. However, the complex interplay of biotic and abiotic factors in a small region also makes these forests distinctly vulnerable to human impacts.5 In Monteverde, Costa Rica, large areas of montane forest are protected to combat biodiversity loss and to preserve habitats. Despite this, the “enigmatic decline” of amphibians persists, more notable in the Neotropics. Enigmatic decline is an elusive type of population decline because it occurs in a supposedly suitable habitat. One speculated driver of enigmatic decline is climate change, among others.1 In Monteverde, the decline in mist frequency and a significant drying trend align with climatic warming and declines in local diversity.6

Amphibians are a taxa that includes frogs, salamanders and caecilians. They are a useful indicator of ecosystem health due to

their peculiar physiological relationship with their environment.7 Amphibians undergo a niche shift as they develop from aquatic to terrestrial life. As such, this group requires multiple types of suitable habitats to maintain healthy populations. In their metamorphosis, amphibians experience a wide range of abiotic stressors. For instance, pollutants in freshwater and heightened UV exposure have been correlated with increased mortality, smaller larvae size, and developmental malformations.2 In addition to their unique evolutionary lineage, amphibians are a sizable fraction of biomass that trickles through the trophic levels of higher predators in food webs. These characteristics make the evaluation of their current status and subsequent conservation efforts pivotal to supporting resilient ecosystems.

Determining the most effective conservation actions hinges on consistent monitoring of amphibian communities. UCEAP student Elizabeth L. McDonald established a recent record of frogs and toads in Monteverde.8 Over the month of May, 2019, McDonald conducted visual and auditory surveys across four Holdridge life zones in five locations to evaluate the species distribution and richness of anurans across an altitudinal gradient. The data was compared to distribution ranges established 20-30 years ago.9,10 McDonald identified four species that inhabited unexpected Holdridge life zones.8

The aim of this study was to determine whether the amphibian species distribution and richness across four Holdridge life zones had shifted in comparison to data collected in 2019 and historical field guides. It was hypothesized that species richness would decrease, that family composition would reflect variability in precipitation, and that species would be driven upslope in response to climate alterations.

MATERIALS AND METHODS

Study Sites

The study sites are located in Monteverde, Costa Rica, in the Cordillera de Tilaran mountain range at elevations between 1200-1700 m. The region of Monteverde encompasses a few urban communities, the Children’s Eternal Rainforest, and the Santa Elena Cloud Forest Reserve on the Pacific and Caribbean slopes.11 Monteverde has seven of the twelve life

Duellmanohyla rufioculis

Table 1. Study sites corresponding to Monteverde Holdridge life zones. Holdridge life zones are characterized by three characteristics of a region: heat, precipitation, and moisture. Evaporation (heat) and precipitation correspond to latitude and longitude, respectively. The selected regions in the Cordillera de Tilaran demonstrate distinctive differences in these three characteristics despite close proximity to each other (Holdridge, 1967).

zones found in Costa Rica.12 This study incorporates four: Tropical Premontane Wet Forest (Zone 2), Lower Montane Wet Forest (Zone 3), Lower Montane Rainforest (Zone 4), and Premontane Rainforest (Zone 5). Tropical Moist Forest (Zone 1) and Tropical Wet Forest (Zone 6) were not included due to limited accessibility. The study sites corresponding to Holdridge life zones were determined by McDonald.8,12 Since their investigation, a thorough verification and update of the zones were conducted (Table 1, Supp Fig 1).13,9 Surveys were conducted on trails and along streams at Santuario Ecológico Monteverde (Zone 2), Café Monteverde (Zone 2), Rachel and Dwight Cramdell Reserve (Zone 2/3), Estación Biológica Monteverde (Zones 3/4/5), and San Gerardo Biological Station (Zone 5). Zones 1 and 6 have no corresponding study site. McDonald conducted a total of 15 survey nights from May 6th, 2019 to May 29th, 2019, with distribution as follows: 3 nights at Estación Biologica, 3 at Instituto de Monteverde, 1 at Café Monteverde, 2 at Santuario Ecológico, and 6 at San Gerardo. All surveys occurred between 7:00 p.m. to 10:00 p.m., accumulating a total surveying time of 21.16 hours. On average, McDonald spent 1.41 hours per night surveying. Felix Salazar, a Monteverde Institute staff working with McDonald, conducted

one survey at an unknown time at an undescribed site called the “Continental Divide.” McDonald also performed one survey night at a man-made pond on the Estación Biológica property. Both the “Continental Divide” and the man-made pond surveys were not included in the 2023 data collection period. Despite these discrepancies between the 2019 and 2023 survey locations, surrogate surveys were conducted. In this study, the “Continental Divide” survey was replaced with one survey at the Estación Biológica S. Principal. McDonald’s surveys at the Estación Biológica’s man-made pond were substituted with the S. Quebrada due to the presence of two small streams.

Amphibian Sampling

Visual and acoustic encounter surveys were used to identify frog species and local abundance. A visual encounter survey(VES) is a simple amphibian survey procedure in which a field technician systematically searches for animals by walking through a habitat for an allocated amount of time, or ‘person-hours.’ The VES technique provides species composition data, especially of forest understory anurans. In turn, this species list can be used to estimate relative abundances.14 An acoustic encounter survey (AES) is a similar survey technique that relies on identifiable differences in male amphibian calls.

at least their family classification.

Surveys were conducted along trails and streams from May 5th, 2023 to May 23rd, 2023 between 7:00 p.m. to 10:00 p.m. Notably, the transition period from wet to dry season occurs in April to May, providing the most significant diversity and abundance of amphibians.15 Survey efforts consisted of 1 to 2 individuals conducting VES and AES whilst disturbing leaf litter, logs, stones, and vegetation. Survey nights were precipitationfree, except for one night, May 19th, 2023. Surveys during rain were preferred because amphibian activity is higher during

Figure 1. Family composition of McDonald and Cornell Anuran surveys. McDonald and Cornell identified 143 frogs from 7 families and 134 frogs from 6 families, respectively. Percentages were calculated based on the number of frogs identified to

precipitation events. But, there were uncharacteristically few rainy nights in May, 2023. Chance encounter visual observations on April 27th, April 30th, May 9th, and May 25th, 2023 were included in the data. Coordinate and altitude data were collected post-observation when possible. A total of 24.85 hours were spent surveying.

In order for species richness and relative abundances to be comparable, it is essential that survey methodology and habitat structure be similar to past surveys. Potential biases and differences in individual expertise can limit comparisons of repeated VES data.14

To combat this, experienced naturalist Eladio Cruz accompanied seven surveys, and ecologist Dr. Moran accompanied 1 survey.

The following data were collected during surveys: date, time, general location, species, temperature (ºC), altitude (m), coordinates, substrate, and other notes (Appendix C). If possible, frogs were photographed and handled for more accurate species identification. These categories are identical to McDonald’s data collection, with the addition of GPS coordinates.8 Coordinate data was added for both ease of replication and geospatial analysis along the altitudinal gradient. The ability to replicate this series of surveys is a foundational component of this study. Coordinates and altitude were recorded with a Garmin global positioning system (GPS), temperature with a traditional thermometer, and photographs with a digital Olympus TG5 camera. Altitude measurements were crossreferenced with Altitude from GPS point Calculator by dCode, an online open-source altitude tool, on a case-by-case basis.

Data Analysis

Species richness and abundance analyses were performed using Google Sheets and R statistical software. A linear regression model was used to describe the relationship between species richness and elevation (Figure 2). Precipitation data was collected by the Joyce-VanDusen household, which is parallel to the Bajo del Tigre Reserve at approximately 1350 meters (Figure 3). Additional precipitation data was acquired from Richard LaVal and the Monteverde Institute. A map of the Holdridge life-zones and study site locations was created using ProCreate, with the aid of previous maps and online geospatial mapping tools such as OpenStreetMaps and AllTrails (Appendix B, Supp. Figure 1).13,9 Missing data were estimated using averages of the adjacent data collected. Adjacent data was utilized to average a missing piece of data if it was collected within 30 minutes of the absent data. Missing altitude data was estimated using this method, as well as referring to previous surveys in the same location. All estimations are denoted by an asterisk (Appendix C).

RESULTS

Composition of Families between Surveys

A total of 134 individuals were observed, classified as 17 species that span six families. 17 individuals were designated as Craugastor, and one individual was classified as Anura due to species ambiguity (Table 2). An analysis of the anuran families and their relative abundance in 2023 displays a community structure similar to that of McDonald’s dataset (Figure 1). McDonald identified 143 frogs to their species classification (Appendix C), including one Bufonidae. The Bufonidae family was not observed in May, 2023. However, this series of surveys encountered 13.52% fewer Centrolidae

Table 2. Abundances and species richness across elevational gradient. Taxonomic classification and number of individuals observed at the following study sites: Santuario Ecológico Monteverde, Rachel and Dwight Cramdell Reserve, Estación Biológica de Monteverde and San Gerardo Biological Station. Elevations correspond to minimum and maximum elevation measured during surveys. Data was obtained from 30 April 2023 to 25 May 2023. *Unidentified Craugastor observations omitted from this count

Figure 2. Species richness across an altitudinal gradient. Species richness observed by McDonald 2019 (green) at elevations 1134-1700 m and Cornell 2023 (black) at elevations 1178-1805 m with trendlines. One chance encounter observation at 1178 m in San Gerardo was omitted from this analysis.

Espadarana prosoblepon

Table 3. Comparison of expected and encountered zonal distributions. Two differences in zonal distributions (C.bransfordii and C.stejnegerianus) were observed in 2023 (X), and 5 differences in zonal distributions were observed in 2019 (O). Amphibian encounters outside of the expected zone are bolded. McDonald characterized expected Holdridge life-zones based on decades older data9,10than this study.15 For data collected in 2023, species distribution elevations were converted from Leenders ranges into the corresponding Holdridge life-zones in Monteverde (Table 1).

and 12.18% more Craugastoridae specimens than McDonald. The following species were observed at relatively high abundances: Rana warszewitschii (13.3%), Diasporus diastema (11.1%), Duellmanohyla rufioculis (10.4%) and Isthmohyla pseudopuma (10.4%).

Amphibian distributions along an elevation gradient

An analysis of anuran species richness displayed a statistically significant negative correlation across an altitudinal gradient of 1178-1805 meters (Figure 2, R2=0.414, p-value≤0.119). Note that this is all identified Anura species data from multiple noncontinuous locations of the Cordillera de Tilaran mountain range. The presented trend contradicts McDonald’s 2019 surveys, which displayed no statistical significance for the same analysis (Figure 2, R2=0.414, p-value≤0.119).

Zonal distribution

In 2019, five species were encountered outside of their historical Holdridge life zones, including P. ridens, I. zeteki, R. taylori, H. talamancae, and C. crassidigitus (Table 3). This study found two different species, C. stejnegerianus and C. bransfordii, outside of their Holdridge life zones, according to more recent amphibian distribution guides. C. stejnegerianus was encountered at a lower elevation than expected. Conversely, C. bransfordii was observed above its expected elevation.

Climate as a driver of shifts in species distribution

The climatic circumstances of the survey periods offer a more rigorous assessment of differences in species richness and zonal distributions between and during respective survey periods. In Monteverde, increasing variability in precipitation and drier days have been demonstrated since the 1980s.6 Since amphibians have unique physiological and reproductive behaviors associated with temperature and rainfall, recording these two abiotic factors allows survey analysis to be tied to large-scale climatic trends.16,17 Precipitation data taken in Monteverde at elevation ~1350 meters reinforces variability in monthly and annual precipitation (Figure 3). The recent surveys conducted between April 30th and May 25th, 2023 saw 2.97 mm of precipitation versus 18.02 mm during the 2019 survey period (Supp. Figure 2).

Figure 3. Average precipitation in Monteverde 2016-2023. At Monteverde, the average monthly precipitation from September 2016 to May 2023. Standard deviations are not displayed, but can be accessed (Appendix D). Precipitation data was provided by the Joyce-VanDusen family at ~1350 m.

Isthmohyla pseudopuma

Craugastor bransfordii

DISCUSSION

Neotropical amphibian communities are shifting and adapting in response to climatic changes. This study aimed to characterize the current Anuran composition and species richness across Holdridge life zones in tropical montane forests. Altogether, the frog abundance and family composition analyses collected in May, 2023 are comparable to McDonald’s May, 2019 surveys. This assessment of the two datasets contrasts the original hypothesis, that frog abundance and family composition would be dramatically different due to weather. However, only minor differences in the Anuran family composition were observed. The 2023 data demonstrated 11.43% more Craugastoridae individuals and 13.52% fewer Centrolidae individuals.

The correlation between frog activity and precipitation has been long established. Neotropical frog communities have diverse modes of reproduction, ranging from year-round to seasonal, and often return to home ranges for breeding. Reproductive patterns influence habitat use in anuran communities, thereby affecting the composition and relative abundance of surveys. In the absence of rain, frogs demonstrate a series of behavioral and morphological adaptations to conserve water and moisture. Anura species have been sighted to make burrows or to retreat to log interiors, rocks, or canopy coverage.18 Tying these characteristics back to differences in family composition, the decrease in Centrolidae encounters is reasonable. The Centrolidae family, known as glass frogs, retreat to high canopy when not courting or breeding on perches near streams.

The high relative abundance of I. pseudopuma and D. diastema in both datasets mimic the upward trend in population size that has been recorded since their initial population crash in 1987 and second decline in 1998.11 The prevalence of I. pseudopuma, a species deemed ‘explosive breeders,’ can be explained by the ephemeral breeding pond observed on April 30th, 2023, post-rainfall. Despite differences in precipitation during the 2019 and 2023 survey periods (Figure 3), both had a relatively high abundance of D.diastema. D.diastema could have a higher abundance in both surveys due to its ease of identification by auditory call.

The two species encountered outside their expected distribution zones did not display a significant trend in upper or lower elevation (Table 3). One C. stejnegerianus individual was identified above its expected zones, along the S. Principal at the Estacion Biologica at 1805 meters (Table 2). Considering that only one survey night was conducted along this ridge of the Pacific slope, more intensive person-hours in this region may reveal a more pronounced relationship. This singular observation of C. stejnegerianus raises concern that deteriorating conditions in its native life zones (Zones 1 and 2) may shift populations upslope. A similar conclusion was drawn from observations of C. bransfordii outside of its expected life zone (Zone 1, Table 2).

Craugastor bransfordii was recorded at Santuario Ecologico (Zone 2) and San Gerardo Station (Zone 5), which are on either side of the Continental Divide. Thus, both frog species display an upslope migration pattern through this culmination of four individuals. Overall, these two observations necessitate further surveys to determine if species migration upslope is occurring on a larger scale. It is common for herpetofaunal studies to describe a decrease in species richness as elevation increases along a gradient.19 The 2019 and 2023 surveys display a similar, weak correlation to this expected trend (Figure 2). However, this conclusion could be influenced by the fewer person-hours spent surveying high elevations as opposed to low to middle elevations. Ideally, future surveys would standardize the amount of hours spent at each study site to reevaluate this trend. It was expected that differences in precipitation amount and pattern would influence the relative abundance and species richness. This was hypothesized because amphibian families rely on different strategies to reproduce, as previously exemplified in Centrolidae. Although a correlation between precipitation and amphibian data was inconclusive between the 2019 and 2023 data, additional robust longitudinal studies may shift this perception. Yet, it is undeniable that the precipitation data reflects inconsistencies uncharacteristic of Neotropical montane forests.

Since past research associated dry periods in Monteverde with amphibian declines, it is necessary that additional surveys be conducted to address gaps identified in this study.6

In future potential studies that utilize Holdridge life zones, an evaluation of the zones could be beneficial. The life zones of Monteverde may have changed in response to the shifts in precipitation patterns and daily temperature extremes that Monteverde has experienced in the past decades.9,6 The annual precipitation between 2016 to 2023 reflects these climatic shifts through precipitation variability on a more recent time scale (Figure 3). As more destructive climatic events transpire with increased frequency, the slope orientation or landmarks delineating the divide of the Holdridge life zones in Monteverde may be reshaped.

The replicability of McDonald’s original surveys could have had an influence on the conclusions drawn between data sets. Although the same study sites were visited for near

Photography by Taryn Cornell. Nine of 17 species observed during 2023 surveys are pictured.

Pristimantis cruentus

Craugastor podiciferus

Craugastor underwoodi

equivalent survey nights, the routes taken and the time spent surveying trails versus nearby streams could have differed. For instance, the 2023 survey trail in San Gerardo was not the identical route taken by McDonald in 2019. The same number of survey nights was not conducted at San Gerardo. As a result of different survey routes, multiple observations in 2019 were made <1200 meters that could not be replicated in 2023. Despite this discrepancy, GPS coordinate data and supplemental maps collected in May, 2023 ensure the replicability of future amphibian surveys in Monteverde. Alternatively, standard visual transect sampling (SVTS) could be utilized to further guide terrestrial amphibian surveys for study sites with varying distances.20 Overall, this continuation of long-term amphibian surveys, such as McDonald’s, provides an invaluable lens into the complex network of factors that are altering the diversity and composition of Neotropical systems. Through future studies at these sites, the management and monitoring of threatened amphibian species must be continued. The survey efforts of this and other researchers in La Selva, San Ramon, and the Cloudbridge Forest Reserve buttress the conservation efforts of biodiversity hotspots such as Costa Rica. With robust data sets on multiple amphibian communities, biologists, policy makers, and advocates can evaluate and prioritize at-risk species and regions efficiently and accurately. As climatic shifts become more dramatic, connectivity in Neotropical communities through biological corridors and continuous forest will be essential to preserving biodiversity. Multi-year monitoring efforts not only contextualize the current state, but can be used by subsequent researchers to model potential outcomes from conservation policies.

ACKNOWLEDGEMENTS

I would like to thank Eladio Cruz, who was willing to accompany and teach me about Monteverde’s herpetofaunal community at a moment’s notice. Without his expertise and patience, this project would not be here. I would also like to thank the naturalists and ecologists who helped me identify frogs: Dr. Matthew Moran, Dr. Alan Pounds, and Geiner Alvarado. To my advisors and mentors, Dr. Frederico Chinchilla and Dr. Frank Joyce. Also, thank you to the Santuario Ecológico for allowing me on their reserve. Finally, thank you to UCEAP and the Monteverde Institute for lending me equipment and endless support.

REFERENCES

[1] Luedtke, Jennifer A., Janice Chanson, Kelsey Neam, Louise Hobin, Adriano O. Maciel, Alessandro Catenazzi, et al. “Ongoing Declines for the World’s Amphibians in the Face of Emerging Threats.” Nature 622, no. 7982 (Oct 2023): 308–14. https://doi.org/10.1038/s41586-023-06578-4

[2] Blaustein, Andrew R. and Joseph M. Kiesecker. “Complexity in conservation: lessons from the global decline of amphibian populations.” Ecology Letters 5, no. 4 (July 2002): 597-608. https://doi.org/10.1046/j.1461-0248.2002.00352.x

[3] IUCN. “The IUCN Red List of Threatened Species.” Version 2022-2; 2023.

[4] Malhi, Yadvinder, M. Silman, Norma Salinas, M. Bush, P. Meir, and Sassan Saatchi. “Introduction: Elevation gradients in the tropics: laboratories for ecosystem ecology and global change research.” Global Change Biology 16, no. 12 (Nov 2010): 3171–3175. https://doi.org/10.1111/j.1365-2486.2010.02323.x

[5] Mata-Guel, Erik O., Malcolm C. K. Soh, Connor W. Butler, Rebecca J. Morris, Orly Razgour, and Kelvin S.-H. Peh. “Impacts of anthropogenic climate change on tropical montane forests: An appraisal of the evidence.” Biological Reviews 98, no. 4 (March 2023): 1200–1224. https://doi.org/10.1111/brv.12950

[6] Pounds, J. Alan, Michael P.M. Fogden, and John H. Campbell. “Biological response to climate change on a tropical mountain.” Nature 398, (April 1999): 611–615. https://doi.org/10.1038/19297

[7] Amarasinghe, A.A. Thasun, Chairunas A. Putra, Sujan M. Henkanaththegedara, Asri A. Dwiyahreni, Nurul L. Winarni, Sunaryo, et al. “Herpetofaunal diversity of West Bali National Park, Indonesia with identification of indicator species for long-term monitoring.” Global Ecology and Conservation 28 (May 2021): e01638. https://doi.org/10.1016/j.gecco.2021.e01638

[8] McDonald, Elizabeth L.. “Anura distribution and richness in a tropical montane forest in Monteverde, Costa Rica.” Tropical Ecology Collection [Monteverde Institute] 206 (June 2019). https://digitalcommons.usf.edu/tropical_ecology/206

[9] Hayes, M. P., Pounds, J. A., and Timmerman, W. W. An annotated list and guide to the amphibians and reptiles of Monteverde Costa Rica. Oxford, Ohio: Miami University; 1989.

[10] Savage, J. M. The amphibians and reptiles of Costa Rica. London: University of Chicago Press; 2002.

[11] Nadkarni, N.M. and Wheelwright, N.T. Monteverde: Ecology and Conservation of a Tropical Cloud Forest - 2014 Updated Chapters. Bowdoin Scholars' Bookshelf, Book 4; 2014. http://digitalcommons.bowdoin.edu/scholars-bookshelf/5

[12] Holdridge, L.R. Life zone ecology, 206. Revised edition. San Jose, Costa Rica: Tropical Science Center; 1967.

[13] Hayes, M. P., Laval, R. The Mammals of Monteverde. San Jose, Costa Rica: Tropical Science Center; 1989.

[14] Heyer W. R., Donnelly, M.A., Roy W. McDiarmid, R. W., Hayek, L. C., and Foster M. S. Measuring and monitoring biological diversity: standard methods for amphibians, 80-88. Washington, DC: Smithsonian Institution Press; 1994.

[15] Leenders, T. Amphibians of Costa Rica. Ithaca, New York: Zona Tropical Press, 6-8: 144-146; 2016.

[16] Hillyard, Stanley D. “Behavioral, molecular and integrative mechanisms of amphibian osmoregulation.” Journal of Experimental Zoology 283, no. 7 (April 1999): 662–674. https://doi.org/10.1002/(SICI)1097010X(19990601)283:7%3C662::AID-JEZ5%3E3.0.CO;2-L

[17] Richter-Boix, Alex, Gustavo A. Llorente, and Albert Montori. “Breeding phenology of an amphibian community in a Mediterranean area.” Amphibia-Reptilia 27, no. 4 (Dec 2006): 549–559. https://doi. org/10.1163/156853806778877149

[18] Duellman, W.E. and Trueb, L. Biology of Amphibians. Baltimore, Maryland: Johns Hopkins University Press; 1994.

[19] Gifford, Matthew E. and Kenneth K. Kozak. “Islands in the sky or squeezed at the top? Ecological causes of elevational range limits in montane salamanders.” Echography 35, no. 3 (Mar 2012): 193-203. https://doi.org/10.1111/j.16000587.2011.06866.x

[20] Veith, Michael, Stefan Lötters, Franco Andreone, and Mark-Oliver Rödel. “Measuring and monitoring amphibian diversity in tropical forests. II. Estimating species richness from standardized transect censing.” Ecotropica 10, (Nov 2004).

SENIOR HONORS THESIS

Red Panda(Ailurus fulgens) yearning for the world outside its enclosure at the San Diego Zoo

UC San Diego's Senior Honors Thesis Program allows undergraduate biology majors to work one-on-one with faculty mentors to pursue independent lab research. These are the abstracts of all the exceptional research projects conducted by honors students this past year.

photo by: Inaya Nicholls

FARID AKHAVAN REZAYAT

Thurgood Marshall College, Human Biology Major

PI: Yuliya Skorobogatko, Ph.D., UCSD School of Medicine, Department of Endocrinology

The Role of Glucose Uptake in Brown Fat in Weight Loss Induced by β3 Adrenergic Agonist

Obesity is widespread and threatens health and longevity. Success of GLP-1 receptor agonists in achieving weight loss created tremendous enthusiasm for obesity research. The compounds reduce food consumption, but have shortcomings, including loss of muscle mass. We explore anti-obesity actions of β3 adrenergic agonist CL316,243 (CL), which acts specifically on adipose tissue to induce heat production, thus dissipating energy generated by increased oxidation of glucose and lipids, instead of storing it. Using metabolic cages, we discovered that CL-stimulated heat production vanishes with repetitive CL injections, causing a plateau in weight loss. We generated mice with increased glucose uptake in brown fat and demonstrated that CL-stimulated heat production and weight loss are improved in these mice. Brown fat is present in 7% of adult population. CL may promote weight loss in this cohort, particularly when glucose uptake in brown fat is stimulated, for example by insulin, which remains to be tested.

SARAH BERGENDAHL

Eleanor Roosevelt College, Molecular and Cell Biology

Major

PI: Trey Ideker, Ph.D., UCSD Department of Medicine Adjunct Professor, Departments of Bioengineering and Computer Science

Validation of Synthetically Lethal Interactions between KDM5C and PARP7 in Cancer Cells

One in every five deaths in the United states is due to cancer, making it the second leading cause of death. The most common treatment today, chemotherapy, is extremely toxic to patients. In synthetic lethality, the newest strategy in treatments tailored to the patient's cancer, two genes must be altered or inhibited to have an effect. From a double CRISPR screen testing potential pairs we identified a histone modifying genes KDM5C and PARP7 to potentially be synthetically lethal. To validate KDM5C and PARP7 synergy, a CRISPR knockout of KDM5C was done to mimic a genomic alteration, and treatment with a PARP7 inhibitor was used to analyze changes in sensitivity to the drug before and after KDM5C knockout. The expectation is that KDM5C knockout sensitizes cancer cells to PARP7 inhibition. These findings would suggest that KDM5C mutational status can be a biomarker for PARP7 since these are a synthetic lethal pair.

Roger Revelle College, Human Biology Major

PI: Aaron F. Carlin, M.D., Ph.D., UCSD School of Medicine, Department of Pathology and Medicine

Role of SREBPs in Human Inflammatory Signaling

Lipid metabolism can alter innate immune responses and is manipulated by some viruses. SREBP (sterol regulatory element-binding protein) transcription factors are master regulators of fatty acid and cholesterol synthesis. We found that Zika virus (ZIKV) infection of human dendritic cells (DCs), a key target of infection, specifically induces lipid biosynthesis gene expression due to increased SREBP recruitment to lipid gene promoters. Additionally, pharmacologic inhibition of SREBPs decreases ZIKV production. Thus, ZIKV infection stimulates lipid biosynthesis in DCs to enhance infection and this pathway can be pharmacologically targeted. However, the mechanisms by which ZIKV activate SREBPs and how this alters innate immune responses are not known. Therefore, we developed a system to exogenously express functionally active and inactive forms of SREBP2 in primary human DCs. We are now using this system to understand how upregulation of lipid biosynthesis via SREBP2 alters human DC functional responses to synthetic ligands and virus infection.

LAWRENCE CHAN

ELSA CHEN

John Muir College, Molecular and Cell Biology Major

PI: Jose Pruneda-Paz, Ph.D., UCSD School of Biological Sciences, Department of Cell and Developmental Biology

Investigating Systemic Transcriptional Changes Induced by a Localized Bacterial Infection in Plants

Plant hosts undergo significant gene expression changes upon pathogen infections to induce immune responses and reduce growth and development. Most transcriptomic analyses characterized gene expression profiles locally at the infection site. However, systemic responses in distal non-infected tissues are key to regulating plant survival. Importantly, our lab has recently found that systemic changes upon infection were distinct depending on the organ (i.e. shootapex or leaves). In this project, I used RNA sequencing to explore gene expression changes in the shoot apex and distal leaves of Arabidopsis thaliana plants infected with the bacterial pathogen Pseudomonas syringae in a single leaf. This analysis revealed that upon infection, several genes are differentially expressed in distal plant organs and that most differentially expressed genes are upregulated and organ-specific (mostly found in the shoot apex rather than in distal non-infected leaves).

YUHAN CHEN

Sixth College, Mathematics Major, Molecular and Cell Biology

Major

PI: Julie Law, Ph.D., Salk Institute for Biological Studies, Plant Molecular and Cellular Biology Laboratory

Multiple Transcriptional Regulators Control CLSY3-Dependent Methylation in Flowering Tissues

DNA Methylation plays key roles in gene regulation and transposon silencing. In the flowering plant A. thaliana, tissue-specific DNA methylation is established by de novo DNA methylation through the RNA Directed DNA Methylation (RdDM) pathway. In the RdDM pathway, RNA POLYMERASE IV (Pol IV) generates small RNA (smRNA) molecules which recruit DNA methylation machinery for site specific methylation. The CLSY family (CLSY1-4) of putative chromatin remodelers are the master regulators of smRNA biogenesis, yet how individual CLSYs are recruited to chromatin remain unclear. Here, we report that several transcriptional regulators, identified through a forward genetics screen, are essential for CLSY3 dependent methylation in flowering tissue. Mutations in these factors display deficient epigenetic patterning in both the maternal and paternal reproductive tissue including loss of tissue specific methylation. Complementation via exogenous transgene rescues mutant phenotype. Altogether, our data suggests a means of epigenetic targeting guided by transcriptional regulators.

Sixth

PI: Binhai Zheng, Ph.D., UCSD School of Medicine, Department of Neuroscience

Assessing the Impact of DLK/ LZK Deletion in Injured Corticospinal Motor Neurons

Corticospinal tract (CST) neurons, like most central nervous system (CNS) neurons, do not spontaneously regenerate following injury. Various signaling pathways play key roles in both injury signaling and pro-death or pro-regeneration post-injury responses. Dual Leucine Zipper Kinase (DLK) and Leucine Zipper Kinase (LZK) are MAP3Ks known to regulate diverse outcomes in injured neurons, including axonal growth and neuronal apoptosis. Additionally, deletion of phosphatase and tensin homolog (PTEN) is known to promote regeneration of CNS neurons. Our lab previously showed that DLK and LZK are required for PTEN-deletion-induced regeneration of CST neurons. Furthermore, preliminary data from our lab indicates that DLK/LZK deletion promotes cell survival in an injury model where CST neurons are injured close to their cell bodies. Here, we use PTEN, DLK/LZK-, and DLK/LZK/ PTEN- deleted mice in combination with this “death model” to assess whether and how these pathways may synergize to regulate CST death.

CATALINA ZARAGOZA FOGG

Eleanor Roosevelt College, Neurobiology Major

PI: Trey Ideker, Ph.D., UCSD Department of Medicine Adjunct Professor, Departments of Bioengineering and Computer Science

Using Prime Editing to Understand Oncogenic Consequences of Nuclear Pore Complex Mutations

The nuclear pore complex (NPC) mediates macromolecular transport in and out of the nucleus. Mutations in the NPC commonly occur in cancers, but the possible oncogenic functions of a majority of NPC mutations remain unknown. Here, we use Prime Editing to introduce specific point mutations observed in cancers in the NPC, which have resisted genetic interrogation due to the lethality of their knockout by conventional CRISPR/Cas9 nuclease editing. To design prime editing guide RNAs (pegRNAs) that can most efficiently introduce each intended NPC edit, we employed a pooled screen to empirically measure the editing efficiency of candidate NPC-editing constructs. Selected pegRNAs were used to make their intended NPC edits in mammalian cells, which were harvested for mRNA sequencing analysis . By comparing transcription profiles between cells that received different NPC mutations, we propose mechanisms of cancer progression that result from disruption of the NPC.

TYLER DIEP

College, Human Biology Major

SAMVEL SARKIS GABOYAN

Eleanor Roosevelt College, Human Biology Major

PI: Laura Crotty Alexander, M.D., UCSD School of Medicine, Department of Medicine, Division of Pulmonary, Critical Care, Sleep Medicine and Physiology

Inflammatory and Anti-inflammatory Effects of Chronic E-cigarette Aerosol Inhalation on Asthma in a Mouse Model

Background: E-cigarette vaping is popular among adolescents, and asthma, characterized by airway inflammation and bronchoconstriction, is common in this population. Cigarette smoking promotes and triggers asthma, but it is unknown whether e-cigarettes will do so. We used an ovalbumin T2-high mouse model of asthma, combined with daily e-cigarette exposures, to assess the impact of e-cigarettes on recruitment of inflammatory cells into the lungs during allergic inflammatory airways disease (AIAD) pathogenesis. Results: Inhalation of flavored and nonflavored nicotine-containing e-cigarette aerosols before and during AIAD induction was associated with reduced lung inflammatory cells. However, nonflavored, nicotine-free e-cigarettes increased lung inflammatory cells. Conclusion: Different chemicals within e-cigarette aerosols have varying effects on the inflammatory state of the lung, leading to altered immunopathology in our mouse model of asthma. These data suggest that while daily inhalation of the base chemicals within e-cigarette aerosols have pro-inflammatory effects, nicotine and some flavorants have anti-inflammatory effects.

JULIA KEUM

Thurgood Marshall College, General Biology Major, Science Education Minor

PI: Andrew Muroyama, Ph.D., UCSD School of Biological Sciences, Department of Cell and Developmental Biology

Measuring the Impact of Stomatal Alignment on Pore Opening in Arabidopsis thaliana

Stomata, microscopic pores on the surface of aerial tissues, enable gas exchange between the plant and its environment. Because stomata are key regulators of water use, understanding how their development impacts agricultural productivity is of paramount importance. We recently identified that stomatal pores are aligned along the proximodistal axis of dicot leaves, but the functional consequences of this remained unknown. To test whether stomatal alignment impacts the efficiency of pore opening in Arabidopsis thaliana, we used confocal microscopy to monitor stomatal apertures following blue light stimulation. Our results showed stomata aligned with the proximodistal leaf axis had smaller apertures compared to unaligned stomata in the same leaf. In ongoing work, we are measuring gas exchange rates in Arabidopsis mutant leaves with hyperaligned stomata. Our results revealed novel links between stomatal alignment and opening that have implications for future engineering efforts to improve stomata efficiency in plants facing increasingly hostile climates.

SOLOMON KIM

Seventh College, General Biology Major

PI: Susan Kaech, Ph.D., Professor & Director of the NOMIS for Immunobiology and Microbial Pathogenesis at the Salk Institute

Elucidating the Role of CXCL16 Isoforms in T-Cell Differentiation

Chemokines are chemoattractants that are crucial for the differentiation and trafficking of immune cells, like T cells. A chemokine called CXCL16 promotes immune cell infiltration, survival, and functionality in tumors, existing in both transmembrane and soluble forms. However, there is limited understanding of these isoform's role in T cell differentiation, critical in the immune response. We hypothesize that structural variation among CXCL16 isoforms contributes to different levels of functionality. To investigate differentiation, we will utilize protein engineering techniques to produce dominantly transmembrane, truncated, and soluble forms of CXL16. Subsequently, we will in-vitro culture tumors and immune cells, expressing various forms of CXCL16, and co-culture them with T cells. This project will be critical for the implication of future studies on CXCL16 involving differentiation, providing foundational insights that could potentially lead to the development of immunotherapies tailored to modulate CXCL16-mediated T-cell differentiation.

RYAN LAM

Sixth College, Neurobiology Major

PI: Pengzhe Lu, Ph.D., UCSD School of Medicine, Department of Neuroscience

Deletion of PTEN/SOCS3 in Neural Progenitor Cell Graft Promotes Graft-derived Axonal Growth after Spinal Cord Injury

More than 300,000 people in the United States suffer from Spinal Cord Injury (SCI). Previous research indicates that deleting PTEN and SOCS3 promotes long-distance axon regeneration after optic nerve injury. This study investigates whether deleting PTEN/SOCS3 in transplanted embryonic spinal cord derived Neural Progenitor Cells (NPCs) enhances graft-derived axonal growth. Using a T8 crush model, we grafted embryonic day 11 spinal cord derived NPCs from PTEN/ SOCS3/Tdtomato floxed mice.

AAVretro-Syn-Cre was delivered to T9 at 4 weeks post SCI/graft, which induced PTEN/SOCS3 knockout (KO) and turned on tdTomato expression in the graft. The PTEN/SOCS3 KO group exhibited significantly increased graft-derived axons compared to controls grafted with NPCs from Tdtomato floxed mice. Next step will examine whether these axons form synapses with host neurons and whether it promotes functional recovery. Results from this project identified a mean to promote axon growth that could enhance functional connectivity between graft and host after SCI.

QUANG MAN LE

Roger Revelle College, Neurobiology Major, Global Health Minor

PI: Karl J. Wahlin, Ph.D., Shiley Eye Institute, Department of Ophthalmology.

Pharmacology-induced Differentiation and Proliferation of Müller Cells in Human-derived Retinal Organoids

Did you know that, in the United States, someone is told that they are going blind every seven minutes? According to the Center for Disease Control and Prevention (CDC), the leading causes of blindness and visual impairment in America include age-related macular degeneration (AMD), glaucoma, and retinitis pigmentosa. The hallmark of these conditions are the loss of retinal photoreceptors. While neurons can regenerate in multiple species, this has never been shown to occur in humans. In the current proposal I seek to activate the molecular pathways that regulate regeneration in other species, except now I will do this in human stem cell derived retinal organoids. Specifically, I will explore the Notch, Jak/Stat3, Wnt/β-catenin, and Yap/Taz signaling pathways alongside the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA). By activating these pathways, I hope to engage the neurogenic potential of human Müller cells to demonstrate that regeneration might be possible in humans.

VERINA HYMNMONG LEUNG

Earl Warren College, Neurobiology Major, Global Health Minor

PI: Li Ye, Ph.D., UCSD Department of Neuroscience & Molecular Medicine, The Scripps Research Institute

3D Visualization of in vivo Drug Targets

Profiling drug-protein interactions is critical to drug discovery. While lysate-based studies of drug-target interactions have revealed molecular targets, they do not inform in vivo cellular and spatial distribution. Recently, the development of Clearing-Assisted Tissue Click Chemistry (CATCH) provides drug-target visualization while preserving cellular and spatial details. Currently, CATCH is limited to thin tissue sections, limiting its throughput. Therefore, we developed volumetric CATCH (vCATCH) to probe drug-target interactions in 3D tissues. Using vCATCH, we evaluated the in vivo engagement of Ibrutinib, a Bruton’s tyrosine kinase inhibitor that has revolutionized B-cell malignancy treatment. Ibrutinib is associated with cardiovascular toxicity that’s not fully understood. We profiled potential Ibrutinib off-targets in intact mouse hearts, especially within rare, non-cardiomyocyte populations. These novel insights may redefine kinase inhibitor toxicity understanding with cellular resolution. vCATCH also establishes a platform to screen and identify in vivo cellular targets in intact organs, extending to whole animals.

YUFEI LIU

Roger

Revelle College, Molecular and Cell Biology Major,

Japanese Studies Minor

PI: Enfu Hui, Ph.D., UCSD School of Biological Sciences, Department of Cell and Developmental Biology

Visualization of Ligand-Dependent signaling of the Inhibitory Immunoreceptor LAG3

The inhibitory immunoreceptor lymphocyte activation gene 3 protein (LAG3) is emerging as a promising target for anti-tumor immunotherapies, but its biochemical mechanism is poorly defined. Moreover, the functional relationship of its two ligands – major histocompatibility complex class II (MHCII) and fibrinogen-like protein 1 (FGL1) – is unknown. To begin addressing these questions, I leveraged total internal reflection fluorescence microcopy to examine how MHCII and FGL1 affects the localization, clustering and dynamics of LAG3 at the immunological synapse. In parallel, I used coculture assays to validate the abilities of MHCII and FGL1 to regulate Lag3 inhibitory function. My results have shed light on the mechanisms of LAG3, with implications on LAG3-targeted therapies.

YANG LIU

Roger Revelle College, Molecular Cell Major, Psychology Minor

PI: Joseph Gleeson, M.D., UCSD School of Medicine, Department of Neuroscience

Deciphering the Role of Somatic Mosaicism in Malformations of Cortical Development: A Genetic Exploration of Focal Cortical Dysplasia

Malformations of Cortical Development (MCD) refers to a group of disorders characterized by abnormal cerebral cortex formation. Focal Cortical Dysplasia (FCD), a specific type of MCD, is a major cause of intractable epilepsy that often requires surgical intervention. While recent studies have suggested a link between brain mosaicism and brain developmental disorders, deciphering how somatic mutations with low allelic fraction contribute to MCD remains a challenge. We aim to identify genetic causes of MCD, combining Amplicon sequencing with panel and TASeq (target amplicon sequencing) applied to ascertained FCD patient brain samples (117 samples). We hope to confirm putative mosaic variants that we discovered using our custom gene panel. Investigation into the role of somatic mosaicism in FCD offers the potential to identify new pathogenesis genetic factors, which could lead to better diagnostic and therapeutic strategies.

Thurgood Marshall College, Human Biology Major, Saltman Quarterly

PI: Michael J. Castle, Ph.D., UCSD School of Medicine, Department of Neurosciences

Enhanced Cortex-Specific Promoters for Gene Therapy in Alzheimer's Disease

Brain-Derived Neurotrophic Factor (BDNF) is a growth factor that can modulate Alzheimer’s Disease (AD) progression. Our study explored novel approaches using Adeno-Associated Virus (AAV) vectors for the delivery of BDNF gene specifically to the cerebral cortex, where AD’s impact is most profound. By employing cortex-specific promoters, we aim to achieve strong and localized gene delivery to the cortex, thereby minimizing adverse effects that may be associated with off-target BDNF expression. We compared five candidate cortex-specific promoters in mice by intravenous injection of AAVPhP.eB vectors carrying the eGFP reporter gene. We found that these promoters effectively directed eGFP expression to cortical neurons, significantly reducing off-target gene expression in cerebellum, brainstem, and thalamus in mouse models. Future research will examine the specificity of these promoters in non-human primate models and evaluate efficacy by delivery of BDNF gene therapy in a mouse model of AD.

HANH YEN NGUYEN

Sixth College, Molecular and Cell Biology Major

PI: Dong-Er Zhang, Ph.D., UCSD, Department of Molecular Biology

Characterizing the Functional Significance of Peptidylarginine Deiminases in t(8;21) Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a malignant blood cancer. Although modern medicine has improved AML management, standards of care have stagnated, necessitating a deeper understanding of the underlying molecular mechanisms. t(8;21) AML, occurring in 5–10% of patients, is characterized by the fusion of AML1 and ETO (AE). To uphold a leukemic phenotype, AE globally dysregulates transcription of key hematopoietic factors. Peptidylarginine deiminases (PADs) are a family of proteins that perform post-translational conversion of arginine to citrulline, inducing protein conformational changes, and mediating a series of citrulline-dependent pathways, such as autoimmune response and transcriptional control of cellular development. By ChIP-seq analysis, we found an AE-dependent super-enhancer at the PAD family locus, suggesting AE-regulated PAD expression sustains the leukemic phenotype. Here, we further clarify the relationship between PAD and AE, identify pathways that uphold its leukemic potential, and characterize prospective PAD targeted therapies to treat t(8;21) AML.

BEIYING LU

LAURA

MARIE NORONHA

John Muir College, Human Biology Major, Music Minor

PI: Nicola J. Allen, Ph.D., Salk Institute, Molecular Neurobiology Laboratory

Dysregulated Cholesterol Metabolism in Alzheimer’s Disease Astrocytes

Astrocytes, non-neuronal glial cells in the brain, play critical roles in blood brain barrier function, synapse development, and lipid metabolism. Alzheimer’s disease (AD), a neurodegenerative disorder, is associated with excess amyloid protein and tau tangles in neurons. Alterations to astrocyte function have been implicated in AD. Previous analyses from our lab revealed significant alterations in the gene and protein expression of AD iPSC-induced astrocytes (iPSC-iAs) and agematched control astrocytes. Specifically, dysregulated cholesterol metabolism and increased production of proinflammatory molecules were observed in AD astrocytes. Thus, we hypothesize changes in cholesterol trafficking in astrocytes may promote an inflammatory state. We pharmacologically targeted a candidate pathway involved in cholesterol metabolism and measured expression of cholesterol metabolic genes and proinflammatory molecules using quantitative PCR. This manipulation resulted in decreased gene expression of proinflammatory cytokines in AD astrocytes. These experiments provide insight into how cholesterol dysregulation and inflammation may be targeted in AD astrocytes.

VEDA PALAPARTY

Sixth College, General Biology

Major, Business and Psychology

Minors, Saltman Quarterly

PI: Karl J. Wahlin, Ph.D., UCSD

Health, Department of Ophthalmology

Developing an Approach for Adeno-Associated Viral Vector Mediated Cellular Reprogramming

Age-related macular degeneration (AMD) is the predominant cause of blindness for individuals above 50 years of age. As it progresses, the degeneration and eventual loss of major cell types, notably photoreceptors and retinal ganglion cells (RGCs), has been extensively documented. Some non-mammalian species, such as zebrafish, have been observed to regenerate retinal neurons by reverting cells into a progenitor-like state from where they can differentiate into mature neuronal cell types of the retina. Packaging selective transgene cassettes required for neuronal cell development into adeno-associated viruses (AAVs), allows us to mimic such regenerative capabilities. AAV provides an ideal method for gene delivery due to its low toxicity, longterm expression of recombinant genes, and potential for cell type specific delivery. By employing the Shh10 helper AAV to target Müller Glia Cells, and performing immunohistochemical tracking of epitope tags coupled with transgene cassettes, we can identify cellular reprogramming mediated by AAV infection.

TEJASVI PATIL

Seventh College, Molecular and Cell Biology Major, Literature/ Writing Minor

PI: Jose Pruneda-Paz, Ph.D., UCSD School of Biological Sciences, Department of Cell and Developmental Biology

Characterizing the Role of TCP1 in Arabidopsis Clock-Controlled Growth and Development

The plant circadian clock is a biological mechanism that influences the ability of a plant to respond to changes in its environment. Controlled by a gene regulatory network, this clock oscillates in synchrony with the 24-hour cycle of the earth’s rotation, and regulates almost every aspect of plant growth and development through yet poorly understood organ-specific mechanisms. In this project, we identified a transcription factor, TCP1, that binds to the promoter of a critical clock component, and found that the expression of TCP1 is restricted to the hypocotyl and leaf petioles (but not leaf blades) in Arabidopsis plants. These findings suggest that TCP1 is an organ specific regulator of the clock function. We hypothesize that through this role, TCP1 controls hypocotyl growth and petiole elongation which are critical plant responses for soil emergence and shade avoidance, respectively. This hypothesis is currently being explored using genetic and molecular approaches.

MADELYN PECK

Thurgood Marshall College, Neurobiology Major

PI: Eiman Azim, Ph.D., Salk Institute for Biological Sciences, Molecular Neurobiology Laboratory

Evaluating the Role of Nucleo-Olivary Projections in Maintaining Internal Models for Skilled Movement

The continuous refinement of skilled movement for smooth and accurate motor execution is driven by error signals, defined as the discrepancy between expected and actualized movement outcomes. These error signals allow for the learned correction of movement errors during adaptation to internal and external changes. It is believed that the inferior olive (IO) sends these error signals to drive cerebellar-dependent sensorimotor adaptation. However, it remains unclear how different inputs to the IO affect the execution or modification of either ongoing or future movements. This study investigates the function of inhibitory nucleo-olivary projections from the anterior interposed cerebellar nuclei to the IO. We hypothesize that this negative feedback pathway regulates IO activity to prevent aberrant changes to movement. The effects of nucleo-olivary neurons on the function of these neurons in execution and maintenance of learned movements was explored by optogenetically perturbing these neurons during skilled goal-directed forelimb reaching.

SHARANYA SRIRAM

Earl Warren College, Molecular and Cell Biology Major

PI: Matthew Wortham, Ph.D., UCSD School of Medicine, Department of Pediatrics

Characterizing Expression of Post-Transcriptionally Regulated Ribosomal Genes in Pancreatic Alpha and Beta Cells

Diabetes results from dysfunction of insulin-producing beta cells within pancreatic islets. Other islet endocrine cells such as glucagon-producing alpha cells work coordinately with beta cells to maintain glucose homeostasis under varying metabolic conditions. Our lab has previously shown that nutrient-stimulated histone acetylation and resulting transcriptional changes underlie adaptive insulin secretion during fasting and feeding. While further analysis yielded few feeding-dependent proteomic changes, notable differences emerged when comparing protein abundances between alpha and beta cells themselves. Intriguingly, several ribosomal proteins exhibited differential abundance between cell types at the protein level but not the mRNA level. We aim to validate this proteomics result through immunofluorescent staining of mouse islet tissue and corroborate ribosomal protein abundance with measurements of translation rates in alpha and beta cells. Understanding fundamental differences in translation machinery between islet cell types should provide insight into differential vulnerabilities of alpha and beta cells to stressors implicated in diabetes pathogenesis.

Roger Revelle College, Molecular and Cell Biology Major, Human and Developmental Sciences Minor

PI: Michael Karin, Ph.D., UCSD School of Medicine, Department of Pharmacology

Identification of Combination Therapies Targeting Macropinocytosis in Pancreatic Ductal Adenocarcinoma (PDAC) Stem Cells

Cancer stem cells (CSCs) exhibit properties such as self-renewal and pluripotency that contribute to the ability of various cancers to evade conventional chemotherapy, resulting in a poor prognosis for patients, especially those whose tumors are CSC enriched. Pancreatic ductal adenocarcinoma (PDAC) is characterized by late detection, resistance to current standard-of-care treatments, and frequent relapse following remission, at least some of which may be due to the presence of CSCs. While there are currently no established therapies that specifically target PDAC CSCs, these cells offer a desirable target for developing novel cancer treatments. To achieve this goal, we established 3D tumor sphere culture system enriched for PDAC CSCs and used this system to investigate the efficacy of combination therapies that include gemcitabine, which is already in clinical use. Among the several combinations of molecular target inhibitors we have tested, a macropinocytosis inhibitor in combination with gemcitabine demonstrated significant tumor sphere growth reduction. We are currently examining how these combination therapies exhibit tumor inhibitory effects.

SONIA STAN

TIMOTHY ANDERS SWAIN

Thurgood Marshall College, Ecology, Behavior, and Evolution Major, Marine Sciences and Ethnic Studies Minors

PI: David Holway, Ph.D, School of Biological Sciences, Department of Ecology, Behavior, and Evolution

Insectivorous Bird Use of Ornamental Vegetation: Tipu Trees, Tipu Psyllids, and Warblers in Suburban San Diego

With expanding urban development, migratory birds increasingly encounter human-modified environments in migration and on their wintering grounds. Urban trees vary in their attractiveness to insectivorous birds, but the basis of this variation remains incompletely understood. Here, we investigate bird use of the tipu tree (Tipuana tipu), which hosts the tipu psyllid (Platycorypha nigrivirga). By estimating psyllid density and bird foraging activity in a sample of tipu trees in La Jolla, we aim to identify (i) the factors contributing to psyllid density, and (ii) the relationships between psyllid density and the foraging activity of insectivorous birds (mostly warblers). Our findings will improve an understanding of how native birds use ornamental vegetation and how urban landscaping might be managed to enhance use by wildlife.

NAYANA TELLAKULA

Eleanor Roosevelt College, General Biology Major

PI: William B. Kristan, Ph.D, Distinguished Professor Emeritus, UCSD Department of Neurobiology, School of Biological Sciences

Planarian Amputation and Regeneration Modify Behavioral Responses

Planarian flatworms regenerate any portion of their body removed by their own behavior, by injury, or by surgical amputation at any location. Intact planarians exhibit three behaviors when stimulated with UV light at different regions: turning (head), elongation (middle), and contraction (tail). Along the animal, the shift in response from turning to elongation occurs at a specific location, one-third back from the head. I found that surgical amputation causes the front third of the back portion of the animal to behave like the normal “head” as stimulating this region elicits turning. I then surgically amputated the animal at different locations and found that the turn-inducing region shifts forward along the animal as the head regenerates over 8 days. These results mean that a region which, in intact animals, produces one response (elongation) to stimulation can switch to produce turning after amputation, then back to elongation as the anterior region regenerates.

JINGYAO WANG

Seventh College, Cell and Molecular Biology Major, Chemistry Minor

PI: Alon Goren, Ph.D., Associate Professor, UCSD School of Medicine.

Western-seq: Transforming Western Blot into Multiplex and High Throughput Protein Detection Method

Western blot (western) is an analytical method that measures protein/epitope abundance within and between samples by antibody-based fluorescent detection. However, western is limited to detecting 1-2 epitopes per assay with low sensitivity. We aim to convert western to a multiplex detection method with increased sensitivity by combining the original approach with next-generation sequencing . Our novel method, western-seq uses antibodies that are conjugated to oligonucleotides, each with a barcode and a unique molecular identifier (UMI, a random sequence), enabling blotting with dozens to hundreds of antibodies at once. Preliminary results with recombinant proteins have demonstrated signal purity and specificity. We are optimizing the method for higher accuracy and specificity on cell lysates. In addition, internal barcoded protein standards are being developed to replace colorimetric ladders. Western-seq would provide a powerful protein quantitative tool for detection of protein variants (e.g., via alternative splicing) while accounting for antibody nonspecific binding.

YUNQING WANG

Thurgood Marshall College, Human Biology & Cognitive Science with Specialization in Neuroscience Major

PI: Alan Saltiel, Ph.D., UCSD School of Medicine, Department of Endocrinology

Investigating the Role of MAVS in the Adipose Tissue

Mitochondrial antiviral signaling protein (MAVS) is a transmembrane protein located on the outer mitochondrial membrane. It participates in the RLR pathway and activates the production of interferons and proinflammatory cytokines essential for innate immunity against double-stranded RNA pathogens. However, its role in adipocytes remains unexplored. We have shown that MAVS expression is dramatically induced upon B3-adrenergic receptor activation during white adipose tissue browning. Our research aims to characterize MAVS behavior and function in adipose tissue regarding energy expenditure and thermogenesis. Potential MAVS binding partners are identified by mass spectrometry in adipose tissue. To validate the binding ability of MAVS and its potential binding partners in vitro, we co-transfected a FLAG-MAVS plasmid and HA-tagged fusion plasmid of its potential binding partners into HEK293T cells. We observed binding of a number of proteins through co-immunoprecipitation. Future research will investigate further downstream activities and provide insights into how MAVS functions in adipocytes.

Earl Warren College, Molecular and Cell Biology Major

PI: Eugene Yeo, Ph.D., UCSD Department of Cellular and Molecular Medicine

The roles of RNA Binding Caprin in Synaptic RNA Within Human iPSC-derived Neurons

RNA Binding Proteins (RBPs) play a crucial role in cellular metabolic regulation via alternative splicing, RNA trafficking, and RNA availability through phase separation. Our research will specifically focus on the RNA Binding Protein Caprin. Caprin is proposed to regulate local translation and synaptic proteins in neurons. Neurons communicate through synapses, and synapses are also essential in maintaining protein homeostasis through RNA splicing, local protein synthesis, and responding to external stimuli. Due to the unique polar structure of neurons, it is difficult to distinguish between localized and overall responses under metabolic stress. We hypothesize that interactions between RBPs and RNA, particularly involving Caprin, are vital in maintaining metabolic homeostasis during stress through the regulation of synaptic proteins. Therefore, we also want to investigate what RNA Caprin is spatially associated with. In this experiment, we will use iPSC-derived neurons and eCLIP to assess the impact of Caprin on neurons under stress by observing changes in RNA transcripts. After generating data, we will produce synaptic transcriptomes by calculating RNA fractions to evaluate the transcripts for spatial enrichment. Our preliminary data suggest that Caprin recruits stress-granule associated RNA to phase-separated granules during metabolic stress.

QINGCHEN ZHOU

Sixth College, Molecular and Cell Biology Major, Sociocultural Anthropology Minor

PI: Qingfei Jiang, Ph.D., UCSD School of Medicine, Division of Regenerative Medicine

Exploring the Role of Matrin-3 in double-stranded RNA sensing in T-cell Acute Lymphoblastic Leukemia

The enhanced survival and self-renewal capacity of leukemia-initiating cells (LICs) is considered to be responsible for T-cell acute lymphoblastic leukemia (T-ALL) relapse and resistance to traditional therapies. Our lab has previously reported that RNA-editing enzyme adenosine deaminase actin on RNA1 (ADAR1) retains endogenous double-stranded RNA (dsRNA) inside the cell nucleus to inhibits cytosolic dsRNA sensing, thereby attenuating innate immune response and contributing to LIC propagation. Moreover, we observed a positive correlation between the expressions of ADAR1 and Matrin-3 (MATR3), an DNA- and RNA-binding nuclear matrix protein, in T-ALL cells. Here, we hypothesize that MATR3 supports T-ALL LIC maintenance by suppressing aberrant dsRNA sensing. We performed immunofluorescence staining and confocal imaging on MATR3-konckout T-ALL cells and observed a lower proportion of nucleus-located dsRNA using fluorescence quantification, suggesting a nucleus-retention function of MATR3. More data will be collected and analyzed to further explore the role of MATR3 in dsRNA-sensing pathway and interferon-mediated signaling.

WINNIE ZHOU

Eleanor Roosevelt College, Molecular and Cell Biology Major, Psychology Minor

PI: Andreas Ernst, Ph.D., UCSD School of Biological Sciences, Department of Cell and Developmental Biology

Characterizing the Mechanisms of TFG-Containing Oncogenic Fusion Proteins

One of the hallmarks of cancer is the ability to sustain proliferative signaling. The MAPK signaling pathway is understood to play a key role in the amplification of signal transduction molecules necessary for cell proliferation and growth. Previous research has shown that certain oncogenic fusion proteins can directly activate the MAPK pathway. TRK-T3 is an oncogenic fusion protein that contains approximately 200 amino acids from the N-terminal domain of the protein TFG and 400 amino acids from the receptor tyrosine kinase TrkA. Our research aims to characterize mechanistically how the TFG moiety in TRK-T3 can lead to activation of the MAPK pathway, and thus contribute to carcinogenesis.

ZHENGYANG ZHU

Seventh College, General Biology Major, Cognitive Science Minor

PI: Nicholas J. Webster, Ph.D., M.A., UCSD School of Medicine, Department of Endocrinology and Metabolism.

Exploring the Role of Alternative Splicing of Insulin Receptor to IR-B in IGF-2 Driven Liver Fibrosis and Inflammation

Hepatocellular carcinoma (HCC) is the most common primary liver cancer that arises after chronic inflammatory liver disease. In humans, 20 to 25% of HCCs showed elevated insulin-like growth factor 2 (IGF2) mRNA expression and protein levels. Recent studies have found that alternative splicing of the insulin receptor mRNA mediated by splicing factor SRSF3 is responsible for the expression of two isoforms, an IR-A that can bind to IGF2 and an IR-B that cannot. At normal SRSF3 expression, IR-B is commonly expressed for regular insulin binding and liver functions, but in liver disease SRSF3 expression decreases, allowing IR-A expression. Our hypothesis is that increased expression of IR-A isoform through alternative splicing creates an IGF2-induced autocrine loop, which potentially causes DNA damage and liver fibrosis that ultimately leads to HCC. Our experimental findings will hopefully shed light on this pathway and the effect of IGF-2 in causing liver fibrosis and HCC.

STAFF

EDITORS-IN-CHIEF

Amoolya Chandrabhatta

Emily White

EDITOR AT LARGE

Chiraag Kambalimath

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Sharanya Sriram

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Jacqueline Cheung

FEATURES DESIGN

EDITOR

Irum Aamer

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Erin Jang

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Shreya Chandrasekhar

RESEARCH FEATURES

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Sannidhi Krovvidi

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Tejasvi Patil

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Kaz Nuckowski

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Kalisa Kang

Sahana Kashyap

Amy Li

Emma Svendsen

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EDITOR

Nicolas Bello

TECHNICAL EDITORS

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Kazune Fei

Megan Hackbarth

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Michelle Liang

Sarah Malko

Samia Rahman

Catherine Zong

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Kristiana Wong

ILLUSTRATORS

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Harnoor Sidhu

PHOTOGRAPHERS

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Kelly Wang

PODCAST TEAM

Shalini Anisetti

Yao Bi

Sarah Callahan

Jorge Valdez Diaz

Jessica Du

Siya Jatia

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Adrian Tayag

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Ritviksiddha Penchala

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Thrisha Praveen

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Jihyun In

BLOGGERS

Monisha Bains

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Syni Solanki

COMMUNITY OUTREACH CHAIR

Caitlyn Santistevan

COMMUNITY OUTREACH COMMITTEE

Onyekachi Ezeokeke

Jennifer Fong

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Meghna Pulacode

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Sahithi Yeturu

HEAD ADVISORS

James Cooke, Ph.D. Assistant Teaching Professor of Neurobiology

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Ralph Greenspan, Ph.D.

Sara Jackrel, Ph.D.

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Alistair Russell, Ph.D.

SPECIAL THANKS TO PATERA DESIGN

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photo by: Harnoor Sidhu