Open Source Solutions in Experimental Design: An Introduction to the Symposium

The Open Science movement has increased dramatically in popularity with deserved calls to action around transparency, access to resources, and inclusion in our field. However, its practical applications within experimental design have been slow to uptake, with researchers unsure where to even start with the dizzying array of open source hardware and software solutions available. The perceived time investment and unknown cost, especially in implementing open source hardware, has stagnated the implementation of inexpensive experimental solutions, but we sought to increase awareness to lower the barrier to participation in this space. While there are countless technical and financial advantages to integrating open source solutions into every biologist\u27s experimental design, we put an emphasis on the “people” part of the equation in our symposium. This symposium championed innovative experimental designs by early career SICB researchers across all fields of biology, from plants to animals, in the lab or in the field, or even virtually engaging with the public and students. The open science movement operates within community norms that champion transparency, continuous development, and collaboration. These values are congruent with the priorities of reducing barriers to participation in science, and we hope our symposium\u27s collection of open source solutions encourages readers to adopt these or other innovative designs into their own experimentation

Virtual Expeditions Facilitated By Open Source Solutions Broaden Student Participation in Natural History Research

From its genesis in the Victorian era as an activity for the elite to today\u27s emphasis on “Big Data” and continuous monitoring, natural history has a prominent role in scientific discoveries for many fields. However, participation in field expeditions is limited by funding, space, accessibility, and safety constraints. Others have detailed the active exclusion of minoritized groups from field expeditions and harm/discrimination faced by the few who do participate, but we provide one solution to broaden opportunities for participation in natural history: Virtual Expeditions. Virtual Expeditions are broadly defined as open source, web-facilitated research activities designed to analyze bulk-collected digital data from field expeditions that require visual human interpretation. We show two examples here of their use: an independent research-based analysis of snake behavior and a course-based identification of invertebrate species. We present a guide to their appropriate design, facilitation, and evaluation to result in research grade data. We highlight the importance of open source technology to allow for longevity in methodology and appropriate quality control measures necessary for projects that include dozens of researchers over multiple years. In this perspective, we specifically emphasize the prominent role that open source technology plays in making these experiences feasible and scalable. Even without explicit design as broadening participation endeavors, Virtual Expeditions allow for more inclusive participation of early career researchers with specific participatory limitations. Not only are Virtual Expeditions integral to the large-scale analysis necessary for field expeditions that generate impossibly enormous datasets, but they can also be effective facilitators of inclusivity in natural history research

Environment-Driven Shifts in Inter-Individual Variation and Phenotypic Integration within Subnetworks of the Mussel Transcriptome and Proteome

The environment can alter the magnitude of phenotypic variation among individuals, potentially influencing evolutionary trajectories. However, environmental influences on variation are complex and remain understudied. Populations in heterogeneous environments might exhibit more variation, the amount of variation could differ between benign and stressful conditions, and/or variation might manifest in different ways among stages of the gene-to-protein expression cascade or among physiological functions. Here, we explore these three issues by quantifying patterns of inter-individual variation in both transcript and protein expression levels among California mussels, Mytilus californianus Conrad. Mussels were exposed to five ecologically relevant treatments that varied in the mean and inter-individual heterogeneity of body temperature. To target a diverse set of physiological functions, we assessed variation within 19 expression subnetworks, including canonical stress-response pathways and empirically derived co-expression clusters that represent a diffuse set of cellular processes. Variation in expression was particularly pronounced in the treatments with high mean and heterogeneous body temperatures. However, with few exceptions, environment-dependent shifts of variation in the transcriptome were not reflected in the proteome. A metric of phenotypic integration provided evidence for a greater degree of constraint on relative expression levels (i.e., stronger correlation) within expression subnetworks in benign, homogeneous environments. Our results suggest that environments that are more stressful on average – and which also tend to be more heterogeneous – can relax these expression constraints and reduce phenotypic integration within biochemical subnetworks. Context-dependent \u27unmasking\u27 of functional variation may contribute to inter-individual differences in physiological phenotype and performance in stressful environments

High Heat Tolerance is Negatively Correlated with Upper Thermal Tolerance Plasticity in North Eastern Pacific Nudibranch Mollusks

Rapid ocean warming may alter habitat suitability and population fitness for marine ectotherms. Susceptibility to thermal perturbations will depend in part on plasticity of a species’ upper thermal limits of performance (CTmax). However, we currently lack data regarding CTmax plasticity for several major marine taxa, including nudibranch mollusks, thus limiting predictive responses to habitat warming for these species. In order to determine relative sensitivity to future warming, we investigated heat tolerance limits (CTmax), heat tolerance plasticity (acclimation response ratio), thermal safety margins, temperature sensitivity of metabolism, and metabolic cost of heat shock in nine species of nudibranchs collected across a thermal gradient along the northeastern Pacific coast of California and held at ambient and elevated temperature for thermal acclimation. Heat tolerance differed significantly among species, ranging from 25.4°±0.5°C to 32.2°±1.8°C (x¯±SD), but did not vary with collection site within species. Thermal plasticity was generally high (0.52±0.06, x¯±SE) and was strongly negatively correlated with CTmax in accordance with the trade-off hypothesis of thermal adaptation. Metabolic costs of thermal challenge were low, with no significant alteration in respiration rate of any species 1 h after exposure to acute heat shock. Thermal safety margins, calculated against maximum habitat temperatures, were negative for nearly all species examined (−8.5°±5.3°C, x¯±CI [confidence interval]). From these data, we conclude that warm adaptation in intertidal nudibranchs constrains plastic responses to acute thermal challenge and that southern warm-adapted species are likely most vulnerable to future warming

Temperature and Salinity Sensitivity of Respiration, Grazing, and Excretion Rates in the Estuarine Eelgrass Sea Hare, Phyllaplysia taylori

Highly dynamic environments such as estuaries are home to organisms accustomed to wide fluctuations in environmental conditions. However, estuarine temperature and salinity conditions are expected to shift with climate change, potentially altering plant and animal physiology and consequently their ecological interactions. Phyllaplysia taylori, a sea hare that lives exclusively in nearshore eelgrass beds in the Eastern Pacific Ocean, is a positive ecological interactor with eelgrass by increasing eelgrass productivity through grazing removal of photosynthesis-blocking epiphytes. The central aim of our study is to understand how increasing temperature and salinity are likely to alter that ecological interaction. First, we determined salinity thresholds for survival of P. taylori at 20 °C (typical summer temperature) for 2 weeks, and found that significant mortality occurs at salinity below 25 ppt. Then, we determined respiration rate, grazing rate, and defecation rate of P. taylori following a crossed 2-week acclimation at typical summer low- and high temperatures (18 and 22 °C) and salinities (27 and 33 ppt). P. taylori respiration and grazing rates were elevated under low salinity and high temperature. To determine how P. taylori responds to very warm and extreme summer temperatures, we measured respiration rates at higher temperatures (26 °C—very warm summer and 30 °C—heat shock) and feeding rates following exposure to the 30 °C heat shock. Irrespective of acclimation salinity, P. taylori acclimated to 18 °C were more sensitive to heat shock, as they had a larger increase in respiration rate at 30 °C, and had reduced feeding rates following the 30 °C exposure, whereas there was no reduction in feeding rate in 22 °C acclimated specimens. This study provides the first data on the salinity and temperature sensitivity and metabolic physiology of P. taylori with relevance to their trophic position in the context of eelgrass ecosystems

Multi-omics Reveals Largely Distinct Transcript- and Protein-Level Responses to the Environment in an Intertidal Mussel

Organismal responses to stressful environments are influenced by numerous transcript- and protein-level mechanisms, and the relationships between expression changes at these levels are not always straightforward. Here, we used paired transcriptomic and proteomic datasets from two previous studies from gill of the California mussel, Mytilus californianus, to explore how simultaneous transcript and protein abundance patterns may diverge under different environmental scenarios. Field-acclimatized mussels were sampled from two disparate intertidal sites; individuals from one site were subjected to three further treatments (common garden, low-intertidal or high-intertidal outplant) that vary in temperature and feeding time. Assessing 1519 genes shared between the two datasets revealed that both transcript and protein expression patterns differentiated the treatments at a global level, despite numerous underlying discrepancies. There were far more instances of differential expression between treatments in transcript only (1451) or protein only (226) than of the two levels shifting expression concordantly (68 instances). Upregulated expression of cilium-associated transcripts (likely related to feeding) was associated with relatively benign field treatments. In the most stressful treatment, transcripts, but not proteins, for several molecular chaperones (including heat shock proteins and endoplasmic reticulum chaperones) were more abundant, consistent with a threshold model for induction of translation of constitutively available mRNAs. Overall, these results suggest that the relative importance of transcript- and protein-level regulation (translation and/or turnover) differs among cellular functions and across specific microhabitats or environmental contexts. Furthermore, the degree of concordance between transcript and protein expression can vary across benign versus acutely stressful environmental conditions

A Push for Inclusive Data Collection in STEM Organizations

Professional organizations in STEM (science, technology, engineering, and mathematics) can use demographic data to quantify recruitment and retention (R&R) of underrepresented groups within their memberships. However, variation in the types of demographic data collected can influence the targeting and perceived impacts of R&R efforts - e.g., giving false signals of R&R for some groups. We obtained demographic surveys from 73 U.S.-affiliated STEM organizations, collectively representing 712,000 members and conference-attendees. We found large differences in the demographic categories surveyed (e.g., disability status, sexual orientation) and the available response options. These discrepancies indicate a lack of consensus regarding the demographic groups that should be recognized and, for groups that are omitted from surveys, an inability of organizations to prioritize and evaluate R&R initiatives. Aligning inclusive demographic surveys across organizations can provide baseline data that can be used to target and evaluate R&R initiatives to better serve underrepresented groups throughout STEM

Kelp Morphology and Herbivory are Maintained Across Latitude Despite Geographic Shift in Kelp-wounding Herbivores

Herbivores can drastically alter the morphology of macroalgae by directly consuming tissue and by inflicting structural wounds. Wounds can result in large amounts of tissue breaking away from macroalgae, amplifying the damage initially caused by herbivores. Herbivores that commonly wound macroalgae often occur over only a portion of a macroalga’s lifespan or geographic range. However, we know little about the influence of these periodic or regional occurrences of herbivores on the large-scale seasonal and geographical patterns of macroalgal morphology. We used the intertidal kelp Egregia menziesii to investigate how the kelp’s morphology and the prevalence of two prominent kelp-wounding herbivores (limpets and amphipods) changed over two seasons (spring and summer) and over the northern extent of the kelp’s geographic range (six sites from central California to northern Washington). Wounds from limpets and amphipods often result in the kelp’s fronds being pruned (intercalary meristem broken away), so we quantified kelp size (combined length of all fronds) and pruning (proportion of broken fronds). We found similar results in each season: herbivores were most likely to occur on large, pruned kelp regardless of site; and limpets were the dominant herbivore at southern sites, while amphipods were dominant at northern sites. Despite the geographic shift in the dominant herbivore, kelp had similar levels of total herbivore prevalence (limpets and/or amphipods) and similar morphologies across sites. Our results suggest that large-scale geographic similarities in macroalgal wounding, despite regional variation in the herbivore community, can maintain similar macroalgal morphologies over large geographic areas

Conference Scheduling Undermines Diversity Efforts

We assessed diversity-focused programming at 29 major biology conferences from 2010 to 2019, noting events tailored to three underrepresented and marginalized groups in biology: women, ethnic and racial minority groups, and the LGBTQ+ community (see Supplementary Information for further methods). Since 2010, diversity-focused events have become more common but frequently address only a subset of URG communities. In general, the percentage of conferences with diversity-focused events increased from 75% in 2019. On average, women were the most frequent focus of these events and the LGBTQ+ community was the least frequent focus (Fig. 1a)

Plasticity of Foot Muscle and Cardiac Thermal Limits in the Limpet \u3cem\u3eLottia limatula\u3c/em\u3e from Locations with Differing Temperatures

Species distributions are shifting in response to increased habitat temperatures as a result of ongoing climate change. Understanding variation in physiological plasticity among species and populations is important for predicting these distribution shifts. Interspecific variation in intertidal ectotherms’ short-term thermal plasticity has been well established. However, intraspecific variation among populations from differing thermal habitats remains a question pertinent to understanding the effects of climate change on species’ ranges. In this study, we explored upper thermal tolerance limits and plasticity of those limits using a foot muscle metric and 2 cardiac metrics (Arrhenius breakpoint temperature, ABT, and flatline temperature, FLT) in adult file limpets Lottia limatula. Limpets were collected from thermally different coastal and inland-estuarine habitats and held for 2 wk at 13, 17 or 21°C prior to thermal performance assays. Compared to limpets from the warm estuary site, limpets from the cold outer coast site had similar foot muscle critical thermal maxima (CTmax; 35.2 vs. 35.6°C) but lower cardiac thermal tolerances (ABT: 30.5 vs. 35.1°C). Limpets from the cold coast site had higher acclimation responses in foot muscle CTmax (0.22°C per 1°C rise in acclimation) than those of the warm estuary site (0.07°C per 1°C rise in acclimation), but lower acclimation responses in cardiac thermal tolerance (ABT: -0.85°C per 1°C rise in acclimation) than those of the estuary site (ABT: 0.10°C per 1°C rise in acclimation). Since outer coast populations had lower cardiac plasticity and higher mortalities in the warm acclimation, we predict L. limatula from colder habitats will be more susceptible to rising temperatures. Our findings illustrate the importance of population-specific variation in short-term thermal plasticity when considering the effects of climate change on ectotherms