Jodi A. Schwarz

Differential gene expression during thermal stress and bleaching in the Caribbean coral Montastraea faveolata

The declining health of coral reefs worldwide is likely to intensify in response to continued anthropogenic disturbance from coastal development, pollution, and climate change. In response to these stresses, reef‐building corals may exhibit bleaching, which marks the breakdown in symbiosis between coral and zooxanthellae. Mass coral bleaching due to elevated water temperature can devastate coral reefs on a large geographical scale. In order to understand the molecular and cellular basis of bleaching in corals, we have measured gene expression changes associated with thermal stress and bleaching using a complementary DNA microarray containing 1310 genes of the Caribbean coral Montastraea faveolata. In a first experiment, we identified differentially expressed genes by comparing experimentally bleached M. faveolata fragments to control non‐heat‐stressed fragments. In a second experiment, we identified differentially expressed genes during a time course experiment with four time points across 9 days. Results suggest that thermal stress and bleaching in M. faveolata affect the following processes: oxidative stress, Ca2+ homeostasis, cytoskeletal organization, cell death, calcification, metabolism, protein synthesis, heat shock protein activity, and transposon activity. These results represent the first medium‐scale transcriptomic study focused on revealing the cellular foundation of thermal stress‐induced coral bleaching. We postulate that oxidative stress in thermal‐stressed corals causes a disruption of Ca2+ homeostasis, which in turn leads to cytoskeletal and cell adhesion changes, decreased calcification, and the initiation of cell death via apoptosis and necrosis.

LATE LARVAL DEVELOPMENT AND ONSET OF SYMBIOSIS IN THE SCLERACTINIAN CORAL FUNGIA SCUTARIA

Many corals that harbor symbiotic algae (zooxanthellae) produce offspring that initially lack zooxanthellae. This study examined late larval development and the acquisition of zooxanthellae in the scleractinian coral Fungia scutaria, which produces planula larvae that lack zooxanthellae. Larvae reared under laboratory conditions developed the ability to feed 3 days after fertilization; feeding behavior was stimulated by homogenized Artemia. Larvae began to settle and metamorphose 5 days after fertilization. In laboratory experiments, larvae acquired experimentally added zooxanthellae by ingesting them while feeding. Zooxanthellae entered the gastric cavity and were phagocytosed by endodermal cells. As early as 1 h after feeding, zooxanthellae were observed in both endodermal and ectodermal cells. Larvae were able to form an association with three genetically distinct strains of zooxanthellae. Both zooxanthellate and azooxanthellate larvae underwent metamorphosis, and azooxanthellate polyps were able to acquire zooxanthellae from the environment. Preliminary evidence suggests that the onset of symbiosis may influence larval development; in one study symbiotic larvae settled earlier than aposymbiotic larvae. Protein profiles of eggs and larvae throughout development revealed a putative yolk protein doublet that was abundant in eggs and 1-day-old larvae and was absent by day 6. This study is the first to examine the onset of symbiosis between a motile cnidarian host and its algal symbiont.

Coral life history and symbiosis: Functional genomic resources for two reef building Caribbean corals, Acropora palmata and Montastraea faveolata

BackgroundScleractinian corals are the foundation of reef ecosystems in tropical marine environments. Their great success is due to interactions with endosymbiotic dinoflagellates (Symbiodinium spp.), with which they are obligately symbiotic. To develop a foundation for studying coral biology and coral symbiosis, we have constructed a set of cDNA libraries and generated and annotated ESTs from two species of corals, Acropora palmata and Montastraea faveolata.ResultsWe generated 14,588 (Ap) and 3,854 (Mf) high quality ESTs from five life history/symbiosis stages (spawned eggs, early-stage planula larvae, late-stage planula larvae either infected with symbionts or uninfected, and adult coral). The ESTs assembled into a set of primarily stage-specific clusters, producing 4,980 (Ap), and 1,732 (Mf) unigenes. The egg stage library, relative to the other developmental stages, was enriched in genes functioning in cell division and proliferation, transcription, signal transduction, and regulation of protein function. Fifteen unigenes were identified as candidate symbiosis-related genes as they were expressed in all libraries constructed from the symbiotic stages and were absent from all of the non symbiotic stages. These include several DNA interacting proteins, and one highly expressed unigene (containing 17 cDNAs) with no significant protein-coding region. A significant number of unigenes (25) encode potential pattern recognition receptors (lectins, scavenger receptors, and others), as well as genes that may function in signaling pathways involved in innate immune responses (toll-like signaling, NFkB p105, and MAP kinases). Comparison between the A. palmata and an A. millepora EST dataset identified ferritin as a highly expressed gene in both datasets that appears to be undergoing adaptive evolution. Five unigenes appear to be restricted to the Scleractinia, as they had no homology to any sequences in the nr databases nor to the non-scleractinian cnidarians Nematostella vectensis and Hydra magnipapillata.ConclusionPartial sequencing of 5 cDNA libraries each for A. palmata and M. faveolata has produced a rich set of candidate genes (4,980 genes from A. palmata, and 1,732 genes from M. faveolata) that we can use as a starting point for examining the life history and symbiosis of these two species, as well as to further expand the dataset of cnidarian genes for comparative genomics and evolutionary studies.

Generation and analysis of transcriptomic resources for a model system on the rise: the sea anemone Aiptasia pallida and its dinoflagellate endosymbiont

BackgroundThe most diverse marine ecosystems, coral reefs, depend upon a functional symbiosis between cnidarian hosts and unicellular dinoflagellate algae. The molecular mechanisms underlying the establishment, maintenance, and breakdown of the symbiotic partnership are, however, not well understood. Efforts to dissect these questions have been slow, as corals are notoriously difficult to work with. In order to expedite this field of research, we generated and analyzed a collection of expressed sequence tags (ESTs) from the sea anemone Aiptasia pallida and its dinoflagellate symbiont (Symbiodinium sp.), a system that is gaining popularity as a model to study cellular, molecular, and genomic questions related to cnidarian-dinoflagellate symbioses.ResultsA set of 4,925 unique sequences (UniSeqs) comprising 1,427 clusters of 2 or more ESTs (contigs) and 3,498 unclustered ESTs (singletons) was generated by analyzing 10,285 high-quality ESTs from a mixed host/symbiont cDNA library. Using a BLAST-based approach to predict which unique sequences derived from the host versus symbiont genomes, we found that the contribution of the symbiont genome to the transcriptome was surprisingly small (1.6–6.4%). This may reflect low levels of gene expression in the symbionts, low coverage of alveolate genes in the sequence databases, a small number of symbiont cells relative to the total cellular content of the anemones, or failure to adequately lyse symbiont cells. Furthermore, we were able to identify groups of genes that are known or likely to play a role in cnidarian-dinoflagellate symbioses, including oxidative stress pathways that emerged as a prominent biological feature of this transcriptome. All ESTs and UniSeqs along with annotation results and other tools have been made accessible through the implementation of a publicly accessible database named AiptasiaBase.ConclusionWe have established the first large-scale transcriptomic resource for Aiptasia pallida and its dinoflagellate symbiont. These data provide researchers with tools to study questions related to cnidarian-dinoflagellate symbioses on a molecular, cellular, and genomic level. This groundwork represents a crucial step towards the establishment of a tractable model system that can be utilized to better understand cnidarian-dinoflagellate symbioses. With the advent of next-generation sequencing methods, the transcriptomic inventory of A. pallida and its symbiont, and thus the extent of AiptasiaBase, should expand dramatically in the near future.

Extensive Differences in Gene Expression Between Symbiotic and Aposymbiotic Cnidarians

Coral reefs provide habitats for a disproportionate number of marine species relative to the small area of the oceans that they occupy. The mutualism between the cnidarian animal hosts and their intracellular dinoflagellate symbionts provides the nutritional foundation for coral growth and formation of reef structures, because algal photosynthesis can provide >90% of the total energy of the host. Disruption of this symbiosis (“coral bleaching”) is occurring on a large scale due primarily to anthropogenic factors and poses a major threat to the future of coral reefs. Despite the importance of this symbiosis, the cellular mechanisms involved in its establishment, maintenance, and breakdown remain largely unknown. We report our continued development of genomic tools to study these mechanisms in Aiptasia, a small sea anemone with great promise as a model system for studies of cnidarian–dinoflagellate symbiosis. Specifically, we have generated de novo assemblies of the transcriptomes of both a clonal line of symbiotic anemones and their endogenous dinoflagellate symbionts. We then compared transcript abundances in animals with and without dinoflagellates. This analysis identified >900 differentially expressed genes and allowed us to generate testable hypotheses about the cellular functions affected by symbiosis establishment. The differentially regulated transcripts include >60 encoding proteins that may play roles in transporting various nutrients between the symbiotic partners; many more encoding proteins functioning in several metabolic pathways, providing clues regarding how the transported nutrients may be used by the partners; and several encoding proteins that may be involved in host recognition and tolerance of the dinoflagellate.

Evolutionary analysis of orthologous cDNA sequences from cultured and symbiotic dinoflagellate symbionts of reef-building corals (Dinophyceae: Symbiodinium)

Dinoflagellates are ubiquitous marine and freshwater protists. The endosymbiotic relationship between dinoflagellates of the genus Symbiodinium (also known as zooxanthellae) and corals forms the basis of coral reefs. We constructed and analyzed a cDNA library from a cultured Symbiodinium species clade A (CassKB8). The majority of annotated ESTs from the Symbiodinium sp. CassKB8 library cover metabolic genes. Most of those belong to either carbohydrate or energy metabolism. In addition, components of extracellular signal transduction pathways and genes that play a role in cell-cell communication were identified. In a subsequent analysis, we determined all orthologous cDNA sequences between this library (1,484 unique sequences) and a library from a Symbiodinium species clade C (C3) (3,336 unique sequences) that was isolated directly from its symbiotic host. A set of 115 orthologs were identified between Symbiodinium sp. CassKB8 and Symbiodinium sp. C3. These orthologs were subdivided into three groups that show different characteristics and functions: conserved across eukaryotes (CE), dinoflagellate-specific (DS) and Symbiodinium-specific (SS). Orthologs conserved across eukaryotes are mainly comprised of housekeeping genes, photosynthesis-related transcripts and metabolic proteins, whereas the function for most of the dinoflagellate-specific orthologs remains unknown. A dN/dS analysis identified the highest ratio in a Symbiodinium-specific ortholog and evidence for positive selection in a dinoflagellate-specific gene. Evolution of genes and pathways in different dinoflagellates seems to be affected by different lifestyles, and a symbiotic lifestyle may affect population structure and strength of selection. This study is the first evolutionary comparative analysis of orthologs from two coral dinoflagellate symbionts.

Comparative Lipid Profiling of the Cnidarian Aiptasia pallida and Its Dinoflagellate Symbiont

Corals and other cnidarians house photosynthetic dinoflagellate symbionts within membrane-bound compartments inside gastrodermal cells. Nutritional interchanges between the partners produce carbohydrates and lipids for metabolism, growth, energy stores, and cellular structures. Although lipids play a central role in the both the energetics and the structural/morphological features of the symbiosis, previous research has primarily focused on the fatty acid and neutral lipid composition of the host and symbiont. In this study we conducted a mass spectrometry-based survey of the lipidomic changes associated with symbiosis in the sea anemone Aiptasia pallida, an important model system for coral symbiosis. Lipid extracts from A. pallida in and out of symbiosis with its symbiont Symbiodinium were prepared and analyzed using negative-ion electrospray ionization quadrupole time-of-flight mass spectrometry. Through this analysis we have identified, by exact mass and collision-induced dissociation mass spectrometry (MS/MS), several classes of glycerophospholipids in A. pallida. Several molecular species of di-acyl phosphatidylinositol and phosphatidylserine as well as 1-alkyl, 2-acyl phosphatidylethanolamine (PE) and phosphatidycholine were identified. The 1-alkyl, 2-acyl PEs are acid sensitive suggestive that they are plasmalogen PEs possessing a double bond at the 1-position of the alkyl linked chain. In addition, we identified several molecular species of phosphonosphingolipids called ceramide aminoethylphosphonates in anemone lipid extracts by the release of a characteristic negative product ion at m/z 124.014 during MS/MS analysis. Sulfoquinovosyldiacylglycerol (SQDG), an anionic lipid often found in photosynthetic organisms, was identified as a prominent component of Symbiodinium lipid extracts. A comparison of anemone lipid profiles revealed a subset of lipids that show dramatic differences in abundance when anemones are in the symbiotic state as compared to the non-symbiotic state. The data generated in this analysis will serve as a resource to further investigate the role of lipids in symbiosis between Symbiodinium and A. pallida.

Integrating Genomics Research throughout the Undergraduate Curriculum: A Collection of Inquiry-Based Genomics Lab Modules

We wish to let CBE—Life Sciences Education readers know about a portal to a set of curricular lab modules designed to integrate genomics and bioinformatics into commonly taught courses at all levels of the undergraduate curriculum. Through a multi-year, collaborative process, we developed, implemented, and peer-reviewed inquiry-based, integrated instructional units (I3Us) adaptable to a range of teaching settings, with a focus on both model and nonmodel systems. Each of the products is built on vetted design principles: 1) they have clear pedagogical objectives; 2) they are integrated with lessons taught in the lecture; 3) they are designed to integrate the learning of science content with learning about the process of science; and 4) they require student reflection and discussion (Figure 1; National Research Council [NRC], 2005). Eleven I3Us were designed and implemented as multi-week modules within the context of an existing biology course (e.g., microbiology, comparative anatomy, introduction to neurobiology), and three I3Us were incorporated into interdisciplinary biology/computer science classes. Our collection of genomics instructional units, together with extensive supporting material for each module, is accessible on a dedicated website (http://serc.carleton.edu/genomics/activities.html) that also provides links to bioinformatics tools and online assessment and pedagogical resources for teaching genomics. Figure 1. Pedagogical elements of the I3U, which was based on the findings of Americas Lab Report (NRC, 2005 ) and was used as the primary curricular design framework for this project. Rapid advances in genome sequencing and analysis offer unparalleled opportunity and challenge for biology educators. More data are being generated than can be analyzed and contextualized in traditional teaching or research models. Indeed, this explosion of data has spawned rapid growth in the discipline of bioinformatics, which is focused on development of the computational tools and approaches for extracting biologically meaningful insights from genomic data. At the same time, access to vast quantities of genomic data stored in publicly available databases can offer educators ways to engage undergraduates in authentic research and to democratize research that was previously possible only at research-intensive universities with massive instrumentation infrastructures. The integration of genomic and bioinformatic approaches into undergraduate curricula represents one response to the national calls for biology teaching that is more quantitative and promotes deeper understanding of biological systems through interdisciplinary analyses (National Academy of Sciences, 2003 ; Association of American Medical Colleges and Howard Hughes Medical Institute [HHMI], 2009 ; NRC, 2009 ; American Association for the Advancement of Science, 2011 ). Yet relatively few faculty members who teach undergraduate biology have expertise in the fields of genomics or bioinformatics, and they may therefore feel inadequately prepared to develop their own new curricular modules capitalizing on this dispersed abundance of available resources. Our Teagle Foundation–funded genomics education initiative, Bringing Big Science to Small Colleges: A Genomics Collaboration, was designed to address the challenges of helping faculty members integrate genome-scale science into the undergraduate classroom. The goal of the project was to create and disseminate self-contained curricular units that stimulate students and faculty alike to think in new ways and at different scales of biological inquiry. To this end, a series of three workshops over 3 yr brought together a total of 34 faculty participants from 19 institutions and a diverse array of disciplines—including biology, computer science, and science education—to develop a set of lab modules containing a substantial genomics component. We believe that these modules are suitable for integration into existing courses in the biology curriculum and are adaptable to a variety of teaching settings. The project website serves as a portal to activity sheets describing each I3U, complete with learning goals, teaching tips, and links to teaching materials, as well as downloadable resources and assessment tools (Figure 2), that can be customized by any interested educator. Each I3U was peer-reviewed by fellow participants, as well as by a professional project consultant who has extensive experience with Web-based description of teaching materials using this format (Manduca et al., 2006 ). The goals of this review process were to ensure that each I3U met the design criteria articulated above, and to evaluate whether the activity sheet provided both an easily accessible overview of the content and enough detailed information for other instructors to adapt and implement the material and its associated assessment strategies. This peer review was complemented by each participants own explicitly framed evaluation of his/her I3U through a formal reflection form (accessible at http://serc.carleton.edu/genomics/workshop09/index.html). Although these I3Us were designed for courses currently taught by the project participants within the specific institutions’ curricula, we propose that they can be inserted into other courses encompassing similar content (Tables 1 and ​and2)2) and/or learning goals (e.g., Figure 2). We have received many communications from colleagues at other institutions who have adapted our I3Us for their courses. Figure 2. Excerpt from an activity sheet from the Genomics Instructional Units Minicollection describing one of the curricular modules developed within the Bringing Big Science to Small Colleges program (for the complete activity sheet, see http://serc.carleton.edu/genomics/units/19163.html ... Table 1. List of I3Us generated in the Bringing Big Science to Small Colleges collaborative project, grouped by the general level in the curriculum in which they were originally taught Table 2. Pedagogical attributes (scale of biological organization, genomic level of analysis, and bioinformatic skills taught) of I3Us developed in this project and disseminated on the projects website One fundamental characteristic of each I3U in our collection is the focus on guided inquiry. The benefits to an undergraduate of hands-on participation in research are well documented (Nagda et al., 1998 ; Gafney, 2001 ; Hunter et al., 2007 ; Kardash et al., 2008 ; Lopatto, 2009 ). Integrating authentic research experiences into the undergraduate curriculum allows this powerful learning model to be scaled from use with only a few students to use with entire laboratory sections (Lopatto 2009 ; Lopatto et al. 2008 ). Like other national participatory genomic teaching initiatives (Campbell et al., 2006 , 2007 ; Ditty et al., 2010 ; Shaffer et al., 2010 ; HHMI, 2011 ), our model for curriculum development in genomics emphasizes synergies between student-centered research and education. However, in contrast with some of these other projects, our grassroots approach leveraged a wealth of existing expertise by providing opportunities for individual faculty members to develop, implement, modify, evaluate, and share undergraduate teaching modules that stem from their own research and/or teaching interests. In this regard, our project most closely resembles the Genome Consortium for Active Teaching, which provides faculty members and their undergraduates access to microarrays from a variety of organisms, allowing participants to define their own research questions in a model system with which they are already familiar (Campbell et al., 2006 , 2007 ). Our collaborative effort among biologists, computer scientists, and science educators has yielded a collection of pedagogical resources that can be adapted for use in a wide variety of educational settings. We invite other biologists to begin building on our work by using and providing feedback on our I3Us. Faculty who have tested materials that exemplify our design principles are encouraged to add them to our collection. For further information, please contact the corresponding author.

Modularity and Sparsity: Evolution of Neural Net Controllers in Physically Embodied Robots

While modularity is thought to be central for the evolution of complexity and evolvability, it remains unclear how systems boot-strap themselves into modularity from random or fully integrated starting conditions. Clune et al. (2013) suggested that a positive correlation between sparsity and modularity is the prime cause of this transition. We sought to test the generality of this modularity-sparsity hypothesis by testing it for the first time in physically embodied robots. A population of ten Tadros — autonomous, surface-swimming robots propelled by a flapping tail — was used. Individuals varied only in the structure of their neural net control, a 2 x 6 x 2 network with recurrence in the hidden layer. Each of the 60 possible connections was coded in the genome, and could achieve one of three states: -1, 0, 1. Inputs were two light-dependent resistors and outputs were two motor control variables to the flapping tail, one for the frequency of the flapping and the other for the turning offset. Each Tadro was tested separately in a circular tank lit by a single overhead light source. Fitness was the amount of light gathered by a vertically oriented sensor that was disconnected from the controller net. Reproduction was asexual, with the top performer cloned and then all individuals entered into a roulette wheel selection process, with genomes mutated to create the offspring. The starting population of networks was randomly generated. Over ten generations, the population’s mean fitness increased two-fold. This evolution occurred in spite of an unintentional integer overflow problem in recurrent nodes in the hidden layer that caused outputs to oscillate. Our investigation of the oscillatory behavior showed that the mutual information of inputs and outputs was sufficient for the reactive behaviors observed. While we had predicted that both modularity and sparsity would follow the same trend as fitness, neither did so. Instead, selection gradients within each generation showed that selection directly targeted sparsity of the connections to the motor outputs. Modularity, while not directly targeted, was correlated with sparsity, and hence was an indirect target of selection, its evolution a “by-product” of its correlation with sparsity.

Keeping an Eye on Biology

Genomics Explorers, an IBI prize–winning module, engages students with bioinformatics and molecular research. Genomic data sets offer opportunities for students to generate original findings without expensive laboratory equipment, extending the benefits of undergraduate research to the classroom (1–4). Yet, working with data sets online can frustrate biology students who may miss connections to fascinating biological questions. Integrating organismal and molecular biology with bioinformatics can enhance student learning. We have developed a program in genomics, adapted for a plant (Chamaecrista fasciculata) and an animal (Aiptasia pallida), designed to support student learning with a Web-based guide (http://serc.carleton.edu/exploring_genomics/index.html) (see the first chart).