Integrating genomic, transcriptomic and developmental approaches to investigate coloniality and life cycle evolution in the Hydractiniidae (Hydrozoa: Cnidaria)

Integrative approaches to evolutionary biology yield rich data through which we can truly begin to understand the marvels of life. This dissertation integrates genomic, transcriptomic, and developmental approaches to understand the evolution of prominent life history characters of the cnidarian class Hydrozoa, including the transition from solitary to colonial forms, an elaboration of coloniality known as polyp polymorphism, and medusae (jellyfish) evolution and loss. While these characters have been repeatedly explored phylogenetically, recognizing interesting and complex evolutionary patterns of character transitions, understanding of these complex patterns of character evolution will ultimately come from insight into their development. In this dissertation, I have developed workflows for analyzing RNA-Seq data in both an intra- and interspecific comparative context. Using next-generation sequencing I not only characterize entire transcriptomic expression profiles in various tissues of two hydractiniid hydrozoans, Hydractinia symbiolongicarpus and Podocoryna carnea, but also assess and accurately characterize intra- and interspecific changes in gene expression. Using these unbiased differential expression analyses, I identify correlated changes in expression and propose candidate genes and gene pathways that are potentially involved in these key transitions. Furthermore, using whole mount in situ hybridization to characterize the spatial expression of various candidates genes, I validated each approach showing expression consistent with their role in the development of a particular tissue or life cycle stage. Results presented in this dissertation suggest that the differential regulation of gene expression, as well as novel gene gain and loss appear to have played an important role in hydrozoan life cycle transitions. Moreover, these results reveal the power of these unbiased genomic/transcriptomic methods over traditional comparative candidate gene approaches to address longstanding questions of hydrozoan morphology and evolution

Racial Microaggressions And Mental Health: Internalized Racism As A Mediator And Black Identity And Social Support As Moderators

Internalized racism, also referred to as appropriated racial oppression, refers to the phenomenon of people of color adopting negative racist messages about their worth and abilities. The internalization of racism by members of the targeted group results in an experience of self-degradation and self-alienation and the assumption of one’s inferiority, which is directly related to issues of self-esteem, self-confidence, shame, depression, and anxiety. This study used structural regression with moderation and mediation to explore the possibility of internalized racism as a mediating variable and black identity and perceived social support as possible moderators. A sample of 639 participants (MAge = 35.29, SDAge = 10.09) who identified as Black and/or African American were included in the study. The findings indicated that internalized racism partially mediated the relationship between racial microaggressions and depression and anxiety, where approximately 50 percent of the relationships were explained by internalized racism. Additionally, the findings indicated that centrality and private regard aspects of Black identity weakened the relationship between racial microaggressions and depression and anxiety. Finally, the findings indicated that social support weakens the relationship between racial microaggressions and depression and anxiety

Interspecific Differential Expression Analysis of RNA-Seq Data Yields Insight into Life Cycle Variation in Hydractiniid Hydrozoans

Hydrozoans are known for their complex life cycles, which can alternate between an asexually reproducing polyp stage and a sexually reproducing medusa stage. Most hydrozoan species, however, lack a free-living medusa stage and instead display a developmentally truncated form, called a medusoid or sporosac, which generally remains attached to the polyp. Although evolutionary transitions in medusa truncation and loss have been investigated phylogenetically, little is known about the genes involved in the development and loss of this life cycle stage. Here, we present a new workflow for evaluating differential expression (DE) between two species using short read Illumina RNA-seq data. Through interspecific DE analyses between two hydractiniid hydrozoans, Hydractinia symbiolongicarpus and Podocoryna carnea, we identified genes potentially involved in the developmental, functional, and morphological differences between the fully developed medusa of P. carnea and reduced sporosac of H. symbiolongicarpus. A total of 10,909 putative orthologs of H. symbiolongicarpus and P. carnea were identified from de novo assemblies of short read Illumina data. DE analysis revealed 938 of these are differentially expressed between P. carnea developing and adult medusa, when compared with H. symbiolongicarpus sporosacs, the majority of which have not been previously characterized in cnidarians. In addition, several genes with no corresponding ortholog in H. symbiolongicarpus were expressed in developing medusa of P. carnea. Results presented here show interspecific DE analyses of RNA-seq data to be a sensitive and reliable method for identifying genes and gene pathways potentially involved in morphological and life cycle differences between species

Influences of functional variation on venom expression in hydractiniid hydrozoans

This presentation was given at the Gordon Research Conference: Venom Evolution, Function and Biomedical Applications – 5-10 August 2018, Mount Snow, VT, USACnidaria (jellyfish, hydra, sea anemones, etc) represent the earliest diverging venomous animal lineage. Cnidarians deploy venom for predation, defense, competition, and digestion. Recent evidence suggests venom composition can be influenced by age, diet, geography, and the presence of predators or prey. Although venom production and maintenance are central to the life history of cnidarians, little is known about their venom composition with respect to biological or ecological function. Hydractiniid hydrozoans are an ideal system for studying venom function and evolution due to their functionally specialized tissue types and complex life cycles. The hydractiniid Hydractinia symbiolongicarpus displays a division of labor among its polyps that comprise the colony: the gastrozooid (feeding and digestion), dactylozooid (defense and predation), and gonozooid (reproduction). Podocoryna carnea, a related hydractiniid hydrozoan, displays a complex life history that includes a benthic colonial stage and a pelagic medusa. Using publicly available RNA-seq data of the functionally specific polyp types of H. symbiolongicarpus and life cycle stages of P. carnea, we characterized the putative venom components and venom expression between these tissues. Based on this analysis, we can determine how the venom arsenal varies for specific tasks and between life cycle stages. Future work will include lineage-based transcriptomics on the stinging cells of H. symbiolongicarpus as well as functional characterization of specific venom components using CRISPR. Understanding how venom composition is influenced by various developmental and ecological factors will lead to a better understanding of venom diversity and function in cnidarians

Differentially expressed venoms in functionally specialized polyps of the colonial hydrozoan Hydractinia symbiolongicarpus

This presentation was given at the 7th European Evolution and Development Conference – 26-29th June, 2018, Galway, Ireland. The poster won third place in the EEDC Student Poster Presentation competition.Cnidaria (jellyfish, hydra, sea anemones, etc.) represent the earliest diverging venomous animal lineage. Venom is deployed in cnidarians for predation, defense, competition, and digestion. Recent evidence suggests venom composition can be influenced by age, diet, geography, and the presence of predators or prey. Although venom production and maintenance are central to the life history of cnidarians, little is known about their venom composition with respect to biological or ecological function. Hydractiniid hydrozoans are an ideal system for studying venom function and evolution due to their functionally specialized tissue types and complex life cycles. The hydractiniid Hydractinia symbiolongicarpus displays a division of labor among its polyps that comprise the colony: dactylozooids (defense and predation), gastrozooids (feeding and digestion), and gonozooids (reproduction). Using an existing transcriptome of the different functional polyp types of H. symbiolongicarpus, we characterized the putative venom components and venom expression between these tissues. By using functionally specific polyps of H. symbiolongicarpus, we can determine how the venom arsenal varies for specific tasks. Understanding how venom composition is influenced by various developmental and ecological factors will lead to a better understanding of venom diversity and function in cnidarians

Differential gene expression between functionally specialized polyps of the colonial hydrozoan Hydractinia symbiolongicarpus (Phylum Cnidaria)

Background: A colony of the hydrozoan Hydractinia symbiolongicarpus comprises genetically identical yet morphologically distinct and functionally specialized polyp types. The main labor divisions are between feeding, reproduction and defense. In H. symbiolongicarpus, the feeding polyp (called a gastrozooid) has elongated tentacles and a mouth, which are absent in the reproductive polyp (gonozooid) and defensive polyp (dactylozooid). Instead, the dactylozooid has an extended body column with an abundance of stinging cells (nematocysts) and the gonozooid bears gonophores on its body column. Morphological differences between polyp types can be attributed to simple changes in their axial patterning during development, and it has long been hypothesized that these specialized polyps arose through evolutionary alterations in oral-aboral patterning of the ancestral gastrozooid. Results: An assembly of 66,508 transcripts (>200 bp) were generated using short-read Illumina RNA-Seq libraries constructed from feeding, reproductive, and defensive polyps of H. symbiolongicarpus. Using several different annotation methods, approximately 54% of the transcripts were annotated. Differential expression analyses were conducted between these three polyp types to isolate genes that may be involved in functional, histological, and pattering differences between polyp types. Nearly 7 K transcripts were differentially expressed in a polyp-specific manner, including members of the homeodomain, myosin, toxin and BMP gene families. We report the spatial expression of a subset of these polyp-specific transcripts to validate our differential expression analyses. Conclusions: While potentially originating through simple changes in patterning, polymorphic polyps in Hydractinia are the result of differentially expressed functional, structural, and patterning genes. The differentially expressed genes identified in our study provide a starting point for future investigations of the developmental patterning and functional differences that are displayed in the different polyp types that confer a division of labor within a colony of H. symbiolongicarpus

Venom system variation and the division of labor in the colonial hydrozoan Hydractinia symbiolongicarpus

Cnidarians (jellyfish, hydroids, sea anemones, and corals) possess a unique method for venom production, maintenance, and deployment through a decentralized system composed of different types of venom-filled stinging structures called nematocysts. In many species, nematocyst types are distributed heterogeneously across functionally distinct tissues. This has led to a prediction that different nematocyst types contain specific venom components. The colonial hydrozoan, Hydractinia symbiolongicarpus, is an ideal system to study the functional distribution of nematocyst types and their venoms, given that they display a division of labor through functionally distinct polyps within the colony. Here, we characterized the composition and distribution of nematocysts (cnidome) in the different polyp types and show that the feeding polyp (gastrozooid) has a distinct cnidome compared to the reproductive (gonozooid) and predatory polyp (dactylozooid). We generated a nematocyst-specific reporter line to track nematocyst development (nematogenesis) in H. symbiolongicarpus, and were able to confirm that nematogenesis primarily occurs in the mid-region of the gastrozooid and throughout stolons (tubes of epithelia that connect the polyps in the colony). This reporter line enabled us to isolate a nematocyst-specific lineage of cells for de novo transcriptome assembly, annotate venom-like genes (VLGs) and determine differential expression (DE) across polyp types. We show that a majority of VLGs are upregulated in gastrozooids, consistent with it being the primary site of active nematogenesis. However, despite gastrozooids producing more nematocysts, we found a number of VLGs significantly upregulated in dactylozooids, suggesting that these VLGs may be important for prey-capture. Our transgenic Hydractinia reporter line provides an opportunity to explore the complex interplay between venom composition, nematocyst diversity, and ecological partitioning in a colonial hydrozoan that displays a division of labor

Frizzled3 Expression and Colony Development in Hydractiniid Hydrozoans

Hydractiniid hydrozoan colonies are comprised of individual polyps connected by tube‐like stolons or a sheet‐like mat. Mat and stolons function to integrate the colony through continuous epithelia and shared gastrovascular cavity. Although mechanisms of hydrozoan polyp development have been well studied, little is known about the signaling processes governing the patterning of colonies. Here we investigate the Wnt receptor family Frizzled. Phylogenetic analysis reveals that hydrozoans possess four Frizzled orthologs. We find that one of these genes, Frizzled3 , shows a spatially restricted expression pattern in colony‐specific tissue in two hydractiniid hydrozoans, Hydractinia symbiolongicarpus and Podocoryna carnea , in a manner that corresponds to their distinct colony forms (stolonal mat in Hydractinia and free stolons in Podocoryna ). Interestingly, Frizzled3 was lost in the genome of Hydra , which is a solitary polyp and thus lacks colony‐specific tissue. Current evidence suggests that the Wnt signaling pathway plays a key role in the evolution of colony diversity and colony loss in Hydrozoa

Coevolution of the Tlx homeobox gene with medusa development (Cnidaria: Medusozoa)

Cnidarians display a wide diversity of life cycles. Among the main cnidarian clades, only Medusozoa possesses a swimming life cycle stage called the medusa, alternating with a benthic polyp stage. The medusa stage was repeatedly lost during medusozoan evolution, notably in the most diverse medusozoan class, Hydrozoa. Here, we show that the presence of the homeobox gene Tlx in Cnidaria is correlated with the presence of the medusa stage, the gene having been lost in clades that ancestrally lack a medusa (anthozoans, endocnidozoans) and in medusozoans that secondarily lost the medusa stage. Our characterization of Tlx expression indicate an upregulation of Tlx during medusa development in three distantly related medusozoans, and spatially restricted expression patterns in developing medusae in two distantly related species, the hydrozoan Podocoryna carnea and the scyphozoan Pelagia noctiluca. These results suggest that Tlx plays a key role in medusa development and that the loss of this gene is likely linked to the repeated loss of the medusa life cycle stage in the evolution of Hydrozoa