Marine cnidarian-microalgal endosymbiosis is an ecologically important intracellular association. However, its underlying molecular mechanisms are essentially unknown. In light of the critical roles of host phagocytosis in intracellular fates of a variety of microbes, and the Rab small GTPases as key mediators of host-symbiont interaction, we set out to investigate the potential involvement of Aiptasia Rab proteins in the model photosynthetic endosymbiosis between the sea anemone, Aiptasia pulchella and the symbiotic dinoflagellate (commonly called zooxanthellae), Symbiodinium spp. Many Aiptasia Rab homologue-encoding cDNA fragments were first cloned through our degenerate RT-PCR and RACE reactions. Significantly, Aiptasia homologues of Rab5 and Rab7 (ApRab5 and ApRab7), two Rabs known to be critical regulators of phagosome maturation were also identified in the screen. The overall sequence identities of ApRab5 and ApRab7 to those of human Rab5C and Rab7 were very extensive, and EGFP reporter, protein fractionation, and immuno-fluorescence studies all suggested that the similarity of the Aiptasia Rabs to their human counterparts extended to the functional levels. Finally, although the phagosomes enclosing latex beads stained positive for ApRab5 and ApRab7 with kinetics characteristics of normal phagosomal maturation, the phagosomes housing zooxanthellae only stained positive for ApRab5. Furthermore, the association of ApRab5 with and the exclusion of ApRab7 from the zooxanthellae-containing phagosomes could be reversed by the heat-killed or photosynthesis-impaired symbionts. Overall, our present study has identified ApRab5 and ApRab7 as potential key regulators of the Aiptasia-Symbiodinium endosymbiosis

Mitochondria and chloroplasts are eukaryotic organelles that were acquired through endosymbiosis. In the case of the mitochondrion, a heterotrophic cell engulfed and retained an alpha-proteobacterium. The engulfed bacterium, or endosymbiont, underwent extensive cellular and genetic integration with its host, thereby becoming an organelle. Chloroplasts are derived from the engulfment and retention of a photosynthetic cyanobacterium that also experienced loss of cellular functions and genetic material. Although mitochondria and chloroplasts retain their own genomes, most of the proteins that function in these organelles are encoded by genes that were transferred to the nucleus in a process known as Endosymbiotic Gene Transfer (EGT). Chloroplasts in plants, green algae and red algae are known as primary plastids. Other photosynthetic organisms have secondary plastids that were acquired by engulfing and retaining a primary plastid-bearing alga. In the process, the nucleus of the engulfed alga underwent EGT (and presumably gene loss) to such an extent that it disappeared completely except in two lineages, cryptophytes and chlorarachniophytes, which retain a highly reduced and miniaturized form known as a nucleomorph. To understand the process of EGT and endosymbiosis in general, the nuclear genomes of the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans were sequenced. In the case of G. theta its nucleomorph is of red algal origin while the nucleomorph of B. natans is derived from a green algal endosymbiont. Prior to the nuclear genome projects the genomes of the three organelles – plastid, mitochondrion, nucleomorph – had already been sequenced. This allowed investigation of recent transfers of organellar DNA to the nucleus. Mitochondrial transfers to the nucleus are still occurring in both organisms but transfers of plastid and nucleomorph DNA are not. The nucleomorph genomes of B. natans and G. theta appear ‘frozen’, unable to undergo EGT and thus unable to disappear as they have in all other lineages with secondary plastids. The creation of a spliceosomal intron from transferred organellar DNA was investigated. I also investigated nuclear genes whose encoded proteins appear to function in the mitochondrion. 833 putatively mitochondrial targeted proteins were identified in G. theta and 720 in B. natans.

Thesis supervisor: Debashish Bhattacharya. Includes bibliographic references (p. 126-142).

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Year: 2016. / Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema have emerged excellent as non-chemical alternatives for control of insect pest population. They have a specific mutualistic symbioses with bacterial symbionts in the genera Photorhabdus and Xenorhabdus, respectively. Native EPN species that are able to tolerate environmental stress including desiccation are of great interest for application. The aim of this study was to isolate indigenous EPN species from soil samples collected from Brits, North West province in South Africa, and to investigate their ability to tolerate desiccation stress. The second aim was to isolate the bacterial symbiont and sequence, assemble and annotate its whole genomic DNA. Insect baiting technique and White trap method proved useful in the recovery of nematodes from collected soil samples and infected cadaver, respectively. Molecular identification based on the amplification of the 18S rDNA and phylogenetic relationships revealed high affinity of the unknown EPN isolate 10 to Steinernema species and due to variation in evolutionary divergence distance, the unknown isolate was identified as Steinernema spp. isolate 10 . Isolates 35 and 42 revealed high similarity to Heterorhabditis zealandica strain Bartow (accession number: GU174009.1), Heterorhabditis zealandica strain NZH3 (accession number: EF530041.1) and the South African isolate Heterorhabditis zealandica strain SF41 (EU699436.1). Both Steinernema spp. isolate 10 and Heterorhabditis species could tolerate desiccation. Steinernema spp. isolate 10 was tolerant up to 11 days of desiccation exposure in loamy sand and up to 9 days of exposure in river sand, causing 26, 6% and 13, 4% cumulative larval mortality after 96 hours, post resuscitation by rehydration, respectively. Heterorhabditis spp. could tolerate desiccation up to 13 days of exposure and induced 26.6% cumulative larval mortality on both loamy and river sand after 96 hours post resuscitation. Swarming, aggregation, coiling and clumping behavioural characteristics were observed when Steinernema spp. isolate 10 was exposed to desiccation and Heterorhabditis species displayed no similar behavioural characteristics associated with desiccation tolerance. Morphological characteristics of the unknown Steinernema spp. isolate 10 have been described, and the thick cuticle and sheath which are both associated with tolerance to desiccation stress have been noted. The bacterial symbiont was isolated from larval hosts infected with Steinernema spp. isolate 10 and molecular identification through NCBI Blastn based on the 16S rDNA revealed high affinity to Xenorhabdus bacterial species. Phylogenetic relationships and evolutionary divergence estimates 16 revealed genetic variation and the species was identified as Xenorhabdus bacterial isolate. The genome assembly of Xenorhabdus bacterial isolate using CLC Bio revealed a total length of 4, 183, 779 bp with 231 contigs (>=400bp), GC content of 44.7% and N50 of 57,901 bp. Annotation of the assembled genome through NCBI PGAAP annotation pipeline revealed 3,950 genes (3,601 protein coding sequences (CDS) and 266 pseudogenes), 12 rRNAs and 70 tRNAs. RAST annotation revealed 55 of virulence, disease and defense subsystem features which are involved in the pathogenicity of Xenorhabdus bacterial isolate. The ability of EPNs to tolerate environmental stress is highly crucial and one of the determining factors for biocontrol potential and successful application, thus the indigenous desiccation tolerant EPN isolate, Steinernema spp. isolate 10 holds great potential as a biological control agent. The genome sequencing and annotation reveals insight to behavioural and physiological attributes of bacterial symbionts and this study will contribute to the understanding of pathogenicity and evolution of the bacteria–nematode complex. / GR 2016

Parasitic reproductive endosymbionts are emerging as formidable threats to insect biodiversity. Wolbachia are prevalent maternally inherited intra-cellular bacteria found in >50% of arthropod species. These symbiotic bacteria interact with their hosts in diverse ways, most often they alter host reproduction causing four conditions that all selectively favor infected females: feminization, male killing, parthenogenesis, and cytoplasmic incompatibility (CI). Furthermore, depending on strain-type and host genetic background, Wolbachia are known to affect insect behavior, expand or shift host thermal tolerance ranges, and confer anti-viral protection to their hosts. Because Wolbachia both reside in and are transmitted with host cell cytoplasm, mitochondria and other cytoplasmically inherited genetic elements become linked with the bacteria. Thus, by enhancing their own transmission, Wolbachia-induced phenotypes can lead to mitochondrial selective sweeps, which may have profound impacts on vulnerable and small insect populations. Elucidating the extent to which endosymbionts influence biological and ecological functions is pivotal to making management decisions regarding imperiled insect species. My dissertation investigates biological and ecological impacts of host-endosymbiont interactions by examining Wolbachia infections in three different host systems. First, I used the federally threatened butterfly species Speyeria zerene hippolyta to determine whether the general reproductive success of local populations was affected by the introduction of CI-inducing Wolbachia-infected butterflies through implemented species recovery programs. Next, by characterizing the Wolbachia infections of parasitoids associated with the Eurema butterfly clade, I analyzed whether host-parasitoid interactions provide a path for interspecies horizontal transmission. Finally, I conducted a laboratory experiment using an isogenic Drosophila melanogaster line to determine whether Wolbachia influence host temperature preference. Together, my research examines how the individual level effects of host-endosymbiont interactions can expand into populations, have broader impacts on insect communities, and potentially impede the conservation and management of insects in nature. In chapter one, I screened S. z. hippolyta samples from three extant populations for Wolbachia infection. To examine the impacts of Wolbachia on small populations, I analyzed and compared infected and uninfected S. z. hippolyta reproductive data and showed that, in a population composed of infected and uninfected S. z. hippolyta, uninfected butterflies had reduced reproductive success (GLMM z = -8.067, P < 0.0001). I then developed a single-population demographic theoretical model using these same reproductive data to simulate and analyze different potential dynamics of small populations resulting from population supplementation with uninfected, CI-Wolbachia infected, or combined uninfected and infected butterflies. Analysis of model simulations revealed that supplementation with CI-inducing butterflies significantly suppressed host-population size (ANOVA F5,593 = 3349, PWolbachia-infected individuals (Tukey's post-hoc test P < 0.0001). In addition, supplementation by multiple releases using a combination of 50 infected and 300 uninfected butterflies has a less severe suppression effect, reducing the population by 75.8%, but the reduction occurs 42.6% faster than with the single release of 50 Wolbachia-infected butterflies (Tukey's post-doc test P < 0.0001). Parasitoid-host interactions have emerged as probable ecological relationships to facilitate horizontal transmission of Wolbachia. In chapter two, I addressed horizontal transmission using Eurema butterflies and their associated parasitoids. From four locations in Northern Queensland, Australia, I collected a total of 404 Eurema hecabe butterfly larvae. Twenty-three parasitoids emerged from the larvae of which 21 were Diptera and two were Hymenoptera. I amplified COI loci fragments from each parasitoid for BLAST query searches and found that 20 individual Diptera parasitoids matched to the genus Exorista and one to the genus Senometopia. One of the Hymenoptera parasitoids matched to the genus Microoplitis and the other to the genus Cotesia. To characterize Wolbachia infections, I used Wolbachia Multi Locus Sequencing Technique (MLST) and discovered that all 20 Exorista parasitoids were infected with an identical Wolbachia strain (ST-41), which is the same strain infecting their Eurema hecabe butterfly hosts. Although, further experiments are necessary to definitively determine that ST-41 Wolbachia are incorporated into germline cells of the parasitoids, this is the first study to provide ecological evidence for inter-ordinal Wolbachia transmission between Lepidoptera and Diptera. Furthermore, this discovery exposes the risk of population augmentation programs that move insects, potentially facilitating the spread of Wolbachia between species within a community through the accidental introduction of new Wolbachia-infected parasitoids. Finally, both Wolbachia and their insect hosts are temperature sensitive organisms. Wolbachia's replication behavior in their hosts is positively-temperature dependent, while environmental variation can have profound effects on insect's immune function, fitness, and fecundity. In chapter three, I conducted a laboratory experiment using a thermal gradient choice assay and an isogenic Drosophila melanogaster line with four different Wolbachia infection statuses -- uninfected, wMel, wMelCS, and wMelPop - to assess whether a relationship existed between Wolbachia infection and host temperature preference. Results from my laboratory experiment revealed that Wolbachia-infected flies preferred cooler temperatures compared to uninfected flies. Moreover, D. melanogaster temperature preferences varied depending on the Wolbachia strain variant with which they were infected; flies infected with the wMel strain had temperature preferences 2°C cooler compared to uninfected flies; flies infected with either wMelCS or wMelPop strains had preferred temperatures 8°C cooler compared to uninfected flies. Wolbachia-associated temperature preference variation within a species can lead to conspecifics occupying different microclimates, genetically adapting to different sets of specific environmental conditions, and may eventually result in ecological and reproductive isolation. While, reproduction isolation is recognized as one of the first stages in speciation, in small populations of endangered and threatened species, the inability to reproduce between conspecifics can drive species to extirpation or extinction. Collectively, the three chapters of my dissertation set precedent for future integration of host-endosymbiont research prior to implementing population supplementation or translocation programs for the conservation of imperiled insects.

Intracellular symbioses between cnidarians and dinoflagellates from the genus Symbiodinium are widespread throughout the marine environment. These associations are ecologically significant, especially in tropical waters where symbiotic interactions between corals and Symbiodinium culminate in the formation of limestone reefs. This thesis focuses on cellular and molecular aspects of the symbiosis, specifically the initiation of the symbiosis and characterization of a host gene, sym32, that is believed to function in the symbiosis. Sym32 was originally identified as a differentially expressed protein in symbiotic vs. aposymbiotic individuals of the sea anemone, Anthopleura elegantissima. Based on its deduced amino acid sequence, sym32 belongs to a family of cell adhesion proteins that play roles in cell recognition in a diverse array of organisms. Chapter 2 examines the process by which a new cnidarian host acquires its first symbionts. Larvae of the scleractinian coral Fungia scutaria, which are initially aposymbiotic, acquired symbionts while feeding. Symbionts that entered the larval gastric cavity with food were subsequently taken into host gastrodermal cells by phagocytosis. Chapter 3 describes immunolocalization of sym32 in A. elegantissima tentacles. In aposymbiotic tentacles, sym32 was localized to vesicles within the host gastrodermal cells. Symbiotic tentacles lacked sym32-containing vesicles. Instead, sym32 was present among the membranes that enclose the symbionts within host cells. Western blots of proteins from Symbiodinium revealed a 45/48kD doublet that cross-reacts with anti-sym32 antiserum. This suggests that homologous proteins are expressed in both host (32kD) and symbiont (45/48 kD). Chapter 4 describes the effects of environmental factors on expression of host sym32. Aposymbiotic and symbiotic anemones maintained in continual darkness for 3 weeks experienced a dramatic decline in sym32 protein levels, relative to anemones maintained on a 12:12 h light:dark cycle. This suggests that light plays a major role in regulating sym32. Exposure of anemones to elevated temperatures for 2 days in the dark caused a mild bleaching response (expulsion of symbionts from the host), but did not affect the levels of sym32 protein. Chapter 5 examines the role of sym32 during the infection process, using antibody interference techniques. F. scutaria larvae and symbionts incubated in sym32 antiserum during the infection process experienced a decline in infection rates. Further, symbionts that were incorporated into host gastroderm appeared to be degenerating in antiserum treatments, but appeared to be healthy in preimmune controls. / Graduation date: 2003

Endosymbiosis in arthropods involves intracellular bacteria that supply an array of benefits to the host. Endosymbionts likely enhance the health of ticks by provisioning amino acids such as cysteine and tyrosine, and cofactors such as biotin and folic acid that are not available in blood--the sole nutrient source of ticks. Endosymbionts of ticks are of special interest due to their close evolutionary relationship with tick-vectored pathogens that impact livestock and human health. For example, ticks typically contain Coxiella-like endosymbionts (CLEs) that are the closest relatives of the human pathogen Coxiella burnetii. In order to understand the evolutionary relationship between the mammalian pathogen Francisella tularensis, which is vectored by ticks, and the Francisella-like endosymbionts (FLEs) present in several ticks, we assembled the genomes of the FLEs in the hard tick Amblyomma maculatum and the soft tick Ornithodoros moubata using high-throughput sequencing. While this project was in progress, another group described the genome of an FLE in the soft tick Argus (Persicargas) arboreus. Utilizing the three genomes, we show that all FLEs evolved from a mammalian pathogen, a relationship that is converse to that of C. burnetii, which likely evolved from a tick-associated non-pathogenic ancestor. Additionally, our analyses indicate that FLEs are horizontally transferred between ticks, and due to their superior metabolic capabilities could replace ancestral endosymbionts with reduced genomes.

My dissertation focuses on genome and functional evolution of photosynthetic eukaryotes and the design and implementation of computational methods and tools to enable genome-wide studies to investigate these taxa. The work described here is grouped into two major topics, 1) endosymbiosis and genome evolution, and 2) harmful algal blooms. I discuss my work related to endosymbiosis and genome evolution in chapters 2-4. Chapters 5-6 cover the work related to harmful algal blooms. In chapter 1, I introduce the state-of-art of what is known about the history of plastids and evolution of photosynthesis in eukaryotes, an overview of marine harmful algae, and the specific aims of my dissertation. In chapter 2, I describe the design and implementation of the phylogenetic sorting tool, PhyloSort and the assembly of a high-throughput phylogenomic pipeline. Together, PhyloSort and the pipeline has become a key tool for multiple subsequent studies. chapter 2 also presents a case study using these tools in which we provide an estimate of the number of cyanobacterial genes that have been transferred to the nuclear genome of Plantae through primary endosymbiotic gene transfer; I use the model unicellular green alga Chlamydomonas reinhardtii for this purpose. In chapter 3, I discuss another case of prokaryotic contribution to the nucleus of photosynthetic eukaryotes. Here, the intriguing relationship of Chlamydiae-like bacteria and plants and algae is examined in a large-scale analysis, in which we scanned all available genomes of the primary photosynthetic organisms for genes of potential Chlamydiae origin. Surprisingly, we identified more than fifty Chlamydiae-derived genes in plants and algae. Here, we propose a model for the role that a Chlamydiae-like symbiont might have played in the establishment of the primary plastid in the common ancestor of Plantae. In chapter 4, I describe a study in which we explored the complete protein models of two diatom organisms as representative for photosynthetic chromalveolates and looked for genes that might have been acquired through endosymbiotic (secondary) or horizontal transfers from red or green algae. In contradiction of the “chromalveolate hypothesis” which states that photosynthesis in chromalveolates originated via the engulfment of a red alga symbiont, our study shows an unexpected green algal contribution that is fourfold greater than that of the canonical red algal symbiont. Our data suggest that the chromalveolate history includes a previously unrecognized green algal endosymbiont that was captured and lost prior to the more recent establishment of the red alga plastid, which is widespread in extant photosynthetic chromalveolates. In chapter 5, I discuss the identification of the phylogenetic origin of the genes involved in the biosynthetic pathway of saxitoxin in cyanobacteria. Here, we used a pyrosequencing approach to sequence de novo genomes of two strains of Anabaena circinalis, one of which is saxitoxin-producing and the other is non-toxic. Using comparative and phylogenetic analyses, I show that, within the saxitoxin gene cluster, genes that encode the key and unique enzymes in the pathway are of foreign origin that originated via horizontal transfer from non-cyanobacterial sources. These genes introduced the ability to produce saxitoxin in the ancestor of the toxic cyanobacterial clade. In chapter 6, I describe a gene expression study in which we used massively parallel signature sequencing (MPSS) to investigate RNA abundance patterns in the toxic dinoflagellate Alexandrium tamarense. This work provides the first clear evidence for the utilization by dinoflagellates of transcriptional to regulation. Moreover, using MPSS, we provide an estimate of the number of the distinct genes in Alexandrium tamarense; i.e., remarkably 40,000 loci. Taken together, our data indicate that dinoflagellates possess a great metabolic flexibility that allows them to efficiently toggle between photoautotrophy and heterotrophy based on the environmental conditions.

Coral reefs ecosystems are some of the most productive and diverse in the world. The symbiotic association between cnidarians and their symbiotic microalgae is of great importance in coral reef ecosystems; however, its underlying molecular mechanism remains unclear even after decades of research. Rab small GTP binding proteins are critical regulators of vesicle trafficking. Here we present the experimental evidence supporting a possible association of ApRab37 with the surface of lipid droplets in the endosymbiosis between the sea anemone, Aiptasia pulchella and the symbiotic dinoflagellate (commonly known as zooxanthellae). ApRab37, a protein of 215 amino acids, displays strong homology with human Rab37. In transfected COS7 cells, EGFP-ApRab37 localized to lipid droplets and clustered in the peri-nuclear region, which stained positive for the ER (endoplasmic reticulum) marker. Immunostaining analysis found ApRab37 associated with symbiosomes and lipid droplets, which was also confirmed by Western blot analysis of in the enrich lipid droplet fraction. Phagocytosis assay showed that ApRab37 involved in late phase of phagocytosis. DCMU treatment indicates symbiosome association of ApRab37 is mediated by zooxanthellae. I propose that ApRab37 plays a pivotal role in the regulation of lipid trafficking from the symbiosomes to the host cell during the Aiptasia-zooxanthellal endosymbiosis.

Thesis--Auburn University, 2007. / Abstract. Includes bibliographic references (p.117-142).