The evolution of lignocellulose digestion in termite-associated protists

Description

The symbiosis between termites and their parabasalid hindgut protists centers around the wood digestion that is needed for both species to acquire the nutrients from wood. One of the important carbohydrate-active proteins required for the wood breakdown are glycoside hydrolase…

The symbiosis between termites and their parabasalid hindgut protists centers around the wood digestion that is needed for both species to acquire the nutrients from wood. One of the important carbohydrate-active proteins required for the wood breakdown are glycoside hydrolase (GH) families. Previous studies have looked at the phylogeny of some of these protein families from a termite whole gut transcriptome or in a different context than lignocellulose digestion. In this study, we attempt to understand the function and evolution of these GH families in the context of protist evolution by using protist single cell transcriptomes. 14 families of interest were chosen to create phylogenetic trees: GH2, GH3, GH5, GH7, GH8, GH9, GH10, GH11, GH26, GH43, GH45, GH55, GH67, GH95 for their interesting expressions across different protists such as being present in all protists or being present in only termite-associated protists. The dbCAN2 (automated Carbohydrate-active enzyme ANnotation) program was used to find GH families in each of the protist single cell transcriptomes and additional characterized sequences registered on the National Center for Biotechnology Information to create phylogenetic trees for each of the GH families of interest. Results show that many of the GH families expressed in protists were acquired through horizontal gene transfer from fungi and bacteria. Additionally, comparison to the parabasalid phylogeny indicates most GH families evolved independently from the protists. Based on the pattern of expression of these GH families throughout different protist orders, conclusions can be made about whether the specific family was vertically or horizontally acquired in the termite symbionts.

Date Created
2023-05
Agent

Discovery, Characterization and Ecological Impact of a Predatory Bacterium of Cyanobacteria Responsible for Epidemics in Biocrusts

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Description
Predatory bacteria are a guild of heterotrophs that feed directly on other living bacteria. They belong to several bacterial lineages that evolved this mode of life independently and occur in many microbiomes and environments. Current knowledge of predatory bacteria is…

Predatory bacteria are a guild of heterotrophs that feed directly on other living bacteria. They belong to several bacterial lineages that evolved this mode of life independently and occur in many microbiomes and environments. Current knowledge of predatory bacteria is based on culture studies and simple detection in natural systems. The ecological consequences of their activity, unlike those of other populational loss factors like viral infection or grazing by protists, are yet to be assessed. During large-scale cultivation of biological soil crusts intended for arid soil rehabilitation, episodes of catastrophic failure were observed in cyanobacterial growth that could be ascribed to the action of an unknown predatory bacterium using bioassays. This predatory bacterium was also present in natural biocrust communities, where it formed clearings (plaques) up to 9 cm in diameter that were visible to the naked eye. Enrichment cultivation and purification by cell-sorting were used to obtain co-cultures of the predator with its cyanobacterial prey, as well as to identify and characterize it genomically, physiologically and ultrastructurally. A Bacteroidetes bacterium, unrelated to any known isolate at the family level, it is endobiotic, non-motile, obligately predatory, displays a complex life cycle and very unusual ultrastructure. Extracellular propagules are small (0.8-1.0 µm) Gram-negative cocci with internal two-membrane-bound compartmentalization. These gain entry to the prey likely using a suite of hydrolytic enzymes, localizing to the cyanobacterial cytoplasm, where growth begins into non-compartmentalized pseudofilaments that undergo secretion of vesicles and simultaneous multiple division to yield new propagules. I formally describe it as Candidatus Cyanoraptor togatus, hereafter Cyanoraptor. Its prey range is restricted to biocrust-forming, filamentous, non-heterocystous, gliding, bundle-making cyanobacteria. Molecular meta-analyses showed its worldwide distribution in biocrusts. Biogeochemical analyses of Cyanoraptor plaques revealed that it causes a complete loss of primary productivity, and significant decreases in other biocrusts properties such as water-retention and dust-trapping capacity. Extensive field surveys in the US Southwest revealed its ubiquity and its dispersal-limited, aggregated spatial distribution and incidence. Overall, its activity reduces biocrust productivity by 10% at the ecosystem scale. My research points to predatory bacteria as a significant, but overlooked, ecological force in shaping soil microbiomes.
Date Created
2022
Agent

The Evolution of Nonsense-Mediated Decay in Parabasalia

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Description

Nonsense mediated decay is a pathway that selectively degrades mRNAs that contain premature termination codons (PTCs). The purpose of this study is to research the evolution of NMD in Parabasalia and infer whether they have a normal, functioning nonsense-mediated decay…

Nonsense mediated decay is a pathway that selectively degrades mRNAs that contain premature termination codons (PTCs). The purpose of this study is to research the evolution of NMD in Parabasalia and infer whether they have a normal, functioning nonsense-mediated decay pathway. Parabasalia are single-celled, flagellated protists that have undergone evolutionary transitions as they become obligate symbionts of termites. The key proteins involved in nonsense-mediated decay, ATM, ATR, UPF1, SMG1, UPF2, UPF3A, UPF3B, were researched and used in order to build phylogenetic trees to analyze what other species of eukaryotes have these same genes and where they branch relative to the nonsense mediated decay proteins present in Parabasalia. The main question being asked in this research is if Parabasalia have enough of the main nonsense mediated decay proteins to have a functional nonsense-mediated decay process and if not, which proteins have been lost over evolutionary history. To carry out this research, phylogenic trees were built using transcriptomes from many different types of eukaryotes that contained the main proteins involved in the nonsense-mediated decay pathway. These transcriptomes were taken from the National Center for Biotechnology Information (NCBI) database using the BLAST algorithm, trimmed using TrimAl, aligned by utilizing AliView which utilizes Muscle. Sequoia was then used to remove redundant species from the trees, and IQ-TREE was used to form the phylogenic trees. This process was repeated four times to create well-rounded trees with various eukaryotic species present. The results of this research found that ATM, ATR, UPF1, SMG1, and UPF2 are present in Parabasalia as well as across many eukaryotic groups, whereas UPF3A and UPF3B were not found in many of the eukaryotes researched. This points to Parabasalia having a normal and functioning nonsense-mediated decay pathway as they have the majority of the essential proteins needed for a functional pathway.

Date Created
2022-05
Agent

Evolutionary History of Eukaryotic Oxysterol Binding Proteins

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Description

Cells have mechanisms in place to maintain the specific lipid composition of distinct organelles including vesicular transport by the endomembrane system and non-vesicular lipid transport by lipid transport proteins. Oxysterol Binding Proteins (OSBPs) are a family of lipid transport proteins…

Cells have mechanisms in place to maintain the specific lipid composition of distinct organelles including vesicular transport by the endomembrane system and non-vesicular lipid transport by lipid transport proteins. Oxysterol Binding Proteins (OSBPs) are a family of lipid transport proteins that transfer lipids at various membrane contact sites (MCSs). OSBPs have been extensively investigated in human and yeast cells where twelve have been identified in Homo sapiens and seven in Saccharomyces cerevisiae. The evolutionary relationship between these well-characterized OSBPs is still unclear. Reconstructed OSBP phylogenies revealed that the ancestral Saccharomycotinan had four OSBPs, the ancestral Holomycotan had five OSBPs, the ancestral Holozoan had six OSBPs, the ancestral Opisthokont had three OSBPs, and the ancestral Eukaroyte had three OSBPs. Our analysis identified three clades of ancient OSBPs not present in animals or fungi.

Date Created
2022-05
Agent

Understanding the Role of Hemocytes in Wing Disc Regeneration in Drosophila melanogaster

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Description
Tissue regeneration is a complex process that activates both developmental and metabolic signaling pathways (Kashio & Miura, 2020). The wing imaginal disc in Drosophila melanogaster has been invaluable in discerning what pathways are activated during tissue regeneration, which is typically…

Tissue regeneration is a complex process that activates both developmental and metabolic signaling pathways (Kashio & Miura, 2020). The wing imaginal disc in Drosophila melanogaster has been invaluable in discerning what pathways are activated during tissue regeneration, which is typically done by genetically or physically wounding the wing disc and using fluorescent markers to track different signals. However, despite its importance in other regeneration contexts (Tafesh-Edwards & Eleftherianos, 2020), immune signaling has not been well studied in this tissue. Furthermore, what we do know about tissue regeneration and immune signaling is specific to apoptotic cellular death, less is known about other types of cellular death, such as necrotic cellular death and the consequent signaling systems that result from necrosis. Drosophila have an open immune system and only possess innate immunity (Pastor-Pareja et al., 2008), making them an ideal model to study hemocyte involvement in tissue regeneration. Hemocytes are equivalent to blood cells in vertebrates, and are involved in immunological response (Kurucz et al., 2003). In this work, we observed hemocyte accumulation during injury-induced regeneration. Cellular damage was induced using a genetic ablation system known as DUAL Control, with hemipterous CA and GluR1 used to induce apoptotic and necrotic cell death respectfully. We have discovered that while hemocytes are recruited to the wing disc upon both apoptotic and necrotic injury, necrotic tissue has more hemocytes adhered than apoptotic tissue. The difference in adherence could be due to basement membrane integrity being damaged more severely in necrotic discs than apoptotic discs. Our results show that hemocytes are attracted to wing discs that have undergone necrotic damage, indicating that the immune system plays some sort of role in necrotic cellular death. Though the immune response to different types of tissue damage in Drosophila is much simpler than in vertebrate models, there are many similarities between the two, and could lead to research involving human immune signaling as it pertains to regeneration.
Date Created
2022-05
Agent

Diversity and Phylogeny of Trichonympha, a Parabasalian Symbiont of Termites

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Description
Trichonympha is a Parabasalian symbiont of lower termites. They are characterized by their spindle-shape, length ranging from 75-150 micrometers, longitudinal flagella, as well as their true rostral tube composed of two plates of parabasal fibers. Ealy analysis through microscopy has…

Trichonympha is a Parabasalian symbiont of lower termites. They are characterized by their spindle-shape, length ranging from 75-150 micrometers, longitudinal flagella, as well as their true rostral tube composed of two plates of parabasal fibers. Ealy analysis through microscopy has revealed that the same species of Trichonympha could be found across multiple species of termites. However, with recent phylogenetic analysis of the 18S region, it has been found that the species is actually genetically distinct and that Trichonympha have coevolved with their termite hosts. In this study, Trichonympha cells from Reticulitermes tibialis, R. flavipes, and R. lucifugus were isolated. DNA from the single cell samples were purified and amplified through a series of polymerase chain reactions (PCR) and gel electrophoresis. The amplified DNA was extracted from the gel and cloning was performed using competent E. coli. The colonies that formed from each sample were collected and those that amplified after a PCR reaction were sent to the ASU Genomics Core for sanger sequencing. The sequences were assembled, cleaned, and aligned and the maximum likelihood and Bayesian phylogenetic trees were constructed. It was found that there is evidence of multiple species of Trichonympha in R. flavipes and that there were multiple species found in R. lucifugus. However, the Trichonympha sequences from R. lucifugus branch in different clades which indicates that they are more distant species than those of R. flavipes. Additionally, Trichonympha cells from R. tibialis branched in the same clade as those from R. flavipes which provides evidence that the same Trichonympha species can be found in multiples species of Reticulitermes. With these pieces of evidence, we can see multiple patterns of diversity of Trichonympha in their termite hosts.
Date Created
2022-05
Agent

The Symbiotic Foundation of Biocrust Microbiomes and its Application in Ecological Restoration

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Description
Biocrusts are microbial communities that inhabit arid soil surfaces, providing essential services to dryland ecosystems. A paradoxical filamentous cyanobacterium, Microcoleus vaginatus, resides within the biocrust. While is often pioneers the colonization of bare, nutrient-poor desert soils worldwide, it cannot fix…

Biocrusts are microbial communities that inhabit arid soil surfaces, providing essential services to dryland ecosystems. A paradoxical filamentous cyanobacterium, Microcoleus vaginatus, resides within the biocrust. While is often pioneers the colonization of bare, nutrient-poor desert soils worldwide, it cannot fix dinitrogen. In nature, M. vaginatus coexists with a unique microbial community, a “cyanosphere”, that is characterized by a high abundance of diazotrophic heterotrophs. This suggests mutualistic relationships wherein nutrients are traded between phototrophs and heterotrophs. To explore these relationships, I performed targeted, pedigreed isolation of cyanosphere members and used co-cultivation to recreate the mutualism in culture. Results showed that, in the absence of fixed nitrogen, M. vaginatus grew well when co-cultured with cyanosphere diazotrophs, but only poorly or not at all when alone or with non-cyanosphere diazotrophs. In agreement with this, the experimental provision of nitrogen to natural populations resulted in a loss of diazotrophs from the cyanosphere compared to controls, but the addition of phosphorus did not. Additionally, the convergence of M. vaginatus trichomes into large bundles held by a common sheath was elicited in culture by the addition of cyanosphere diazotrophs, pointing to a role of cyanobacterial motility responses in the development of mutualistic interactions. I then demonstrated that the tendency of M. vaginatus to stay within bundles and close to the sheath-dwelling cyanosphere was dependent on the cyanosphere population size. This effect was likely mediated by glutamate that acted as a signaling molecule rather than as a N source and impacted the gliding speed and negative chemophobic responses on the cyanobacterium. Glutamate seems to be used as a cue to spatially optimize cyanobacterium-cyanosphere mutualistic exchanges. My findings have potential practical applications in restoration ecology, which I further pursued experimentally. Co-inoculation of soil with cyanosphere diazotrophs resulted in swifter development of biocrusts over inoculation with the cyanobacterium only. Further, their addition to disturbed native soils containing traces of cyanobacteria sufficed for the formation of cohesive biocrusts without cyanobacterial inoculation. The inclusion of such “biocrust probiotics” in biocrust restoration is recommended. Overall, this body of work elucidates the hitherto unknown role of beneficial heterotrophic bacteria in the initial formation and development of biocrusts.
Date Created
2021
Agent

Genome Size Estimation of the Protist Symbionts of Coptotermes formosanus

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Description
Parabasalia is a phylum of flagellated protists with a large range of cell sizes, spanning from as little as 7 µm in length (e.g. Pentatrichomonas hominis) to well over 300 µm (e.g. Pseudotrichonympha grassii). Many Parabasalia are associated with…

Parabasalia is a phylum of flagellated protists with a large range of cell sizes, spanning from as little as 7 µm in length (e.g. Pentatrichomonas hominis) to well over 300 µm (e.g. Pseudotrichonympha grassii). Many Parabasalia are associated with animals in mutualistic, parasitic, or commensal relationships. The largest Parabasalia species are obligate mutualists of termites, which help to digest lignocellulose. While the specific digestive roles of different protist species are mostly unknown, Parabasalia with different cell sizes are known to inhabit different regions of the termite hindgut. It is currently unclear whether these size differences are driven by selection or drift, but it is well known that cell size correlates with genome size in eukaryotes. Therefore, in order to gain insight into possible selection pressures or mechanisms for cell size increase, genome sizes were estimated for the five Parabasalia species that inhabit the hindgut of Coptotermes formosanus Shiraki. The cell volumes and C-values for the five protist species are 89,190 µm3 and 147 pg in Pseudotrichonympha grassii, 26,679 µm3 and 56 pg in Holomastigotoides hartmanni, 8,985 µm3 and 29 pg in Holomastigotoides minor, 1,996 µm3 and 12 pg in Cononympha leidyi , and 386 µm3 and 6 pg in Cononympha koidzumii. The positive correlation between genome size and cell size was maintained in this group (R2 = 0.76). These genome sizes are much larger than the previously estimated genome sizes of non-termite associated Parabasalia, which spanned 2-fold ranging from 0.088 pg (in Tetratrichomonas gallinarum) to 0.181 pg (in Trichomonas foetus). With these new estimates, the range now spans over 1,500-fold from 0.088 pg to 147 pg in P. grassii, implying potential differences in the level of selective pressures for genome size in termite-associated Parabasalia compared to other protists.
Date Created
2021
Agent

Plasmodium Cost of Resistance and Life Stage Development within the Mosquito Vector

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Description

Hundreds of thousands of people die annually from malaria; a protozoan of the genus Plasmodium is responsible for this mortality. The Plasmodium parasite undergoes several life stages within the mosquito vector, the transition between which require passage across the lumen…

Hundreds of thousands of people die annually from malaria; a protozoan of the genus Plasmodium is responsible for this mortality. The Plasmodium parasite undergoes several life stages within the mosquito vector, the transition between which require passage across the lumen of the mosquito midgut. It has been observed that in about 15% of parasites that develop ookinetes in the mosquito abdomen, sporozoites never develop in the salivary glands, indicating that passage across the midgut lumen is a significant barrier in parasite development (Gamage-Mendis et al., 1993). We aim to investigate a possible correlation between passage through the midgut lumen and drug-resistance trends in Plasmodium falciparum parasites. This study contains a total of 1024 Anopheles mosquitoes: 187 Anopheles gambiae and 837 Anopheles funestus samples collected in high malaria transmission areas of Mozambique between March and June of 2016. Sanger sequencing will be used to determine the prevalence of known resistance alleles for anti-malarial drugs: chloroquine resistance transporter (pfcrt), multidrug resistance (pfmdr1) gene, dihydropteroate synthase (pfdhps) and dihydrofolate reductase (pfdhfr). We compare prevalence of resistance between abdomen and head/thorax in order to determine whether drug resistant parasites are disproportionately hindered during their passage through the midgut lumen. A statistically significant difference between resistance alleles in the two studied body sections supports the efficacy of new anti-malarial gene surveillance strategies in areas of high malaria transmission.

Date Created
2021-05
Agent

Reverse Fountain Cytoplasmic Streaming in Rhizopus Oryzae

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Description
The intracellular motility seen in the cytoplasm of angiosperm plant pollen tubes is known as reverse fountain cytoplasmic streaming (i.e., cyclosis). This effect occurs when organelles move anterograde along the cortex of the cell and retrograde down the center of…

The intracellular motility seen in the cytoplasm of angiosperm plant pollen tubes is known as reverse fountain cytoplasmic streaming (i.e., cyclosis). This effect occurs when organelles move anterograde along the cortex of the cell and retrograde down the center of the cell. The result is a displacement of cytoplasmic volume causing a cyclic motion of organelles and bulk liquid. Visually, the organelles appear to be traveling in a backwards fountain hence the name. The use of light microscopy bioimaging in this study has documented reverse fountain cytoplasmic streaming for the first time in fungal hyphae of Rhizopus oryzae and other members in the order Mucorales (Mucoromycota). This is a unique characteristic of the mucoralean fungi, with other fungal phyla (e.g., Ascomycota, Basidiomycota) exhibiting unidirectional cytoplasmic behavior that lacks rhythmic streaming (i.e., sleeve-like streaming). The mechanism of reverse fountain cytoplasmic streaming in filamentous fungi is currently unknown. However, in angiosperm plant pollen tubes it’s correlated with the arrangement and activity of the actin cytoskeleton. Thus, the current work assumes that filamentous actin and associated proteins are directly involved with the cytoplasmic behavior in Mucorales hyphae. From an evolutionary perspective, fungi in the Mucorales may have developed reverse fountain cytoplasmic streaming as a method to transport various organelles over long and short distances. In addition, the mechanism is likely to facilitate driving of polarized hyphal growth.
Date Created
2020
Agent
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