Shell morphological diversification patterns and molecular systematics of the testate amoebae orders Arcellinida and Euglyphida

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de Lectura: 09-03-2023Para inferir los patrones generales que rigen la biodiversidad es necesario tener una buena representación de los taxones que la componen, y esto incluye también a los organismos más pequeños. Si bien se puede argumentar que el conocimiento de ciertos grupos de plantas y animales puede ser insuficiente, existe un claro vacío de conocimiento en los protistas, especialmente en el suelo y agua dulce. Para resolver esta “laguna” de conocimiento, esta tesis propone centrarse en un grupo particular de protistas que viven principalmente en ecosistemas continentales, las amebas tecadas. Pero para ello, es necesario resolver algunas faltas de conocimiento y desarrollar protocolos específicos para el estudio rápido y eficiente de la biodiversidad en estos taxones. La ausencia de tales protocolos limita enormemente su estudio, así como sus potenciales aplicaciones. Las amebas tecadas son un grupo parafilético de protistas ameboides que tienen en común un “caparazón” o teca autoconstruida. Estos organismos constituyen órdenes dentro de "supergrupos" eucariotas muy poco relacionados; Arcellinida en Amebozoa, Euglyphida y Thecofilosea en Rhizaria y Amphitremida en Stramenopiles (=Heterokonta). Dentro de cada grupo, estos organismos difieren en la composición y forma de las tecas, que constituyen la base de su taxonomía y sistemática. Intuitivamente, los investigadores han clasificado a los organismos asumiendo que morfologías de la teca similares deberían compartir un ancestro común. Esta suposición se basa en la hipótesis de que las tecas están sometidas a una selección neutral, y descarta la posibilidad de convergencias evolutivas entre especies o clados. Sin embargo, el “barcoding” molecular ha desafiado la sistemática y la taxonomía clásicas basadas en la morfología, mostrando patrones de diversificación morfológica de las tecas mucho más complejos y enmarañadas de lo que se pensaba. Estos resultados subrayan la necesidad de aplicar un enfoque molecular para caracterizar los taxones y establecer las relaciones entre ellos. Sin embargo, por el momento, casi todos los datos moleculares disponibles son de un único infraorden dentro de Arcellinida, los Hyalospheniformes. En Euglyphida, sólo el infraorden Euglyphina ha sido (relativamente) bien muestreado molecularmente. El primer objetivo de esta tesis es aumentar la base de datos molecular de las amebas tecadas, centrándose en Arcellinida y Euglyphida, recuperando las regiones genéticas 18S rRNA, COI y NADH. Dentro de estos genes que se han utilizado, el gen nuclear 18S rRNA fue el más conservado. También ha sido el más útil para la reconstrucción de relaciones más profundas, aunque demasiado conservado para discriminar entre especies. Por este motivo, nos centramos en el gen mitocondrial COI, de rápida evolución, que a su vez permite una buena resolución a nivel de especie. Siguiendo los principios de la taxonomía integrativa, también obtuvimos (además de las secuencias moleculares) datos sobre su localización, ecología y morfología de la teca. Esta tesis incluye los primeros datos moleculares para amebas tecadas de la Península Ibérica, tanto en ambientes de agua dulce, suelos, como de sedimentos marinos. También incluyen los primeros datos moleculares para géneros como Plagiopyxis o Trigonopyxis . Estas bases de datos servirán de antecedente para futuros estudios, y serán fundamentales para responder a dos preguntas que estructuran esta tesis: 1) "¿Cómo evoluciona la morfología de la teca en las amebas tecadas?": Entender los patrones de diversificación en las amebas tecadas es esencial para aclarar su taxonomía y sistemática, así como la aplicación de sus rasgos funcionales en los análisis ecológicos. Aquí nos centramos en la familia Cyphoderiidae (Euglyphida), Arcellidae (Arcellinida) y otros taxones de Arcellinida. Evaluamos las relaciones filogenéticas entre los taxones basándonos en datos moleculares y “mapeamos” las morfologías de las tecas y la ecología de los organismos en los árboles filogenéticos. Nuestros resultados muestran correlaciones entre ambientes y morfotipos, aportando varios casos de patrones convergentes. Esto sugiere que algunos rasgos de la teca pueden estar bajo selección positiva. 2) "¿Cómo generar datos moleculares de forma rápida y eficiente en Arcellinida?": La obtención de datos moleculares en amebas tecadas siempre ha sido un problema importante, debido a las dificultades de trabajar con estos organismos (en su mayoría) no cultivables. En consecuencia, la obtención de datos moleculares sobre las amebas tecadas es costosa en términos de tiempo y dinero, lo que explica en gran medida que sigan siendo relativamente poco estudiadas en comparación con otros grupos de protistas. Para resolver este problema, diseñamos un protocolo específico para obtener datos de ADN ambiental de Arcellinida, basado en los datos disponibles. Con este protocolo molecular específico de Arcellinida, se espera obtener cientos de secuencias ambientales mediante técnicas de “secuenciación de alto rendimiento”. Esto permitirá realizar experimentos ecológicos y biogeográficos de gran tamaño, así como estudios de bioindicación, todo lo cual requiere cantidades considerables de datos que eran imposibles de obtener en el pasado. Esta tesis aporta una nueva perspectiva integral de la historia evolutiva y la diversificación morfológica de las tecas de los órdenes Arcellinida y Euglyphida existentes; destacando la importancia de incorporar a los protistas, como las amebas tecadas, a la hora de sacar conclusiones generales que se apliquen a los eucariotas o a la biodiversidad en genera

Foregut development: an act of balance

Esophageal atresia (EA) is a relatively rare congenital anomaly in which there is no connection between the proximal esophagus and the stomach. In more than 90% of patients, the distal esophagus has an abnormal connection to the trachea; this is called a trachea-esophageal fistula (TEF). Approximately two thirds of patients also have other major malformations, mostly one or more of the types of defects included in the so-called VACTERL association: vertebral, anorectal, cardiac, tracheo-esophageal, renal or urinary tract, and limbs malformations). In the Erasmus MC-Sophia cohort of trachea-esophageal anomalies (TE) nine percent of patients have a known genetic syndrome and another 1-2% the condition is strongly associated with an environmental factor. This leaves almost 90% of TE disease burden unexplained. Using two relatively new techniques, SNP-array and whole exome sequencing we aimed to explore genetic variation in EA/TEF and VACTERL association

<i>Acartia spp.</i> (Copepoda: Calanoida) as model organisms to evaluate the toxicity of emerging contaminants: an ecotoxicogenomic approach

Copepods are small crustaceans of great ecological and ecotoxicogenomic importance. Among them, Acartia tonsa, invasive species in the Mediterranean Sea, is a model species in ecotoxicology. Acartia clausi, native of the Mediterranean, is a candidate alternative model species in this field. In this PhD project, we compared physiological (naupliar immobilisation, egg hatching success, egg production, faecal pellet production, adult survival) and molecular (quantitative gene expression) responses of both copepod species exposed to nickel chloride (NiCl2) and nickel nanoparticles (NiNPs), through ecotoxicological bioassays and RT-qPCR. A de novo transcriptome of A. clausi females exposed to elutriates of polluted sediments was also generated. We were able to keep multi-generational cultures of A. clausi, fed the optimal diet of Rhinomonas reticulata, for up to four months, with 26 days from eggs to F2 generation. Bioassays confirmed that nauplii of A. clausi were more sensitive than those of A. tonsa, approximately 6 (NiNPs) and 95 times (NiCl2). Adults of both species were less affected by these toxicants, with mild effects mostly on egg production. Effects of both toxicants on egg hatching success was mainly due to Ni ions, which are continuously released from NiNPs. The first de novo assembled transcriptome for A. clausi exposed to elutriates of polluted sediments revealed 1,000 differentially expressed genes (743 up-, 257 down-regulated). The response was mostly in up-regulation of proteolysis and detoxification, and down-regulation of ribosomal proteins. Comparative gene expression in copepod females showed greater responses in A. clausi, especially with NiCl2, mostly with a down-regulation of genes involved in detoxification, indicating an inhibition of molecular defences. For A. clausi, down-regulation of vitellogenin seems correlated with low egg production. Overall, we provided novel information on the physiology and molecular biology of A. clausi and on NiNPs behaviour, which could threaten marine environment in the near future

Network-based identification of driver pathways in clonal systems

Highly ethanol-tolerant bacteria for the production of biofuels, bacterial pathogenes which are resistant to antibiotics and cancer cells are examples of phenotypes that are of importance to society and are currently being studied. In order to better understand these phenotypes and their underlying genotype-phenotype relationships it is now commonplace to investigate DNA and expression profiles using next generation sequencing (NGS) and microarray techniques. These techniques generate large amounts of omics data which result in lists of genes that have mutations or expression profiles which potentially contribute to the phenotype. These lists often include a multitude of genes and are troublesome to verify manually as performing literature studies and wet-lab experiments for a large number of genes is very time and resources consuming. Therefore, (computational) methods are required which can narrow these gene lists down by removing generally abundant false positives from these lists and can ideally provide additional information on the relationships between the selected genes. Other high-throughput techniques such as yeast two-hybrid (Y2H), ChIP-Seq and Chip-Chip but also a myriad of small-scale experiments and predictive computational methods have generated a treasure of interactomics data over the last decade, most of which is now publicly available. By combining this data into a biological interaction network, which contains all molecular pathways that an organisms can utilize and thus is the equivalent of the blueprint of an organisms, it is possible to integrate the omics data obtained from experiments with these biological interaction networks. Biological interaction networks are key to the computational methods presented in this thesis as they enables methods to account for important relations between genes (and gene products). Doing so it is possible to not only identify interesting genes but also to uncover molecular processes important to the phenotype. As the best way to analyze omics data from an interesting phenotype varies widely based on the experimental setup and the available data, multiple methods were developed and applied in the context of this thesis: In a first approach, an existing method (PheNetic) was applied to a consortium of three bacterial species that together are able to efficiently degrade a herbicide but none of the species are able to efficiently degrade the herbicide on their own. For each of the species expression data (RNA-seq) was generated for the consortium and the species in isolation. PheNetic identified molecular pathways which were differentially expressed and likely contribute to a cross-feeding mechanism between the species in the consortium. Having obtained proof-of-concept, PheNetic was adapted to cope with experimental evolution datasets in which, in addition to expression data, genomics data was also available. Two publicly available datasets were analyzed: Amikacin resistance in E. coli and coexisting ecotypes in E.coli. The results allowed to elicit well-known and newly found molecular pathways involved in these phenotypes. Experimental evolution sometimes generates datasets consisting of mutator phenotypes which have high mutation rates. These datasets are hard to analyze due to the large amount of noise (most mutations have no effect on the phenotype). To this end IAMBEE was developed. IAMBEE is able to analyze genomic datasets from evolution experiments even if they contain mutator phenotypes. IAMBEE was tested using an E. coli evolution experiment in which cells were exposed to increasing concentrations of ethanol. The results were validated in the wet-lab. In addition to methods for analysis of causal mutations and mechanisms in bacteria, a method for the identification of causal molecular pathways in cancer was developed. As bacteria and cancerous cells are both clonal, they can be treated similar in this context. The big differences are the amount of data available (many more samples are available in cancer) and the fact that cancer is a complex and heterogenic phenotype. Therefore we developed SSA-ME, which makes use of the concept that a causal molecular pathway has at most one mutation in a cancerous cell (mutual exclusivity). However, enforcing this criterion is computationally hard. SSA-ME is designed to cope with this problem and search for mutual exclusive patterns in relatively large datasets. SSA-ME was tested on cancer data from the TCGA PAN-cancer dataset. From the results we could, in addition to already known molecular pathways and mutated genes, predict the involvement of few rarely mutated genes.nrpages: 246status: publishe

It takes three to tango: tripartite interaction between cabbage root flies, their gut microbiome and host plant defense compounds

Plants are constantly attacked by herbivores. To defend themselves, they produce chemical compounds. The plant family Brassicaceae synthesizes glucosinolates (GSL) that are hydrolyzed to toxic isothiocyanates (ITCs) and other compounds during herbivory. Several insect herbivores adapted to their host plant defense system. Microbial communities can play a crucial role in allowing insects to thrive in recalcitrant habitats and feed on toxic food sources. However, adaptation mechanisms in belowground herbivores and the role of gut bacterial communities (GBCs) in this adaptation process remained uninvestigated so far. This thesis aims to understand the molecular mechanisms underpinning the adaptation of the cabbage root fly D. radicum and the turnip root fly D. floralis larvae to the GSL-ITC defense system in their host plants. Biochemical response of roots to D. radicum and D. floralis infestation indicated that the larvae of both Delia species are exposed to the GSL-ITC system while feeding. To elucidate molecular mechanisms that underlie the adaptation processes of Delia species to their host plant&#39;s defense system, the genome of D. radicum was assembled and annotated. A combination of metabolomic and transcriptomic analyses revealed that the larvae possess the enzymatic machinery to detoxify ITCs by activating the mercapturic acid conjugation pathway and a hydrolytic pathway. Both mechanisms are likely the results of co-evolutionary processes with their host plants. In addition, 16S amplicon sequencing revealed that the larval GBCs responded to ITCs and likely express their own detoxification mechanism (hydrolytic pathway). How essential the GBCs are for the larvae to detoxify ITCs remains an unanswered question. The results of this thesis provide a knowledge base for understanding mechanisms that underpin co-evolutionary processes between host plants and belowground herbivores

Identity and function of key bacterial groups in Arctic deep-sea surface sediments

The deep-sea floor covers about 65% of the Earth s surface and benthic biomass is dominated by highly diverse bacterial communities. Bacterial carbon cycling in deep-sea sediments plays a crucial role in global biogeochemical cycles, and remineralization efficiency of organic carbon can be more than 97%. However, key bacteria relevant for carbon turnover and ecosystem functioning remain unknown. Benthic bacteria mainly depend on organic carbon supply from the surface ocean, and will therefore likely be affected by changing surface ocean conditions. The Arctic Ocean is already impacted by environmental changes more rapidly here than in any other ocean region and will be impacted even more in the future. This turns the Arctic Ocean into an important study site to understand the effects of environmental changes on bacterial communities and ecosystem functioning, such as carbon cycling. At the same time, the Arctic Ocean remains to a large extent understudied, and little is known about the identity of key bacterial groups, which could be useful as indicators to describe the state of the ecosystem and to monitor community response to changing environmental conditions. Consequently, the goals of this thesis include the identification of indigenous key bacteria in deep-sea sediments and their metabolic potential, as well as the development of a better understanding of the specific response of Arctic deep-sea bacterial communities to changes in the supply of organic matter. The Long-Term Ecological Research site HAUSGARTEN (HG) is one out of two open ocean, long-term observatories in a polar region, and therefore provided a unique opportunity to study key bacterial groups from Arctic deep-sea sediments. Chapters I and II present one of the first characterizations of a globally sequence-abundant sediment bacterial group, the JTB255 marine benthic group (JTB255). Cell counts with newly designed probes evidenced high cell abundances in coastal (Chapter I) and deep-sea sediments (Chapter II). Labeling experiments together with metatranscriptomic data suggested a chemolithoautotrophic lifestyle, with a potential high importance for sulfur-based carbon fixation in coastal sediments (Chapter II). Furthermore, genomic analyses of single cells emerged as a powerful means to provide first insights into the metabolic potential of JTB255 representatives in deep-sea sediments, suggesting a heterotrophic lifestyle with oxygen as terminal electron acceptor (Chapter II). Genomic analysis showed that JTB255 encode enzymes for the oxidative degradation of polymeric cell material such as membranes and cell walls, suggesting recalcitrant organic carbon sources in marine sediments. Therefore, it is hypothesized for the first time that some representatives of JTB255 might be involved in the cycling of a major class of refractory sediment organic matter, potentially explaining their global ecological success. In an ex situ experimental approach, the response of Arctic benthic bacterial deep-sea communities at HG to different types of detritus was explored (Chapter III). This is the first experimental study investigating the response of bacterial deep-sea communities to the addition of natural food sources by combining measurements of community function with the analysis of high resolution taxonomic community structure. Our results provide evidence that differences in organic matter composition lead to significant changes in bacterial community structure and function at the seafloor, which can affect carbon turnover and retention in the deep sea. In addition, opportunistic groups of bacteria were identified that may serve as indicator taxa for different organic matter sources at this site. In Chapter IV, a pilot study is presented which addresses an issue often discussed in deep-sea research, i.e. the unknown effects of sample retrieval from high-pressure environments on bacterial communities. Therefore, the influence of de- and recompression on deep-sea sediment bacteria, as inherently imposed during sediment retrieval and subsequent laboratory experiments, was studied in a small-scale experiment. Results indicated few effects of de- and recompression on bacterial community structure within the experimental time frame, but contained evidence for changes in the metabolic activity of specific taxa, after the retrieval of decompressed samples from the seafloor. These observations remain to be verified with further sample replication. In summary, this thesis contributes to the identification of candidate key bacterial groups. It further provides valuable insights into bacterial diversity and function in Arc-tic deep-sea sediments and will help to assess impacts of future climate scenarios on pelago-benthic coupling in the Arctic

From metacommunity dynamics to rapid biodiversity assessment: DNA-based approaches expand horizons in both fundamental and applied ecology

Molecular tools have long been recognised as having enormous potential to expand horizons in ecology, but the promise remains substantially unfulfilled. In this thesis, I apply genetic approaches to two ecological problems that have proved difficult to solve using traditional techniques. Chapters 1 and 2 apply molecular tools to a community ecology problem to ask what mechanisms govern the persistence of an ant-plant metacommunity. I first use molecular data to clarify the number of coexisting ant species, and then employ population genetic techniques to investigate dispersal scale and other elements of life-history in the three most common species. Where hostplant density is high, a clear dispersal hierarchy is detected, which correlates positively with ant body size and negatively with fecundity, consistent with the hypothesis of a dispersal-fecundity trade-off. The hierarchy is less clear when hostplant density is low because one species shows dispersal plasticity, dispersing longer distances when hostplants are scarce. Results are discussed in the context of mechanisms that allow the coexistence of multiple symbionts with a single plant host. Chapters 3 to 8 address the use of molecular tools for informing decision-making in environmental management and biodiversity conservation. COI metabarcoding data are used to analyse patterns of arthropod diversity in the contexts of sustainable forest management (Chapter 5), agricultural management (Chapter 6), and habitat restoration (Chapter 7). It is shown that this potentially revolutionary technique can detect even fine-scale environmental changes, accurately characterise the biodiversity response to management variables, and be used to test the usefulness of convenient indicator variables. COI data is shown to outperform 18S data in recovering alpha and beta diversity information, and reference-based OTU-picking is demonstrated to be a useful approach where there is interest in the responses of a particular set of species. Potential applications and current limitations are discussed in Chapter 8