Phylogenetic and Transcriptomic Analyses of Vision in Two Cave Adapted Crustaceans, Asellus aquaticus (Isopoda: Asellidae) and Niphargus hrabei (Amphipoda: Niphargidae).

The unique characteristics of aquatic caves and of their predominantly crustacean biodiversity nominate them as ideal study subjects for evolutionary biology. The present dissertation capitalizes on a perfect natural experiment, the Molnar Janos thermal cave system in Budapest, Hungary. This intricate freshwater cave system and the immediately adjacent Malom Lake present the ideal opportunity to address questions of colonization, adaptation, and evolution. Despite marked environmental differences between the cave and surface waters, both localities are inhabited by natural populations of two emerging model cave species, the isopod Asellus aquaticus and the amphipod Niphargus hrabei. In the present dissertation, I first conduct an extensive literature review to examine and discuss the role that molecular methodologies have played in the study of cave biology. Additionally, I discuss the potential of “speleogenomic” methodologies to address long-standing questions in cave and evolutionary biology in fields such as biodiversity, phylogeography, and evolution. I then investigate the phylogeographic patterns and divergence-time estimates between surface and cave populations of the aforementioned species to elucidate mechanisms and processes driving the colonization of subterranean environments. These populations’ phylogenies then serve as robust frameworks on which to evaluate the transcriptional basis behind the divergence of traits involved in troglomorphy, namely vision. RNA sequencing approaches are used to identify and evaluate differences in the transcription of photoreception genes and pathways to in subterranean vs. surface populations. To achieve so, in a scalable manner suitable for modern sequencing technologies, here I produce a bioinformatics pipeline that allows for an accurate and efficient identification of genes present in a transcriptome that are involved in photoreception and visual pathways. I then use this bioinformatics pipeline to depict, in a phylogenetically informed context, the transcriptional basis behind photoreception and vision in A. aquaticus and N. hrabei, and the role these traits play in cave adaptation, and in the evolution of troglomorphy in the subphylum Crustacea. With the findings herein, the present dissertation aims to provide a framework for the discovery of evolutionarily significant molecular mechanisms that permit the survival and evolution of life in caves and other extreme environments

Phototransduction Components in the Visual System of Hard-bodied Ticks

Ticks are terrestrial invertebrate parasites that attach to their hosts to feed on their blood. Ticks are composed of three families: Ixodidae, Argasidae, and Nuttalliellidae. Ixodid ticks include members of the genera Amblyomma, Ixodes, Haemaphysalis, Hyalomma, Dermacentor, and Rhipicephalus. Ticks can transmit diseases to animals and humans, making them an important organism to study. Current tick-bite mitigation strategies include acaricides (harmful if misused), and CO2 traps (effective in decreasing tick abundance, but costly to use). Since the visual system of ticks has not been studied extensively, I expect that by studying their visual system, alternative tick-bite mitigation strategies could be developed. In this study, I used the Phylogenetically Informed Annotation (PIA) workflow to analyze the genes present in the phototransduction pathway of hard-bodied ticks from the transcriptome sequences and the whole-genome sequences (WGS). All six of the genera listed above are included. My analyses document the occurrence of opsin proteins, r_opsin, c_opsin, Gq subunits, lark, and ovo genes. These results imply that ticks possess the proteins necessary to sense light, since many of these components enable light-detection in the retinas of animals

Light Organ Photosensitivity in Deep-Sea Shrimp May Suggest a Novel Role in Counterillumination

Extraocular photoreception, the ability to detect and respond to light outside of the eye, has not been previously described in deep-sea invertebrates. Here, we investigate photosensitivity in the bioluminescent light organs (photophores) of deep-sea shrimp, an autogenic system in which the organism possesses the substrates and enzymes to produce light. Through the integration of transcriptomics, in situ hybridization and immunohistochemistry we find evidence for the expression of opsins and phototransduction genes known to play a role in light detection in most animals. Subsequent shipboard light exposure experiments showed ultrastructural changes in the photophore similar to those seen in crustacean eyes, providing further evidence that photophores are light sensitive. In many deep-sea species, it has long been documented that photophores emit light to aid in counterillumination – a dynamic form of camouflage that requires adjusting the organ’s light intensity to “hide” their silhouettes from predators below. However, it remains a mystery how animals fine-tune their photophore luminescence to match the intensity of downwelling light. Photophore photosensitivity allows us to reconsider the organ’s role in counterillumination - not only in light emission but also light detection and regulation

<i>De novo</i> transcriptome analyses provide insights into opsin-based photoreception in the lanternshark <i>Etmopterus spinax</i>

The velvet belly lanternshark (Etmopterus spinax) is a small deep-sea shark commonly found in the Eastern Atlantic and the Mediterranean Sea. This bioluminescent species is able to emit a blue-green ventral glow used in counter-illumination camouflage, mainly. In this study, paired-end Illumina HiSeqTM technology has been employed to generate transcriptome data from eye and ventral skin tissues of the lanternshark. About 64 and 49 million Illumina reads were generated from skin and eye tissues respectively. The assembly allowed us to predict 119,749 total unigenes including 94,569 for the skin transcriptome and 94,365 for the eye transcriptome while 74,753 were commonly found in both transcriptomes. A taxonomy filtering was applied to extract a reference transcriptome containing 104,390 unigenes among which 38,836 showed significant similarities to known sequences in NCBI non-redundant protein sequences database. Around 58% of the annotated unigenes match with predicted genes from the Elephant shark (Callorhinchus milii) genome. The transcriptome completeness has been evaluated by successfully capturing around 98% of orthologous genes of the « Core eukaryotic gene dataset » within the E. spinax reference transcriptome. We identified potential “light-interacting toolkit” genes including multiple genes related to ocular and extraocular light perception processes such as opsins, phototransduction actors or crystallins. Comparative gene expression analysis reveals eye-specific expression of opsins, ciliary phototransduction actors, crystallins and vertebrate retinoid pathway actors. In particular, mRNAs from a single rhodopsin gene and its potentially associated peropsin were detected in the eye transcriptome, only, confirming a monochromatic vision of the lanternshark. Encephalopsin mRNAs were mainly detected in the ventral skin transcriptome. In parallel, immunolocalization of the encephalopsin within the ventral skin of the shark suggests a functional relation with the photophores, i.e. epidermal light-producing organs. We hypothesize that extraocular photoreception might be involved in the bioluminescence control possibly acting on the shutter opening and/or the photocyte activity itself. The newly generated reference transcriptome provides a valuable resource for further understanding of the shark biology

Opsin Diversity and Molecular Characterization of the Phototransduction Cascade in Modified Crustacean Eye Types with a Focus on Alima pacifica, Neocalanus finmarchicus, and Labidocera madurae

M.S. University of Hawaii at Manoa 2016.Includes bibliographical references.Evolution of the crustacean visual system is complex and cryptic; unusual eye types coupled with an abundance of visual pigments depicts a history of rapid diversification. Using transcriptomic analysis, visual phototransduction genes were identified from whole copepods of Neocalanus flemingeri, (naupliar eye), and Labidocera madurae (transformed naupliar eyes), and from the double retina compound eye of last-stage stomatopod larvae (Alima pacifica). Similar components to the visual phototransduction cascade of Drosophila melanogaster were identified in all species. Identified visual opsins clustered with long-wavelength sensitive, middle-wavelength sensitive (MWS) and ultraviolet sensitive opsins in A. pacifica, while both copepods expressed only MWS opsins. Non-visual pteropsins were identified in all three species, and one peropsin was found in A. pacifica. Developmental shifts in opsin expression of larval versus adult transcriptomes were identified in both L. madurae and A. pacifica. The molecular components of these unusual eye types supplement the history of crustacean visual evolution

Comparative analysis of gene expression associations between mammalian hosts and Plasmodium

Artenübergreifende Interaktionen helfen uns, Krankheitsmechanismen zu verstehen und Targets für Therapien zu finden. Die Koexpression von Genen, gemessen an der mRNA-Häufigkeit, kann Interaktionen zwischen Wirt und Pathogen aufzeigen. Die RNA-Sequenzierung von Wirt und Pathogen wird als "duale RNA-Sequenzierung" bezeichnet. Malaria ist eine der am besten untersuchten parasitären Krankheiten, so dass eine Fülle von RNA-seq-Datensätzen öffentlich zugänglich ist. Die Autoren führen entweder duale RNA-seq durch, um den Wirt und den Parasiten gleichzeitig zu untersuchen, oder sie erhalten kontaminierende Sequenzierungs-Reads aus dem Nicht-Zielorganismus. Ich habe eine Meta-Analyse durchgeführt, bei diese beiden Arten von RNA-seq-Studien verwendet wurden, um über korrelierte Genexpression auf Wirt-Parasit-Interaktionen zu schließen. Ich habe Studien mit Homo sapiens, Mus musculus und Macaca mulatta als Wirte und ihre Plasmodium-Parasiten einbezogen. Ich benutzte orthologe Einzelkopien von Genen, um ein Repertoire von Interaktionen bei Malaria und in diesen Modellsystemen zu erstellen. Ich verknüpfte die Daten von 63 Plasmodium-Phasen-spezifischen Studien und reduzierte die Zahl der Interaktionen von potenziell 56 Millionen auf eine kleinere, relevantere Menge. Die Zentralität in den Netzwerken der Blutphasen konnte die Essentialität der Plasmodium-Gene erklären. Das aus den verketteten Daten sagte die Genessenzialität besser vor als die einzelnen Studien - ein Vorteil der Meta-Analyse. Neutrophile und Monozyten Immunmarkergene waren überrepräsentiert, was auf eine Fülle von phagozytären und respiratorischen Reaktionen hindeutet. Die Analyse der Leberphase ergab Wirts- und Parasitenprozesse in frühen und späten Entwicklungsphasen. Ich fand bekannte Wirt-Parasit-Interaktionen, die für beide Phasen gleich sind, sowie bisher unbekannte Interaktionen. Dieses Prinzip lässt sich auch auf andere Krankheiten anwenden, um Mechanismen und therapeutische Ziele zu verstehen.Cross-species interactions help us understand disease mechanisms and find targets for therapy. Gene co-expression, measured by mRNA abundance, can identify host-pathogen interactions. The RNA-sequencing of host and pathogen is termed “dual RNA-sequencing”. Malaria is one of the most studied eukayotic parasitic diseases, making an abundance of RNA-seq data sets publicly available. Authors either perform dual RNA-seq to study the host and parasite simultaneously or acquire contaminant sequencing reads from the non-target organism. I performed a meta-analysis using these two kinds of RNA-seq studies to infer host-parasite interactions using correlated gene expression. I included studies of Homo sapiens, Mus musculus and Macaca mulatta as hosts and their corresponding Plasmodium parasites. I used single-copy orthologous genes to generate a repertoire of interactions in human malaria and in these model systems. I found 63 malaria RNA-seq studies. I concatenated sequencing runs from Plasmodium stage-specific studies and reduced the number of interactions from a potential 56 million to a smaller, more relevant set. Centrality in the blood stage networks was able to explain Plasmodium gene essentiality. The network from the concatenated data predicted gene essentiality better than the individual studies, indicating a benefit of the meta-analysis. Immune marker genes for neutrophils and monocytes were over-represented, suggesting an abundance of phagocytic and respiratory burst-related responses. The liver stage analysis revealed linked host and parasite processes at early stages until late developmental stages. I found linked host and parasite processes that are common to the two stages, e.g. parasite cell gliding and invasion and host response to hypoxia and immune response. I showed that existing data can be explored for new information. This principle can be applied to other diseases to understand mechanisms and therapeutic targets

Octopus skin ‘sight’ and the evolution of dispersed, dermal light sensing in Mollusca

We now know that co-option, or reuse of ancestral components, plays a prominent role in the evolution of emergent systems, like the reuse of gene regulatory networks in the evolution of developmental programs for morphology. Do the evolutionary origins of animal behaviors show evidence of modular reuse that we find at other levels of biological organization? I found that the skin of Octopus bimaculoides is intrinsically light sensitive, and that bright light causes colored chromatophore organs in octopus skin to expand, even without input from the central brain or eyes. Because this Light-Activated Chromatophore Expansion (or LACE) behavior relies on evolutionary novel chromatophore organs, LACE is also an evolutionary novelty. As such, I can pinpoint its origin in evolutionary time and ask whether the ability of mollusc skin to sense light existed prior to the evolution of cephalopod chromatophores and LACE. I found expression of the same r-opsin based phototransduction genes in both O. bimaculoides eyes and skin, and the spectral sensitivity of LACE closely matches that of the r-opsin in octopus eyes, consistent with the hypothesis that r-opsin phototransduction underlies LACE. The r-opsin phototransduction cascade can be traced back to at least the last common ancestor of bilaterians, so did the reuse of the cascade in octopus skin arise before, in time with, or after the evolution of cephalopod chromatophores? After surveying 28 mollusc mantle transcriptomes for opsins, I found that r-opsin cascade genes are expressed across the molluscs, from multiple species in each of the major mollusc classes, and an ancestral state reconstruction suggests that the last common ancestor of molluscs expressed r-opsin in its mantle. Taken together, these results suggest that the evolution of LACE required co-option of an ancient phototransduction module, and that like the evolution of development and other emergent systems, reuse may play a fundamental role in the macroevolution of animal behaviors