Design, Validation and Annotation of Transcriptome-Wide Oligonucleotide Probes for the Oligochaete Annelid Eisenia fetida

High density oligonucleotide probe arrays have increasingly become an important tool in genomics studies. In organisms with incomplete genome sequence, one strategy for oligo probe design is to reduce the number of unique probes that target every non-redundant transcript through bioinformatic analysis and experimental testing. Here we adopted this strategy in making oligo probes for the earthworm Eisenia fetida, a species for which we have sequenced transcriptome-scale expressed sequence tags (ESTs). Our objectives were to identify unique transcripts as targets, to select an optimal and non-redundant oligo probe for each of these target ESTs, and to annotate the selected target sequences. We developed a streamlined and easy-to-follow approach to the design, validation and annotation of species-specific array probes. Four 244K-formatted oligo arrays were designed using eArray and were hybridized to a pooled E. fetida cRNA sample. We identified 63,541 probes with unsaturated signal intensities consistently above the background level. Target transcripts of these probes were annotated using several sequence alignment algorithms. Significant hits were obtained for 37,439 (59%) probed targets. We validated and made publicly available 63.5K oligo probes so the earthworm research community can use them to pursue ecological, toxicological, and other functional genomics questions. Our approach is efficient, cost-effective and robust because it (1) does not require a major genomics core facility; (2) allows new probes to be easily added and old probes modified or eliminated when new sequence information becomes available, (3) is not bioinformatics-intensive upfront but does provide opportunities for more in-depth annotation of biological functions for target genes; and (4) if desired, EST orthologs to the UniGene clusters of a reference genome can be identified and selected in order to improve the target gene specificity of designed probes. This approach is particularly applicable to organisms with a wealth of EST sequences but unfinished genome

FUNCTIONAL ANALYSIS OF MEDICINAL PLANTS USING SYSTEMS BIOLOGY APPROACHES

Plant derived medicine is an important source of life saving drugs, but the genome information of most important medicinal plants is still unavailable. The need of the hour is to identify more functional genes and enzymes that control secondary metabolite production in medical plants, develop new methods for systematics, engineer resistance to number of biotic and abiotic stresses, and develop new conservation strategies, more genomics, proteomics and metabolomics information needs to be produced. In this review, a brief overview of various omic technologies and its applications to medicinal and aromatic plants are discussed.Â

PHYSELLA ACUTA, COMPARATIVE IMMUNOLOGY AND EVOLUTIONARY ASPECTS OF GASTROPOD IMMUNE FUNCTION

Gastropod immunobiology has benefitted from investigations focused on the planorbid snail Biomphalaria glabrata, intermediate host for the human parasite Schistosoma mansoni. Though such concentrated efforts have elucidated fascinating aspects of invertebrate immunity, they have not provided full knowledge regarding the evolution of immune function among other gastropod species. This dissertation presents the importance of making strategic choices regarding which organisms to select for comparative immunology. Herein, the choice was made to investigate the immunobiology of Physella acuta, a freshwater snail species of the Physidae, a sister family to Planorbidae to which B. glabrata belongs. Benefiting greatly from the use of next-generation sequencing (NGS), the immunobiology of P. acuta was studied using 454 pyrosequencing, Illumina RNA-seq, experimental infections with Echinostoma paraensei (trematode parasite), and other molecular techniques. These analyses revealed that many components of gastropod immunity have been conserved among physid and planorbid snails. Also, P. acuta displays differences in immune function, such as the use of fibrinogen-related proteins in response to trematode parasite exposure. Remarkably, P. acuta differentially expressed relatively large immune-relevant gene families (CD109/TEP, dermatopin, GTPase IMAP, among others) after exposure to E. paraenesi. Inspection of the individual members that represent these gene families demonstrated complex transcriptional profiles that suggest parasite influence on host immune function and the capacity of a host to maintain homeostasis while supporting parasite development, an extended phenotype of E. paraenesi. These lab-based studies represent the first large scale characterizations of P. acuta immune function. The immune factors described through NGS approaches enable investigations of the ecoimmunology of P. acuta snails collected from the field. This approach uncovered many sequences that are differentially expressed by P. acuta naturally in the field relative to the lab environment. There is variation in the expression of certain antimicrobial factors and genes governing biological processes. Overall, this dissertation has expanded the scope of gastropod immunity and provides resources and insights that are accessible for continued development and understanding of evolutionary and comparative immunology concepts

A Molecular and Phylogenetic Analysis of Cryobiosis in Nematodes of the Genus Panagrolaimus

Many organisms are able to survive freezing temperatures through the development of biochemical and physiological adaptations. These biochemical adaptations may include the synthesis of proteins such as antifreeze proteins or cryoprotectants such as trehalose, the elimination of ice nucleators, and the expression of stress associated proteins (such as molecular chaperones, antioxidants, late embryogenesis abundant (LEA) proteins). Physiological adaptations include the ability to undergo cryoprotective dehydration. The molecular mechanisms underlying freezing stress tolerance are poorly understood. One of the main aims of this project was to employ phylogenetic, proteomic, and transcriptomic approaches to gain insights into the adaptations that aid the survival of the free- living cryotolerant nematode Panagrolaimus superbus. Panagrolaimus sp. from temperate, subpolar, polar and continental geographic regions show a range of freezing ability from strains that show high survival upon direct exposure to -80 oC to those that are freezing sensitive. Acclimation significantly improves the freezing survival of temperate, subpolar, polar and continental Panagrolaimus strains and species. They also undergo anhydrobiosis and a correlation exists between desiccation tolerance and freezing tolerance. A phylogenetic study did not find a relationship between freezing phenotype, biogeography and phylogeny. The freezing and desiccation tolerance of ten new tropical Panagrolaimus strains was investigated. These strains are desiccation tolerant but not freezing tolerant, suggesting that freezing survival requires some specialised adaptations. A phylogenetic study of all the Panagrolaimus strains used in this study showed that the desiccation tolerant tropical Panagrolaimus strains are more divergent from the other strains and species in this study. Protein extracts from freezing tolerant Panagrolaimus sp. can inhibit the growth of ice along specific planes of an ice crystal, resulting in hexagonal bipyrimidal ice crystals. This ice faceting capacity was considered most likely to be due to the presence of ice binding proteins. An ice affinity purification method was implemented to purify ice-binding proteins from P. superbus. Several proteins found to be enriched in the ice fraction were identified by mass spectrometry. As none of the identified proteins was an obvious ice binding protein, it was not possible to determine whether these proteins had ice-binding protein activity. The divergence times for five Panagrolaimus strains and species were estimated using the relaxed molecular clock approach. The Panagrolaimus sp. were found to have diverged from other nematodes 70.12 million years ago. The Antarctic nematode P. davidi diverged from its Californian sister species PS1579 approximately 17.18 million years ago and the Arctic nematode P. superbus diverged from its Pennsylvanian sister species Panagrolaimus sp. AF36 9.97 million years ago. The genes that are differentially expressed in response to a period of cold acclimation were determined using the next generation sequencing method RNA-seq. A large number of novel genes were significantly up-regulated (P-value <0.01) including those involved in the oxidative stress response, transporting, membrane modification, metabolism, signalling and cytoskeleton remodelling