Multifaceted biological insights from a draft genome sequence of the tobacco hornworm moth, \u3cem\u3eManduca sexta\u3c/em\u3e

Manduca sexta, known as the tobacco hornworm or Carolina sphinx moth, is a lepidopteran insect that is used extensively as a model system for research in insect biochemistry, physiology, neurobiology, development, and immunity. One important benefit of this species as an experimental model is its extremely large size, reaching more than 10 g in the larval stage. M. sexta larvae feed on solanaceous plants and thus must tolerate a substantial challenge from plant allelochemicals, including nicotine. We report the sequence and annotation of the M. sexta genome, and a survey of gene expression in various tissues and developmental stages. The Msex_1.0 genome assembly resulted in a total genome size of 419.4 Mbp. Repetitive sequences accounted for 25.8% of the assembled genome. The official gene set is comprised of 15,451 protein-coding genes, of which 2498 were manually curated. Extensive RNA-seq data from many tissues and developmental stages were used to improve gene models and for insights into gene expression patterns. Genome wide synteny analysis indicated a high level of macrosynteny in the Lepidoptera. Annotation and analyses were carried out for gene families involved in a wide spectrum of biological processes, including apoptosis, vacuole sorting, growth and development, structures of exoskeleton, egg shells, and muscle, vision, chemosensation, ion channels, signal transduction, neuropeptide signaling, neurotransmitter synthesis and transport, nicotine tolerance, lipid metabolism, and immunity. This genome sequence, annotation, and analysis provide an important new resource from a well-studied model insect species and will facilitate further biochemical and mechanistic experimental studies of many biological systems in insects

Multifaceted biological insights from a draft genome sequence of the tobacco hornworm moth, Manduca sexta

Manduca sexta, known as the tobacco hornworm or Carolina sphinx moth, is a lepidopteran insect that is used extensively as a model system for research in insect biochemistry, physiology, neurobiology, development, and immunity. One important benefit of this species as an experimental model is its extremely large size, reaching more than 10 g in the larval stage. M. sexta larvae feed on solanaceous plants and thus must tolerate a substantial challenge from plant allelochemicals, including nicotine. We report the sequence and annotation of the M. sexta genome, and a survey of gene expression in various tissues and developmental stages. The Msex_1.0 genome assembly resulted in a total genome size of 419.4 Mbp. Repetitive sequences accounted for 25.8% of the assembled genome. The official gene set is comprised of 15,451 protein-coding genes, of which 2498 were manually curated. Extensive RNA-seq data from many tissues and developmental stages were used to improve gene models and for insights into gene expression patterns. Genome wide synteny analysis indicated a high level of macrosynteny in the Lepidoptera. Annotation and analyses were carried out for gene families involved in a wide spectrum of biological processes, including apoptosis, vacuole sorting, growth and development, structures of exoskeleton, egg shells, and muscle, vision, chemosensation, ion channels, signal transduction, neuropeptide signaling, neurotransmitter synthesis and transport, nicotine tolerance, lipid metabolism, and immunity. This genome sequence, annotation, and analysis provide an important new resource from a well-studied model insect species and will facilitate further biochemical and mechanistic experimental studies of many biological systems in insect

Dormancy awakened: aminergic control of diapause in Drosophila

Coping with adverse environmental conditions is one of the most crucial challenges for all living beings. The coupling between external cues and hormonal signaling is key to allow survivorship of individuals and insects in particular have been intensively studied to better understand this connection. Although the hormonal cascade that promotes insect development and reproduction is well known (insulin signaling - juvenile hormone – 20-Ecdysone), how this neuroendocrine axis is modulated by environmental stimuli remains still largely elusive. To deepen the molecular features of IIS-JH-20E axis regulation, we focused our attention on one of the best examples of physiological strategies triggered by environmental stimuli, diapause. Diapause is an inducible developmental arrest, which characterizes the life cycle of several species, from Caenorhabditis elegans to mammals. Our results shed new light on the regulation of key neuroendocrine pathways for growth and development, and suggest how organisms couple environmental conditions with inner hormonal physiology

Design, synthesis and use of chiral pheromone-based probes to study pheromone enantiomer discrimination in the pheromone binding proteins from the gypsy moth, Lymantria dispar

The gypsy moth is a widespread and harmful pest causing extensive damage to the Canada’s forest and orchard ecosystems. It uses (+)-disparlure as a sex pheromone. Discovery of the pheromone, including its absolute configuration, has enabled monitoring of gypsy moth populations. Disparlure of low enantiopurity is not attractive to the moths and, for this reason, enantiopure (+)-disparlure has been a synthetic target for many years. To access (+)-disparlure of high enantiopurity we have used a diastereoselective nucleophilic addition reaction with the enantiopure α-chloroaldehyde (2-chlorododecanal) that yields a stereocontrolled access to the 1,2-anti chlorohydrin core. The (+)-disparlure was prepared through a series of transformations that include a Mitsunobu inversion. We have successfully completed the synthesis of (+)-disparlure in 5 steps as compared to Iwaki’s first synthesis in 12 steps and Sharpless’s widely used synthesis in 6 steps. The same approach was used to produce 18-hydroxydisparlure enantiomers, which were coupled to a linker with an alkyne moiety at the end. The alkyne was then coupled to azide-based commercial fluorescent probes, to furnish fluorescent disparlure-based probes for physical studies. The gypsy moth has two different pheromone binding proteins, LdisPBP1 and LdisPBP2. Previously, our group has addressed the enantiomer selectivity of these two PBPs and found that PBP1 binds (-)-disparlure more strongly than (+)-disparlure, while PBP2 binds (+)-disparlure more strongly. Despite several binding assays, the interaction and discrimination of gypsy moth PBPs towards disparlure enantiomers are not fully understood due to lack of binding interaction and kinetic studies, which are technically demanding, due to the hydrophobicity of the pheromone. In this thesis, we have studied the binding interaction of deuterium-labelled (+)-disparlure and (-)-disparlure with LdisPBPs by 2H NMR spectroscopy. The results from NMR studies were correlated with the results from docking simulations of (+)-disparlure and (-)-disparlure bound to one internal site and multiple external sites of LdisPBP1 and LdisPBP2. These results indicated that (+)-disparlure and (-)-disparlure adopt different conformations and orientations in the binding pockets of LdisPBP1 and LdisPBP2. Most of the reported work on PBPs focuses on the pheromone binding affinities of PBPs. However, the pheromone-PBP interactions require more than half an hour to establish equilibrium, whereas male moths respond to female pheromones in milliseconds. Therefore, the interactions between pheromones and olfactory components such as PBPs and pheromone receptors may not be under thermodynamic control. In this thesis, we aimed to provide a dynamic perspective of pheromone-PBP interactions and to link these to the functions of PBPs. We have studied thermodynamic (Kd) and kinetic properties (kon and koff) of LdisPBPs-disparlure enantiomer interaction by fluorescence binding assays and kinetic experiments using fluorophore-tagged disparlure enantiomers. The result indicated that the binding preference of disparlure enantiomers to LdisPBPs. Based on the kinetic data of LdisPBPs with fluorophore-tagged disparlure enantiomers, we propose a kinetic model that includes a two-step binding process. Each of these two steps may contribute to a different function of the LdisPBPs

Silk cocoon: structure and properties

This study investigated the silk cocoon structure and its protective roles. It focussed mainly on three protection roles, viz mechanical protection (indentation), bacterial and UV protection. Silk cocoon absorbed UV-A and UV-B radiations and had the potential to be used as a photoprotective agent in sunscreens

Emerging model spedies driven by transciptomics

This work is focused on 'emerging model species', i.e. question-driven model species which have sufficient molecular resources to investigate a specific phenomenon in molecular biology, developmental biology, molecular ecology and evolution or related molecular fields. This thesis shows how transcriptomic data can be generated, analyzed, and used to investigate such phenomena of interest even in species lacking a reference genome. The initial ButterflyBase resource has proven to be useful to researchers of species without a reference genome but is limited to the Lepidoptera and supports only the older Sanger sequencing technologies. Thanks to Next Generation Sequencing, transcriptome sequencing is more cost effective but the bottleneck of transcriptomic projects is now the bioinformatic analysis and data mining/dissemination. Therefore, this work continues with presenting novel and innovative approaches which effectively overcome this bottleneck. The est2assembly software produces deeply annotated reference transcriptomes stored in the Chado database. The Drupal Bioinformatic Server Framework and genes4all provide species-neutral and an innovative approach in building standardized online databases and associated web services. All public insect mRNA data were analyzed with est2assembly and genes4all to produce the InsectaCentral. With InsectaCentral, a powerful resource is now available to assist molecular biology in any question-driven model insect species. The software presented here was developed according to specifications of the General Model Organism Database (GMOD) community. All software specifications are species-neutral and can be seamlessly deployed to assist any research community. Further through a case studies chapter, it becomes apparent that the transcriptomic approach is more cost-effective than a genomic approach and therefore sequence-driven evolutionary biology will benefit faster with this field

Ecological risk assessment of transgenic conditional lethality systems for genetic biocontrol strategies

Genetic strategies of insect pest control are receiving increased interest due to recent advances in genetic engineering. By introducing alleles that make males functionally sterile, genetically modified (GM) insects can be used in area-wide pest management programs similar to the Sterile Insect Technique (SIT). Genetic control strategies carry several potential benefits, including improved efficacy and specificity, lower operation costs, and reduced dependence on chemical insecticides, but their use is still controversial despite extensive technical documentation indicating their innocuity to humans and the environment. This hesitation is likely due to the novel nature of the approach. While the first field applications of genetic biocontrol programs have provided promising evidence on the efficacy to suppress field pest populations, the limited number of studies leaves uncertainty about the potential environmental risks associated with the use of GM insects. Thus, prospective risk assessment studies provide the best evidence for understanding the potential hazards of these innovative strategies. In this dissertation, I use the common fruit fly Drosophila melanogaster as a model to assess the performance of conditional lethality (CL) expression systems under various ecologically relevant scenarios. Specifically, we examine the phenotypic variation of the CL system under variable abiotic conditions and the evolutionary response of experimental populations under sublethal exposure to GM male flies. Our results indicate that the CL system in GM insects is robust under most abiotic conditions experienced directly or indirectly along their lifecycle, but stressful conditions may reduce their effectiveness. Additionally, our results indicate that exposure of experimental populations to sublethal ratios of GM insects carrying CL expression systems results in minimal changes in embryonic susceptibility, but in some cases, changes in susceptibility may arise due to prezygotic changes in adult mate preference that lead to assortative mating. Overall, our results provide evidence of the robustness of the CL expression systems in GM insects and highlight potential sources of evolutionary change that may impact the efficacy of genetic biocontrol strategies. In addition to these primary studies on conditional lethality, in a side project I applied my computational skills to develop a novel algorithm for automatically detecting motion in insects for behavioral and physiological studie