INTERNATIONAL LEPIDOPTERAN GENOME
PROJECT
Contents:
1. Project objective
Rationale
Plan for action
2. Why lepidopteran genomics ?
Evolution and comparative genomics
Insects and Agriculture
3. Specific issues for lepidopteran genomics
The heliothines
Complex interactions
Sericulture
Neurobiology and olfaction
Insecticide resistance
Evolution and conservation
Bioengineering/biotechnology
4. Current state of knowledge
Facts and figures about lepidopteran genomes
Mutants
Cytogenetics
Sequence databases
5. Resources in lepidopteran genomics
A broad-based scientific community
Current status and future plans
6. Postgenomic tools for identification of gene function
Bioinformatics
Transformation
Disruption of gene function
Transcriptome analysis
Expression systems
7. Expected outcomes from lepidopteran genomics
8. References
1. Project Objective
The objective of the International Lepidopteran Genome Project is to promote international cooperation to sequence the genome of Bombyx mori and undertake comparative genomics of other economically and scientifically important Lepidoptera. This cooperation will foster knowledge both in the basic and applied aspects of insect science.
1.1. Rationale
The project first addresses the problem of insect pests of agriculture. Insects consume a large share of food and fiber destined for humans (pre and post harvest). Among these pests, Lepidoptera represent a diverse and important group. Agricultural development is particularly relevant to developing nations in which growing populations need to ensure and protect their food supply. Knowledge of lepidopteran genomics will create new methods of insect pest management and will contribute to sustainable agriculture, protection of the environment and the maintenance of biodiversity.
A second role of the project is to provide another insect model for comparative genomics and functional studies. This aspect will complement the Drosophila and Anopheles projects. Often the scientific questions that one can answer are highly dependent on the choice of an experimentally tractable model system, and the natural phenomena they exhibit. As many basic physiological processes of insects are conserved through evolution, their study in lepidopteran models will help further elucidate the function of gene homologues. The large body size, accessible genetics, and extreme diversity of lepidopteran species are important experimental advantages. Much of our knowledge of endocrinology, reproduction, behaviour, and immunity derives from studies in Lepidoptera.
The project will build on the existing lepidopteran genome project on Bombyx mori. It is open to all interested parties worldwide and solicits support of its goals through new grants from national and international agencies. Considering the impact of this project on world food production and national economies, the project is committed to open access to all data and resources. As substantial industrial outputs (biotechnology, agrochemistry) will be derived, we encourage the private sector to contribute to the project.
1.2. A plan for action
The strength of lepidopteran genomics lies in the diversity of the group as a whole. Although there are clear insect models for genetic analysis (Drosophila) or disease vectors (Anopheles), the Lepidoptera are rich in diverse model systems for a variety of biological processes. In particular, the variety of interactions between plants and Lepidoptera, and between Lepidoptera and their pathogens, necessitates an array of approaches in their control. We encourage the pursuit of diverse genomic projects on lepidopteran species, and the definition of a set of common goals for the scientific community. These goals are:
1) Complete sequence of the Bombyx mori genome.
The silkworm Bombyx mori will provide the first complete sequence of a lepidopteran and will serve as a comparative reference for all other work. This insect has the most advanced status in terms of mutants, linkage maps, BAC-based physical maps, and EST collections.
2) BAC-based physical maps and deep EST collections for other Lepidoptera.
These tools facilitate the use of B. mori as the relay species of genomic information to other lepidopterans of economic importance (e.g., heliothines, Heliothis, Helicoverpa) and model systems (e.g., hornworms, Manduca sexta).
3) Development of postgenomic tools.
Transgenesis, microarrays for functional genomics, mutant analysis, bioinformatics, and other postgenomic tools provide the link between genomic information and biology of Lepidoptera.
These goals lend themselves to a concerted effort, where different participants can choose to support one or the other depending on their own research priorities, and yet contribute significantly to the overall objective.
2. Why lepidopteran genomics?
2.1. Evolution
and comparative genomics:
The complete sequence of a lepidopteran genome will be an important contribution to comparative genomics. Development of wings (380 MYA) and complete metamorphosis are keys to insects' remarkable biodiversity. The split between branches leading to Diptera and Lepidoptera occurred 290-340 MYA, a time of separation earlier than that between mammals and birds. Thus Drosophila and Anopheles are distant evolutionarily from Lepidoptera, yet genome analysis of a species closely related to Lepidoptera has not been performed. Nonetheless, conservation of some regulatory elements between Drosophila and Bombyx is evident, (e.g., for chorion gene expression, Mitsialis and Kafatos, 1985), and a wealth of new knowledge can be gained through comparative analysis of animal genomes. The silkworm Bombyx mori has been domesticated for silk production for about 5,000 years and intensively studied. As a result, knowledge of its biology and genetics is the most advanced of any lepidopteran species. Comparison with its wild ancestor B. mandarina provides the opportunity to examine the effects of artificial selection leading to domestication at the genome level.
The order Lepidoptera is represented by more than 160,000 species--after Coleoptera, the most biodiverse group of animals. The family Noctuidae is the largest in the order, and it includes some of the most devastating pests of agriculture, particularly the Heliothines (Helicoverpa armigera, H. zea, Heliothis virescens) and other genera (Mamestra, Spodoptera , Trichoplusia etc.). Although lepidopteran pests of agriculture, forestry and food storage are found in diverse families (Noctuidae, Pyralidae, Arctiidae, Sphingidae, Tortricidae, Lymantridae, Yponomeutidae) they all belong to the same suborder, Ditrysia, and are not very divergent phylogenetically. The Lepidoptera, as a distinct taxonomic group appear recently in the fossil record (130 MYA), about the same time as flowering plants. Therefore, the genomic information of the model species Bombyx mori should be applicable to most economically important species in Lepidoptera.
Comparative studies on genome sequences between Lepidoptera, Drosophila, C. elegans, and vertebrates can point to lepidopteran-specific genes. As well as illustrating at the molecular level what makes Lepidoptera different from other insects, many of these newly identified genes are potential candidates for targets of lepidopteran-selective insecticides.
2.2. Insects and Agriculture:
Agriculture feeds 6 billion humans daily and within 50 years will have to feed 9 billion (UN forecast). It is estimated that one-third of the world's agricultural production is lost to insect pests, pathogens and weeds. The control of agricultural pest populations is achieved mainly by the application of chemical insecticides. The global insecticide market represents a 12 billion dollar yearly expenditure. Biological control methods (parasites, parasitoids and entomopathogens such as bacteria and viruses) are also part of integrated crop protection strategies. Genetically modified crops producing their own insecticides have more recently been deployed, with lepidopteran pests as the most common target.
These various strategies of control all have limitations of efficacy or public acceptance. The utility of broad-spectrum insecticides, both synthetic and natural, is limited in time by the development of resistance. Concerns about food and environmental contamination have lowered public acceptance of insecticides in developed countries. Research is needed to identify new targets for insecticides that are intrinsically selective and therefore potentially safer. Biological control methods are well adapted to high value crops or confined environments, but are often costly. Research is needed to facilitate the integration of biological control methods in pest management systems and to increase their efficacy. Despite environmental benefits of the reduced use of conventional pesticides, public acceptance of transgenic crops is low. Research is needed to develop new ways to enhance pest resistance of crops and allay concerns about genetically modified organisms.
3.Specific
Issues for Lepidopteran Genomics
3.1 The
heliothines
The heliothines, Helicoverpa armigera in Africa, Southern Europe, Asia, Australia and the Pacific, and H. zea and Heliothis virescens in the Americas, are three of the world's major crop pests. Orthologous genes of the three species are extremely similar, so that genomic information on one can readily be transfered to the other. These species cause damage amounting to billions of dollars yearly. Where they cannot be controlled, they affect the livelihood and cultural practices of entire regions. Heliothine pests infest maize, sorghum, soybean, cotton, sunflower, pulses, and many horticultural crops. They are characterized by their polyphagy, high mobility, high fecundity, and facultative diapause. These traits contribute to their status as major agricultural pests (Fitt, 1989). Because larvae feed on plant structures rich in nitrogen (flowers and fruits) they influence yield directly. This is of particular concern in high value crops where there is a low damage threshold.
3.2 Complex
interactions
Programs in plant, microbial or viral genomics can be productively interfaced with lepidopteran genomics. The "triangle" of human, Plasmodium and mosquito interactions has been a strong argument in favor of the A. gambiae genome project. Lepidoptera are involved in an even broader range of interactions with other organisms. Plant-herbivore interactions can be studied on model plant species (e.g., Arabidopsis) and on crop species for which extensive genomic information is at hand. Tri-trophic interactions between plants, Lepidoptera and their parasitoids offer new paradigms for environmentally safe pest management (see, e.g., De Moraes et al., 2001). Interactions between Lepidoptera, entomopathogenic nematodes and their bacterial symbionts offer new ways to study factors involved in virulence and symbiosis. Interactions between Lepidoptera and baculoviruses have led to important biotechnological advances in recombinant protein production, and point towards enhancing the efficacy of these environmentally benign biological control agents. Other viruses (polydnaviruses) are able to modulate the lepidopteran endocrine and immune systems, and are vectored by hymenopteran parasites used in biological control.
3.3
Sericulture
World silk production has approximately doubled during the last 30 years in spite of man-made fibers replacing silk for some uses. Silk production also plays an important role in local economies of developing nations. Three million people in India alone are involved in sericulture, which is very labor intensive and provides a key to improving local quality of life. However, disease resistance and other improved traits are needed. Applying methods of crop improvement, genes affecting growth rate, yield and fiber quality, can be tagged with molecular markers for rapid construction of genetically improved strains. Production of heterologous (e.g., spider) silk and other biomaterials in silkworms would be facilitated by knowledge of the complete genome and the full suite of biochemical, developmental, and physiological processes that make the silk gland such an efficient bioreactor.
3.4 Neurobiology
and olfaction:
Pheromones are chemicals emitted by living organisms to send messages to individuals of the same species. The class most widely explored are the sex pheromones produced by female moths which are used to attract conspecific males for mating (http://www-pherolist.slu.se). The discovery of pheromones and their binding proteins were achievements of lepidopteran biology. The neural encoding, processing and integration of olfactory signals from mates and hosts is also an area in which Lepidoptera continue to serve as important models. Genetic variance in the biosynthesis, reception or response to pheromones between closely and distantly related lepidopteran species is an area that genomics is uniquely suited to exploit.
3.5 Insecticide
resistance:
Several of the most important lepidopteran pests are particularly troublesome because of their ability to adapt rapidly to insecticides by evolving resistance. For example, diamondback moth generally develops resistance to new insecticides within two years of introduction. Many other species develop resistance on cotton, the most intensively sprayed crop. Although Bt-producing transgenic cotton is currently effective in controlling pest damage while reducing chemical sprays, Bt-resistance threatens to force a return to the "pesticide treadmill". Only recently (Gahan et al, 2001) was the first Bt-resistance gene cloned from an insect (H. virescens), and it will be a great challenge to apply this new knowledge soon enough to forestall or delay the appearance of resistance in the field.
3.6 Evolution and Conservation:
The discovery of mimicry in butterflies provided one of the most important early pieces of evidence for Darwin's theory of evolution by natural selection. Industrial melanism in Biston betularia is the most widely cited example of natural selection in evolution textbooks. More recently, butterfly wings have re-emerged as a model system in developmental genetics, as the mechanistic control of the complex development of wing color patterns has been dissected and analyzed in terms of evolutionary conserved signal transduction pathways (McMillan et al., 2002). Butterflies and moths figure as ecologically important and aesthetically appealing components of biodiversity and conservation projects worldwide.
3.7
Bioengineering/biotechnology:
The exploitation of insects as biofactories has been enabled by the development of baculovirus expression system to produce high value proteins either in cell lines, or in whole silkworms. A complete knowledge of the lepidopteran genome enables further optimization of this system, for instance by providing a complete catalog of the enzymes used in post-translational processing of the recombinant products.
4. Current State of Knowledge
4.1. Facts and figures about lepidopteran genomes: When compared to the Drosophila genome of 180 Mb (120 Mb euchromatin) in 4 chromosomes, and the 260Mb of A. gambiae in three chromosomes, the genome of Lepidoptera is characterized by a larger size and higher chromosome number, typically about 30. The genome size of Bombyx mori is 530 Mb, of Manduca sexta 500 Mb, and of Heliothines is approximately 400 Mb, comparable to the rice genome. Haploid chromosome numbers in these species range from 31 (H. virescens) to 28 (B. mori). Each represents the equivalent of 2-3 polytene chromosome bands of Drosophila or 3% of the genome, which makes them relatively small packets for construction of physical maps (contigs). Genetic crosses are routinely accomplished in Lepidoptera. Achiasmatic oogenesis in Lepidoptera implies that there is no crossing over in the heterogametic (ZW) females (males are ZZ). Thus linkage is all or none, synteny is easy to detect, and recombination means that genes are on different chromosomes. The GC content of lepidopteran DNA is about 35-40%. A variety of transposable elements are known from Lepidoptera, including some that have proved useful in insect transformation (e.g. piggyBac). The occurrence of repeated sequences has not posed a problem in current sequencing efforts.
4.2 Mutants: Modern genomics is complemented by a strong foundation of classical genetics. The silkworm is second only to the fruitfly as an insect model for genetic studies, with more than 400 visible mutations identified and mapped to over 200 loci. These mutations affect many fundamental aspects of the insect's life cycle, including egg and eggshell formation, early embryonic development and pattern formation, larval feeding behavior, molting, and embryonic diapause. Hundreds of practical breeding stocks maintained for sericulture represent a second, largely untapped resource for quantitative or polygenic traits affecting important biological characters such as body size, silk quality, fecundity, pathogen and insecticide resistance, and heat tolerance. B. mandarina, which produces fertile hybrids with B. mori, provides an additional source of variants lost during domestication and important for survival in the wild. H. virescens and H. subflexa represent another pair of lepidopteran congeners which differ significantly in their ecology (one is a pest but the other is not because of its restricted food source) but can be hybridized in the laboratory. The many important behavioral and developmental features which distinguish them, such as pheromone blend, olfaction, egglaying, host plant and virus resistance, can be mapped experimentally as quantitative traits even moved from one species to another (Sheck and Gould, 1996), leading to the discovery of genes affecting adaptation under conditions of sexual and natural selection.
4.3 Cytogenetics: Lepidopteran chromosomes are holocentric, meaning that their centromeres are spread over at least 70% of their length. In addition they generally have a relatively small size. This makes it difficult to distinguish the different chromosomes. To circumvent this problem much effort has been devoted to in-situ techniques (hybridization, fiber FISH analysis, and primed synthesis) and this has enabled the display of repeated DNA (rDNA, telomeric repeats) or single copy genes in various species, including B. mori and Spodoptera. At least two types of chromosomal sex determination exist (Z/ZZ and WZ/ZZ), and the structure of the W (female-specific chromosome) is under intense study by direct sequence analysis (Abe et al., 2002).
4.4 Sequence Databases: In GenBank there are currently more than 23,000 entries for Lepidoptera. The Bombyx "SilkBase" database (www.ab.a.u-tokyo.ac.jp/silkbase/) contains information on 32 cDNA libraries with 26,000 entries of which 9500 are non-redundant. The larger body size of Lepidoptera when compared to Diptera makes organ dissection easier, providing a higher coverage of expressed genes through tissue-specific EST collections.
5. Resources in
Lepidopteran Genomics
5.1 A broad-based scientific community: Lepidopteran genomics with efforts on Bombyx mori and on Noctuidae brings together research groups from East and West, and from North to South. In addition to sericulture-related work in Japan, China, India and more than 60 other developing countries, Bombyx is used as a biological model throughout the world, with strong research groups in Europe, Japan, and the United States. Work on lepidopteran pests of agriculture is a worldwide activity, one in which technology transfer between North and South is crucial. "Research and development addressing specific problems facing poor people have proved time and time again how technology can be not just a reward for development but a critical tool for achieving it." (Brown, 2001 - UNDP Human Development Report 2001). Research and training opportunities for developing countries using lepidopteran pest genomics as a framework will be a high priority for this project.
In the United States alone, the CRIS database from USDA lists well over 350 research projects on Lepidoptera, with the majority on Heliothis virescens / Helicoverpa zea. Over the last 25 years, CAB abstracts lists over 10,000 papers on heliothines and an estimated 1,000 papers per year on Noctuidae. These range from very applied aspects of control to basic and molecular studies. In addition, NIH funds more than 40 laboratories for work on Manduca sexta.
5.2 Current status and future plans: A full scale project has been initiated in Japan on sequencing the B. mori genome through large-scale ESTs, BAC contig construction, anchoring genetic and physical maps, and sequencing of selected chromosomes. The potential of the whole genome shotgun approach is being discussed, as well as the need for a comprehensive BAC-end sequencing effort.
Contributors from around the world are generating EST collections from a variety of lepidopteran species, many of which are major pests. Many of these EST libraries are from specific tissues such as midgut, pheromone glands, antenna and endocrine organs. NSF in the United States and INRA in France have funded the construction of BAC libraries from key lepidopteran species. Table 1 shows the current status and projected goals of these and related projects.
6. Postgenomic tools for identification of gene
function.
6.1 Bioinformatics. Interconnected databases for
the Bombyx and Noctuid genomes are planned to facilitate access
to the information generated during the project. In addition,
SilkBase is anticipated to be greatly expanded in the near future.
For complete annotation of the Bombyx genome, additional tools need to be developed.
6.2 Transformation: Stable germline transformation of Bombyx mori by insertion of a recombinant piggyBac transposable element has been achieved (Tamura et al., 2000). This transposon was initially discovered in Trichoplusia ni and subsequently engineered for use as transformation vector. Transformation of the pink bollworm with this transposable element has also been reported (Peloquin et al., 2000) and it is likely that transformation using piggyBac or other transposon-derived vectors will become a widespread postgenomic tool in Lepidoptera. The high frequency of transformation, multiple insertions and the random targeting suggest that piggyBac can be used to generate gene knockouts if a large scale effort is invested.
6.3 Disruption of gene function: Transient RNAi is a valuable tool for the validation of gene identification in insects and has been achieved in in several lepidopteran insects, includingHeliothis virescens (Hajos et al, 1999), Bombyx mori (Quan et al., 2002), Helicoverpa armigera (Whyard et al., 2001), Hyalophora cecropia (Bettencourt et al., 2002) and Manduca sexta (Vermeheren et al., 2001). Many laboratories have the capabilities of using this technology in lepidopteran systems, and significant progress is anticipated in the near future.
6.4 Transcriptome analysis: EST-based DNA microarrays for the analysis of gene expression are an essential tool to exploit genomic data. Drawing on the Bombyx EST collection, a microarray containing 6,000 independent ESTs has been prepared in Japan, which will be supplied to any researcher at cost price. Other groups worldwide are also developing microarrays for analysis of lepidopteran gene expression(e.g., INRA-France, Spodoptera array). A SAGE analysis of gene expression in silk gland is being conducted in France and Japan
6.5 Expression systems: Other tools are also available, including many lepidopteran cell lines, which are widely used for production of recombinant proteins by baculovirus-mediated expression or following stable transformation.
7. Expected
outcomes from lepidopteran genomes.
The diverse expertise of the lepidopteran research community will lead to many new developments and discoveries, affecting a number of areas:
*
insecticide
targets
lepidopteran-specific and safe to mammals and nontarget
organisms
*bioactive
molecules
antimicrobials, enzymes, antagonists of new targets, antifungals, antifreeze proteins
*protein
expression
cell culture systems
silk gland, novel silks
*green
chemistry
enzymes for chemical synthesis and bioremediation biosensors
*biosensors