AM-CC Posters

Abstract: Cryopreservation is a practical strategy for conserving and managing invertebrate diversity. Degraded nucleic acids present the single greatest obstacle to successful molecular research programs. Since storage at cryogenic temperatures essentially stops biological degradation, it is likely that long-term cryopreservation will be a suitable method for storing tissues for current scientific applications and for future genomic studies. Although some damage may occur, for most genetic techniques cryopreservation is a valid option for long-term tissue storage. Other commonly used tissue preservation methods, such as storage in ethanol, are increasingly revealed to be inadequate even for present applications. A study undertaken by the Ambrose Monell Collection for Molecular and Microbial Research compared the quality of DNA isolates from Drosophila melanogaster frozen under various conditions. Samples were frozen with 6 cryoprotectants in combination with 4 freeze/thaw strategies. The Comet Assay was used to visualize the extent of DNA fragmentation. Spot applications of insect DNA are subjected to gel electrophoresis and a comet-like pattern of fragment migration is observed. The longer the tail, the greater the DNA damage. Results showed that DMSO, glycerol, and ethylene glycol were better than ethanol in protecting DNA from freeze/thaw damage. The slow freeze/slow thaw method caused the least amount of DNA damage with all cryoprotectants. Slow freezing with no cryoprotectant was found to be the best method for preserving DNA. The study was expanded to include a comparison of the effects of various combinations of temperatures and storage media on the preservation of DNA in beetles, which resist permeation by cryoprotectants. Samples were stored in buffer or ethanol, dry, or pretreated with a commercial preservative, RNAlater (Qiagen, Inc.), and frozen at -20oC and -160oC. DNA extractions were subjected to gel electrophoresis, and DNA was qualified and quantified. This study demonstrates the critical nature of specimen storage for biomolecule preservation.

AMCC Lab Senior Curatorial Assistant, Alva Wright, standing by her poster at the CBC Spring Symposium

PRESERVING INSECT DIVERSITY: The online Tissue Specimen Database of the Ambrose Monell Cryo-Collection (AMCC) at the American Museum of Natural History

A. Corthals, J. Feinstein

Abstract: In a time of massive species loss, natural history museums should lead the way in providing digital access to biodiversity information, especially for biomaterial collections used in modern genetic, genomic and taxonomic studies. The American Museum of Natural History (AMNH) established the Ambrose Monell Cryo Collection (AMCC) to help meet the demand for properly documented frozen tissue specimens required by scientific researchers. The rapidly growing AMCC collection houses over 18,000 specimens. Arthropods comprise more than 10% of the collection. Over 1000 specimens of Drosophilidae make this the 2nd largest repository of fruit flies in the world. The AMCC continues to acquire and bank biomaterials, while actively developing and improving protocols for long-term storage of biomolecules. The collection maintains an online database (see: www.research.amnh.org/amcc/) which runs on the MySQL relational database management system. The database is integrated with the National Center for Biotechnology Information. This allows for AMCC records with nucleotide sequence accession numbers to link out to corresponding pages on the NCBI Genbank and Taxonomy sites. Conversely, records of genetic sequences on the GenBank website that are housed at the AMCC are linked to that specimen's details on the AMCC website. The AMCC database also links specimen records to digital images (e-vouchers), making for a complete connection between sequence data and the visual identity of the specimen examined. Consequently, specimen records in the collection may be located by barcode number, NCBI taxonomy number, GenBank sequence number, or taxon name, or browsed by taxonomic hierarchy. Tissue specimen collection records will ultimately be linked to bibliographic citations, alternate taxonomic determinations, and geospatial referencing information. The AMCC represents a unique effort in biodiversity preservation that can serve as a model facility for centralized biomaterial banking.

Schistosomiasis in Ancient and Modern Egypt: the ecology of an infectious disease from Ancient to Modern times.

A. Corthals

Historically, the study of schistosomiasis on the banks of the river Nile has addressed ancient and modern populations and their environment separately. A co-evolutionary study of the schistosome worms and its human host from ancient to modern times, coupled with an examination of ancient sources and both their description of the pathology and the remedy they offered for the infection would seem a valuable addition, both for its historical illumination and its potential medical and ecological usefulness. Thus I propose to illustrate this study which combines Egyptology and Ecology with modern microbiology.

1. The study of ancient sources: will illustrate the study centred on documents, such as the Edwin and the Ebers papyri (Nunn, 1996), and the findings of patterns of pathologies associated with parasitic infections as well as the nature of the infection depending on the type of water environment in which the parasite was acquired. Modern sources on the subject will also be illustrated as collected in the field through interviews to be compared to these ancient texts in order to assess any difference in the virulence of the infection or of the pathology based on the ecology, the population and the nature of the infection.

2. Fieldwork: will illustrate consist in getting samples from both parasite and host, both ancient and modern. Sampling of both ancient and modern human will be illustrated as well as the techniques of molecular biology used in order to produce the sought results.

3. Finally, the culprits of the disease S. mansoni and S. haematobium will be shown in the study of establishing a phylogeny of schistosome species within the area studied and the evolution of the parasite in conjunction with that of its host, the human population of the Nile banks.

The poster will also illustrate the goal of this study which is to track waves of schistosome species which are responsible for infection cases and observe patterns of evolution in the ecology of schistosomiasis within a particular region of Egypt. The implications of such an analysis can be expected to go well beyond a simple evolutionary study and have important medical and ecological implications and will point out to environmental measures on the banks of the river Nile in order to prevent this infection.

Identification of animal tissue samples using cytochrome b DNA sequences (mitochondrial DNA)

M. Subran; Mentor: A. Corthals

Abstract: The Ambrose Monell Collection, better known as the frozen tissue lab, houses many different types of animal tissues. The samples that have all their data recorded are stored in the permanent vats but sometimes the data is questionable. No one really has time to check these samples to see if they correspond with the information sent by the collectors.

Our project was centered around the taxonomical identification of tissues which were held temporarily at the AMCC following a freezer meltdown in one of the museum's collections. After contacting the relevant researchers, it became clear that all that was left of the data associated to those tissues were a mere 4 pages of scribbled field notes and whatever was written hastily on the vials and containers.

The project was designed to verify the taxonomic identity of the tissues. Queries on GenBank helped us determine which region of the gene was to be sequenced and which tissue could potentially be selected. We chose to isolate and sequence cytochrome b of 13 tissues. The sequences were then compared to those registered in GenBank to positively identify the species.

The result of the sequencing verified some of the taxonomical identification. More importantly, the sequence obtained for one specimen determined its species, which was previously unknown.

This project has stressed the importance of having a sequencing protocol as part of the routine quality control of the specimens being accessioned at the AMCC.

Salvage of genetically valuable tissues following a catastrophic mechanical

freezer failure and subsequent prolonged specimen meltdown

R. Hanner, N. Smilowitz, B. Webster, A. Corthals, C. J. Cole and H. C. Dessauer

Abstract: Standard protocols following meltdown of frozen tissue collections have yet to be established. Currently, most rare biological specimen collections are maintained in mechanical freezers where they are at risk of freezer failure or power disruptions. This study reports on an ultracold mechanical freezer failure in New Orleans that exposed tissues to room temperature for a period of approximately five days prior to detection. Many of the specimens had liquefied during this time and appeared highly degraded. The collection involved a diverse assemblage of reptilian tissues. As the majority of the specimens had already been used in a variety of studies, this presented an opportunity to test the stability of heat stressed macromolecules in the degraded specimens. To that effect, we ran tests on individual proteins and extracted nucleic acids from homogenates of liver, kidney, heart, skeletal muscle and red cell hemolysates, in addition to other tissues, to examine the degree to which any macromolecules might have survived.

How much DNA? Genome size quantification as an important application of biodiversity repositories

T. Ryan Gregory and Robert Hanner

ABSTRACT: The collection and appropriate storage of tissues will determine the future course of comparative genome biology, and for this reason biodiversity repositories are of great importance to genetics. While many efforts are being taken to ensure that genome sequencing, and even protein or RNA analyses, remain feasible with stored specimens, little attention has yet been paid to the important question of genome size diversity. Genome sizes vary more than 200,000-fold among eukaryotes, and correlate with many important cytological and organismal traits. Nuclear DNA content is also a primary consideration in the choice of future large-scale sequencing projects. Here we outline the case for including genome size quantifications as part of standard repository protocols, and describe some methods by which this can be accomplished efficiently. If implemented, such a program will allow the collection of genome size data on an unprecedented scale, and will permit the utility of tissue repositories to genome biology to be fully realized.

The online tissue specimen database of the Ambrose Monell Cryo-Collection (AMCC) at the American Museum of Natural History (AMNH)

Robert Hanner, Angélique Corthals and Mark Breedlove

With the advent of the "genomic revolution" and the rapid refinement of techniques of molecular biology, natural history museums are at the forefront of digital access to information about specimens, taxa and organismal biology. In a time of massive species loss, efforts to offer the most comprehensive, exhaustive and widely accessible record of the earth's biodiversity are essential.

Stemming from the need of referencing tissue samples and their associated data, AMNH's Frozen Tissue Collection, the Ambrose Monell Cryo-collection (AMCC), launched a website with an online database making its holdings accessible to the world's scientific community.

The AMCC's on-line database contains taxonomy, image, and sequence information for nearly 5,000 frozen specimens, and is integrated with the NCBI's taxonomy and genetic sequence databases. Specimens can be located by barcode number, NCBI taxonomy number, or NCBI GenBank number, if these are known, or may be searched for by taxon or browsed by taxonomic hierarchy. Records of specimens with NCBI taxonomy or sequence numbers refer to corresponding pages on NCBI's website. Likewise, records of genetic sequences in the GenBank website that are kept at the AMCC are linked to the details page of the AMCC's site. The database uses a flexible model for storing and displaying external links, and is designed to be able to link out to any other website without modification of the front-end's code.

The AMCC's web database runs on the MySQL relational database management system and its front-end is written in PHP and served by an Apache webserver.

The data available on the AMCC website constitute a subset of data from the facilities' relational database Freezerworks Unlimited, loaded into the MySQL database. This allows the AMCC to have better control of the amount of data published on the web. The database is updated every three months during which Datacheck software is run to make the process faster and to align to standards of database information.

Modern bioinformatics initiatives will ultimately link collections with taxonomic determinations, bibliographic citations, geospatial referencing information, genetic data, and much much more…

Sustaining Seascapes: The Science and Policy of Marine Resource Management

A. Corthals, P. Doukakis

The Oceans were once thought to be so vast as to be immune from human harms. We now know that they are not. Although oil spills and trash washed up on beaches may be the most obvious signs of humanity's toll on the seas, anthropogenic threats to marine biodiversity are much more widespread. Most alarming, the web of connections among diverse species, and the ecosystem functions that these species provide, actually appear to be stretched beyond their limits in many places. These stressed systems now exist in highly reduced versions, providing less productivity (for both humans and other species) and less resilience to major disturbances such as epidemic disease, hurricanes, and climate change.

As evidence of these broad changes increases, so do marine conservation and restoration activities around the world. Although it remains to be see whether the patterns of marine degradation can be reversed, progress is being made in certain areas of marine management.

One of these areas is the expanding use of marine protected areas (MPAs) and particularly reserves that exclude extractive activities. Although the concept of MPAs is not new (and is arguably even ancient in some cultural traditions), the last ten years have witnessed accelerating interest in their design and implementation. Because marine systems are relatively open, however, even larger reserves can't conserve biodiversity and ecosystem function as surrounding areas continue to degrade. In other words, protected areas, in the sea even more so than on land, do not function in isolation. Consequently, conservationists have increasingly turned to designing networks of reserves and other site-based protective measures, and are confronting a host of new technical and policy issues in the process.

As interest has grown in scaling up to larger seascape levels, many conservationists have realized that much better communication and collaboration is needed between natural and social scientists, and between these professionals and stakeholders. Whether at the level of local communities or international institutions, the management of seascapes involves managing people. Only through an increased understanding of how humans engage with the natural world - culturally, economically, and politically - can we develop adaptive mechanisms to better manage our resources, and approach sustainability.

While addressing coupled natural and human systems is arguably one of the critical next steps in marine conservation science, we must not forget some of the more straightforward tasks at hand. Better communication with the public about the wonders of marine life, its radical decline due to heedless human activities, and its potential for recovery is essential. We must covince more people that, along with being consumers of marine resources, we must become caretakers: the days of oceanic invulnerability are long gone.

AMCC and the ANTONGIL BAY Fisheries project

Beginning in August 2000, the Wildlife Conservation Society (WCS) established the Antongil Bay Fisheries Initiative in eastern Madagascar . This initiative aims to monitor and sustainably manage the use of this important yet heavily exploited marine ecosystem. By involving local fishermen as collectors, WCS can catalogue the shark population of the Bay in order to assess the impact of the Asian shark fin soup driven fishery. A DNA based monitoring system is used to accurately identify shark species and field research has been undertaken in order to map and physically characterize the fishing grounds in the Bay. This will allow us, with the help of a GIS system under development, to accomplish a full identification of critical fisheries habitat serving as potential candidates for a new marine protected area.

The Antongil Bay shark specimens are sent to the Ambrose Monell Collection for Molecular and Microbial Research at the American Museum of Natural History (AMNH). Archiving tissue samples allows for subsequent multipurpose genetic assays to be done on the material without duplicating unnecessary collecting effort. When the long-term use of such a resource is undefined, then a good archival paradigm is that "colder is better". Thus the samples, originally collected in ethanol, are transferred into bar-coded 1.8ml cryo-vials at AMNH where they are archived at cryogenic temperatures (below -150¼ C) in liquid Nitrogen cooled freezers. The specimens are frequently accompanied by digital files and photographs Such information, in addition to GenBank accession numbers and bibliographic citations, is archived and then cross-referenced with the appropriate tissue specimen(s) in the collection.

Insect Genomic Symposium

R. Hanner

The U.S. systems of renewable resource production and land stewardship face formidable challenges in the new millennium. Among the most exacting challenges is successfully adapting these systems to the accelerating rates of change in factors affecting agricultural productivity. Climatic extremes may now occur more frequently due to human activities. Water and soils are being depleted more rapidly. As global agricultural production incorporates more fertilizers, herbicides, or pesticides, water and soils are also increasingly threatened by pollution. Unless new technologies are developed and utilized, regulations and other proposed remedies for those phenomena might rapidly complicate resource management, food and fiber production and processing. These complications, in turn, may result in more rapidly increasing prices paid by consumers, more volatile commercial markets, reduced profits for producers, and a narrower competitive edge for U.S. products in world markets. More costly food may result in less nutritious diets for the poor.

Natural plant communities and landscapes that contain potentially useful plants are disappearing at an accelerated rate. Honeybees, other insect pollinators, as well as insects potentially beneficial as natural enemies of introduced weeds or pests, are endangered in their native habitat from habitat destruction, including excessive use of pesticides. Microbes that produce industrial products, serve as biological control agents, degrade and recycle minerals, cellulose, proteins, and are indispensable for cycling nitrogen, carbon, oxygen, sulfur, and other elements, are imperiled by human disturbances of the ecosystem. Burgeoning human populations worldwide are increasingly urban, with cities now occupying ever more hectares of formerly productive agricultural land. Industrial activity also exacerbates soil, water, and air pollution and contamination. As a result, rates of agricultural productivity can be raised only if the remaining land under cultivation yields more agricultural production.

New, more intensive production practices implemented throughout the Nation, (e.g., higher density plantings, reduced tillage, and chemical inputs) place new demands on crops. Formerly minor pathogens are now economically important because of changing production practices. New, more virulent genetic variants of already important pathogens and pests are cause for grave concern. These provide an impetus to continue searching for new sources of host-plant resistance. Economic constraints to agricultural profitability underscore the immediate need for value-added and alternative crops for increasing the monetary return to producers (especially in rural areas), and for efficiently diversifying the productive capacity of the U.S. system of renewable resource production.

The high societal costs associated with rapid destruction of natural habitats and agricultural productive capacity may be most extreme in the developing countries of the tropics, where a wealth of genetic resources vital to U.S. agriculture is endangered. Greater emphasis should be placed on conservation of germplasm through international cooperation. Development and maintenance of stable biological communities in the natural environment should be a high priority goal worldwide. Essentially all the major microbes, beneficial insects, and crops we grow and use originated in other parts of the world. Consequently, the stability of the vast agricultural system of the United States is based primarily on organisms that were imported long ago, and on their continual genetic improvement via more recently acquired genes conserved in germplasm collections. Extinction of these resources, or inaccessibility caused by lack of financial support for genebanks and preserves, changing regulations governing international germplasm exchange, or changing intellectual property rights regimes, may increase the genetic vulnerability of agriculture to rapidly evolving pests, pathogens, environmental changes, and to competitive market demands, the latter, which changes continually and rapidly according to consumer preferences and advances in processing technology.

The demands placed on the national system of renewable resource production by a rapidly changing world can only be met by technologies that optimally harness the inherent genetic potential of insect, plant, and microbial germplasm. Production systems that optimally preserve and harness that genetic potential will maximize profits, security of supply, price stability, market competitiveness, and avoid crop losses from genetic vulnerability. More rapid and efficient methods for identifying useful properties of genes and genomes, and for manipulating genetic and genomic material and information, are required. These new methods will include more effective breeding strategies, and more comprehensive knowledge of microbial, insect, and crop genomic structures. More cost-effective molecular markers must be developed, so as to improve the efficiency of gene identification and mapping. More rapid gene analyses and mapping methods and strategies are needed, as are the means for determining the function of particular genomic segments. The new scientific approaches of genomics and biotechnology are critical for developing improved crops and microbes that enable producers to maximize yields of high-quality products, but minimize chemical input, water and soil depletion, water and soil contamination, as well as production costs.

Paradoxically, sole reliance on the preceding methods of genetic improvement may lead to superior but excessively narrow genetic bases for crop, honeybee (and other beneficial insect species), or microbial gene pools. As a result, the Nation's future food, fiber, feed, ornamental, and industrial product supply may be more vulnerable to rapidly changing pathogens, pests, or environmental extremes. It may be less abundant, nutritious, and diverse, hence less capable of adapting to changing regulatory concerns or to global environmental change and changing commercial markets. Consequently, the national program in Plant, Microbial, and Insect Genetic Resources, Genomics, and Genetic Improvement (which encompasses conserving, enhancing and improving genetic resources, managing and analyzing genomic data, in addition to developing novel approaches to breeding and genetic improvement) is crucial to developing safer, more secure, and more efficient agricultural systems.

Genetic raw materials, water, air, soil, and management practices comprise the agricultural production system that sustains humanity, and provides the United States with an affordable, highly diverse, and nutritious diet. The plant, microbial, and insect genetic resources managed by this national program are the bases of the U.S. systems of renewable resource production and land stewardship. Germplasm conservation is expensive and complicated. It requires that species or variety- specific procedures be developed (e.g., methods to culture beneficial microbial symbionts of plants, or to efficiently maintain standard check strains of plant pathogens for germplasm evaluations). The utility and integrity of those resources must be conserved and enhanced by improved, more effective methods such as genomics. Genomics exploit cross-organismal similarities in genetic repertoires to efficiently identify, characterize, and map genes, elucidate their function, and deploy them in genetic enhancement of crops. With these new scientific tools and processes, and traditional methods for genetic improvement, germplasm introduced from genebanks will be adapted to particular production niches, or its utility will be otherwise enhanced.

Ambrose Monell Cryo Collection (AMCC)

Contents

Poster Sessions and AMCC posters

FREEZING INSECTS: The effects of cryopreservation protocols on nucleic acid degradation.

Alva Wright, Joann Mercedes, Miriam Delarosa, J. Feinstein, A. Corthals