Title: THE COCCIDIA OF THE WORLD

PROJECT SUMMARY
The protozoan phylum Apicomplexa Levine, 1970, comprises a large and heterogeneous assemblage of obligate intracellular parasites of medical and veterinary significance (e.g., Eimeria, Cryptosporidium, Plasmodium, Toxoplasma). The largest subgroup of the phylum, the suborder Eimeriorina Leger, 1911, contains organisms referred to collectively as the coccidia (Levine et al., 1980). Predominately intestinal parasites, they infect most phyla of invertebrates and all vertebrate classes. The disease they cause, coccidiosis, is recognized as the major health hazard in domestic animal husbandry, in zoo environments and in wild animal populations when habitat is lost and overcrowding occurs. At present, the Eimeriorina has 10 Families, at lease 42 genera and over 1800 named species.
Working within the taxonomy of the coccidia is difficult: (1) The literature is vast and widely scattered, much of it appearing in obscure journals that have limited circulation. The only definitive work on the group has been Pellerdy's Coccidia And Coccidiosis (1974); although Levine (1988a,b) authored a 2 volume checklist, The Protozoan Phylum Apicomplexa, these volumes are useful as lists, but they are incomplete, not cross-referenced by host, parasite, locality, author or any other way and, thus, have limited value. In consequence, literally hundreds of species descriptions remain unarchived and/or uncatalogued. (2) The coccidia have a complex life cycle that includes 3 sequential stages: endogenous merogony and gamogony followed by sporogony which is exogenous. This complexity resulted in various stages of the same coccidian species being described as different species, or even placed in higher taxa (genera to suborders), before their basic life history was understood. (3) the endogenous (intracellular) developmental stages in a coccidian life cycle are unknown in >99% of all described species and are impossible to find or identify under field conditions, so these characters have no present taxonomic value. (4) the exogenous stage (the oocyst), upon which the majority of all species descriptions are based, is highly resistant to all known fixation techniques and, to date, no satisfactory method is known to permanently preserve its structural features; thus, the taxonomy of coccidians has been non-specimen-based. In result, most species are described solely on measurements of different structures in the sporulated oocyst, some additional key qualitative features, and line drawings. Even though both the Botanical and Bacteriological Codes accept line drawings as type specimens for organisms that cannot be preserved, the Zoological Code has not yet addressed this issue adequately.
These problems make sorting through the taxonomic literature of coccidians difficult and differentiating species can be guesswork even for experienced coccidian taxonomists. Thus, instead of studying curated specimens in accredited museums, all original literature for a particular species must be obtained and interpreted on a species-by-species basis.
The PI and subcontract PI collectively have more taxonomic papers on the coccidia than anyone worldwide. We propose to combine these collections and, after obtaining (virtually) all of the world's remaining literature on the group, produce a definitive monograph. This "magnum opus" will be available both in electronic and hard copy formats, and both will be expandable in perpetuity. We also will train 3 graduate students in modern coccidian taxonomy and systematics, and several related disciplines to be able to place the coccidia in a co-evolutionary context with their hosts which, in turn, may shed some light on the evolution (or lack thereof) of the presumed strict host-specificity of these enigmatic, poorly-known, but economically and important protozoan parasites.

PROJECT DESCRIPTION

Historical perspective, taxonomic breadth, importance.
The protozoan phylum Apicomplexa Levine, 1970, comprises a large and heterogeneous group of obligate, intracellular parasites, including many species of medical and veterinary significance (e.g., Eimeria, Cryptosporidium, Plasmodium, Toxoplasma). The history of the coccidia dates to 1674 when Leeuwenhoek saw the oocysts of Eimeria stiedai in rabbit bile and partially described them in his unpublished 7th letter addressed to the Secretary of the Royal Society (Dobell 1922; Corliss 1975; Wenyon 1926). The first published description of a coccidian stage was in 1839 by Hake, who thought the unsporulated oocysts of E. stiedai were pus globules in the liver of rabbits (Levine 1973). When coccidians were first recognized to be protozoans (Dobell 1922), it was not known they had complicated life cycles involving merogony, gamogony and sporogony. As a result, early workers placed the exogenous transmission stage, the oocyst, in one genus and the endogenous tissue stages (meronts, gamonts) into different genera, families or even suborders (Levine 1982)! With the advent of electron microscopy, however, the relationship of the coccidia to other protozoans, and to themeslves, became evident. We now know that organisms placed within the Apicomplexa had various subcellular features (e.g., polar ring, conoid, etc.) that separated them from other protozoans (e.g., Microsporidia, Myxozoa) that lack these structures, and which once were put into the same group (the old "Sporozoa") with the apicomplexans. The phylum now contains five principle groups: coccidians, gregarines, haemogregarines, malarial organisms and piroplasms. In the last definitive taxonomic work done by the Committee on Systematics and Evolution of the Society of Protozoologists (Levine et al. 1980), the "true" coccidia (Levine 1982) were placed into the suborder, Eimeriorina Leger, 1911. Since 1980, several genera and many species have been added to the coccidia. Today, this group consists of 10 Families, 42 genera and 1800+ named species.
Coccidians and their relatives parasitize most phyla of invertebrates and all classes of vertebrates. The disease which they cause, coccidiosis, is recognized as the major health hazard both in the intensive husbandry of all domestic animals and in wild animal populations when habitat is lost, and crowding occurs, usually due to human intervention. For example, the modern poultry industry, among others, could not have developed without adequate means to control coccidiosis with anti-coccidial drugs required in all commercial poultry feed, at considerable expense (McDougald et al. 1986). Consequently, the importance of the coccidia has stimulated research in many directions (immunology, physiology, molecular biology, genetics, biochemistry, etc.), with the major exception being that of their taxonomy.
Coccidians may have either direct (homoxenous) or indirect (heteroxenous, two or more hosts) life cycles that include both asexual (merogony) and sexual (gamogony) reproduction; both types of development occur within the epithelial or endothelial cells of the gastrointestinal tract, or related structures (e.g., bile duct, renal tubular epithelium, etc.), of the host animals. In either cycle, the end-product of gamogony is a resistant structure, an oocyst, the only stage in the cycle to leave the host, usually via the feces. The Eimeriidae is comprised of strictly homoxenous coccidia and contains the majority of species in the suborder within two genera, Eimeria (ca. 1,200 spp.) and Isospora (ca. 300 spp.); most of these species described solely from their sporulated oocyst. The Sarcocystidae accomodates most heteroxenous coccidia and contains six genera with about 150 species, most (125 spp.) in the genus Sarcocystis. In these taxa, gamogony and oocyst formation occur in one host while merogony and formation of dormant merogonous cysts occur in an intermediate host. For proper taxonomic placement, the tissue stages need be correlated with the oocyst although many species are known only from the encysted parasites in the intermediate host.
It is unfortunate that the tissue stages and oocysts of most coccidia (>99%) have never been correlated. Oocysts usually are discharged from their host in an undeveloped (=unsporulated) state. Once outside the host oxygen and moisture are key elements needed for the "sporoplasm" within the oocyst to divide several times (meiotically and mitotically) to form smaller cyst structures (sporocysts), which contain two or more sporozoites (the actual infective units of the coccidian). This sporulation process produces an oocyst which is highly resistant to environmental extremes and immediately infective to the next host that chances to ingest it. Because of the difficulty of finding and isolating the tissue stages, and because study of tissue stages would require killing the (sometimes exotic or valuable) host animal, it is the structure of the sporulated oocyst upon which the taxonomy of the coccidia is based.

Objectives of Proposed Research.

Monography. The cornerstone of taxonomy is the type specimen which is intended to be unchanging and objective, whereas the limits of the nominal species are recognized to be subjective and transient. The type specimen serves as the anchor for the name and to some extent, it is the name (Mayr et al., 1953). Without the type specimen there is no "species." Coccidians present a serious problem when it comes to collecting type specimens for two major reasons: 1) their endogenous stages are intracellular, transient, difficult to collect and impossible to identify under field conditions; and 2) no standardized methods have been developed to permanently preserve oocysts. Unlike helminths that stay in the gut or body cavity for lengthy periods (and easily can be preserved and deposited into museums for later retrieval), coccidian sporozoites penetrate epithelial cells, reproduce via merogony a genetically-determined number of times (e.g., 2-4) within a few days and finally form gametes that unite and pass out of the host via the feces as the (unsporulated) oocyst. Once outside, the oocyst needs access to moisture, oxygen and usually a temperature less than host body temperature to undergo development (=sporulation). Thus, the oocyst is the most readily available stage in the coccidian life cycle. It can be easily collected (in feces), and once it has sporulated, it can be identified. Unfortunately, although the oocyst is a fairly resistant stage and can remain viable in the soil for a year or more or in aqueous potassium dichromate at refrigerator temperatures for up to five years (personal observation), traditional methods of fixation do not preserve oocysts in perpetuity (Duszynski & Gardner, 1991). Only Marchiondo and Duszynski (1978, 1988) have reported a technique to preserve sporulated oocysts over time, but the method is time-consuming and expensive and has not been adopted by others who work with the coccidia.
Historically, those who have described coccidian species generally followed the same methods: isolate oocysts from the feces of infected hosts, allow them to sporulate in aqueous potassium dichromate, measure the many structural characters in a few to a few hundred oocysts, then describe a new species based on quantitative and qualitative observations of the oocyst, on the host and its locality, and by using a drawing as the type specimen. The latter procedure is allowed by the General Assembly of the International Union of Biological Sciences. Both the Botanical Code (Voss et al., 1983, Article 9.3) and the Bacteriological Code (Lapage et al., 1975, Rule 18A) have made provisions for organisms that cannot be permanently preserved: drawings are acceptable as type specimens. Previous editions of the Zoological Code have allowed illustrations to serve as lectotypes, but have not addressed the issue of whether illustrations may serve as holotypes. The most recent version of the Code (Ride et al., 1985, Article 73 [a] [iv], p. 149) states, "Designation of an illustration of a single specimen as a holotype is to be treated as designation of the specimen illustrated; the fact that the specimen cannot be traced does not of itself invalidate the designation (my emphasis)." Although it may seem obvious that line drawings are merely cartoons, the tradition of written descriptions documenting quantitative/qualitative structural features of a sporulated oocyst, accompanied by a line drawing of that oocyst, has perpetuated itself almost to the present as the only method of describing and documenting new coccidian species. Even as little as 10-15 years ago, I was criticized by referees and journal editors for attempting to publish photomicrographs of sporulated oocysts (i.e., a permanent record of a real specimen) as a waste of journal space.
Only recently have coccidian biologists begun to accept the importance of documenting actual specimens and Bandoni and Duszynski (1988) finally were able to start a "type tradition" for those who study the coccidia, standard practice among helminthologists for more than a century. Several journal editors also have recognized that photomicrographs can produce lasting specimens (phototypes) not available by various fixation techniques (Duszynski and Gardner 1991), and their journals (e.g., Journal of Parasitology, Journal of Eukaryotic Microbiology, and others) now will not publish descriptions of new coccidian species unless phototypes of sporulated oocysts, deposited and accessioned into permanent national collections, accompany the description. The result of this long-standing tradition in coccidian taxonomy is that only since 1988 have taxonomists begun to deposit phototype specimens with national museums, and most of these have been our own work (e.g., Lambert et al., 1988; Upton et al., 1995). Therefore, unlike most taxonomists who need to visit many natural history museums worldwide to review specimens, we need to go to major libraries, especially in key cities abroad, to retrieve original literature to collect our "specimens." It is not until we collect all these "specimens" and the basic biological information that goes with them that we can begin to answer some of the most fundamental questions about these unique animals, the coccidia: How many species exist? How many different species live in each host species? How are the species in one genus related to those in another genus (e.g., Eimeria spp. vs. Isospora spp.)? Why are members of some genera never found in certain host species (e.g., no eimerians are known to infect humans although they are the most common parasites of most other vertebrates)? Often, the most basic questions are the most difficult to answer and the problem is compounded because sporulated oocysts of the same species of coccidian can vary greatly in size and appearance (Duszynski, 1971; Parker and Duszynski, 1986; Gardner and Duszynski, 1990). In other hosts, however, the oocysts of eimerian species are so similar that they cannot be reliably differentiated by morphology and size alone (Joyner, 1982). Such issues cannot be dealt with until all of the literature is in hand, a formidable task, but one within reach of this project. Only then can we use the available information to answer these basic questions and the answers that are forthcoming should allow us to frame hypotheses that can be tested. For example, when all the coccidians from a host genus are known it will be possible using a variety of modern systematic procedures to address historical relationships that may demonstrate coevolutionary processes (for character states to be used see Reduker et al., 1987). Finally, the take-home message is that there are very few coccidian taxonomists, not more than three or four in the U.S. and perhaps a dozen more throughout the rest of the world. Therefore, the monograph which we propose to develop (see below) will require a very basic approach, but when completed, it should provide the basis and the stimulus for many avenues of future research: modes of speciation, co-evolutionary patterns, advances in histological and preservation techniques, especially for oocysts, biogeographical analyses, etc.

Training of students. In our opinion, the training of graduate students in taxonomy is one of the most important parts of this project, especially because of the dwindling number of individuals left in the world who actually can identify a coccidian species. The graduate students who work in this program will be in a unique position in that virtually no other lab in the country is now training protozoan taxonomists with so many creative twists available for their students. The students will be involved in all aspects of the scientific process including literature survey, development of their individual projects, training in the identification, description and characterization of protozoans in general and coccidian taxa in particular, various laboratory techniques (e.g., host inoculation, parasite isolation, sucrose gradient separation of oocysts, LM, TEM, SEM & Confocal Microscopy of parasite stages, etc.), modern systematic, phylogenetic and cladistic techniques (see Gardner, 1991) and, when appropriate, various molecular techniques (see Reduker et al., 1987), data analysis, presentation of results at regional, national and international scientific meetings, submission of dissertation improvement proposals to federal agencies, and submission of their work for publication to refereed journals. All students-in-training will be required to complete a series of formal courses at UNM that will be tailored to their individual programs, talents and needs, but that will include some substantial subset of the following: biometrics (401), scientific communication (405), ecological genetics (418), herpetology (488), mammalogy (489), ornithology (486), ichthyology (487), invertebrate biology (371), parasitology (382), genetics of speciation (510), molecular evolution (522), principles of systematic biology (523) and perhaps others. These students have the added advantage that at UNM we enjoy a long-standing, close, working relationship with invertebrate biologists (e.g., Drs. Corliss, Crawford, Loker, Stricker), vertebrate biologists (e.g., Drs. Bogan, Molles, Ligon,Yates, Snell, Conant,), ecologists (Brown, Milne), LTER (Parmenter) and museum (Frey, Yates) personnel and other members of this Department who can and will give these students many different, and important, perspectives in which to frame their taxonomic work including, but not limited to, development of curatorial skills, systematic/taxonomic methodologies, biogeographic interpretations, etc.
For examples of publications in which student taxonomic work was developed with and by various supportive data sets see Aquino-Shuster et al., 1990; Bandoni and Duszynski, 1986; Couch et al., 1993; Eastham et al., 1982; Frey et al., 1992; Lambert et al., 1988; Patrick et al., 1995; Reduker et al., 1987; Upton et al., 1983; Wash et al., 1985, 1990). Former students cited in the previous sentence all worked in my laboratory at one time, either as an undergraduate, as my own graduate student, or as the graduate student of another professor while I served on their Committee on Studies. The topics of their taxonimically-related projects and related data sets are evident from the Literature Cited section at the end of this proposal. Their present positions and locations are: Aquino-Shuster: Director, Inventario Biologico Nacional, Museo de Historia Natural de Paraguay, Ministerio de Agricultura y Ganaderia, San Lorenzo, Paraguay; Bandoni: Assistant Professor of Biology, SUNY, Genesseo NY; Couch: Chairperson of Biology, Albuquerque TVI Community College; Eastham: Optomistrist, Pacific Northwest; Frey: Postdoctoral Associate Hantanvirus Project and Museum of Southwestern Biology, UNM; Lambert: Diagnostician, State of New Mexico Scientific Laboratory, Albuqueruqe; Patrick: completed Ph.D. in December, 1994, now applying for jobs; Reduker (deceased) was Associate Professor of Pathology, Colorado State University, Ft. Collins CO; Upton: Associate Professor of Biology, Kansas State University, Manhattan KS; Wash: M.D., Ft. Worth TX). We are convinced that this very broad base to their academic training is the best way to prepare our students for careers in biology and to make them competitive for positions in academia, government or industry.
Two students who are interested in participating in this long-term project are Mr. Brett Pickering (Western State College, Gunnison CO) and Mr. Wade Wilson (UNM), both undergraduates who will complete their B.S. degrees in May 1995. Both are good students who have participated in NSF-sponsored REU summer programs and thus already have a modest amount of research experience as undergraduates. Also, both have presented papers at regional and/or national scientific meetings and each has one refereed publication as a co-author. If this grant is funded, additional students will be recruited at the joint meeting of the American Society of Parasitologists (ASP) and the American Association of Veterinary Parasitologists (AAVP) to be held 6-10 July, 1995. It is not anticipated that it will be difficult to recruit another first-rate student to fill this program. Finally, there is a "method to our madness" of filling our taxonomists-in-training slots with first-year graduate students. First, there are no Ph.D. programs now turning out M.S. or Ph.D. level coccidian taxonomists in the U.S. so there are no Masters/Ph.D.-level individuals to enter Ph.D/postdoctoral positions as experts-in-training. And second, starting a cohort of students at the very beginning of their programs allows us the rare opportunity to train them in every aspect of this program that we view essential to their development as competent and broadly trained taxonomists.

Computer infrastructure. We are in the unique position to take advantage of an extensive infrastructure developed especially for long-term projects at UNM. The approximately 7.5 Gb of imagery that we estimate will be archived in the course of this project will be integrated into the Sevilleta LTER Information Management System (SIMS). SIMS is comprised of personnel, protocols, and software that together provide for the quality assurance of data entry, security, access, and analytical support (data analysis for presentation of results) for researchers involved with the Sevilleta LTER Program. This includes database management systems (dbms), geographic information systems (GIS), and a variety of network information servers. SIMS is implemented on a cluster of DEC and SUN UNIX workstations and servers located on the UNM campus and at the Sevilleta Research Station. The workstations are networked via fiber-optic technology using standardized protocols. Through this collaborative effort, the SIMS infrastructure, when networked with the 586 PCs of the PI and the subcontract PI, can guarantee support and security for the project data. Further description of the system can be found in Brundt (1994). Information from this effort can be made available to the scientific community via GOPHER and MOSAIC information servers maintained by SIMS. These servers are accessible directly over the internet or via other national and international information servers. SIMS staff will assist with preparation of the archive (parasite and host taxa, unique species characters, specimen information, images [line drawings, photomicrographs], identification keys, literature, etc.) for transfer to and storage on CD-ROM.

"P" is for Partnership. In my opinion, the only way a project of this magnitude can be completed in an acceptable manner--given the unique characteristics and historical constraints of the coccidia--is with cooperation from (1) at least one other expert in the taxonomic area under consideration and (2) interested scientists willing to help from other continents, where the original literature in this area may be significant, but has never reached the rest of the world's mainstream reference sources. The partnership we have developed in this regard is strong: (1) Dr. Upton, the subcontract PI, is the only other coccidian taxonomist in the U.S. who continues to publish prolifically in this area; (2) Drs. Filatov (Russia), Molnar (Hungarian Academy of Sciences), Kamiya (Japan), Patterson (Australia) and Shaw and Lainson (Brazil) all have a strong interest either in coccidian biology or parasitology, are all interested in and committed to this project, and have all agreed to support our visits to their countries and work with us to retrieve most/all of the taxonomic literature dealing with the coccidia in their countries (see letters of support and invitation); (3) the curators of the two largest parasitology museums in the U.S. have agreed to receive, curate and make available (electronically and in "hard copy") all specimens we will send to them (see letters of support); (4) UNM's Museum of Southwestern Biology is on site and available to curate all symbiotype host specimens that we would care to deposit in the various divisions (mammals, birds, amphibians and reptiles, fishes, invertebrates); (5) Dr. Norman D. Levine, the "Father of Coccidiology" has given us his support in this effort (letter attached); (6) Dr. Leo Margolis has entrusted Dr. Paul Fitzgerald's entire library on fish coccidia of the world to our care so that it can be incorporated into this effort (letter attached); (7) the editors of the 15 primary journals in the world that publish new species descriptions of coccidians (and other parasites) have already published announcements alerting the community of coccidian biologists to our proposed endeavor to settle this problem once and for ever (see copy of announcement and list of journals following letters of support); and (8) we have a strong commitment from the UNM administration, at all levels within its infrastructure (the Biology Department, including its associated Long Term Ecological Research [LTER] program and the five divisions of it's Museum of Southwestern Biology [MSB], the College of Arts and Sciences, and the Office of Research Administration), of both intellectual and financial (cost-sharing) support.

Project Management Plan.

Collection of original literature. In the case of the coccidia, as noted above, the actual species descriptions of coccidians, for better or for worse, are the specimens/species. For this project to be definitive and complete, it is necessary for the PI and the subcontract PI to combine their personal libraries and to search the libraries of specific other countries (Australia, Brazil, Japan, Russia/Hungry) where, to our knowledge, the majority of work is being done on the coccidia by our international colleagues. These contacts and collection trips should secure virtually all of the needed, including generally obscure, literature from Australia, South America, Asia and the Eastern Block countries of Europe. Still undone will be the literature of China, India, Africa and Madagascar, which at this point are beyond the financial scope of this proposal. One way in which funds could be secured for trips to these remaining areas would be for us to seek supplemental support from the NSF International Programs, should this request be successful. Once the collected literature, i.e., the "specimens," are in hand, species descriptions, information on the biology, line drawings, photomicrographs (when available), etc. can be scanned into the data base, stored, and later manipulated as needed to complete this project with the level of thoroughness we desire and have tried to document in this proposal.

Reference collections/deposition of specimens. Phototypes of new coccidians (Bandoni and Duszynski, 1988) will be deposited in the U.S. National Museum Parasite Collection (USNMPC) and additional specimens will be deposited in the University of Nebraska State Museum Parasite Collection (see letters of support, Drs. J.R. Lichtenfels, S.L. Gardner). During the course of this project both the PI and the subcontract PI will continue their work with other colleagues who continually collect new host specimens. When such specimens also contain new coccidian species, as often is the case, host symbiotypes for new parasite species (see Frey et al., 1992; Brooks, 1993) will be deposited, along with parasite paratypes and/or phototypes, in the unique and separate symbiotype collection of the MSB-UNM.

Electronic database creation, management, access. It is important that this be the last time that these data are collected and it is our intention that we establish procedures to archive in perpetuity. As noted (3.3, above), the archival databases for this project will be integrated into the LTER Sevilleta Information Management System (SIMS). But storage and accessibility are separate issues. As is the case with all LTER research data, data from this compilation of coccidian species and their hosts will be made available for legitimate scientific purpose via network query to qualified researchers after they are archived. Information derived from this survey will be made available to the public via GOPHER and MOSAIC information servers.
Database/Data Model. We have not yet determined the preferred database we will use to record/store the information we will collect on the Coccidia of the World. In September 1994, The Symposium and Workshop on Taxonomic Data Resources held in Chambery, France, during the 14th CODATA Conference presented a forum centered on the questions of access to taxonomic data. The group was unanimous in its view that many problems must be solved in providing access to accurate and comprehensive taxonomic data, and in the process reached consensus on three points: 1) the most critical resources are the species name databases; 2) much critical work must be done by taxonomists to provide the baseline data on species names and authorities for these names; and 3) database producers need to better serve the user communities by providing systems to link directly to the appropriate databases or to the appropriate panel of experts. In the latter case, one database, DELTA, was noted to have gained wide acceptance because both the World Virus List and the World List of Legumes are being prepared with it. Although we are not yet familiar with DELTA, it is a system we will consider using, after consultation with Dr. Jeff Shaw and others who presently are more closely involved with the "Master Species Database" being supported by the International Union of Biological Societies (IUBS) (see Jeff's letter of support for our proposed project).
A model that we envision for the Coccidia of the World is a host-based relational model. The primary key for all observations will be the phylum of the host. The model can be represented by various tables which will be implemented at first in /rdb software and then translated to Sybase for network accessibility. The model is expandable at all levels. The number of sub-taxonomic attributes can be increased as needed and there are no limits on the number of new tables that can be associated with the primary key.
Data will be archived in the Intersite Archive File format (see Conley and Brunt 1991) along with complete documentation and meta-data. Entry will be made via data entry screens that will be developed using SAS AF to provide the context based quality assurance necessary to verify the accuracy of the data by providing host table feedback and various levels of range and field checking. Entry screens also will reflect the temporal sequence of data collection.
Authority Tables. There will be authority tables maintained both for host and parasite species and will contain, at minimum, the following fields: PHYLUM, CLASS, ORDER, FAMILY, GENUS, SPECIES, PARASITE-AUTHORITY, HOST-AUTHORITY, COLLECTION DATE, COLLECTION LOCALITY, OOCYST STRUCTURAL INFORMATION (for example, see p. 13), LIFE HISTORY INFORMATION, SPECIMEN DEPOSIT LOCALITIES & NUMBERS, COMMENT - free form text.
Locations will be stored as part of the supporting metadata for the dataset and will contain RMS error calculations associated with GPS location that are available for each host species or as can best be estimated from information provided in the original description (unfortunately, in many cases this will not be possible).

Hard copy database, the monograph. The Coccidia of the World will consist of Volume I, A Primer For Coccidiologists and Volume II, The Species Account. Volume I has 13 chapters, Literature Cited, Glossary, and Index. This volume is intended to introduce beginning parasitologists to the coccidia and to outline the techniques of specimen collection, species identification, the concept of "type" specimens and the methods and necessity of depositing specimens into accredited museums, the value and need for using symbiotypes of host species, the basic classification of the coccidia and a broad overview of the coccidia of invertebrates and all classes of vertebrates. Following the overview chapters of species accounts from the various host groups will be a final chapter that discusses the value, the need, and the necessity of this monograph. This chapter will contain sections/discussions of the broad ecological and evolutionary overview of the coccidia: From which ancestors did they evolve? Why are they all so biologically and structurally similar? Does strict host specificity really exist or is it an artifict of the way the data have been collected over historical time? Why did some species evolve heteroxenous while others have maintained homoxenous life histories? How can the genus Eimeria have so many species and is it really monophyletic? Do the coccidian oocysts of certain host groups (e.g., Artidactyla) have suites of characters (e.g., micropyle and micropylar cap) that are unique to that host group and can be predictive? Given the nature of the direct life cycle of most coccidians, will they be important pathogens of endangered species (sensu Dobson and Carper, 1992) if habitat diminishes due to global warming and species become crowded? These and other discussions that may arise from the production of Volume I should help both current and future workers in this field to begin with a more thorough understanding of the distribution of coccidians throughout the world in both space and time and give them some hint of the evolutionary relationships between these unique and ubiquitous parasites and their hosts. The outline and details of the chapters are presented on pp. 11-13 of this proposal.
Volume II will be the actual species account of the coccidia and will be organized by host Phylum, Class, Order, Family, Genus, Species. Within each Host Species, the coccidian parasites recorded from that host will be ordered chronologically, i.e, the history of our knowledge of the coccidia will be detailed in the order it occurred by Family, Genus, Species. The reason for this method (vs. alphabetical listing of species, for example) is so the taxonomic history of coccidians from each host species can be reconstructed over time to better understand when and how mistakes (e.g., possible synonyms) may have occurred within each group; this will aid us in correcting such mistakes and in deciding priorities as needed. For example, perhaps two species were described from the same host species, but from different geographic localities, one in November, 1950 and the second in January, 1951. The two parasites may be the same species, but because the host group either is obscure or has had very little work done with it since those descriptions, they both will remain as distinct species in the literature in perpetuity until their history is reconstructed and the names are synonymized.
In Volume II, each coccidian species will be allotted one page (two-sided) of space and each page will include, along with a line drawing and a photomicrograph (if available), the following: hosts (type-host and others), detailed description of the sporulated oocyst, information on the basic biology (anything known: biochemistry, genetics, excystation, sporulation, life history stages, pathology, phylogeny, biogeography, host specificity, ecological data, etc.), remarks about that species that separate it from all others, where specimens (both coccidian and its host) have been deposited and their accession numbers, type and other localities where the parasite has been recorded (vs. distribution maps which in the majority of cases would not be time/space effective), prevalence information (if known), and one to ten of the most pertinent references for this coccidian species. A sample page of a coccidian species account is given on p. 14, 15 of this proposal. Following each Host Family account will be identification keys to all coccidian species described from that Host Family and, if enough information is known about both the hosts and the coccidians, we will attempt to generate host-parasite phylogenies as has been done only once with the coccidia to date (Reduker et al., 1987), but that has recently shed some light on the coevolutionary relationships of other host-parasite associations. Following all the species accounts of Volume II will be sections that cross-references all coccidian species by host, locality (county/district, state, country) and author.

Timetable of proposed work.

Years 1 & 2. Combine the UNM and KSU personal reprint libraries. Get all articles scanned into the database and backed up on CD-ROM. Begin to organize literature (species) into groups by host and then parasite. Travel to Russia, Hungary and Japan, collect literature, get translations (as necessary), and scan, archive, and organize these literature (species) as they become available. Get graduate students started on their individual projects as soon as possible and get them through their required course work and course work needed to support/supplement their individual programs. Make sure the students stay on target, make adequate progress towards their degrees and during Year 2 that they begin to develop presentations (at least) for regional parasitology meetings. Begin initial series of publications in the primary literature, mostly new species descriptions, combinations, synonymies, host summaries. At the end of Year 2, begin to write the chapters in Volume I, Primer.
Years 3 & 4. Travel to Australia and Brazil to collect literature. Continue to do literature searches, photocopy, scan, archive, backup, interpret, organize literature, make species decisions, etc. As graduate students finish their M.S. and begin their Ph.D. programs, they should begin preparing dissertation improvement grant proposals during 3rd or 4th year of their program. Take students to Annual Meetings of the American Society of Parasitologists to present papers on their work to date. Continue to publish species descriptions, but as the literature becomes more complete on various host groups, more concentration will be devoted to major review articles, probably by host group. Begin to work with science editor(s) at Cambridge University Press, complete and submit Volume I by the end of Year 4 (see letter of interest in the monograph from Dr. Allen Crowden, Editorial Director: Science, Technology & Medicine, Cambridge University Press).
Year 5. Continue work with graduate students as above; those who started on Ph.D. in Year 1 should be nearing completion. Continue to collect/enter data as new information is produced. Begin final compilation of species descriptions with Cambridge University Press.
Write and submit final review articles as various host groups become completed. Travel to Annual Meeting of the ASP with graduate students. Write Final Report to NSF.

Volume 1, Primer for Coccidiologists

Chapter 1: Introduction

What are coccidia? Where are coccidia found in an animal and in nature? Why are coccidia important? When are coccidia important or are they? Why do we study coccidia? Why don't others study coccidia? Where is most of our knowledge on coccidia and: 1) Is the data real? 2) What host groups for coccidia is the data most real? Where/why is knowledge lacking? Goals of Vol. I: 1) Not to duplicate world literature; 2) Provide parasitologists with a primer to interest them in coccidia; 3) List coccidia from a host chronologically, not alphabetically, and give reasons for this decision.

Chapter 2: Collection of Specimens (Host and Parasite)

Why is feces so important and not tissues? Pros and cons of different chemical solutions for collecting and storing fecal material. Storage conditions (field and lab) over time. Processing in lab. Concentration techniques--separating feces and concentrating oocysts: a) salt, b) sugar, c) wet mounts, d) sedimentation, e) types of vials/bottles that are useful, f) preservation of host tissues in field (pros & cons) methods.

Chapter 3: Identification

Morphology of oocysts: size and shape; wall thickness, layers, striations, color, texture, knobs, etc.; residua; polar granule; micropyle; micropylar cap; sporocyst shape and size; sporocyst wall; Stieda body; substieda body; parastieda body; membranes, veils, sporopodia, sutures, sporocyst residua; sporozoite size both in situ and excysted; refractile bodies; position of nucleus; striations; arrangement. What to record and how to record it: outline or page showing how to record typical data; measurements and sketches; photomicrographs; line drawings; record as much as possible; take lots of pictures; measure oocysts from multiple hosts (i.e. Duszynski, 1971); minimum number of measurements, etc.

Chapter 4: Literature Review and Decision Making

Sources: Index Catalogue of Medical and Veterinary Zoology, Pellerdy (1965, 1969, 1974), Levine (1988a,b) and Levine and Ivens (1965, 1990a, b), Advances in Parasitology Vols. 1-present (1963-present), Biological Abstracts, Zoological Record, Individual papers and recent review articles on major groups, Protozoology Abstracts (Vol. 1-present, 1977-present). What to look for in sources: 1) historical errors, 2) species, genus, family specificity, 3) all coccidia from the same family, 4) pseudoparasites. Secure types, if available, if not distinctly different from what you see.
Base decisions on: 1) quantitative and qualitative features, 2) consider evidence in primary description, 3) remember that things may have been missed, 4) be careful to be complete, 5) don't clutter literature with needless new species and genera when not sure.

Chapter 5: Deposition and Storage of Specimens

Parasites: 1) history, 2) methods available: fixation, storage, etc., 3) national museums (Beltsville & Manter lab), but not private collections. Hosts: 1) Symbiotypes, voucher specimens, 2) accredited museums.

Chapter 6: Classification Scheme of the Eimeriorina/Eimeriidae

Mention other family reviews (i.e., Sarcocystidae, Cryptosporidiidae). Give classification characteristics of genera covered. Note type species for each genus. Cite approximate numbers of species in each genus and number of species in each major class or phylum group to be considered.

Chapter 7: Eimeriidae of Invertebrates

General statements (introduction) and general overview. List species and synonyms of genera, perhaps in a table. This probably will be a special chapter since much revision is needed and most species may be /are pseudoparasites.

Chapter 8: Eimeriidae of Fish

General overview. List whose/which host classification scheme to be used and why; talk about number of families, genera, and species with described species of coccidia. Go through genus-by-genus account of coccidians from these host groups. Are there any recent coccidian reviews on these host groups (e.g., Dykova & Lom, 1983; Davies & Ball, 1993)?. What about Calyptospora? Talk about undescribed species, synonyms, homonyms, nomen nuda, lapsus, etc.

Chapter 9: Eimeriidae of Amphibia

General overview. List whose/which host classification scheme to be used and why; talk about number of families, genera, and species with described species of coccidia. Go through genus-by-genus account of coccidians from these host groups. Are there any recent coccidian reviews on these host groups (e.g., Upton & McAllister, 1988; Upton et al., 1993)? Talk about undescribed species, synonyms, homonyms, nomen nuda, lapsus, etc.

Chapter 10: Eimeriidae of Reptilia

General overview. List whose/which host classification scheme to be used and why; talk about number of families, genera, and species with described species of coccidia. Go through genus-by-genus account of coccidians from these host groups. Are there any recent coccidian reviews on these host groups (e.g., Upton & McAllister, 1990)? Talk about undescribed species, synonyms, homonyms, nomen nuda, lapsus, etc.

Chapter 11: Eimeriidae of Aves

General overview. List whose/which host classification scheme to be used and why; talk about number of families, genera, and species with described species of coccidia. Go through genus-by-genus account of coccidians from these host groups. Are there any recent coccidian reviews on these host groups? Talk about undescribed species, synonyms, homonyms, nomen nuda, lapsus, etc.

Chapter 12: Eimeriidae of Mammalia

General overview. List whose/which host classification scheme to be used and why; talk about number of families, genera, and species with described species of coccidia. Go through genus-by-genus account of coccidians from these host groups. Are there any recent coccidian reviews on these host groups? Talk about undescribed species, synonyms, homonyms, nomen nuda, lapsus, etc.

Chapter 13: What Does It All Mean?

How does this monograph (Vols. I & II) add value to our knowledge of the coccidia? What can be learned from the compilation of all these data/species descriptions? Can we answer questions--or at least gain some insight--on the following topics that have teased, but eluded, coccidian biologists to date? Is there any sense to the evolution of form and function in the coccidia? Is host-specificity in this group fact or fiction? What are the advantages of two-host vs. one-host life cycles, or are there advantages? Can the genus Eimeria really be monophyletic? Why are there so many eimerian species? Is the biology of the coccidian biology relevant to such seemingly unrelated events as global warming or loss of biodiversity?

Chapter 14: Literature Cited for Volume 1

Chapter 15: Glossary of terms used (including synonyms)

Chapter 16 - Index





D. LITERATURE CITED

Aquino-Shuster, A.L., D.W. Duszynski & H.L. Snell. 1990. Three new coccidia (Apicomplexa) from the Hood Island lizard, Tropidurus delanonis, from the Galapagos archipelago. Journal of Parasitology 76:313-318.
Bandoni, S.M. & D.W. Duszynski. 1988. A plea for improved presentation of type material for coccidia. Invited Critical Comment. Journal of Parasitology 74:519-523.
Brooks, D.R. 1993. Extending the symbiotype concept to host voucher specimens. Invited Critical Comment. Journal of Parasitology 79:631-633.
Brunt, J.W. 1994. Research Data Management in Ecology: A Practical Approach for Long-term Projects. In, Proceedings of the Seventh International Working Conference on Scientific and Statistical Database Management. IEEE Computer Society Press. pp. 272-275.
Conley, W. & J.W. Brunt. 1991. An Institute for Theoretical Ecology. Part V: Practical data management for cross-site analysis and synthesis of ecological information. Coenosis 6:173-180.
Couch, L., D.W. Duszynski & E. Nevo. 1993. Coccidia (Apicomplexa), genetic diversity and environmental unpredictability of four chromosomal species of the subterranean superspecies Spalax ehrenbergi Nehring (mole-rat) in Israel. Journal of Parasitology 79:181-189.
Davies, A.J. & S.J. Ball. 1993. The biology of fish coccidia. In, Advances in parasitology, Vol. 32. Academic Press, Inc., NY. pp. 293-366.
Dobson, A. & R. Carper. 1992. Global warming and potential changes in host-parasite and disease-vector relationships (Ch. 16). In, Global warming and biological diversity (R.L. Peters, T.E. Lovejoy, eds.). Yale University Press, New Haven, CT. pp. 201-217.
Duszynski, D.W. 1971. Increase in size of Eimeria separata during patency. Journal of Parasitology 57:948-952.
______ & S.L. Gardner. 1991. Fixing coccidian oocysts is not an adequate solution to the problem of preserving protozoan type material. Journal of Parasitology 77:52-57.
Dykova, I. & J. Lom. 1983. Fish coccidia: An annotated list of described species. Folia Parasitologica (Praha) 30:193-208.
Eastham, G., D.W. Duszynski & T.L. Yates. 1982. Eimeria from jumping mice (Zapus spp.): A new species and genetic and geographic features of Z. hudsonius luteus. Journal of Parasitology 68:1146-1148.
Frey, J.K., T.L. Yates, D.W. Duszynski, W.L. Gannon & S.L. Gardner. 1992. Designation and curatorial management of type host specimens (Symbiotypes) for new parasite species. Invited Critical Comment. Journal of Parasitology 78:930-932.
Gardner, S.L. 1991. Phyletic coevolution between subterranean rodents of the genus Ctenomys (Rodentia: Hystricognathi) and nematodes of the genus Paraspidodera (Heterakoidea: Aspidoderidae) in the Neotropics: temporal and evolutionary implications. Zoological Journal of the Linnean Society 102:169-201.
______ & D.W. Duszynski. 1990. Polymorphism of eimerian oocysts can be a problem in naturally infected hosts: An example from subterranean rodents in Bolivia. Journal of Parasitology 76:805-811.
Hake, T.G. 1839. A treatise on varicose capillaries, as constituting the structure of carcinoma of the hepatic ducts, and developing the law and treatment of morbid growths. With an account of a new form of pus globule. London
Joyner, L.P. 1982. Host and site specificity. In, The biology of the coccidia, P.L. Long (ed.). University Park Press, Baltimore MD. pp. 35-62.
Lambert, C.R., S.L. Gardner & D.W. Duszynski. 1988. Coccidia (Apicomplexa: Eimeriidae) from the subterranean rodent Ctenomys opimus Wagner (Ctenomyidae) from Bolivia, South America. Journal of Parasitology 74:1018-1022.
Lapage, S.P., P.H.A. Sneath, E.F. Lessel, V.B.D. Skerman, H.P.R. Seeliger & W.A. Clark (eds.). 1975. International code of nomenclature of bacteria and statutes of the International Committee on Systematic Bacteriology and statutes of the Bacteriology Section of the International Association of Microbiological Societies. The American Society for Microbiology, Washington, D.C., 180 p.
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______. 1982. Taxonomy and life cycles of coccidia. In, The biology of the coccidia (Long, P.L., ed.). University Park Press, Baltimore MD. pp. 1-62.
______. 1988a. The protozoan phylum Apicomplexa, Volume I. CRC Press, Inc., Boca Raton, FL. 302 p.
______. 1988b. The protozoan phylum Apicomplexa, Volume II. CRC Press, Inc., Boca Raton, FL. 154 p.
______, J.O. Corliss, F.E.G. Cox, G. Deroux, J. Grain, B.M. Honigberg, G.F. Leedale, A.R. Loeblich III, J. Lom, D. Lynn, E.G. Merinfeld, F.C. Page, G. Poljansky, V. Sprague, J. Vavra & F.G. Wallace. 1980. A new revised classification of the Protozoa. Journal of Protozoology 27:37-58.
______ & V. Ivens. 1965. The coccidian parasites (Protozoa, Sporozoa) of rodents. Illinois Biological Monograph Number 33, University of Illinois Press, Urbana IL. 356 p.
______ & ______. 1990. The coccidian parasites of rodents. CRC Press, Inc., Boca Raton, FL. 228 p.
______ & ______. 1970. The coccidian parasites (Protozoa, Sporozoa) of ruminants. Illinois Biological Monograph Number 44, University of Illinois Press, Urbana IL. 278 p.
Marchiondo, A.A. & D.W. Duszynski. 1978. Permanent light microscopy slides of Eimeria nieschulzi oocysts. Journal of Parasitology 64:163-164.
______ & ______. 1988. On the status of Eimeria nieschulzi oocysts embedded in resin eleven years ago: A permanent method for preserving coccidian oocysts. Journal of Parasitology 74:737-739.
Mayr, E., E.G. Linsley & R.L. Usinger. 1953. Methods and principles of systematic zoology. McGraw-Hill Book Company, Inc., NY.
McDougald, L.R., L.P. Joyner & P.L. Long. 1986. Research in avian coccidiosis: Proceedings of the Georgia coccidiosis conference. University of Georgia, Athens, GA. 642 p.
Palma, R.E. 1995a. Historical biogeography of Thylamys. Journal of Mammalogy. In Review.
______. 1995b. Systematic analysis of Thylamys spp. using chromosomes, allozymes and mtDNA sequences. Biological Journal of the Linnean Society. In Review.
______ & T.L. Yates. 1995. The karyotypes of two South American mouse opossums of the genus Thylamys (Marsupialia: Didelphidae), from the Andes and eastern Paraguay. Proceedings of the Biological Society of Washington. Accepted.
Parker, B.B. & D.W. Duszynski. 1986. Polymorphism of eimerian oocysts: A dilemma posed by working with some naturally-infected hosts. Journal of Parasitology 72:602-604.
Patrick, M.J., A. Johnson & D.W. Duszynski. 1995. Parasites of mammals on the Sevilleta National Wildlife Refuge, Socorro, New Mexico: Markovian chain analysis of parasite prevalence in the kangaroo rat, Dipodomys merriami. Journal of Ecology. Submitted.
Pellerdy, L.P. 1965. Coccidia and coccidiosis (1st ed.). Akademiai Kiado, Hungarian Academy of Sciences, Budapest. 657 p.
______. 1969. Catalogue of Eimeriidae (Protozoa, Sporozoa). Supplement I. Akademiai Kiado, Hungarian Academy of Sciences, Budapest. 80 p.
______. 1974. Coccidia and coccidiosis (2nd ed.). Verlag Paul Parey, Berlin. 959 p.
Reduker, D.W, D.W. Duszynski & T.L. Yates. 1987. Evolutionary relationships among Eimeria spp. (Apicomplexa) infecting Cricetid rodents. Canadian Journal of Zoology 65:722-735.
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Upton, S.J. & C.T. McAllister. 1988. The coccidia (Apicomplexa: Eimeriidae) of Anura, with descriptions of four new species. Canadian Journal of Zoology 66:1822-1830.
______, C.S. Crawford & R.L. Hoffman. 1983. A new species of thelastomatid (Nematoda: Thelastomatidae) from the desert millipede, Orthoporus ornatus (Diplopoda: Spirostreptidae). Proceedings of the Helminthological Society of Washington 50:69-82.
______, C.T. McAllister & S.E. Trauth. 1993. The coccidia (Apicomplexa: Eimeriidae) of Caudata (Amphibia), with descriptions of two new species from North America. Canadian Journal of Zoology 71:2410-2418.
______, M.A. Stamper & B.R. Whitaker. 1995. Isospora bellicosa sp. n. (Apicomplexa) from a Peruvian red-breasted meadowlark, Sturnella bellicosa (Passeriformes: Icteridae). Archiv fur Protistenkunde 145:132-134.
Voss, E.G., H.M. Burdet, W.G. Chaloner, V. Demoulin, P. Hiepko, J. McNeill, R.D. Meikle, D.H. Nicholson, R.C. Rollins, P.C. Silva & N. Greuter. 1983. International code of botanical nomenclature. Bohn, Scheltema & Holkema, Utrecht/Antwerpen, Dr. W. Junk, Publishers, The Hague/Boston.
Wash, C.D., D.W. Duszynski & T.L. Yates. 1985. Eimerians from different karyotypes of the Japanese wood mouse (Apodemus spp.) with descriptions of two new species and a redescription of Eimeria montgomeryae Lewis and Ball, 1983. Journal of Parasitology 71:808-814.
______, ______ & ______. 1990. Enzyme variation of Eimeria arizonensis from Peromyscus truei and E. boylii. Journal of Protozoology 37:536-540.
Wenyon, C.M. 1926. Protozoology. Bailliere, Tyndall and Cox. London. pp. 803-869.
Wilber, P.G., B. Hanlet, B. Van Horne & D.W. Duszynski. 1994. Two new species and temporal changes in the prevalence of Eimerians in a free-living population of Townsend's ground squirrel (Spermophilus townsendii) in Idaho. Journal of Parasitology 80:251-259.