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~ l . Definition anc! Description of Experimental Amphibians , . . A. INTRODUCTION Standardization of amphibians for experimental use demands that a class) fication be established to permit investigators to select animals most ap- propriate to their needs, communicate effectively with suppliers, and accurately report their data. The following standard categories are recom- mended: 1. Wild 2. Wild Caught a. Wild-Caught Nonconditioned (1) Wild-caught nonconditioned nontreated (2) Wild-caught nonconditioned treated (3) Wild-caught nonconditioned miscellaneous b. Wild~aught Conditioned ( 1) Wild-caught conditioned larvae (2) Wild-caught conditioned juveniles or adults (3) Wild-caught conditioned miscellaneous 3. Laboratory Reared a. Laboratory-reared standard b. Laboratory-reared miscellaneous 4. Laboratory Bred a. Laboratory-bred standard b. Laboratory-bred miscellaneous Among laboratory-reared and laboratory-bred animals the following types of populations and lines may be designated: 27

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28 B. DEFINITION 1. Wild (1) Random matinglines (2) Heterozygous isogenic clones (3) Heterozygous marked lines (4) Mutant lines (5) Inbred lines (6) Gynogenetic diploid lines (7) Homozygouslines (8) Haploid animals (9) Polyploid animals These are pre- and postmetamorphic amphibians in nature on which exper,- ments are conducted in nature, e.g., experiments on such questions as mi- gration, population characteristics, and physiological ecology. The investi- gator should record the location, time, temperature, and other relevant observations concerning the collection. 2. Wild Caught See Chapter V, Section C.2 for a special category of this classification. a. Nonconditioned (1) Non trea ted (a) General Description Wild caught nonconditioned non- treated pre- or postmetamorphic amphibians refers to those collected in nature and shipped to the user with no handling or treatment other than that involved in catching, shipping to distribution points, holding between capture and sale and sorting, etc. (Gibbs et al., 1971~; these animals re- ceive no disease treatment or maintenance under regularly standardized procedures. The characteristics of these animals will vary with the region from which they were captured and with the season of the year. These animals may have been maintained under a variety of environmental con- ditions; are usually provided no food; and, although normally shipped to buyers within a week, are often held in bulk pens for much longer periods. It is mandatory that dealers who wish recognition for meeting "standards" with respect to wild-caught amphibians specify the following: species to the lowest recognized taxonomic level. 2. geographic origin of the parent known to the closest possible geo- graphic unit,

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29 3. date and method (see Chapter III, Section C) of reproduction known and the date of metamorphosis recorded to within 1 month, 4. method and period of holding, and 5. the environmental conditions to which the animals were exposed from fertilization to shipment to the user and, if possible, the env~ron- mental conditions during shipment. A subdivision of this classification is "northern frogs" or R. pipiens capable of being ovulated during the winter months. Generally, they are collected north of the line separating ice-free from ice-covered ponds and streams. They may also occur at altitudes above the ice line. In contrast, "southern frogs" are R. pipiens that cannot be ovulated in this season and are collected south of the ice line. Dealers must be most cautious con cerning admixture of these animals; their reproductive cycle is not the only physiological difference between them. Shipments of amphibians may contain variants. Thus, while northern collections of R. pipiens, with the exception of areas in Minnesota and part of Wisconsin, are reasonably uniform, some collections of R. p. pipiens may contain the Kandiyohi or Burnsi mutants. However, neither of these mutants constitutes more than 5 percent of the population, even in areas where they are most abundant. Other variants may also occur, for example, admixture of frogs belong- ing to different segments of the R. pipiens complex (Brown, 1973), or from populations with lower or higher incidence of the Lucke renal adenocarcinoma. Although R. catesbeiana, R. clamitans, R. pipiens, R. palustris, and R. sylvatica occur in the same areas where northern col- lections are obtained, dealers seldom include these species in shipments of R. pipiens. Caution should be exercised, however, since rapid sorting of animals occasionally results in admixtures of R. catesbeiana and R. clamitans and of R. pipiens, R. palustris, and juvenile R. clamitans. Southern collections include frogs from the southern states of the United States and from Mexico. Such collections may contain mixtures of R. p. pipiens and of R. p. sphenocephala if made in the central states and of R. p. berlandieri if from the southern states through Mexico and Central America. The systematics of these species, however, has not yet been fully resolved. Among the other species, the nature of variations within collections has not been well defined. Physiological variants must be expected where the species range extends longitudinally. Among other physiological variants sexually "differentiated" and "undifferentiated" populations of R. catesbeiana have been identified (Witschi, 1930), but the geographic co- ordinates of these populations have not been defined, which exemplifies the need for specification of geographic origin.

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30 These wild~caught nonconditioned nontreated anunals must fulfill other criteria before they can be placed under a higher classification. (b) Embryos or Larvae Embryos or larvae are, of course, premetamorphic stages. There is no opportunity to treat or condition wild-caught embryos, commonly known as egg clutches, or young larvae prior to shipment. Thus, they are classified under this category. Larvae held for longer periods may qualify for classification under another category. (2) Treated Wild~aught nonconditioned treated are animals that meet all of the criteria for the wild-caught nonconditioned nontreated classification and usually are provided some food prior to use or shipment to a buyer. A unique character of this classification is that attempts have been made to treat the anunals for disease or parasites, although it must be recognized that standard treatments remain undefined. It is important that the investigator know the treatments to which the animals have been exposed. Normally, these animals are shipped within a few days after being received by the supplier but may, in fact, be held for much longer periods. Dealers, in addition to meeting the pee specifications listed above, must also specify 6. treatments to which the animals have been exposed. (3) Miscellaneous This category includes treated or nontreated em- bryos, larvae or adults for which two or more of the specifications listed under nontreated are not fulfilled. b. Conditioned Wild-caught conditioned amphibians not only fulfill the six criteria estab- lished for nonconditioned animals [see Section III.B.2.a(1),(2~] but also meet the specification that records are available, indicating: 7. their treatment, if any, and length of exposure to each laboratory environment, 8. pathological and general physiological state (e.g., whether or not in hibernation, known diseases, feeding behavior, activity), and 9. approximate age (if known). (1) Larvae Wild~aught conditioned larvae are wild-caught am- phibian larvae or larvae from eggs collected in the wild. They should be

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31 maintained under standard laboratory conditions for a period sufficient to demonstrate that their mortality rate is not appreciably greater than is normal for laboratory-reared representatives of the species or mutant in question. If the larvae are maintained through metamorphosis, they should fulfill the nine requirements for conditioned juveniles and adults (2) Juveniles or Adults Wild~aught conditioned juveniles or adults are wild-caught amphibians maintained under standard laboratory condi- tions for a sufficient time to demonstrate for the species in question the absence of symptoms of disease or physiological disorders. For example, R. catesbeiana adults should be held for a minimum of 6 weeks, during the last 2 weeks no symptoms of disease or physiological disorder should be in evidence. Newly metamorphosed R. catesbeiana should be maintained under stand card conditions for a minimum of 10 weeks. This assures that they have survived the 2-month period of high Mediate postmetamorphic mortality when disease symptoms are not easily recognized and death is too rapid for medication to be administered. (3) Miscellaneous Wild-caught amphibians maintained under the conditions needed to assure freedom from symptoms of disease or physio- logical disorder, but lacking in the other requirements for conditioned amphibians, are classified as wild-caught conditioned miscellaneous. 3. Laboratory Reared a. Standard Laboratory-reared standard are amphibians reproduced in the laboratory with at least one wild~caught parent or have been fed living food items col- lected from nature or living food items exposed to intermediate parasite hosts isee Chapter V, Section C.2 and Chapter VI, Section B.1 .b(2~] . The offspring must fulfill the nine criteria required of wild-caught conditioned amphibians. The parents may be collected at any stage of development. This classification recognizes the possibility of disease or parasite trans- mission from a parent or from food exposed to intermediate hosts. b. Miscellaneous Laboratory-reared miscellaneous amphibians meet the requirements for laboratory-reared standard with at least one field-collected parent but lack such pertinent information for proper classification as geographical origin

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32 of parents, laboratory conditions, disease treatment, and age (see Chapter III, Section B.2~. 4. Laboratory Bred a. Standard Laboratory-bred standard amphibians are those produced by reproductive events that did not occur in nature and whose parents were not field col- lected; they are fed processed food items or living food items bred under laboratory conditions isolated from the amphibian population. Thus, laboratory-bred amphibians must fulfill all the criteria required of labora- tory-reared amphibians and must be at least of the F2 generation. Laboratory-bred standard amphibians are free of parasites and sym- bionts that require intermediate hosts; they may, however, possess those parasites and symbionts that are regularly transmitted vertically and without intermediate hosts. b. Miscellaneous Laboratory-bred miscellaneous amphibians are those fulfilling all labora- tory-bred requirements except for maintenance on a diet of food isolated from the natural environment. For example, in some areas, R. catesbeiana is maintained in the laboratory predominantly on a diet of living fish from outdoor ponds [see Chapter VI, Section B.1 .b(2~] . Suppliers of laboratory- bred R. catesbeiana are reminded that this classification requires documen- tation of the types of food being used. C. DESCRIPTION OF LABORATORY-REARED AND LABORATORY-BRED AMPHIBIANS 1. Types of Populations and Lines Amphibians are unique in that no other laboratory animals are capable of reproduction by as many significantly different procedures. They can be reproduced by natural biparental mating, natural parthenogenesis, artifi- cial insemination, various types of artificial parthenogenesis, or by nuclear transplantation. Since each procedure has significantly different genetic consequences, the method of reproduction must be specified when defin- ing laboratory-reared or laboratory-bred amphibians (Asher, 1970, in press a,b; Nace et al., 1970; Adler and Nace, 1971~. The following is an out- line of the various lines that may result from the application of these sev

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33 oral methods of reproduction. It is simplified in that terms are not sug- gested for many of the types of genealogies that could result from combinations of the several methods of reproduction. a. Random Mating Lines Random mating lines refer to genealogies resulting from the bisexual mat- ing of random animals within a population. Reproduction may be by natural mating in season, by hormonally induced mating, or by artificial insemination in or out of season. As a first approximation the progeny of such matings will be as genetically heterogeneous as the population from which the parents were chosen. b. Heterozygous Isogenic Clones Groups of animals produced from wild-caught animals or random mating lines by the technique of nuclear transplantation (Chapter VII, Section A.9) comprise Heterozygous isogenic clones if the nuclei used in the trans- plantations are all from a single individual. Each member of such a clone is genetically identical to its clone mates. Because they are isogenic, each will accept grafts from the others (Volpe and McKinnell, 1966~. However, since the nuclear transfer frogs are heterozygous, biparental progeny of members of a clone are as genetically heterogeneous as the progeny of matings within any set of identical siblings. Clones may be produced by the nuclear transplantation technique from animals in any of the populations or lines described below. In such cases, clone members with the genetic properties of the parental line are pro- duced. These may be more or less Heterozygous depending on the state of the parental line. c. Heterozygous Marked Lines Increasing numbers of mutations in amphibians are being described. Many produce characteristics that are significant to investigators (Briggs, in press; Malacinski and Brothers, in press). Some of the phenotypes are apparent from external examination; others are biochemical or developmental mu- tants that can only be detected by special techniques. It is advantageous to link biochemical and developmental mutants to the externally visible mutants to allow ready laboratory manipulation. Consequently, in certain lines, pigmentation or pattern mutations are being selected without regard to the status of the remaining genome. Such lines are characterized as containing Heterozygous marked animals into which less evident mutations can subsequently be introduced.

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34 d. Mutant Lines Mutant lines may include heterozygous marked lines or lines of animals se- lected for any specific mutations with the remainder of the genome spe- cif~ed to varying degrees. e. Inbred Lines Inbred lines may arise from any of the previously defined lines and are characterized by varying degrees of genetic homogeneity. They may be pro- duced either by a sequence of selected biparental matings or by any of the various techniques of parthenogenesis. Sequential biparental matings of amphibians yield inbred lines com- parable to those common to inbreeding within over kinds of organism. Brother-sister or parent-child matings are utilized to minimize the genetic diversity among the progeny. A sufficient number of generations of such breedings can result in specified degrees of genetic homozygosity with the limitations imposed by low viability, genetic drift, and gene fixation that are well known from inbreeding within other organisms. f. Gynogenetic Diploid Lines Gynogenetic diploid lines are special inbred lines produced by a modifica- tion of parthenogenesis, which, as generally applied, is genetically equiva- lent to fertilizing the egg with its own second polar body (see Chapter VII, Sections A.5 and A.6~. This technique does not immediately produce ani- mals homozygous at all gene loci. The first generation is homozygous for those loci located close to the kinetochore, but crossing-over results in in- creasing heterozygosity as the gene-kinetochore distance increases. Never- theless, three generations of gynogenetic reproduction of R. pipiens is genetically equivalent to 22 generations of biparental inbreeding in mice (Nace et al., 1970; Asher and Nace, 1971~. The limitations of low viability, genetic drift, and gene fixation apply. g. Homozygous Lines Homozygous lines refer to those lines in which most or all gene loci are in the homozygous state. "Standard" homozygous lines refers to the former; "absolute" homozygous lines refers to the latter. Lines produced by three or more generations of diploid gynogenesis may be considered standard homozygous lines win the limitations noted above. Absolute homozygous lines of several types may be produced, e.g., gynogenetic, androgenetic, or nuclear transplant recipients. In gynogenetic

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35 lines, the genome is totally Mat of the female progenitor; in androgenetic lines, it is totally that of the male. The process of producing gynogenetic diploids is initiated but without the step that results in the retention of the second polar body. Such animals would be haploid if left untreated. At the time of first cleavage, such eggs are exposed to hydrostatic pres- sure of 5,000 psi (A 3.5 X 107 Pa). This suppresses cytoplasmic division but allows nuclear division and the reconstitution of the diploid state from the original haploid set of maternal chromosomes. In the case of androgenetic homozygous animals, the female pronucleus of artificially inseminated eggs is removed before union win the male pro- nucleus. The diploid state is reconstituted in the progeny from the haploid paternal set of chromosomes by exposure to high pressure at the time of first cleavage, as in the case of the gynogenetic animals. Animals in abso- lute homozygous lines produced from animals other than those in standard homozygous lines show extremely low viability. Those from standard homozygous lines show higher viability because of the selection involved in the production of the parental lines. Homozygous diploids may be produced by transplanting haploid nuclei to enucleated eggs (see Chapter V'II, Section Am. A delay in cytokinesis occurs spontaneously in only a few eggs receiving transplanted nuclei. The delay in cytokinesis can also be produced by pressure treatment. The hap- loid nucleus undergoes mitosis, and the daughter nuclei fuse to produce a homozygous diploid. Normal cytokinesis follows after the delay of one cleavage interval. Nuclear transfer homozygous diploids, as absolute ho- mozygous lines, have low viability (Subtelny, 1958~. h. Haploid Animals Death as a result of the haploid syndrome, which usually expresses itself at early morphogenesis (tailbud stage), prevents the development of haploid lines (Porter, 1939~. Haploid individuals, however, may be produced by the same technique used for the production of gynogenetic or androgenetic dip- loid animals, except that the step resulting in the retention of the second polar body or suppression of the first cleavage is omitted. In spite of their poor viability, such haploid animals are potentially useful as sources of hap- loid lines of cells for tissue culture (Freed and Mezger-Freed, 1970) or other experimental procedures. i. Polyploid Animals Polyploid animals and polyploid lines of almost any specified type may be produced by the combination of several techniques (Kawamura and Nishioka, 1963, 1967, 1972, 1973~. Thus, normal biparental mating fol

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36 lowed by retention of the second polar body, as in the production of gyno- genetic diploids, results in triploid animals with two chromosomal sets of maternal origin (Dasgupta, 1962~. The production of diploid animals either by gynogenetic techniques or by normal biparental mating followed by the suppression of the first cleavage division results in Me production of-tetra- ploids. In Me second case, two sets of chromosomes are of maternal origin and two are of paternal origin. By using this technique with diploid, trip- loid, or tetraploid parents, many other varieties of ploidy are possible (Fankhauser, 1945~. Frogs of several ploidy classes are routinely observed in nuclear transplantation experiments (hlcKinnell, 1964) (see Chapter VII, Section A.9~. 2. Sex Determination and Its Manipulation The sex determination of wild or wild-caught amphibians does not con- stitute a problem because it usually follows expectations. However, among laboratory-reared or laboratory-bred amphibians unusual sex ratios may be observed. Because sex determination may be a critical variable to the investigator, sex determination must be considered here. Sex determination in amphibians follows either the XX-XY or ZW- ZZ form of genetic control (see Chapter II, Section B). The former is typical of Ranidae; the latter is typical of the urodeles. Note that Xenopus differs from the Ranidae as it is of the ZW-ZZ type (see Chapter II, Sec- tion B.2.a). Thus it would be expected that in the case of Ranidae, diploid gynogenesis, in which the genome is totally of maternal origin, should re- sult in the production of 100 percent females. In actual practice, however, it has been found (Richards and Nace, unpublished) that males may be pro- duced in unexpectedly high frequencies, both in certain gynogenetic re- productions (3.6 9: 1 ~ among 1,234 progeny of 106 females in 4 years) and in the biparental matings of frogs from different geographical areas (1 9: 17.4 ~ from northern females X Mexican amelanoid males). As a result, it is the practice in some laboratories to administer 50 ,ug/liter of ,B-estradiol or testosterone to larval stages to control the numbers of males and females needed for breeding stock. Thus, animals may be pheno- typically female but genetically male or vice versa. Untreated biparental progeny of such sex-reversed animals develop in accordance with the sex specified by their genetic composition. The above facts, plus the normal lability of sex determination in certain species of amphibians, should warn the user to exercise caution in inter- pretations of experimental results that may vary as a consequence of a disparity between the genetic and phenotypic sex of the animals in the experimental groups.

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37 3. Species of Laboratory-Reared or Laboratory-Bred Amphibians Available in the United States The number of species of amphibians that are available as laboratory-reared or laboratory-bred animals remains limited. Most extensively bred is the A. mexicanum, the axolotl. Laboratory-bred X. Iaevis are now available in a number of laboratories. R. pipiens and B. orientalis have been bred and are becoming available. a. Anurans (1) R. pipiens Random mating and heterozygous marked lines are available. Other lines are in production or may be started by the investigator using the random mating or marked lines. The heterozygous marked lines are being developed using the best known mutants of R. pipiens [e.g., burns) (Moore, 1942), kandiyohi (Volpe, 1955), and amelanoid (albino) (Smith-Gill et al., 1972~] . The melanoid mutant (Richards et al., 1969) is still under analysis but may soon yield a marked line. A few biochemi- cal and other mutants that require special testing are also being produced as mutant lines. Inbred lines should be available within several years. At the time of this writing, arrangements can be made with the Amphibian Facility at the University of Michigan to obtain some animals from these lines. (2) R. catesbeiana 6'Differentiated race"-sex stable, "undifferen- tiated race"-reversal from female to male is common after metamorphosis (Witschi, 1930; Hsu and Liang, 1970~. Laboratory-reared and laboratory- bred animals are now being produced by Dudley D. Culley (School of Forestry and Wildlife Management, Louisiana State University, Baton Rouge, Louisiana) and other suppliers are initiating production efforts (Nace etal., 1971~. (3) B. orientalis Random mating lines of this species are now avail- able as laboratory-bred animals from the Amphibian Facility at the Uni- versity of Michigan or the Laboratory for Amphibian Biology at the Uni- versity of Hiroshima. (4) X. Iaevis Animals of this species can be obtained from a variety of sources. None routinely provide laboratory-bred animals, although these are available on occasion from the Amphibian Facility at the University of Michigan and from other private investigators in the United States. The largest colony is in the laboratory of M. Fischberg of the University of

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38 Geneva, who maintains random mating and mutant lines of X. Iaevis and a number of other Yenopus species. A. mexicanum The Mexican axolotl is available from a number of laboratories. However, the Indiana University Axolotl Colony of R. R. b. Urodeles A. mexicanum The Mexican axolotl is available from a number of laboratories. However, the Indiana University Axolotl Colony of R. R. Humphrey possesses these in largest number and has developed the largest inventory of mutants (Briggs, in press; Malacinski and Brothers, in press). Heterozygous marked lines, mutant lines, and inbred lines have been developed. No other species of urodeles are normally available as laboratory-bred animals.