III Definition and
Description of
Experimental Amphibians
A. INTRODUCTION
Standardization of amphibians for experimental use demands that
a classification be established to permit investigators to select
animals most appropriate to their needs, communicate effectively
with suppliers, and accurately report their data. The following
standard categories are recommended:
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-Caught 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:
(1) Random mating lines
(2) Heterozygous isogenic clones
(3) Heterozygous marked lines
(4) Mutant lines
(5) Inbred lines
(6) Gynogenetic diploid lines
(7) Homozygous lines
(8) Haploid animals
(9) Polyploid animals
B. DEFINITION
1. Wild
These are pre- and postmetamorphic amphibians in nature on
which experiments are conducted in nature, e.g., experiments
on such questions as migration, population characteristics, and
physiological ecology. The investigator 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) Nontreated
(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 receive 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 conditions; 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:
1. species to the lowest recognized taxonomic level,
2. geographic origin of the parent known to the closest possible geographic unit,
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 environ-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 concerning 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 Bumsi mutants. However, neither of these mutants constitutes more than S percent of the population, even in areas where they are most abundant.
Other variants may also occur, for example, admixture of frogs belonging 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. ciamitans, R. pipiens, R. palustris, and R. sylvatica occur in the same areas where northern collections 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. catesbetana and R. ciamitans and of R. pipiens, R. palustris, and juvenile R. ciamitans.
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. catesbeinna have been identified (Witschi,
1930), but the geographic coordinates of these populations have
not been defined, which exemplifies the need for specification
of geographic origin.
These wild-caught nonconditioned nontreated animals 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-caught 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 animals 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 five specifications listed above, must also specify
6. treatments to which the animals have been exposed.
(3) Miscellaneous This category includes treated or nontreated embryos, 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 established for nonconditioned animals [see Section III.B.2.a(l ),(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-caught conditioned larvae are wild-caught amphibian larvae or larvae from eggs collected in the wild. They should be
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-caught conditioned juveniles or adults are wild-caught amphibians maintained under standard laboratory conditions 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 standard conditions for a minimum of 10 weeks. This assures that they have survived the 2-month period of high immediate 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 physiological 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 collected from nature or living food items exposed to intermediate parasite hosts [see Chapter V, Section C.2 and Chapter VI, Section B. i .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 transmission 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
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 collected; 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 laboratory-reared amphibians and must be at least of the F2 generation.
Laboratory-bred standard amphibians are free of parasites
and symbionts 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 laboratory-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. catesbetana are reminded that this classification requires
documentation 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,
artificial 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 defining laboratory-reared or laboratory-bred
amphibians (Asher, 1970, in press a,b; Nace et al., 1970;
Asher and Nace, 1971). The following is an outline of the various
lines that may result from the application of these sev-
eral methods of reproduction. It is simplified in that terms are
not suggested 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
mating 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 transplantations 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 produced. 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 mutants 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.
d. Mutant Lines
Mutant lines may include heterozygous marked lines or lines of
animals selected for any specific mutations with the remainder
of the genome specified 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 produced 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
comparable to those common to inbreeding within other 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 modification of parthenogenesis, which, as generally applied,
is genetically equivalent 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 animals 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. Nevertheless,
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 with the limitations
noted above. Absolute homozygous lines of several types
may be produced, e.g., gynogenetic, androgenetic, or nuclear transplant
recipients. In gynogenetic
lines, the genome is totally that 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 frrst cleavage, such eggs are exposed to hydrostatic pressure of 5,000 psi (~ 3.5 X 10 7 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 with 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 absolute 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 VII, Section A.9). 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 hapbid 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 homozygous 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 dipbid 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 haploid
lines of cells for tissue culture (Freed and Meager-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-
lowed by retention of the second polar body, as in the production
of gynogenetic 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 the production of tetraploids. In the second
case, two sets of chromosomes are of maternal origin and two are
of paternal origin. By using this technique with diploid, tripbid,
or tetraploid parents, many other varieties of ploidy are possible
(Fankilauser, 1945). Frogs of several ploidy classes are routinely
observed in nuclear transplantation experiments McKinnell, 1964)
(see Chapter VII, Section A.9).
2. Sex Determination and Its Manipulation
The sex determination of wild or wild-caught amphibians does not constitute 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 ZWZZ 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, Section 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 result in the production of 100 percent females. In actual practice, however, it has been found (Richards and Nace, unpublished) that males may be produced in unexpectedly high frequencies, both in certain gynogenetic reproductions (3.6 9:1 d 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 arnelanoid males). As a result, it is the practice in some laboratories to administer 50 g(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 phenotypically 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 interpretations 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.
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. laevis
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., burnsi (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 biochemical 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. catesbejana "Differentiated race"-sex stable, "undifferentiated 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 et al., 1971,).
(3) B. orientalls Random mating lines of this species are now avail-able as laboratory-bred animals from the Amphibian Facility at the University of Michigan or the Laboratory for Amphibian Biology at the University of Hiroshima.
(4) X. laevis 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
Geneva, who maintains random mating and mutant lines of X. laevis
and a number of other Xenopus species.
A. mexicanum The Mexican axoloti is available from s number of laboratories. However, the lndiana University Axolotl Colony of R. R.
b. Urodeles
A. mexicanum The Mexican axoloti 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.
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