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12. ZOOLOGY

1

A long-time interest in collecting, identifying and studying the geographic range of mollusks led a Maryland boy, Frank Wayne Grimm of Catonsville, first to sharing his questions and specimens with the curators of the Smithsonian Institu­tion in Washington, D.C., and then to writing a thoroughgo­ing report on six snails he discovered in the Maryland Piedmont, far from their normal habitat in the mountains of Pennsylvania. He submitted the paper as part of his entry in the Eighteenth Science Talent Search and was not only judged one of the top winners but was awarded the $6000 scholarship because of his outstanding promise as a research scientist. Pictures and maps illustrated the paper, "Unusual Land Snails in Maryland's Susquehanna Valley," a portion of which is quoted here.

"Distribution  Records

On March 2, 1958, I collected a small series of snails in the valley of the Susquehanna. In this series were three species which I had found previously in the mountains of Maryland but which I had never seen in the Piedmont. Following this discovery, I became determined to explore the entire length of the valley below the Mason-Dixon line in search of additional records. This search proved quite fruitful, for it revealed the presence of three additional mountain species in the vicinity of the river.

The following compilation includes all of the species in Maryland's Susquehanna Valley which were collected in a period of roughly eight months. In addition to the specific records for the Susquehanna area, I include the over-all range of each species in the United States, plus its occurrence elsewhere in Maryland. I obtained these statewide records over a period of approximately four years, in which I con­centrated heavily upon the Piedmont and Appalachian prov­inces.

Conclusions  and  Questions  Raised

A.        A Definite Conclusion Which Raises Several Questions
The preceding data indicate the presence of six snails not heretofore recorded from Maryland's Piedmont: Triodopsis tridentata, T. notata, Ventridens intertextus, Mesomphix cupreus, Discus patulus and Cionella morseana. Comparison of the ranges of these snails shows both that they are found in the Appalachian Mountains, generally, and that (with the exception of Cionella morseana) they range westward through the Ohio-Mississippi drainage region in varying climatic zones. Obviously, they have a wide climatic toleration.

From the data I conclude that the snails must have floated downstream from the Pennsylvania mountains and established themselves in the hills above the river. There they occupy a climatic zone quite warmer than that of the mountains.

After extensive and thorough collecting, I have found none of these species elsewhere east of the Appalachians in Maryland. Since they are distributed widely in the moun­tains and westward on the Mississippi slope, why are they not found outside the river valley in the Piedmont? Are they blocked by an ecologic barrier which prevents automotive dispersal, or is the Piedmont outside their present peripheries of automotive dispersal?

B.         A Tentative Conclusion Which Presents a Challenge

Consideration of the data presented leads me to conclude tentatively that the Piedmont is merely outside their present peripheries of automotive dispersal. This means that the Susquehanna acts as a highway down which the snails can travel rapidly, escaping the time limits set by automotive dispersal. In other words, by means of automotive dispersal the snails may take thousands of years to spread a few miles, whereas by means of adventitious dispersal they may be distributed very quickly. On one hand automotive dispersal is sure to be successful if it does not meet with a barrier. On the other hand, adventitious dispersal, although rapid, does not insure survival. Therefore, the presence of the six snails in Maryland's Piedmont is purely a result of luck. They are there because a chance flood or series of floods have deposited them nearby. Chance determined that they would land safely, and chance determined that they would land in a place which could support them.

In summary, two important facts support the above state­ments. First, to my knowledge there are no ecologic or geo­graphic barriers east of the mountains which would prevent the snails from colonizing the Piedmont. All of these snails are found on the Appalachian Plateau as well as in the true mountains. All are found in the lower southern Appalachians, which have a climate almost identical to that of the northern Piedmont. Second, already they have begun to colonize the Piedmont by means of adventitious dispersal.

However, in order to prove my theory conclusively I must find the existing periphery of automotive dispersal for each species. Are any of these snails found elsewhere in the Piedmont, removed from the direct influence of a river or stream?

In the future I hope to answer this question. Meanwhile, I should like to continue exploring the Susquehanna Valley and to expand my field of observation to include the valleys of other major rivers. In this way I may gain a more com­plete knowledge of the situation elsewhere."

2

William Albert Dunson, of Atlanta, Georgia, was a finalist in the Ninth National Science Fair. His project was called "Radioiodine in Guppies."

"The purpose of this project was to determine iodine distribution in guppies by means of tracer techniques, using radioactive iodine131. Through the use of autoradiographs, it was found that the thyroid and the gut of guppies (Lebistes reticulatus) definitely concentrated radioiodine. The concen­tration areas apparently were not affected by the sex of the guppies. The eye pigment and the teeth of the guppies did not concentrate radioiodine, as others have noted in tadpoles. Some guppies were treated prior to their exposure to radio­iodine with thiouracil, which reduces the action of the thyroid. Within the time limits of this study, the concentra­tion of radioiodine by the thiouracil-treated guppies was not appreciably different from the normal fish.

This investigation was carried out at the Emory University biology radiolaboratory, under the supervision of an asso­ciate professor of zoology. About 53 hours were spent in the laboratory, from July through December, 1957. A total of 69 guppies were taken from an indoor pool in the Emory greenhouse and acclimated in small bowls. Thiouracil (.05%) was placed in the bowls with 23 of these guppies for 1 week. Then 9 normal guppies and 4 thiouracil-treated guppies were exposed to 1 microcurie per milliliter of radioiodine131 in their bowls for 24 hours. I fixed and embedded 7 of these guppies (4 normal, 3 thiouracil-treated) in paraffin. The guppies were microtomed into 20-micron sections, and 537 selected sections were mounted on 22 slides. The slides were measured for radioactivity with a Beta counter, and then clamped on photographic plates. After 7 days the autoradio­graphs were developed. The 22 slides were stained, fitted with cover glasses and dried. Photographs were made of the slides of guppy sections with their corresponding autoradiographs. The sections were examined under the microscope, and compared with their autoradiographs. Tables were compiled to show the distribution of radioiodine131 in these guppies. This experiment demonstrated that radioiodine131 is a safe and useful tracer isotope. Autoradiographs are effective in measuring uptake and distribution of radioisotopes. For valid conclusions, zoology research must test large numbers of animals under controlled conditions."

3

"Reproduction and Concentration of Mutations in Guppies" was the project of Ninth National Science Fair finalist Carol McColm, of Farmington, New Mexico.

"Introduction

About fifty-five years have passed since the small vivipar­ous tropical fish, Lebistes reticulatus, was first employed for genetic studies. Lebistes reticulatus. or "guppy," is well known to all aquarium fanciers. It is characterized by very con­spicuous sex differences. The slender male is about 2 cm. long and brightly hued on its lateral surfaces and sometimes on the dorsal and caudal fins. Furthermore, its anal fin is modified into a copulating organ. The female is normally about 4 cm. long and her color is generally an inconspicuous grayish brown.

Although the female normally presents no color pattern, it can transmit its sex-limited genes for color and other traits.

Before the advent of sex hormones, it was almost impossible to differentiate between sex-limited and sex-linked inheritance. With the introduction of these hormones, some of the con­clusions of early geneticists must be discarded. To read the early works of Winge and Ditlevsen, one might conclude that only one pair of chromosomes is of much consequence in guppies—the X and Y, but there are twenty-two other pairs to reckon with.

Modifying genes doubtless are present to affect the amount of color shown, the size of spots, the size and shape of fins and the size of the guppy itself. Their number or dominance or recessiveness have not been determined.

The purpose of my experiments was to determine the inheritance of several guppy mutations, some of which have been previously described by some geneticists as sex-linked and located on the Y chromosome. Also, it was to be de­termined how these mutations could be concentrated, two or three into the same pure line, with four lines in all.

Preliminary Problems Solved

The hormone methyl testosterone (Gordon) can be used to bring out sex-limited color traits in female guppies. Even more effective is pregnenolone (Regnier). The use of .0008 mg. pregnenolone per liter of water will produce an even more intense effect than 1 mg. of methyl testosterone per liter.

Before beginning my study of mutations in guppies, I experimented with guppy environmental factors such as tem­perature, water quality and food. Guppies proved to be omnivorous, surviving best at 75°-80° F. and at a pH of from 7.1 to 7.5. Acid water produced loss of color and cessation of breeding activity.

Guppies could tolerate a saline water solution of up to one tablespoon sodium chloride per gallon but reproduction was inhibited and the mortality rate increased. This is to be expected, because a sodium chloride solution is known to dissolve the DNA out of the nucleus of the cell by breaking the bonds that link DNA molecules to protein molecules and to one another. Since DNA is the basic hereditary material, it is obvious why reproduction is inhibited by sodium chloride solutions.

In genetic studies, it is imperative that all progeny of the parents be saved and counted to reach an accurate conclusion. Adult guppies, if given the chance, consume their young as soon as they are born. I found two effective solutions to this problem.

Method 1: A fry "trap" based on a mechanical principle can be constructed from a sheet of plastic, bent into a cone and fastened. A small hole is left at the bottom and an "X" of thread sewn over it to prevent the escape of the mother. The cone, placed in a jar of water, houses the mother prior to delivery. The fry, when born, are heavy and sink through the hole to safety.

Method 2: Virgin female guppies can be placed in a tank in which is suspended a small bowl of baby guppies. The females will dash themselves madly against the glass bowl, attempting to get at the baby guppies. But, probably due to unpleasant effects, a conditioned response is induced which causes them to completely ignore baby guppies after from one to three weeks of treatment. This conditioned response remains in effect for the life of the fish. This is the most effective method of preserving guppy offspring from parental cannibalism.

After the guppies are born, the problem of sexing them arises. This must be done before they are sexually mature, to prevent random fertilization. Sexing baby guppies has been considered impossible because of undeveloped sex charac­teristics at this age. Ordinarily the selective breeder is forced to raise each fish separately, requiring a vast number of containers. This obstacle was overcome by observing that some day-old guppies, when held under a bright light, showed a dark anal spot, while others did not. They were separated and those showing the dark anal spots ultimately developed into females. Sexing based upon this discovery was employed throughout the following experiments.

Materials and Methods

Mutant guppies used in these experiments were obtained from a number of aquarists in Farmington, N. M., as well as from my own aquaria. These mutations consisted of variations in basic body coloring (melanin formation) and tail shape.

An original pedigree system was devised which facilitated identification of each fish with its genotype, parents and offspring.

Ten male mutations for tail shape were isolated with two or  more  guppies  showing  the  same  mutation  if possible.

These all were tested for dominance by mating them with females of gold round-tail stock. All males showing muta­tions for tail shape showed the common gray coloration. All offspring from this mating of gold round tail to gray mutant tail were gray round tail. This proves three im­portant facts about guppy inheritance.

1. The  round-tail  shape  is dominant to  all the  mutant shapes illustrated below.
   
2. Gray is dominant to gold body color.
   
3. Guppies follow the Mendelian Principles of Unit Char­acters and Dominance.
   

To ascertain whether guppies would follow the Mendelian Principle of Segregation, I mated each Fi generation of three of the groups (lyre tail, sword tail & pin tail) brother to sister. The characters segregated as expected according to typical Mendelian inheritance, giving the types and quantities shown below.

The foregoing results proved that guppies also follow the Mendelian Principles of Segregation and Purity of Characters.

Males of the F2 generation which showed two recessive characters were backcrossed to their gold sisters to determine which carried the sex-limited genes for the mutant tail shapes. The females which proved to carry the sex-limited recessive genes were used to breed pure lines of gold lyre tail, sword tail and pin tail guppies.

With the foregoing information proved about guppy hered­ity, I planned and conducted matings which concentrated three mutations into one pure line. The types of offspring and quantities obtained are shown in the accompanying diagram. As before, the backcross was used to separate the desired females.

Summary

1. Inheritance of guppy mutation in these experiments followed the Mendelian Principles of Independent Unit Char­ acters,  Dominance   and   egregation,   Purity  of  Characters and Random Fertilization.
   
2. No X or Y linkage was evident in the inheritance of any of the mutations studied in these experiments. Earlier investigators, principally Winge, claimed to find such linkage in their experiments. It is possible that guppy mutations, although similar in appearance, are not always inherited alike. This may account for the differences in inheritance of these mutations.

CONCENTRATION OF THREE RECESSIVES

3. Mutations for tail shape and basic body color can be concentrated in almost any combination the breeder desires by applying Mendelian Principles of Heredity with correct environmental conditions.

Bibliography Research Papers:

Regnier, M. T., 1942. "Masculination des Femmelles de Lebistes Reticulatus sous l'lnfluence de la Pregneginolone." Compt. Rend. Soc. Biol., vol. 136, pp. 202-203.

Singe, O., 1922. "One-sided Masculine and Sex-linked In­heritance in Lebistes Reticulatus." Genetics, vol. 12, pp. 145-162.

1923. "Crossing Over between the X and Y Chromo­somes in Lebistes." Genetics, vol. 1, pp. 201-217.

1927. "The Location of Eighteen Genes in Lebistes Reti­culatus." Genetics, vol. 18, pp. 1-42.

Smith, D. C. and G. M. Everett, 1943. "The Effect of Thyroid Hormones on the Growth Rate, Time of Sexual Differenti­ation and Oxygen Consumption in the Fish Lebistes Re­ticulatus." Jour. Exp. Zool., vol. 94, pp. 229-240.

Tuchmann, H.,  1936. "Action de l'Hypophyse sur la Mor-phogenese et la Differention Sexuelle de Girardinus Guppi." C. R. Soc. Biol., vol. 122, pp. 162-164.

Winge, O. and Eshem Ditlevsen, 1947. "Color Inheritance and Sex Determination in Lebistes." Heredity, vol. 1, pp. 65-83.

Books:

Abbot, C. G. Cold-blooded Vertebrates, Smithsonian Scienti­fic Series, 1934.

Gordon, Myron. Guppies as Pets, New Jersey: TFH Publi­cations, Inc, 1955.

Milne & Milne. The Biotic World and Man, New York: Prentice-Hall, Inc., 1952.

Srb and Owen. General Genetics, California: W. H. Freeman & Company,  1957.

Whitney, Leon F. All about Guppies, Connecticut: Practical Science Publishing Company, 1952."

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