INDUCT I Oil OP VISIBLE 1IUTATICNS IN MOEMONIELLA

BY USE OP LOW FREQUENCY ULTRASONIC ENERGY

APPROVED:

US * Major Professor

roife

Director^ of the De artrae Biology

Dean of tne Graduate School

Grubbs, Steven C., Induction of Visible Mutations

in Mormoniella by Use of Low Frequency "Ultrasonic Energy#

Master of Arts (Biology), August, 1972, 31 PP»» ^ tables,

3 illustrations, bibliography, 3b titles#

Low-frequency ultrasonic energy was utilized in an

attempt to induce visible mutations in the parasitoid Wasp

Mormoniella vitripennis (Walker). Ultrasound exposure at

a frequency of 20,000 cycles per second was accomplished

in aqueous medium with a commercially obtained energy

source#

Sixty-three phenotypically changed wasps were re-

covered among 22,396 progeny of exposed males and females#

Three of these changes from exposed males and two from

exposed females proved to be genetically transmissible#

Ho transmissible changes were found among £,179 control

progeny#

This study demonstrates that low frequency ultra-

sound may be used as an effective mutagenic agent in this

organism, and suggests that it may have applications to

other genetic systems#

INDUCTION OP VISIBLE MUTATIONS IN MORMONIELIA

BY USE OP LOW FREQUENCY ULTRASONIC ENERGY

THESIS

Presented to the Graduate Council of the

North Texas State University in Partial

Fulfillment of the Requirements

For the Degree of

.MASTER OF ARTS

By

Steven C, Grubbs, B. A,

Denton, Texas

August, 1972

4) " §

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• TABLE OP CONTENTS

LIST OP TABLES . . . . . . . . . . . . . Iv

LIST OP ILLUSTRATIONS . . . . . . . . V

Chapter

I. "INTRODUCTION ; • • • • 1

II. MATERIALS AND METHODS 11

III, RESULTS « . . . 16

IV. DISCUSSION . 22

V. SUMMARY 28

BIBLIOGRAPHY 29

4 44

LIST OP TABLES

Table Pago

I. Fp Male Progeny from Males Exposed to Ultrasound • • • • • • • • • . . • • • • • 17

II# Male Progeny from Females Exposed to Ultrasound • 18

III. Results of Mutant Transmissibility Tests of Phenotypically Clianged Progeny from Ultrasound-Exposed Males . . . . . . 19

IV. Results of Mutant Transmissibility Tests of Phenotypically Changed Progeny from Ultrasound-Exposed Females • • • • • 21

•f tr

LIST OP ILLUSTRATIONS

Figure Page

1. Method of Examining the Progeny of Exposed Females for Possible Mutants • • « • • • • « 12

2. Method of Examining the Progeny of Exposed . Males for Possible Mutants • • • » • • • • • 13

3« Procedure to Determine if Mutant Character is Transmissible • • • • • • • • » • • • • • llj.

CHAPTER I

INTRODUCTION

Momoniella vitripermis (Walker) is a parasitoid

wasp belonging to the Family Pteromalidae. Males and

females of this species average 2.5 mm and 2.0 mm, re-

spectively, and normally complete their holoraetabolic

life cycle in 10-1I}. days at 26-30° C (22). As is

characteristic of the Order Hjpmenoptera, this genus

exhibits male haploidy. Adult females deposit their

eggs "within the puparia of any of a variety of dipterans,

after themselves feeding on the host's body fluids. Two

types of eggs are left by the female: fertilized eggs,

containing ten chromosomes and normally yielding diploid

females, and five-chromosome unfertilized eggs which

develop into haploid males (18).

Following the placing of the egg by the female, a

young larva emerges and begins to feed. The larva punctures

the host's skin with its mandibles and, firmly attached,

feeds on the host's body fluids. It remains in the same

position through four larval instars until full grown.

With cessation of feeding the larva enters a resting stage,

at the middle of which defecation takes place. After

defecation, there is a continuation of the resting stage,

followed by the last larval molt which discloses the pupa#

The light-colored pupa gradually develops eye pigmentation,

after which the body darkens from anterior to posterior#

Adults mature within the puparium and remain there for

several hours before emerging (23)#

Mated females yield about eighty-five percent female

progeny# In the dark pupal stage they may be identified

by a light streak on the median ventral surface of the

abdomen, indicating the developing ovipositor (26), and

by larger wing sacs than in the male# This sexual dimor-

phism enables one to isolate and identify virgin females

in the dark pupal stage, eliminating the necessity of

using an anesthetic when making crosses# Adult males

may be distinguished from females by their shorter wings,

brighter body coloration, and lighter colored antennae#

The wings of the female reach approximately to the tip

of the abdomen, while the male has very short wings and is

unable to fly# Although capable of flight, the females

prefer to run actively about and jump for considerable

distances, aided by their wings (21}.)# Additional infor-

mation concerning the biology of Mormoniella may be found-

in the monograph of A# R. Whiting (23)#

The potential of using this wasp for genetic research

was realized in April, 19^8, when it was brought to the

attention of P# W# Whiting as a pest infesting cultures

of the green bottle fly Lucilia, used in connection with

osteomyelitis treatment at Johns Hopkins University (2J?)»

A dose-action curve for dominant X.~ ray-induced lethal

mutations was worked out during the summer of 19l|8 by

David T. Ray (16 )• During his study, the first visible

©ye color mutations* oyster-DR and scarlet-DR, were isolated

from the progeny of irradiated wild type females. These

visible mutations were induced at a complex chromosomal

region, the R locus, named after Ray. Subsequently, other

X-irradiation studies produced dose-action curves for

visible mutations (12, 13, 17). Genetic analysis thus

has proceeded rapidly with the aid of many radiation-in-

duced and a.number of spontaneous mutations.

Genetic mutations have also been produced by exposing

various organisms to ultrasonic energy. Most humans cannot

hear corapressional waves in the air that have a frequency

higher than 20,000 cycles per second. Sound waves of this

and higher frequencies are called ultrasonic waves, meaning

above or beyond sound in the sense of frequency (l)»

Ultrasound has a variety of medical and theraputic

applications, A review of the action of ultrasonic waves

on biomacromolecules, microorganisms, viruses, and bacterio-

phages has been compiled by El'piner (6).

Ultrasonic energy was first used in an unsuccessful

attempt to produce mutations by Hersch, Karrer and Loomis (8),

Unanaesthetized Drosophila melanoeaster were treated in a • I •»»IvtMrnrnrtmnmi* II«14m tmtf» m ttkmtrm tiHra'wwii'T" Will

glass exposure chamber immersed in an oil bath over a

quartz crystal oscillating at 285*000 vibrations per

second. The study was restricted to the Bar locus on the

X chromosome, and no mutations were found among a total of

26,135 progeny from l\29 treated males. The occurrence of

the rare character for mottled eye among the progeny of

three treated males was not attributed to the ultrasound

treatment because., due to their very close genetic rela-

tionship, it was felt that the mutation may have existed

prior to treatment#

Bushnell and Wallace (2) found that preliminary tests

of male Droaophila exposed to ultrasonic vibrations in

glass test tubes produced mutations at twice the sponta-

neous rate. However, when young males were etherized and

exposed in plastic or tin metal containers, higher mutation

rates were observed. The C1B method was used to detect

induced mutations, and among 23l\. treated chromosomes, 17

(7.2 percent) proved to be lethal and 37 (15.9 percent)

were semi-lethal. Controls yielded one lethal and one

semi-lethal among 181). tested chromosomes. In mapping the

new lethals, four proved to be long inversions. Other'

effects observed were phenocopy induction, visible mutations,

sterility, or death, depending upon the time and technique

of exposure#

Wallace, Bushnell, and Newcomer (21) reported the first

evidence of mutations produced by ultrasound in plants.

They observed phenotypic changes in Hellanthus shoots

after exposure to a frequency of ij.00,000 vibrations per

second. Chromosomal examination of root tip smears and

sections revealed frequent breakage of whole chromosomes

and individual chromatids. Newcomer and Wallace (15)

found chromosomal abnormalities in root tip cells of

ffarcissus which had been exposed to ultrasonic vibrations*

Fritz-lliggli and BSni (7) examined imagoes derived

from eggs, larvae, and pupae of Drosophila melanogastep

which had been exposed to supersonic vibrations at

intensities of 0.3, 0.71, and 1.75 watts per square

centimeter and a frequency of 800,000 vibrations per

second. After treatment the adults were used in breeding

experiments to detect any recessive or dominant mutations#

The C1B method was applied for the X chromosome, and of the

lj.73 chromosomes tested, one lethal xras found#

Spencer (19) observed that agitation by ultrasound

at a frequency of 500 kilocycles per second and intensity

of ten or twenty watts per square centimeter induced

cytological and histological aberrations of growth in the

root tip of Pisum. Further experiments with the same

organism (20) revealed that intense ultrasonic vibrations

of similar frequency and intensity induced an apparent

dauermodifxcation in growth. These maternally transmitted

morphogenetic variations included increased length of root

and shoot" axes, first manifested as ari accelerated develop-

ment of the radicle#

Newcomer (ll.) observed root tips of Narcissus which

had been exposed to ultrasonic vibrations at an intensity

of thirteen watts per square centimeter, and found no

chromosomal rearrangements. At the intensity used, chromo- .

some breaks, and nuclear disorganizations were abundant,

but neither recombination nor restitution of these frag-

ments could be verified cyt©logically. However, Dubow (5)

reported structural rearrangements of chromosomes in

Drosophila. Carpio (3) and Carpio and Orellana (Ij.) also

recovered chromosomal rearrangements in plants after

ultrasonic exposures.

Kato studied the effect on Drosophila melanomaster

of ultrasonic vibrations at a frequency of £60 kilocycles

per second. Dominant and recessive lethal mutations were

recovered (9, 10). Thirty-day-old males of the Tokyo

wild-type strain were exposed to ultrasound for thirty

minutes and visible mutations were induced with a ratio

of 1.17.:l (new mutants: wild flies). The most common

modifications encountered were a brighter red-eye mutant

of the vermillion-like eye and a bobbed-like mutant. The

inducing effect of ultrasound in producing mutations was

much more striking than that of X-irradiation as reported

by Kato (11).

The present investigation was undertaken to determine

if low-frequency* ultrasonic energy is capable of producing

visible genetic mutations in Mormoniella, The optimal

conditions for producing mutations were sought by varying

the intensity and length of exposure to ultrasound.

CHAPTER BIBLIOGRAPHY

1, Bueche, P., Principles of Physics, New York, McGraw-Hill Book Co., 19S£T

2# Bushnell, Ralph J«, and Raymond H. Wallace, "Induction . of Sex-Linked Mutations in Drosophila with Ultra-sonic Treatment," Anatomical Record, 101 (August, 191)5), 690. ' . '

3» Carpio, M. D. A,, "Aportsciones para el Estudio de Variaciories Chromosomicas Indue 1 da s p or Ultra-sonidos," Genetlea Iberica, 3 (1951)# 113-128.

Ij.. Carpio, M. D. A., and E. Orellana, "Aportaciones para el Estudio de las Variaciones Chromosomicas In-due Idas por Ultrasonidos," Genetics Iberica, 3 (1951), 3-20. ~ " "

5. Dubow, R, J., "Mutagenic Effects of Ultrasonic Vibra-tions on Drosophila melanogaster." unpublished thesis, University of Connecticut, Storrs, Connecticut, 19ij-9»

6. Elfpiner Isaak E,, Ultrasound Physical,. Chemical, and Biological Effects, Hew York, Consultants Bureau, 196lj.#

7» Pritz-Higgli, H«, and A, Boni, "Biological Experiments on Drosophila melanogaster with Supersonic Vibra-tions/' Science. 112 (July. 1950), 120-122.

8. Ilersch, A. H., E. Karrer and A. L. Loomis, "An Attempt to Induce Mutation in Drosophila melanogaster by Means of Supersonic Vibrations," American Naturalist. 6i|. (January-February, 1930), 552-j£5% ;

9. Eato, Mikio, "Induction of Dominant Lethal Mutations by Ultrasonxc Vibration in Drosophila melanogaster," Osaka Medical School Bullet jnTT2*~nT96' .' 10?-107."

10» -> "Inductivity of Recessive Lethal Mutations by Ultrasonic Vibrations in Drosophila melanogaster." £§§i£a Medical School Bulletin. 12 (19<5"6T, idB-113.

8

11. a "Visible Mutation Induced in Drosophila by Ultrasonic Vibration," Osaka Medical School Bulletin, 12 (1966), llii-llF:

12. Kayhart, Marion, "A Comparative Study of Dose-Action Curves for Visible Eye-Color Mutations Induced by X-Rays, Thermal Neutrons and Past Neutrons in Morraoniella vitripennis," Radiation Research# irnsnsspjT — —

13. Kayhart, Clarion E. and P. W, Whiting, "X-Ray Muta-tions and Fecundity of Mormoniella." Biological Bulletin. 97 (December, 19WT7*355«

lJ!|.. Newcomer, Earl H», "Observation on Dosage, the Mech-anism of Action and the Recovery of Cells Exposed to Ultrasonic Vibrations," American Journal of Botany. Ip. (May, 3%~3B9l

15. Newcomer, E. H., and R. H. Wallace, "Chromosomal and Nuclear Aberrations Induced by Ultrasonic Vibra-tions," American Journal of Botany. 36 (February, 19l"-9), 230-236.~~

16* Ray, D. T,, "Dominant Lethals Induced by X-Rays in Sperm of the Chalcidoid Wasp Nasonia, brevicomia Ashmead = Mormoniella vitripennTs^TWalker) fide Muesebeck in lit.Biolo/rical Bulletin, 95 (October, 19l{F),

17* Kay, D. T„, and P. W, Whiting, "An X-Ray Dose-Action Curve for Eye-Color Mutations in Mormoniella," Biological Bulletin. 10.6 (February, 195^), ;00~106.

18® Saul, G. B,, and M. E. Kayhart, "Mutants and Linkage in Mormoniella," Genetics, ILI (November, 1956), 930-937#

19. Spencer, John L., "Effects of Intense Ultrasonic Vibrations on Pisum. I. On Root Meristems," Growth', 16 (March-December, 1952), 2i].3-25i|..

, Effects of Intense Ultrasonic Vibrations on Pisum. II. Effects on Growth and their Inheritance," Growth, 16 (March-December. 1952), 255-277.

21. Wallace, R. H«, R. J. Bushnell and Earl H. Newcomer• The Induction of Cytogenetic Variations by Ultra-sonic Waves," Science. 107 (May, 19I4.8), 577-578.

10

22. Whiting, A. R„, "The Complex locus R in Mormoniella vitripennis (Walker), Advances in Genetics. 1*5 13^77351-358.

23. .j "The Biology of the Parasitic Wasp Mormoniella vitripennis (Walker),n Quarterly Review of Biology, lj2 (September. 1967), 333-i|-06.

2if.. Whiting, P. W#, "A Parasitic Wasp and its Host for Genetics Instruction and for Biology Courses,'* Carolina Tips. 18 (April, 1955), 13-16.

25* • , "Drosophila, Eabrobracon, Mormoniella," Carolina Tips. 13 (December, 1955), 37-39.

26. , "Mormoniella and the Nature of the Gene':' Mormoniella vitripennis (Walker) (H^menoptera t Pteromalidae)V*' Proceedings Tenth International Congress of Entomology. Vol, II, Ottawa, Canada, Mortimer ETmi ted,' l'95o.

CHAPTER II

MATERIALS AND METHODS

Materials

A Woods Hole #2 stock of Mormoniella was obtained

from the Mormoniella Stock Center, Middlebury College,

Middlebury, Vermont. White-eyed host pupae of the dipteran

blowfly Sarcophap;a bullata (Parker) were procured from the

Carolina Biological Supply Company# The host pupae were

stored in a refrigerator until they were needed, to prevent

further development.

The source of ultrasonic vibrations used in these

experiments was equipment manufactured by Branson Sonic

Power, Danbury, Connecticut, The power.unit was a Sonifier-

Cell Disrupt or,. Model ¥185>D, and the probe for vibration

administration was a Sonifier Converter, Model L# The

unit operated at a frequency of twenty kilocycles per

second.

The effect on mutation rate by heat increase alone,

without accompanying ultrasonic vibrations, was measured

by use of a hot plate and magnetic stirrer. The stirrer

mimicked the agitation produced by ultrasound, and a

thermometer was used to measure the temperature increase»

comparable with that produced by ultrasonic treatment.

11

. 12

Methods

Wild type wasps were exposed to ultrasonic vibrations

while in the light pupal stage. Groups of ten pupae were

placed into a fifty-milliliter beaker which contained

twenty milliliters of distilled water. The tip of the

Sonifier Converter was immersed four to five millimeters

below the surface of the distilled water, and the initial

temperature was taken. The wasps then were euqposed to

ultrasonic vibrations for varying times and the final

temperature was recorded immediately after the insects

were removed from the beaker# The exposed wasps then were

placed in eight-dram shell vials plugged with gauze-covered

cotton, and were allowed to eclose. Two methods were

utilized in the search for possible mutants# (See Figure 1

and Figure 2).

ultrasound N females (211) set unmated

exposure 4 males (N) examined for possible

mutations

Fig. 1—Method of examining the progeny of exposed females for possible mutants.

In the procedure outlined in Figure 2, virgin females,

one for each male, were added to the shell vial after

eclosion and allowed to mate for twenty-four hours before

hosts were added, one for each female. During the breeding

13

procedures the shell vials were stored in an incubator at

27 i 1° C.

ultrasound ^ males (N) X + virgin females (2N)

exposure

V P, females (2N) set unmated

F- males (discard)

. Fp males examined for possible mutations

Fig, 2—Method of examining the progeny of exposed males for possible mutants®

Any possible mutations found by the abovementioned

methods were tested to determine if the mutation could be

passed on to the next generation. This was accomplished

by the procedure outlined in Figure 3*

If all wild type Fg males were produced,then the

mutation x-ras assumed to. be non-transmissible. If equal

numbers of wild and mutant Fg males were produced, the

mutations could be assumed to be transmissible. True

breeding mutant stocks were obtained by crossing hetero-

zygous females with mutant males, then selecting pheno-

typically mutant females for crossing with mutant males.

. 3 4

suspected mutant rriale (N) X + virgin female (2N)

females (2N) set unmated

males (discard)

<4? Pg males (N) examined for

occurrence and ratio of mutant types

Fig» 3—Procedure to determine if mutant character is transmissible.

The power of the acoustic field in which the wasps were

exposed was 'approximated by the following equation from

Lloyd (1).

P a MJS (dT/dt)Q

M is the mass of absorbing material in the beaker, J the

mechanical equivalent of heat, S the specific heat of the

absorbing material and tthe time necessary for a change

in temperature T» The subscript o represents the slope

of the line of the graph of T versus t very near the

origin,so the possibility of a heat dissipation error is

minimized.

CHAPTER BIBLIOGRAPHY

1, Lloyd, E. A,, "Energy Measurement," Ultrasonic Tech-niques in Biology and Medicine, edited by B# Brown and D, Gordon, London, London Iliffe Books, 1967»

15

CHAPTER III

RESULTS

The results of the search for possible mutations

among the progeny of ultrasound exposed males and females

are summarized in Table I and Table II, respectively. In

all experiments some diapausing larvae and other individuals

were produced *fhich did not complete development as far as

the dark pupal stage. These individuals were not included

in the tabulations.

After possible mutations were discovered among the

progeny of wasps exposed to ultrasound, these individuals

were tested to determine if the traits were transmissible

to the succeeding generations. Table III indicates the •

results of such tests on possible mutant progeny from

exposed males. Many deviations from the wild type Irri-

doscent copper-green body color and dark brown eyes were

observed. Body color changes included blues, greens,

purples, reds,and combinations thereof. Eye color dif-

ferences were varying shades of red or reddish brown.

Some morphological irregularities also were noted. Among

the fifty-one possible mutants found, three were confirmed

as transmissible. A Chi Square test calculated on the

actual numbers of mutant and wild type wasps produced,as

16

17

compared to.the expected 1:1 ratio, gave probability values

of greater than 0.30 in each case#

TABLE I

Fp MALE PROGENY PROM MALES EXPOSED TO ULTRASOUND

Group Intensity (Watts/cm2)

Length of Exposure (min.)

dT <°c.)

Total Progeny?:-#

Possible Mutants-

1-* - wm mm 1,900 1

2 0.26 3 5 .4 908 11 3 0.60 3 10.0 922 6

k 0.7k 3 15.3 1,611* 5 5 1.16 3 20.0 1,006 4 6 1.1*6 3 25.5 193 1 7 0.26 5 12.6 1,654 2 8 0.60 5 16.0 1,427 1 9 0.60 5 17.5 . 1,551 2

10 0.7*1. 5 25*5 1,2*62 2 11 0.74 5 25.6 1,695 1 12 0.22 10 14.5 2,172 7 13 0.26 10 21.3 1,974 5 14 OJ4.3 10 25.8 2,130 4 Total

18,708 51

•--Control group which was not added to the total.

-"""-Diapause larvae were not counted in this tabulation.

o o u n t e T t a 1 t h S ° c o l C ? i b l e m U t a n t S W h i ° h K 8 P e a l l T e w e r e

TABLE II

MALE PROGENY PROM FEMALES EXPOSED TO ULTRASOUND

18

Group . Intensity (Watts/cm^)

Length of Exposure (min.)

dT i ° c . )

Total Progeny-;:-*

Possible Mutants

1* - - - 460 0

2 0.71}- 3 13.5 485 5.

3 0.95 3 16.5 145 0

4 1.16 3 20.2 145 1

5 1.16 3 20.3 149 1

6 0.26 5 9.7 477 0

7 0.60 5 17.5 530 0

8 0.74 • 5 24.3 549 2

9 0.26 10 17.0 206 0

10 0.26 10 16.4 242 0 •

11 0.26 10 20.3 452 3

12 0.35 10 23.0 308 0

Total 3,688 12

-"•Control group which was not added to the total•

-"-^Diapause larvae were not counted in this tabulation,

•JHKfrOnly those possible mutants which were alive were counted in this column*

19

TABLE III

RESULTS OP MUTANT TRANSMISSIBILITY TESTS OP PHENOTYPICALLY CHANGED PROGENY PROM

ULTRA SOUND-EXP OSED MALES

Source From. Table I Possible Mutant

Description Progeny

P2 Progeny

Group Possible Mutant Description Progeny •r Mutant

2 . Copperless head •fsi 121 0 same + 63 0 same + 70 0 same + $ 0 0 same + 3-74 160 same' + •u li|5 129 same Mb - -

same M — tmmm

same + 79 0 same + 99 0 same + 83 0

3 Blue front + 332 0 Copper front + 3 |1 0 Purple head + 18Z| 0

same ' + 203 0 Blue front + $ 0 Blue green front + 353 0

k Small head + 150 0 Blue purple front + 271 0 Blue red front + 325 0 Bar eye 16 \ 0 Purple front + k42 0

5 Blue body color NP° Red thorax + 101 0 Bar eye + 106 0

6 Purple body + 93 0

6 Green front M mm mm

7 Green purple front + 330 0

8 Blue green front M mm mm mm am

8 Copper front D<* *» mm —

aThis denotes that viable F^ females were produced#

*>A11 progeny in the were male

CThe host pupae were either not viable or were not parasitized by the wasp and eclosed.

^All F-j_ progeny were diapause larvae#

TABLE III—Continued

20

Source Prom Table I Group

Possible Mutant Description

_ F1 Progeny

f2 Progeny

* TftrEant

9 Small head D *»w W W

Reddish eyes + 353 0 10 Copperless front + 317 0

11 • Dahlia eyes + 195 20k

11 • Blue front + i4o 0 12 Green front D «>»»» W W

same HP w w W W

Pour legs M •mm W W

Copper red front + 111 0 Large eye M W W W W

13 Green purple front D mm w W W

One eye +, one eye dull red D w w mat M

Blue purple front D W W M W

One eye D W W W W

ik Green front + 128 0

ik Green red front HP W W W w

Green front, dark red eyes + l$k 0

Green purple front D w » W W

Bar eye HP W W - -

Table 17 shows the results of the-transmissibility

tests on possible mutant progeny from ultrasound exposed

females, From the twelve groups in Table II twelve possible

mutants were observed. Among these two proved to be trans-

missible, and gave Chi Square probabilities greater than

0.30.

In the control group of females exposed to heat in-

crease without accompanying ultrasonic energy, no possible

mutants were observed among 776 male progeny® One dead

TABLE IV

RESULTS OP MUTANT TRAITSMIS SIBILITY TESTS OF PHENOTYPICALLY CHANG-ED PROGENY PROM

ULTRASOUND-EXPOSED FEMALES

21

Source Prom Table II Group

Possible Mutant Description

pl Progeny

* P 2

Progeny W a n t ' . '

2 * Blue green front •f«3! 75 0

Copperless front + 135 0

Purple head + 2 3 0 0

Blue front + 2 3 6 0

Copper front + 0

k Blue front + % 0

5 Red thorax + 118 0

8 Reddish eyes M b * » « »

Dark head + 289 0

l l Copper head * 118 10$ Dahlia eyes X

4 121 1 0 6

Green front, reddish eyes + 22^ 0

aViable P^ females were produced#

bAll progeny in the Fj were male#

scarlet eye mutant was found among 2,043 F2 progeny

from male wasps exposed to heat increases#

CHAPTER IV

DISCUSSIOH

The observations recorded in this investigation would

indicate that low frequency ultrasound is a valid mutagenic

agent, and that Morraonlella is susceptible to genetic change

when exposed to ultrasonic energy. The previous investiga-

tions employing ultrasonic energy as a mutagenic agent used

frequencies many times greater than the frequency used in

this work,- The use of Mormoniella as a test organism with

ultrasonic energy has not been heretofore reported. However*

earlier laboratory experiments (10) have produced phenotypic

changes in progeny of ultrasound-exposed Mormoniella,

According to A, R. Whiting (12), the rate of spontaneous

eye color mutation from wild type stocks of Mormoniella is

probably somewhat less than O.OOlf percent# (Caspari (3) has

reported an X-ray-induced mutation rate as high as 1,8 per-

cent,) Low frequency ultrasound at the level utilized in

this investigation produced a mutation rate of 0,023 percent

transmissible'mutations. However, higher ultrasonic inten-

sities and longer lengths of exposure might raise the

mutation rate in Mormoniella to the levels reported by

Kato (6) and Bushnell and Wallace (l).

22

23

In addition to the transmitted visibles, other mutations

also may have occurred. Some of the phenotypically changed

males crossed with, wild type virgin females produced an Pi

generation without females* This implies that the male did

not mate, it was inviable, or that its sperm was ineffec-

tive# A mutation to male sterility may have resulted in

the third possibility.

Since ultrasound is capable of increasing the mutation

rate in organisms, the mode of action of ultrasound in

producing the changes is of interest. Carlin (2) and

Gordon {$) agree on three physical characteristics of ultra-

sound which determine its biological action. It appears

likely that the results are due to one or all of the

following characteristics: (l) high pressures and accel-

erations which cause motions within the cells, (2) locally

generated heat, and (3) cavitation.

Newcomer and Wallace (8) concluded that mechanical

vibrations were responsible for observed chromosomal aber-

rations. Gordon (5) also has suggested that the mechanical

effect of ultrasound may be responsible for its action.

Selman (11) carried out tests under increased pressure

to eliminate cavitation and was unable to obtain similar

chromosomal aberrations obtained with cavitation. He

therefore favored the view that cavitation was responsible

for the biological effects of ultrasound.

2ij.

Ultrasound has been shoxm to "be capable of depoly-

merizing macromolecules. Laland, Overend, and Stacy (?)

reported evidence that some disaggregation of DM is

induced by ultrasonic exposure. El*piner (ij.) presented

evidence of the breakdown of biomacromolecules by ultra-

sound* Since all the previously mentioned characteristics .

of ultrasound were noted during these experiments it is not

possible to label one as the most effective# Possibly all

the characteristics are involved to a greater or a lesser

degree#

The mutations produced in Mormoniella as a result of

ultrasonic treatment could have been caused by either

chromosomal breakage or point mutation. Both types of

mutation previously have been reported as a result of

ultrasonic exposure. In order to determine which occurred

in this investigation, a cytological study of the mutant

stocks would be required#

The heating effect of ultrasonic exposure does not

seem to cause the increase in mutation rate. With one

exception, which was dead when found, wasps that were

exposed to a heat increase without accompanying ultra-

sonic vibrations did not show aberrant progeny# Among the

sixty-three possible mutant progeny tested in this investi-

gation,no phenotypic changes to scarlet eye color were

observed# Thus it would seem that the scarlet eye color

25

change was due to a spontaneous mutation rather than the

heat Increase#

Further investigations as to the nature of the muta-

tions produced in these experiments is indicated. Using

appropriate marker genes in a test cross with mutant stocks*

it would be possible to determine if the eye color muta-

tions produced were R-locus or non R-loeus. Since the most

frequent R-locus eye color mutations are either oyster or

scarlet (12), the dahlia eye mutants, if they are R-locus,

might provide valuable information concerning this complex

chromosome region. Roozen and Conner (9) suggested that

the R-locus mutations might be caused by chromosomal re-

arrangements within the factors of this locus.

The body color mutations produced in this investiga-

tion also should be investigated to determine their position

among the linkage groups. Appropriate marker genes from

each of the linkage groups could be used in reciprocal

crosses with the mutant body color stocks. After deter-

mining the linkage group to which the mutations belonged,

further test crosses with marker genes along the linkage

group could establish the mutant gene's position relative

to the other genes belonging to this linkage group* After

being mapped, these body color mutations could provide a

useful tool in future recombination studies with eye color

mutants, since the recombinant types can easily be

26

distinguished by use of a known body color mutant stock

and an unknown eye color mutant stock#

CHAPTER BIBLIOGRAPHY

1. Bushnell, Ralph J. and Raymond H. Wallace, "Induction of Sex-Linked Mutations in Drosophila with Ultra-sonic Treatment," Anatomical Record, 101 (August, 19^8), 690#

2. Carlin, Benson, Ultrasonics, New York, McGraw-Hill Book Company, Inc., 19&0

3. Caspari, S. B,, "An X-Ray Sperm-Dose-Action Curve for Mutations at a Single locus in Mormoniella," Radiation Research, 8 {March, 1958), 273-2o3#

l}.« El'piner, Xsaak E., Ultrasound Physical, Chemical, and Biological Effects, New York, Consultants Bureau®

5# Gordon, A. G., "Mutations and Ultrasound," Ultrasonic Techniques in Biology and Medicine, edited by B, Brown and D. Gordon, London, London Iliffe Books, 1967.

6. Kato, Mikio, "Visible Mutation Induced in Drosophila by Ultrasonic Vibration," Osaka Medical School Bulletin, 12 (1966), llI|-lT87~~

7. Laland, S., W. G. Overend and M. Stacy, "Some Effects of the Ultrasonic Irradiation of Deoxyribonucleic Acids," Research, 3 (August, 19j?0)> 306-387•

8. Newcomer, E. H. and R. H. Wallace, "Chromosomal and Nuclear Aberrations Induced by Ultrasonic Vibra-tions," American Journal of Botany, 36 (February, 1949)# 230-2J6V

9. Roozen, Kenneth J. and George W. Conner, "Genetic Analysis of the R-locus in Mormoniella," Journal of Heredity, 60 TApril, 1969), 269-271.

10. Saul, George B,, 2nd, Personal Communication.

11. Selman, G. C., "The Effect of Ultrasonics on Mitosis," Experimental Cell Research. 3 (1952), 656-67^.

12. Whiting, A. R., "The Complex locus R in Mormoniella vitripennis (Walker)," Advances"~in Genetics, 13 (1965), 3U-358. — — ^

27

CHAPTER V

SUMMARY

Low frequency ultrasonic energy was utilized in an

attempt to induce visible mutations in the parasitoid wasp

Mormoniella vitripennis (Walker). Ultrasound exposure at

a frequency of 20,000 cycles per second was accomplished

in aqueous medium with a commercially obtained energy

source.

Sixty-three phenotypically changed xmsps were re-

covered among 22,396 progeny of exposed males and females#

Three of these changes from exposed males and two from

exposed females proved to be genetically transmissible.

No transmissible changes were found among £,179 control

progeny.

This study demonstrates that low frequency ultrasound

may be used as an effective mutagenic agent in this organism,

and suggests that it may have applications to other .genetic

systems.

28

BIBLIOGRAPHY

Books

Bueche, F., Principles of Physics, Hew York, McGraw-Hill Book Co., 1965.

Carlin, Benson, Ultrasonics, New York, McGraw-Hill Book Company, Inc.'," 19&0. """

El,piner, Isaak E., Ultrasound Physical, Chemical, and Biological Effects,' iJew York, Consultants Bureau, X9<r ~

Gordon, A. G., "Mutations and Ultrasound," Ultrasonic Techniques in Biology and Medicine, edited by B. Brown and D. Gordon," London, London Iliffe Books', 1967.

Lloyd, E. A., "Energy Measurement," Ultrasonic Techi in Biology and Medicine, edited by B. Brown and D. Gordon, London, London Iliffe Books, 1967*

Articles

Bushnell, Ralph J. and Raymond H. Wallace, "Induction of Sex-Linked Mutations in Drosophila with Ultrasonic Treatment," Anatomical Record. 101 (August, 19JJ.8), 690.

Carpio, M. D. A., "Aportaciones para el Estudio de Variaciones Chromosomicas Inducidas por Ultrasonidos," C-enetica Iberica. 3 (1951), 113-128.

Carpio M. D. A# and E, Orellana, "Aportaciones para el Estudio de las Variaciones Chromosomicas Inducidas por Ultrasonidos," Genetica Iberica. 3 (1951), 3-20.

Caspari, S. B., "An X-Ray Sperm-Dose-Action Curve for Mutations at a Single Locus in Mormoniella," Radiation Research. 8 (March, 1955), 273-2o3#

Prioz-Liggli, H. and A. Boni, "Biological Experiments on Drosophila melanogaster with Supersonic Vibrations." Science, 112 (July, 19^0), 120-122.

29

30

Hersch, A. H«, B. Karrer and A. L, Loomis, "An Attempt to Induce Mutation in Drosophila melanogasteg by Means of Supersonic Vibrations, American Naturalist , 64 (January-February, 1930), 552-559#

Kato, Mikio, "Induction of Dominant Lethal Mutations by Ultrasonic Vibration in Drosophila melanomaster, Osaka Medical School Bulletin, 12 (1966),102-107•

, "Inductivity of Recessive Lethal Mutations m by Ultrasonic Vibrations in Drosophila melanogaster, Osaka Medical School Bulletin!12(19o6T> 108-113#

, "Visible Mutation Induced in Drosophila by Ultrasonic Vibration," Osaka Medical School Bulletin, 12 (1966), 111J.-118.

Kayhart, Marion, "A Comparative Study of Dose-Action Curves for Visible Eye-Color Mutations Induced by X-Rays, Thermal Neutrons and Past Neutrons in Mormoniella vitripennis," Radiation Research, If. '(January, 1956), 65J-76Y

Kayhart, Marion and P. W. Whiting, "X-Ray Mutations and Recundity of Mormoniella," Biological Bulletin, 97 (December, 19^9), 3M-1-*

Laland, S., ¥. G. Oyerend and'M. Stacy, "Some Effects of The Ultrasonic Irradiation of Deoxyribonucleic Acids, Research, 3 (August, 1950), 386-387•

Netircomer, Earl H,, "Observation on Dosage, the Mechanism of Action and the Recovery of Cells Exposed to Ultra-sonic Vibrations," American Journal of Botany, Ip. (May, 19$L), 38^-389.

Newcomer, Earl H# and R„ H. Wallace, "Chromosomal and Nuclear Aberrations Induced by Ultrasonic Vibrations," American Journal of Botany, 36 (February, 19li-9)» 230-236.

Ray, D. T,, "Dominant Lethals Induced by X-Rays in Sperm of the Chalcidoid Wasp Nasonia brevicornis Ashmead -Mormoniella vitripennis (Wlkerl^fide Muesebeck in litBiological Bulletin, 95 (October, 191*8), 2^7-258•

Roozen, Kenneth J. and George W, Conner, "Genetic Analysis of the R-locus in Mormoniella," Journal of Heredity, 60 (April, 1969), 269-271.

31

Saul, G. B. and M. E. Kayhart, "Mutants and Linkage in Mormoniella," Genetics, ijJL (November, 1956), 930-937*

Selman, G. C., "The Effect of Ultrasonics on Mitosis," Experimental Cell Research, 3 (1952), 656~67i|-.

Spencer, John L., "Effects of Intense Ultrasonic Vibrations on Pisum. I. On Root Meristems," Growth, 16 (March-December, 1952), 2Ji3-35i|..

, "Effects of Intense Ultrasonic Vibrations on Pisuni. II. Effects on Growth and their Inheritance," Growth, 16 (March-December, 1952), 255-277#

Wallace, R. H»., R. J. Bushnell and Earl H. Newcomer, "The Induction of Cytogenetic Variations by Ultrasonic Waves," Science, 107 (May, 19ij.8), 577-578.

Whiting, A, R., "The Complex locus R in Mormoniella vitripennis (Walker)," Advance's in.Genetics, 13 7WT7151-358.

, "The Biology of the Parasitic Wasp Mormoriiel'la vitripennis (Walker)," Quarterly Review of Biology, 1|2. (September, 1967), 333-W&*

Siting, P. W,, "A Parasitic Wasp and its Host for Genetics Instruction and for Biology Courses," Carolina Tips, 18 Ckpril, 1955), 13-16.

"Drosophila, Habrobracon, Mormoniella," Carolina Tips, 13 (December, 1955)> 37-39.

Publications of Learned Organizations

Whiting, P. W,,. "Monsioniella and the Nature of the Gene: Mormoniella vitripennis (Walker) (Hymenoptera: P t eroriia 11 dae)V11 Pr oc e e din,g;s Tenth International Q-Ongress. of .Entomology, Vol. II, Ottawa, Canada, Mortimer Tdmited, i$5o.

Unpublished Materials

Dubow, R. J., "Mutagenic Effects of Ultrasonic Vibrations on Drosophila melanomaster," unpublished thesis University of Connecticut, Storrs, Connecticut, I9I4.9•

Saul, George B,, 2nd, personal communication.