SOME EFFECTS OF PENICILLIN

ON THE GERMAN COCKROACH, BLATTELLA GERMANICA (LINN.)

ORTHOPTERA: BLATTIDAE

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By

JAMES NEIL LILES, B. A., M. Sc.

The Ohio State University 1956

Approved by:

Adviser Department of Zoology and Entomology ACKNOWLEDGEMENTS

Thla work was accomplished while the writer was serving as a University Fellow under a grant supported with fund3 from The Ohio State University for aid in fundamental research. This project was supervised by

Dr. R. H. Davidson and Dr. F. W. Fisk.

I wish to especially thank my adviser, Dr. F. W.

Fisk for his suggestions and criticisms throughout this and other work.

I wish to thank my wife, Dale, whose encouragement and sacrifices helped make this work both enjoyable and possible; and for her assistance in the preparation of the manuscript.

Special thanks are also due George Hrubant for suggestions concerning microbiological assays, and to

Dr. E. L. Green and Edwin P. Les for information con­ cerning the statistical analyses.

Thanks are also extended to Professors Carl Venard,

Ralph H. Davidson, and Frank W. Fisk who read and criti­ cized the manuscript.

ii TABLE OP CONTENTS

Pace INTRODUCTION

PART Is E PFECTS OF DIETARY PENICILLIN ON THE EIONOMICS OP THE GERMAN COCKROACH...... 5 LITERATURE REVIEW...... 5

MATERIALS AND METHODS,

Teat Insects ...... 9

Tea t Rearing Methods...... 9

Rearing Conditions...... 11

Weekly Weighings...... 11

Diets...... • 12

Test Insects Used in Vitamin Assays and in Second Generation Studies ..... 14

Penicillin Used...... 15

EXPERIMENTS AND RESULTS...... 18

Studies of Effect of Penicillin on Growth Rates, First Generation...... 18

Time to nAdult Stage”...... 34 I Mortality...... 36 j Reproductive Capacity...... 36 ! Studies of Effect of Penicillin on Growth Rates, Second Generation...... 40 j Tiijie to "Adult Stage" ...... 55

Mortality...... 55 | Reproductive Capacity...... 60

DISCU$SION AND CONCLUSIONS...... 62

PART II: EFFECTS OF DIETARY PENICILLIN ON CONCEN­ TRATION OF B COMPLEX VITAMINS IN THE ROACH 70 iii iv Page LITERATURE REVIEW...... ~70

MATERIALS AND METHODS...... 74

General Methods of Assay. 74

General Procedure for the Microbiological Assay of a Vitamin with Riboflavin as the Spec ific Example...... 75

Preparation of Sample...... 75

Preparation of Stock Cultures. 77 Preparation of Standard Tubes. 77 Preparation of Assay Tubes.... 78 Sterilization...... 78 Preparation of Inoculum...... ^...... 79

Inoculation and Incubation.....'•...... 80 Determination of Amounts of Growth in the Sample ...... 80 Construction of a Standard Curve with Known Solutions...... 81 Calculation of the Quantity of yitamin in the Sample ...... 81 Niacin...... 83 Biotin...... 83 Folic Acid ...... 84 Insects Used...... 85 EXPERIMENTS AND RESULTS...... |...... 91 j DISCUSSION AND CONCLUSIONS...... |...... 98 SUMMARY...... |...... 104 REFERENCES...... 107 AUTOBIOGRAPHY...... !...... 114 LIST OP FIGURES

Figure Page 1 Growth curves produced by the check and first seven diets in first generation studies 25

2 Growth curves produced by the check and last eight diets in first generation studies 27

3 Growth of the German roach at four weeks on a diet containing different amounts of peni­ cillin 28

4 Growth of the German roach at five weeks on a diet containing different amounts of peni­ cillin 29

5 Growth of the German roach at nine weeks on a diet containing different amounts of peni­ cillin 30

6 Time in which the German roach reached the adult stage, when reared on a diet contain­ ing different amounts of penicillin 35

7 Mortality in the various levels, first generation, ninth week 38

8 Normal and deformed egg capsules 39

9 The results of rearing on the check diet the offspring from first generation cock­ roaches fed initial diets 1-8 46

10 The results of rearing on the diets of their respective parents the offspring from first generation cockroaches fed initial diets 1-8 48

11 Growth of the second generation roaches at two weeks on the control and parental diets 50

12 Growth of the second generation roaches at four weeks on the control and parental diets 51

13 Growth of the second generation roaches at eight weeks on the control and parental diets 52

V vi

Figure Page 14 Time In which second generation roaches reached the adult stage, when reared on the control and parental diets 56

15 Mortality In the various levels at eight weeks, second generation 59

16 Standard riboflavin curve 82

17 Quantity of riboflavin present in male and female roaches reared on the various diets 93

18 Quantity of niacin present In male and female roaches reared on the various diets 94

19 Quantity of biotin present in male and female roaches reared on the various diets 95

20 Quantity of folic acid present In male roaches reared on the various diets 96

21 Quantity of folic acid present in female roaches reared on the various diets 97 LIST OP TABLES

Page 1 Composition of the diets employed in the rearing experiments- 13

2 Weights of first generation roaches on test diets 19

3 Regression coefficients of the sixteen treatments, each coefficient calculated on the entire growth period 31

4 Analysis of variance of the growth rates with the sixteen diets. Growth time equals the entire growth period 32

5 Regression coefficients of the sixteen treat­ ments calculated from zero through four weeks of growth 33

6 Analysis of variance of the growth rates with the sixteen diets. Growth time equals four weeks 34

7 Mortality in the first generation, through nine weeks 37 8 Weights of second generation roaches on test diets 41

9 Regression coefficients of the fifteen second generation treatments. Each coefficient calculated on the entire growth period 53

10 Analysis of variance of the growth rates with second generation diets. Growth time equals the entire growth period 54

11 Effect of various test diets on second gener­ ation roaches whose parents were fed test diets 1-8 55

12 Second generation mortality through eight weeks 57

13 Weights and numbers of cockroaches used for riboflavin determinations 87

vii viii

Table Page 14 Weights and numbers of cockroaches used for niacin determinations 88

15 Weights and numbers of cockroaches used for blotin determinations 89

16 Weights and numbers of cockroaches used for folic acid determinations 90

17 Total quantity of four vitamins in the test cockroaches 92 INTRODUCTION

The purpose of this In vestigation was to deter- mine some of the effects of various concentrations of

penicillin administered ora lly to the German cockroach,

Blattella germanica (L.), (Orthoptera: Blattidae).

Penicillin was developed during World War II, at a time when the demand for chemotherapeutic agents was

great. Although it had beeb discovered by Fleming in

1929, it was of little more than academic interest through 1938. In 1940, the studies of Chain, Florey, et al., reestablished interest in the material, and by

1944, It was being produced on a large scale. Many other antibiotics were soon discovered and eventually

produced and used commercially.

Waksman (1951) defined an antibiotic as follows:

n An antibiotic Is a chemical substance, produced by microorganisms, which has the capacity to Inhibit the growth and even destroy bacteria and other microorganisms, in diluted solutions.” i At first, the most important use of antibiotics was In the treatment of hun^an and domestic animal dis­

eases of bacterial origin.

Later, it was discovered that addition to the diet

of domestic animals of sma]j.l amounts of several of the more common antibiotics of1?en Increased the growth rate 2

This appeared to be especially true when the animal was not reared under optimal conditions of nutrition and

sanitation. Now, antibiotics are also being used to

some extent to stimulate crop production, and to control

plant diseases incited by microorganisms. Work is in

progress to determine their potentialities in food preser­ vation.

One of the most important properties of many anti­ biotics, and especially penicillin, is its apparent non­

toxicity both to animals and to cells in tissue culture.

This was noted even with the earliest and crudest prepar­ ations of penicillin. Up to two grams per kilogram of

crystalline sodium penicillin G have been injected into mice without mortality or ill effect. Guinea-pigs are much more susceptible, the LD50 being about forty milli­

grams per kilogram.

In most species of animals tested, there has been

little evidence that penicillin produces a cumulative

toxic effect. Dosages of over ten grams per day, given

to humans by Intravenous or intramuscular Injection over

a period of sixteen days, and over sixty grams per day

for periods of a week have been reported to produce no

ill effects in the patient. Penicillin apparently has

little or no effect on leucocytes and normally does not

interfere with phagocytosis. In the past, the only tissues known to be damaged by contact with excessive penicillin dosage were nerve tissues and tissues of the eye. However, with the more widespread use of penicillin, many other deleterious effects have been reported. These include allergy, serum sickness, anaphylactic shock, cardiovascular and renal difficulties, and irritation of mucous membranes. Evi­ dence points toward penicillin as a cause of a variety of disorders including agranulocytosis, periarteritis nodosa, anorexia, and the destruction of certain vitamins or the production of symptoms of hypovitaminoses. Most of these adverse conditions arise only after prolonged treatment at high dosage levels.

Thus, penicillin has been found to incite a variety of effects on many biological organisms. However, since very little such work has been done with insects, it was decided to determine if there were effects at both high and low concentrations on a common insect, the German cockroach.

This research was divided into two major parts.

Part I Involved the effects of fifteen different dietary concentrations (6-100,000 ppm.) of penicillin to the growth rate, time to maturity, fecundity, and mortality

In two successive generations of the cockroach. The insects were placed on the test diets soon after hatching, and, therefore, received the antibiotic all of their lives. The first generation was studied to ascertain immediate effects, and the second generation for latent or other possible effects.

Part II involved the assay of the total quantity of four B complex vitamins within the cockroaches. This was done to ascertain whether the various concentrations of penicillin effected them through loss or sparing action.

The B vitamins tested were biotin, riboflavin, folic acid, and niacin. Determinations were done on whole roach tissue by microbiological assay. PART Is EFFECTS OF DIETARY PENICILLIN ON THE BIONOMICS OF THE GERMAN COCKROACH

LITERATURE REVIEW

Recently, a great number of papers have appeared In the literature on accelerated growth Induced by the ad­ dition of small amounts of antibiotics to the diets of domestic and laboratory animals. Penicillin was one of the antibiotics used. These papers have been excellently reviewed by Jukes and Williams (1953), Stokstad (1954), and

Jukes (1955). Extensive reference lists accompany each of these reviews.

Adverse effects Induced by antibiotics in humans and domestic animals are discussed by Pratt and Dufrenoy (1953), and Flippin and Eisenberg (1955).

In comparison with the numerous studies on domestic animals, very little work has been reported on the effects of antibiotics on insects.

Some insect diseases have been controlled by the use of antibiotics. Haseman and Childers (1944) reported successful control of American foulbrood, a bacterial in­ cited disease of the common honeybee, with sulfa drugs.

Katznelson, et al. (1952) studied the possibility of con­ trolling European foulbrood, a similar bee disease, with several antibiotics. They found streptomycin, aureomycin, and terramycin prevented the disease while Chloromycetin 6- appeared to increase its severity. Moffett (1953) fed nine different materials to European foulbrood diseased colonies to determine their effect on the disease. The colonies fed dihydrostreptomycin and streptomycin showed a highly significant recovery rate over the untreated colonies. Terramycin was also effective. The use of the other six materials (penicillin, sulfone, diamidin, promin, aureomycin, and aureofac) resulted in no significant

improvement.

Some effects of antibiotics on intracellular symbi­

onts have been studied in cockroaches. Brues and Dunn

(1945) attempted to destroy the intracellular bacteroids

of the Florida cockroach, Blaberus cranilfer Burmeister, by injection of sublethal doses of penicillin and certain

sulfa drugs. The penicillin, in large doses, killed many

of the bacteroids, but also killed the cockroaches. Since

death of the cockroaches did not occur for several days,

the authors concluded that death of the bacteroids was

responsible for the death of the cockroaches, rather than

a direct effect of the antibiotic itself on the insect.

Glaser (1946) reported lethal effects on the American cock­

roach, Periplaneta amerlcana (L.), when penicillin was in­

jected into the body cavity or when the cockroach was held

at 39°C. for several days. These lethal effects were not

observed during prolonged treatment with sodium sulfa- thiazole in the cockroaches1 drinking water. The author concluded that the penicillin and heat exerted a direct toxic effect on the insect, although the bacteriods were killed by all three treatments.

Brooks and Richards (1955) obtained bacteroid free nymphs of the German cockroach by feeding their parents a dog food diet containing 1,000 ppm. of aureomycin all of their lives. The bacteroids were not eliminated from the parents, but were absent from their offspring. The

Maposymbiotic" (without intracellular bacteroids) nymphs were practically incapable of growth on a natural diet which was adequate for nymphs containing bacteroids.

Higher concentrations of aureomycin resulted in excessive mortality.

Steinhaus and Bell (1953), in attempting to obtain intracellular bacteroid-free stored grain beetles by adding antibiotics to the grain, found Chloromycetin, penicillin G, polymixin B, streptomycin, and terramycin to be highly toxic. At levels of 20,000-36,000 ppm., 100 percent of the beetles were dead in 30-60 days. De (1956) reported that streptomycin administered in the diet of stored grain beetles at 200-1,000 ppm. had no apparent effect. If anything, it extended the length of life.

Liles and Fisk (1955) reported the toxicity of sever­ al antibiotics to the German cockroach by injection. There 8 was a wide range of effects with polymyxin the most toxic and penicillin the least.

Mengle and Fisk (in press) fed adult female German cockroaches high concentrations of 11 antibiotics, five of which produced little or no mortality even at 20 per­ cent of the weight of the diet. The other six produced significant mortality when fed at the same level. MATERIALS AND METHODS

Test Insects

The insects used in this work were from a normal

(non-insecticide-resistant) strain of the German cock­ roach, Blattella germanioa (L.). The strain has been reared for several years in the insect rearing room of the Department of Zoology and Entomology, The Ohio State

University, Columbus, Ohio. During this period the cock­ roaches were reared in large, cut off, glass jars. They received tap water from plastic cups with dental wicks inserted through holes in the lids, and micromixed pulver­ ized Purina dog checkers for food.

Test Rearing Methods

Because it was desired to feed the cockroaches the special diets all their lives, it was necessary to obtain first instar nymphs. To obtain these first instar nymphs, approximately 25 capsulated female cockroaches were placed in each of two large glass jars.

These jars were prepared from five gallon jars by cutting off the tops about one-half the way up with an electrically heated wire. The top two inches of each jar was coated oh the inner surface with a mixture of petrole­ um jelly and mineral oil to prevent escape of the females

and their offspring. In addition, the jars were covered with two layers of cheesecloth and secured with rubber 10 bands to prevent escape or contamination with other cock­ roaches.

The cockroaches in these jars were provided with dis­ tilled water from a large vial plugged with absorbent cotton and a diet consisting of equal parts of vitamin free casein, dextrose, and powdered non-nutritive cellulose.

The first instar nymphs received no other nutrients until placed on the test diets.

In three or four days, when most of the eggs from these females hatched, the jars were placed in a deep freeze until the cockroaches were inactive. The adult females were then removed and destroyed, and the young were removed by use of a wet camel’s hair brush. Groups of approximately 35 were placed in individual pyrex crystal­ lizing dishes.

These dishes were five and three-quarters inches in diameter and three inches deep. The top one inch of the

inner surface of these dishes was also coated with the

petroleum jelly-mineral oil mixture. The dishes were then covered with cheese cloth secured with rubber bands.

A few hours later, the cockroaches were again chilled

and each group was weighed. This was done to provide uni­

form groups of cockroaches as to initial weight. Weighings were performed with a Roller-Smith Torsion Balance graduated

from 1-1,500 milligrams. In these and further weighings, I

11.

the cockroaches were picked up individually by the an­

tennae with a fine forceps, and placed on the weighing

pan. In addition, each group was reduced to 30 individuals.

Following the weighings, distilled water and one of the

prepared diets were added to each dish. Two replicates were

used with each test diet. The distilled water container was

a vial three inches long and one inch in diameter, plugged

at the open end with absorbent cotton. The food container

was a vial two inches long and one-half inch in diameter.

Rearing Conditions

The dishes were placed in a large constant tempera­

ture -humidity box set at 27±1° C. and approximately 73

percent relative humidity. The humidity was controlled

by placing two three-quart capacity jars filled with satu­

rated sodium chloride solution into the cabinet. Water

was added to the jars as necessary. The insects were kept

in the dark except when subsequent weekly weighings were

made.

Weekly Weighings

The insects were weighed at weekly intervals from the

first instar to the adult stage. When weighed after the

initial weighing, the insects were anesthetized with carbon

dioxide. New food and water were added each week at the

time of weighing. 12

Diets

The basic diet used throughout this study wa3 micro- •1 mixed, pulverized "Purina Dog Chow Checkers", manufactured by the Ralston Purina Company of St. Louis 2, Missouri.

Sixteen diets were prepared, fifteen of which contained a different concentration of penicillin. The quantity of penicillin in the diets ranged from six to 100,000 parts per million of the total diet.

The preparation of the diets was as follows: (1) approximately 500 grams of the dog food was placed in a large beaker and mixed thoroughly; (2 ) for each diet a definite quantity (see Table l) of the dog food was weighed and placed into a small, wide mouth bottle to which was also added a small but definite quantity of Alphaeel

(powdered, non-nutritive cellulose) (see Table 1) purchased from the Nutritional Biochemical Corporation, Cleveland,

Ohio; (3) the appropriate quantity of sodium penicillin G

(see Table 1) was dissolved in five milliliters of water

■** Analysis of "Purina Dog Chow Checkers"-

1. Crude protein ----- not lessthan 24 percent 2. Crude fat ------not less than 5 percent 3. Crude fi b r e ----not more than 5 percent 4. Non-fattening matter------not less than 46 percent 5. Ash ------not more than 10 percent

Tiiis is the analysis of the dog food as reported by the manufacturer. 13 and added to each diet* Dilutions of a stock solution were used in the small concentrations for greater accuracy and convenience; (4) after the addition of penicillin, each diet was mixed thoroughly and dried in a vacuum oven for

24 hours* The oven was operated at room temperature to avoid destruction of the antibiotic. (5) After drying, each of the various diets was again thoroughly mixed by grinding in a mortar and pestle. The prepared diets were stored in a deep freeze until needed.

Table 1 Composition of the diets employed in the rearing experiments.

Diet Grams Grams Grams Ppm. Grams No. Dog Pood Alphacel Penicillin Pen./Diet Total

l(ck) 18.0 2.000 0.000000 0 20.000 2 18.0 2.000 0.000122 6 20.000 3 18.0 2.000 0.000244 12 20.000 4 18.0 2.000 0.000488 25 20.000 5 18.0 2.000 0.000976 50 20.001 6 18.0 1.990 0.001953 100 19.991 7 18.0 1.990 0.003906 195 19.994 8 18.0 1.990 0.007812 390 19.998 9 18.0 1.980 0.015625 781 19.995 10 18.0 1.968 0.031250 1562 19.999 11 18.0 1.938 0.062500 3125 20.000 12 18.0 1.875 0.125000 6250 20.000 13 18.0 1.750 0.250000 12500 20.000 14 18.0 1.500 0.500000 25000 20.000 15 18.0 1.000 1.000000 50000 20.000 16 18.0 0.000 2.000000 100000 20.000 14

Teat Insects Used In Vitamin Assays and In Second Generation Studies

For vitamin assay work, the first ten to fifteen male

and female cockroaches to reach the adult stage on each

particular diet were collected, and immediately preserved

in a deep freezer at -15° C. until the assays were per­ formed.

In preparing the test for the second generation

studies, three principal difficulties were encountered:

1 ) the first generation female cockroaches reared on any

particular test diet matured at different times and there­

fore produced egg capsules at different times; 2 ) the egg

capsules produced by these females contained varying

numbers of viable embryos; 5) as a result of the foregoing

the numbers of second generation first instar nymphs of the

same age available at any given time in any particular diet

group was limited. This influenced the number and repli­

cations of some (diet levels 8-15) second generation tests.

The offspring from each first generation diet were

reared on both the control diet and the diet of their re­

spective parents. It was desired to have two replicates

of these two diets. In all cases, it was not possible to

obtain enough suitable insects for two replicates per diet.

In such cases, only one was employed.

The following method was employed to obtain first

instar nymphal cockroaches for the second generation 15

studies. After the samples for vitamin assay work were

removed, the remaining cockroaches, which reached the adult

stage on the test diets, were allowed to mate and produce

egg capsules, if any. Then, first generation female cock­

roaches with darkly pigmented egg capsules were removed a3

they became available in each test group. The dark pigmen­

tation indicated that the eggs would soon hateh. Two to

four such females from each diet were placed in each of

four clean crystallizing dishes; two containing the check

diet and two containing the respective parental diet on which the young were to be reared.

When the eggs hatched, the adult females were removed,

and the number of offspring in any one dish was reduced to

thirty. The initial weights of the young were then re­

corded.

The remainder of the second generation rearing pro­

cedures were the same as those used for first generation

studies. However, fresh diets, identical to those employed

for the first generation were prepared for the second gener­

ation studies.

Penicillin Used

"Penicillin,” as produced by customary mold culture

with Penicillium notatum Westling and P. chrysogenum Thom

(Ascomycetes: Aspergillaceae), is not a single compound,

but consists of a mixture of about twelve different com- 16 pounds of closely related structure. However, in nature, only five or 3ix of these are produced in abundance.

The structure of a penicillin consists of a mono- carboxylic acid with beta lactam and thiazolidine rings attached through a -CONH- linkage to a prosthetic group designated as R. The R group differs in the various peni­ cillins. The six most common are named F, G, X, dihydro F, and K. Penicillin G has become recognized as the most satisfactory for clinical U3e. The salts of penicillin G are more stable than the acid itself, and the sodium salt has proved to be the most satisfactory and least toxic.

The generalized structure of a penicillin is given below, along with the formulae of the various commonly encountered penicillins.

6H3

C — N C—COOH

0 H

Penicillin

In the various penicillins, R is:

Penicillin F: CH3-CHg = CH-CHg-, 2 pentenyl

Penicillin dihydro F: CH^CHg-CHg- CHg-CHg-N- amyl

Penicillin G benzyl

Penicillin K: CH3 (CHg)6H!I- heptyl 17

Penicillin X: H0--CHgrP- hydroxybenzyl.

The crystalline sodium salt of penicillin G, used in these experiments, was produced by the Sharp and Dohme

Division of Merck and Company, Inc., Philadelphia. The formula for this salt is given below:

H 0 H H ^ 1 II i 1 _ C— C— N— C— C— H C— CH

H C — N C— COONa

Na Penicillin G. EXPERIMENTS AND RESULTS

Studies of Effect of Penicillin on Growth Rates, First Generation

As previously stated, fifteen diets containing differ­ ent quantities of penicillin plus a check containing no penicillin were prepared. Two replicates of 30 German cockroaches each, were set up on each test diet. The insects were weighed each week from the initial weighing to the adult stage.

Cockroaches, even from the same egg capsule and reared under apparently identical conditions, reach the adult stage in different lengths of time. Because record­ ings of the weights of replicates containing both nymphs and adults would be of little value in determining the growth rate during the developmental period, it was neces­ sary to designate a particular point as the ”adult stage”.

Therefore, when 10 percent of the individuals in a test had actually reached the adult stage, or when the average weight per roach exceeded 42 mg., the whole group was con­ sidered to have reached the ”adult stage”. In following this procedure, it was found that approximately 40-50 per­ cent of the insects would actually reach the adult stage in the following week.

The results of this experiment in average weight in milligrams per cockroach per week, are recorded in Table 2.

18 Table 2 Weights of First Generation Roaches on Test Diets.

Week . Avg.. Wt. in Mg./ Roach Diet Number

One (check) Two Three Four A B Avg. A B Avg. A B Avg. A B Avg.

Initial 1.44 1.30 1.37 1.30 1.36 1.33 1.33 1.30 1.32 1.28 1.40 1.34 1 2.81 2.38 2.59 2.34 2.77 2.56 2.73 2.20 2.47 2.35 2.57 2.46 2 5.07 4.50 4.78 4.62 4.93 4.78 4.74 4.53 4.67 4.42 5.00 4.72 3 7.00 7.36 7.19 6.93 6.83 6.88 6.93 6.65 6.80 7.23 7.81 7.53 4 8.23 7.82 8.01 7.82 8.63 8.24 8.17 7.85 8.01 7.35 8.41 7.88 5 14.50 14.07 14.27 13.81 14.57 14.20 13.90 13.75 13.83 14.20 14.92 14.56 6 20.79 20.59 20.68 19.92 20.54 20.24 20.69 17.13 19.07 19.72 19.31 19.50 7 25.50 27.33 26.47 24.88 26.96 25.94 27.50 23.88 25.86 27.44 26.62 27.01 8 41.25 41.48 41.37 39.65 38.69 39.17 38.62 36.83 37.81 44.00 42.58 43.27 9 46.71 50.15 48.52 46.65 44.61 45.63 49.46 37.79 44.07 49.04 47.46 48.23 10 # ■w V V' 55 * W 55 * V 55 V * X 55 * V & 55 * 55 X 55 11 V V W if W V * 55 A* ■if V w V 12 V 13 ■x- v V V 55 55 55 VT ■Vf w w # 14 * 55 55 55 55 V 55 * 'a" V w u «/ V V 15 "W V* 55 55 A* w 55 A 55 55 # 16 * 55 55 55 V 55 55 55 55 55 V 17 55 -51 55 55 55 55 ■55* 55 55 55

& No records because the cockroaches had reached the ttadult stage”. Table 2 Weights of First Generation Roaches on Test Diets Continued

Week Avg,. Wt. :in Mg./ Roach Diet Number

Five Six Seven Eight A B Avg. A B Avg. . A B Avg. A B Avg.

Initial 1.36 1.33 1.35 1.34 1.33 1.34 1.27 1.39 1.33 1.33 1.33 1.33 1 2.60 2.63 2.62 2.58 2.63 2.61 2.22 2.61 2.43 2.43 2.43 2.43 2 4.80 4.40 4.60 4.71 4.79 4.75 4.31 4.62 4.47 4.57 4.55 4.56 3 7.31 6.83 7.07 6.96 7.18 7.07 6.73 6.68 6.70 7.46 6.90 7.18 4 8.52 7.43 7.96 8.57 8.07 8.32 7.65 7.86 7.75 8.25 8.45 8.35 5 14.41 13.21 13.78 14.00 14.67 14.33 13.96 13.85 13.90 14.52 14.11 14.31 6 17.37 20.38 18.92 20.15 19.89 20.01 18.08 19.04 18.57 19.07 19.30 19.18 7 28.26 27.62 27.92 26.93 25.26 26.09 26.84 25.89 26.34 27.30 25.30 26.29 8 37.89 25.59 31.52 40.67 38.59 39.62 38.04 39.26 38.67 41.04 36.37 38.70 9 45.70 42.00 43.53 46.59 46.96 46.77 47.04 43.19 45.03 42.78 42.64 42.71 10 it Vc if «■ it * it V a if * it At At 11 if V it it V * it V* a- it Vf it it it if it it -> it w # it it it 12 At 13 if it if it it * V V ■v w it 14 if it VT it w ye it it it 15 it * a it * if it yr * if it if M At 16 it * 7* "W it it V w VT if 17 it m&m V V it it * V it V it

No records because the cockroaches had reached the "adult stage". Table 2 Weights of First Generation Roaches on Test Diets Continued • Week Avg. c+ in Mg./ Roach Diet Number

Nine Ten Eleven Twelve A B Avg. A B . Avg. A B Avg. A B Avg.

Initial 1.40 1.33 1.37 1.33 1.30 1.32 1.37 1.29 1.33 1.27 1.40 1.33 1 2.83 2.41 2.62 2.47 2.41 2.44 2.52 2.25 2.38 2.27 2.69 2.47 2 4.46 4.41 4.44 4.69 4.72 4.70 4.37 4.14 4.25 4.53 4.57 4.55 3 6.81 6.83 6.82 6. S3 6.21 6.52 6.56 6 . 32 6.44 6.57 6.71 6.64 4 7.67 7.59 7.62 7.83 7.97 7.89 7.11 7.29 7.20 7.50 7.89 7.68 5 13.15 13.15 13.15 13.75 12.79 13.26 11.46 12.26 11.86 12.89 12.70 12.80 6 19.96 14.00 17.52 16.57 15.07 15.32 13.96 13.70 13.82 13.82 15.33 14.56 7 25.31 21.56 23.77 23.21 22.33 22.78 17.76 18.89 18.34 20.21 19.26 19.74 8 17*46 24.77 20.45 34.14 27.30 30.78 25.50 23.33 24.35 25.93 27.30 26.60 9 16.83 33.22 25.03 38.63 32.78 35.70 29.04 27.11 28.01 29.46 30.93 30.18 10 36.75 37.38 37.08 44.33 38.93 41.62 35.17 32.44 33.72 35.43 36.00 35.70 11 54.50 49.78 52.00 50.81 42.22 46.43 40.30 37.38 38.75 40.85 39.64 40.26 12 n- it it it it it V V it ,w. it 13 a it * it it it it a it it it it 14 a a V it a it & it it it a */ 15 it it # it it % a it it it it «/ 16 it a V it a it it it it it it »/. 17 it it V* it it V it a it it it

W No records because the cockroaches had reached the “adult stagen. Table 2 Weights of First Generation Roaches on Test Diets Continued

Week Avg.. Wt. in Mg./ Roach Diet Number

Thirteen Fourteen Fifteen Sixteen A B Avg. A B Avg. A B Avg. A B Avg.

Initial 1.33 1.33 1.33 1.33 1.34 1.34 1.37 1.37 1.37 1.41 1.36 1.39 1 2.53 2.79 2.66 2.45 2.45 2.45 2.48 2.41 2.44 2.79 2.48 2.64 2 4.30 4.17 4-24 4.45 4.61 4.53 4.57 4.31 4.42 4.24 4.10 4.17 3 7.00 7.00 7.00 6.48 6.14 6.31 6.43 6.03 6.20 6.15 6.25 6.20 4 7.83 7.76 7.79 7.62 7.59 7.60 7.61 7.50 7.54 7.15 7.14 7.14 5 12.55 12.25 12.40 11.70 11.54 11.61 11.91 10.42 11.03 10.35 10.36 10.36 6 14.28 14.71 14.49 13.73 13.46 13.59 13.64 11.94 12.64 11.40 11.32 11.36 7 19.38 20.50 19.92 18.62 19.68 19.16 18.14 18.38 18.28 15.60 14.24 14.92 8 24.61 26.32 25.46 22.50 22.74 22.62 21.00 18.83 19.71 16.64 17.36 17.00 9 28.00 29.21 28.61 26.73 27.11 26.92 22.38 23.45 23.01 18.84 20.32 19.58 10 31.19 34.36 32.80 31.31 31.12 31.21 26.95 27.67 27.38 20.36 20.84 20.60 11 36.33 37.15 36.76 33.73 32.27 33.00 31.01 30.65 30.82 23.68 22.92 23.30 12 V * * 40.96 36.65 38.76 33.80 35.34 34.70 26.48 26.08 26.28 13 V it * 46.36 41.52 44.05 39.52 44.66 42.50 29.72 30.24 30.00 14 # * * * V it it it it 34.79 32.00 33.36 15 * it a a it w it 37.43 33.84 35.56 16 a- * ■it *- * a it 34.00 38.00 36.00 V V 17 0* * * it a * V* * it 35.42 39.29 37.50

No records because the cockroaches had reached the "adult stage”. 23

The growth curves at the various levels of dietary penicillin concentration are presented in Figures 1-5.

Figure 1 compares the check with the first half of the test diets, while Figure 2 compares the check with the last half of the test diets. From Figures 3 and 4, it may be seen that, between the fourth and fifth weeks of rearing, there was apparently a beginning of inhi­ bition of growth in the cockroaches fed diets containing the higher levels of penicillin. Figure 5 represents the differences in weight at nine weeks. At this time, the cockroaches reared on the check diet reached the "adult stage".

At this point, a statistical analysis of the growth rates of the cockroaches on the different diets was per­ formed to answer this question: were the growth rates of the cockroaches on all the various diets equal or not equal?

By plotting the average weight in milligrams per cockroach against week, it was possible to calculate

the regression coefficient of each of the growth curves.

However, since the growth curves did not produce straight

lines on the arithmetic 3cale, a transformation which

would produce straighter lines was sought. A log-log

transformation was found to produce the desired results.

(continued on page 32) 24

Figure 1 Growth curves produced by the check and first seven diets in the first generation studies. Be­ cause the lines are close together and overlap consider­ ably until the eighth week, the range covered by the curves up to this point is filled in to avoid confusion. l-CK. ROACH AVG. AVG. WT. INI MG.

WEEKS 26

Figure 2 Growth curves produced by the check and the last eight diets in first generation studies. Be­ cause the lines are close together and overlap consider­ ably until the fourth week, the range covered by the curves up to this point is filled in to avoid confusion. AVG. WT. IN MG. / ROACH o

OJ

cn

O)

o d

(O o ru

CD Ol Avg. Vt. in mg./Roech 10^ C. Levels (Ck.) L 4 6 8 1 1 I I 1 1 16 15 14 IS IP 11 10 9 8 7 6 5 4 o L 1 GROWTH OF THE GERMAN ROACH AT 4 WEEKS, ON A ON WEEKS, 4 AT ROACH GERMAN THE OF GROWTH IT OTIIGDFEET AMOUNTS DIFFERENT CONTAINING DIET OF PENICILLIN OF 28

GROWTH OF THE GERMAN ROACH AT 5 WEEKS, ON A DIET CONTAINING DIFFERENT AMOUNTS OF PENICILLIN

1 2 '6 4 5 6 7 6 9 10 11 12 12 14 15 16 (Ck.) Levels Avg. Wt. in ing./Roech C. Levels (Ck.) GROWTH OF THE GERMAN ROACH AT 9 WEEKS, ON A ON WEEKS, 9 AT ROACH GERMAN THE OF GROWTH IT OTIIGDFEET AMOUNTS DIFFERENT CONTAINING DIET OF PENICILLIN OF iue 5 Figure 30

31

Table 3

Regression Coefficients of the Sixteen Treatments Each Coefficient Calculated on the Entire Growth Period (Log Wt. on Log Week)

Diet No.______Replicate A.______Replicate B______

1 1.5340 1.6087

2 1.5886 1.5465

3 1.5758 1.5372

4 1.6296 1.5642

5 1.5480 1.5054

6 1.5776 1.5610

7 1.6083 1.5379

8 1.5761 1.5517

9 1.3789 1.4808

10 1.5493 1.4792

11 1.4075 1.4191

12 1.4567 1.4100

13 1.3867 1.4035

14 1.3997 1.3738

15 1.3182 1.3455

16 1.1785 1.2211 32

The regression coefficients for the whole growth

curves were then calculated from the values obtained by

plotting the logarithm of the average weight in each week

against the logarithm of the week. These regression coef­

ficients are an estimate of the rate of gain per week on

each diet. The results are listed in Table 3.

Next, an analysis of variance was used to estimate whether the rates of gain produced by all the treatments were equal or not equal. See Table 4. The results of the

test indicated that the growth rates were not the same.

Table 4 Analysis of variance of the growth rates with the sixteen diets. Growth time equals the entire growth period. Test based on two replications of 30 roaches each, per treatment.

Source SS DF MS F F - 5 < j Z F-1J&

Treatments .3758 15 .02505 9.1221** 2.35 3.41

Error .0209 16 .00131

Total .3967 31 *# Significant at 1% level.

Because the raw data indicated that growth in all the

diets was approximately equivalent through four weeks of

rearing, regression coefficients were also calculated for

this period, alone. These are listed in Table 5.

An analysis of variance was then performed to see if

the growth rates were equal through four weeks. See Table

6. (continued on page 34) 33

Table 5

Regression Coefficients of the Sixteen Treatments Calculated from Zero through Four Weeks of Growth (Log Wt. on Log Week)

Diet No.______Replicate A______Replicate B_____

1 1.1234 1.1936

2 1.1824 1.1306

3 1.1634 1.1824

4 1.1732 1.1913

5 1.1880 1.1214

6 1.1886 1.1751

7 1.1834 1.1188

8 1.2030 1.1916

9 1.0923 1.1444

10 1.1585 1.1581

11 1.0814 1.1335

12 1.1702 1.1099

13 1.1532 1.1309

14 1.1299 1.1125

15 1.1102 1.0869

16 1.0305 1.0719 Table 6 Analysis of variance of the growth rates with the sixteen diets. Growth time equals four weeks. Test based on two replications of 30 roaches each, per treatment*____

Source SS DP MS F F-5# F-lg

Treatments .0410 15 .002733 3.016* 2.35 3.41

Error .0145 16 .000906

Total______.0555___51______* Significant at 5$.

The results of the test indicated that the growth

rates were not the same at four weeks. However, the sig­ nificant difference was small, and present only at the

five percent level. Therefore it is concluded that, as

shown graphically in Figures 4, 5, and 6, the growth rates

did differ throughout, but most noticeably from the fifth week on.

The differences between the individual treatments and

the check were tested for significance by the method of

Least Significant Difference (L.S.D.). Difference was not

encountered with diets 2-8, but was encountered with diets

9-16. From this, it was concluded that growth was in­

hibited if the diet contained 780 or more ppm. of peni­

cillin (levels 9-16).

Time to "Adult Stage”

Figure 6 represents graphically the time in weeks in

which the cockroaches reached the "adult stage”, when

reared on the different diets. (continued on page 36) 35

TIME IN WHICH THE GERMAN ROACH REACHED THE ADULT STAGE, WHEN REARED ON A DIET CONTAINING DIFFERENT AMOUNTS OF PENICILLIN 36

Again, no increase In time occurred unless the diet con­

tained 780 or more ppm. of penicillin.

Mortality

The mortality in the various levels is given in

Table 7 and Figure 7.

There appeared to be little difference in mortality

in the various treatments with the exception of diet 9.

It appeared as if the cockroaches in that treatment became

diseased. Considering that the insects were anesthetized

and handled once a week from the time of hatching to the

adult stage, mortality appeared to be low throughout the

experiments.

Reproductive Capacity

Reproduction appeared to be decreased by all test

diets containing 200 or more ppm. of penicillin (diet3 7-

16). This reduction was estimated to be around 40-50 per­

cent at 200 ppm. (diet 7), and Increased to about 99 per­

cent at 50,000 (diet 15).

The egg capsules produced by the females at these

levels were smaller than normal, and commonly misshapen.

Many egg capsules were dropped prematurely, with none of

the eggs hatching. In addition to being shriveled, some

of the capsules were blackened and the embryos inside were

dead. Figure 8 illustrates several of the deformed egg

capsules compared with normal ones. (continued on page 40) Table 7 Mortality in the First Generation, through Nine Weeks

Initial Nos. Total Alive % Mortality Avg. % Mortality Level No. A B____ A B A B

1 27 30 25 28 7.4 6. 6 7.0 2 30 30 27 27 10.0 10.0 10.0 3 30 30 29 25 3.3 16.7 10.0 4 29 30 26 27 10.3 10.0 10.2 5 30 30 28 30 6.7 0.0 3.3 6 29 30 28 28 3.5 6.7 5.1 7 30 31 26 28 13.3 9.7 11.5 8 30 30 28 26 6.7 13.3 10.0 9 30 30 19 19 36.6 36.6 36.6 10 30 30 28 28 6.7 6.7 6.7 11 27 28 25 28 7.4 0.0 3.6 12 30 30 29 28 3.3 6.7 5.0 13 30 30 28 29 6.7 3.3 5.0 14 30 29 27 28 10.0 3.5 6.8 15 30 30 24 30 20.0 0.0 10.0 16 29 30 26 26 10.3 13.3 11.9 % Mortality 10 15 20 25 30 35 40 0 5 CK. 2 1

4 6 7 6 5 4 3 MORTALITY IN VARIOUS THE MORTALITY EES 1t GENERATION, 1st.LEVELS, 58 9th. WEEK iue 7 Figure Levels 8

1 11 31 5 161514131211 10 9

39

Figure 8 Normal egg capsules on the left. Deformed egg capsules on the right. 40

No egg capsules were produced "by females receiving

diet 16, containing 100,000 ppm. of penicillin.

Studies of Effect of Penicillin on Growth Rates, Second Generation

All rearing procedures used in the second generation were the same as those used in the first generation studies.

However, the second generation nymphs employed in these

tests were not all of the same age, because, due to differ­

ences in the growth rate of first generation cockroaches,

the egg capsules matured and nymphs hatched at different

times. Because of this variation in time of hatching, the

first recorded weighings are for approximately one week old

insects.

Some offspring of adults reared on diets 1-8 (0-400

ppm. penicillin) were reared on the check diet, while

others were fed the diet of their respective parents. The

results of this experiment in average weight in milligrams

per cockroach per week, are given in Table 8.

The growth curves of second generation nymphs are

presented in Figures 9-13. Figure 9 represents, graphically,

the results of rearing on the check diet the offspring from

first generation cockroaches fed initial diets 1-8. Figure

10 represents, graphically, the results of rearing on the

diets of their respective parents the offspring from first

generation cockroaches fed initial diets 1-8. (continued on page 49) Table 8 Weights of Second Generation Roaches on Test Diets

Week Avg. Wt . in Mg./ Roach Diet Number

Onei (check) Two Twof A B Avg. A B Avg. A B Avg. Initial « 1 2.30 2.57 2.43 2.30 2.10 2.20 2.35 2.45 2.40 2 3.63 3.63 3.63 3.56 3.35 3.46 3.21 3.34 3.32 3 6.75 6.82 6.80 8.00 7.92 7.96 8.10 7.90 8.00 4 9.79 11.21 10.50 12.56 11.59 12.06 10.05 10.69 10.37 5 17.46 15.00 16.23 16.24 18.02 17.13 14.60 16.16 15.38 6 24.05 25.35 24.70 23.27 24.97 24.12 22.30 24.08 23.44 7 31.68 35.84 33.76 32.31 33.75 33.03 30.71 33.79 32.25 8 42.00 47.72 44.86 43.40 42.10 42.75 40.00 41.80 40.90 9 ■Z if ■if it # * * ij- «• 10 * it if * it it ■* if 11 * if if if it if it * 12 * * V it * if i* V if « * w it if # it * 13 ** 14 •it V if * * * 15 'S it * if V if if V*

» Indicates use of parental diet. The check diet was used for the others. * No records because the cockroaches had reached the ”adult stage”. Table 8 Weights of Second Generation Roaches on Test Diets Continued

Week Avg.. Wt. :in Mg./ Roach Diet Number

Three Three » Four Four t ' A B Avg. A B Avg. A B Avg. A B Avg.

Initial «■» «• mm mm 1 2.73 2.43 2.58 2.50 2.73 2.61 2.40 2.78 2.60 2.11 1.97 2. 03 2 3*67 3.45 3.55 3.55 3.38 3.46 3.08 3.08 3.08 2.68 2.67 2.68 3 7.47 5.37 6.41 6.93 6.00 6.45 4.45 4.17 4.30 6.33 8.40 7.42 4 9.90 9.83 9.86 10.10 9.47 9.77 10.55 11.60 11.08 7.89 12.60 10.36 5 13.87 14.27 14.06 17.45 15.23 16.32 16.03 16.20 16.11 12.33 18.30 15.47 6 23 * 13 21.47 22.30 21.90 21.33 21.61 21.38 20.02 20.69 20.22 26.10 23.31 7 29.23 29.53 29.38 32.86 28.24 30.55 34.31 32.60 33.44 26.96 33.93 20.57 8 41.55 34.07 37.74 37.07 34.17 35.62 48.72 47.00 47.80 35.85 46.76 41.50 9 46.72 45.33 46.01 53.57 47.83 50.65 2- * a it « V V 10 a VV 7* * .y.n at it • if v 11 it a * V w it * it it V u u V Kf 12 it V V V it V it V V V V M 13 V it V_v * V* it ** it V 14 V* * w V it * W it * u \A ** 15 w it w V it it V * a" *

» Indicates use of parental diet. The check diet was used for the others. * No records because the cockroaches had reached the nadult stage”. Table 8 Weights of Second Generation Roaches on Test Diets Continued

Week______Avg. Wt. In Mg./ Roach Diet Number

Five Five 1 Six Six* A B_ ___ Avg. A B Avg. A B Avg. A B Avg.

Initial ------1.13 1.16 1.14 mm - - 1 1.86 1.86 1.86 2.10 2.10 2.10 2.06 2.76 2.48 2.23 1.4 6 1.87 2 2.57 2.33 2.70 2.93 3.19 3.06 3.94 4.34 4.19 3.20 2.77 2.98 3 4.46 4.69 4.57 4.72 4.42 4t. 56 6.33 6.14 6.21 4.28 5.04 4.66 4 7.50 7.76 7.63 7.97 7.73 7.34 8.44 7.79 8.04 6.17 7.81 7.04 5 8.82 9.45 9.14 7.93 8.23 8.08 12.39 12.22 12.28 7.70 8.15 7.93 6 12.14 14.14 13.14 12.79 11.67 12.20 17.39 16.89 17.08 10.26 13.27 11.85 7 16.29 19.10 17.71 15.79 13.87 14.79 11.33 21.30 17.31 11.87 13.50 12.73 8 20.04 23.83 21.96 21.36 20.60 21.20 22.22 28.30 25.86 15.57 18.46 17.10 9 30.68 32.31 31.50 31.54 28.37 29.88 31.94 36.30 34.55 18.96 19.27 19.12 10 37.25 37.66 37.27 38.21 35. 60 36.86 48.17 46.03 46.38 25.22 23.08 24.08 44.15 46.07 43.10 44.53 it * it 27.83 32.42 30.26 11 43.20 45.11 •s it it * V it V 37.74 38.69 38.24 12 a a u 13 it a a- i1 V it it 45.00 43.85 44.38 it 14 V '5 it V it it * a it W V H r 7 » * it 15 V it W it it it

* Indicates use of parental diet. The check diet was used for the others. # Ho records because the cockroaches had reached the ttadult stage”. Table 8 Weights of Second Generation Roaches on Test Diets Continued

Week Avg* Wt. in Mg./ Roach Diet Number

Seven Seven * Eight Eight » A B Avg. A B Avg. A B Avg. A B Avg.

Initial m ------1 1.71 2* 23 2.02 1.56 1.82 1.69 2.13 1.87 2.00 2.00 2.00 2.00 2 2.83 3.46 3.26 2.53 2.59 2.56 3.03 2.60 2.81 3.47 3.65 3.54 3 4.63 4.32 4.42 8.23 4.46 6.35 4.48 3.52 4.01 5.25 4.63 5.00 4 6.72 6.40 6.50 8.53 6.00 7.27 6.24 4.03 5.03 6.54 6.05 6.34 5 9.81 7.08 7.92 11.53 6.92 9.23 7.92 6.33 7.05 8.96 7.74 8.46 6 11.18 10.04 10.39 12.15 8.46 10.31 9.50 7.30 8.27 10.93 8.79 10.06 7 15.27 10.44 11.92 15.65 5.96 10.81 12.33 9.20 10.59 13.18 11.42 12.46 8 15.72 14.51 14.89 17.88 13.50 15.73 15.13 11.43 13.07 14.79 11.63 13.50 9 25.20 14.92 18.08 19.20 18.98 18.99 18.79 13.80 10.02 22.61 17.00 20.34 10 28.18 19.60 22.22 27.92 24.17 26.08 21.33 16.67 18.74 24.07 17.26 21.32 11 37.81 22.16 26.94 36.10 28.96 32.61 30.58 19.47 24.41 31.89 21.05 27.40 12 40.72 24.79 29.80 38.30 38.30 38.57 34.79 24.00 28.78 36.54 27.11 32.55 13 42.00 35.40 37.48 41.10 47.00 44.04 42.50 27.40 34.11 42.31 28.00 36.26 14 44.00 43.75 44.60 V- * * 38.13 29.40 33.27 50.31 33.95 43.40 15 ** * * % *■ 53.33 36.67 44.07 50.50 35.40 44.67

1 Indicates use of parental diet. The check diet was U3ed for the others. # No records because the cockroaches had reached the ”adult stage”. 45

Figure 9 The results of rearing on the check diet the offspring from first generation cockroaches fed initial diets 1-8* O) AVG. WT. AVG. IN MG. / ROACH

WEEKS Figure 10 The results of rearing on the diets of their respective parents the offspring from first gener­ ation cockroaches fed initial diets 1-8. 50

45

40 49

Figure 11 represents the growth of the second gener­ ation cockroaches at two weeks, on the control and the parental diets. Figure 12 represents the growth of the

second generation cockroaches at four week3, on the con­ trol and parental diets. A comparison of these two graphs

shows that inhibition possibly began between the second and fourth weeks of rearing.

Figure 13 represents the differences in weight at eight weeks of the second generation nymphs reared on the various diets. At that time, the second generation cock­

roaches which were reared on the check diet and whose

parents were also reared on the check diet reached the

”adult stage”.

In Figure 13, it appears that second generation cock­

roaches whose parents received 50 or more ppm. of peni­

cillin (diets 5-8), were inhibited in growth.

To test for the inhibition apparent in Figure 13, a

statistical analysis comparable to that performed on first

generation results was used on the second generation re­

sults. It was desired to answer this question: were the

growth rates of the second generation cockroaches on all

the diets equal of unequal?

As before, the regression coefficients of the growth

curves were computed prior to use of the analysis of

variance. (continued on page 54) Avg. Wt. in mg ./Roach 10 Figure 11 Slashed bars, represent cockroaches reared reared bars, cockroaches Slashed represent 11 Figure ersn ccrahs reai. cockroaches ’represent o;idlet» control the on the diets of their reap o — . parents. Solid bars bars Solid parents. o . reap their — of diets the on RWHO H n. EEAINRAHSA 2 AT ROACHES GENERATION 2nd. THE OF GROWTH CK. I I1 I1 1I 11 1 7. 1 I 1 1 6 i 1 5 II I4 1 3 1 2 I 1 WEEKS, ON THE CONTROL AND PARENTAL DIETS PARENTAL AND CONTROL THE ON WEEKS, 50 Levels _

B __ 1 Avg. Vt. in mg ./Roach 15 bars represent cockroaches reared on the control control the on reared cockroaches represent bars Figure 12 Slashed bars represent cockroaches reared reared cockroaches represent bars Slashed 12 Figure diet. on the diets of their respective parents. Solid Solid parents. respective their of diets the on GROWTH OF THE 2nd. GENERATION ROACHES AT 4 AT ROACHES GENERATION 2nd. THE OF GROWTH . 2 11 I 1.12 3 l II 4 5 ,1 | 1 6 WEEKS, ON THE CONTROL AND PARENTAL DIETS PARENTAL AND CONTROL THE ON WEEKS, 51 Levels I —1 1 1 7—

Avg. Wt. In mg ./'Roach 10 SO 40 20 50 0 ■ Figure 15 Slashed bare represent cockroaches reared reared cockroaches represent bare Slashed 15 Figure on the diets of their respective parents. Solid bars bars Solid parents. respective their of diets the on represent cockroaches reared on the control diet. control the on reared cockroaches represent (Ck.) , I 1 GROWTH OF THE 2nd. GENERATION ROACHES AT AT ROACHES GENERATION 2nd. THE OF GROWTH WEEKS, ON THE CONTROL AND PARENTAL DIETS PARENTAL AND CONTROL THE ON WEEKS, »2 I 5 I I I .4. 52 Levels | 1 5 i 6 i i 7 I I 8 I 8

53

Table 9

Regression Coefficients of the Fifteen Second Generation Treatments Each Coefficient Calculated on the Entire Growth Period

______(Log Wt. on Log.Week)______

Diet on Which Diet on Which Parents Reared Offspring Reared Replicate A Replicate B

l(ck.) 1 1.4352 1.4596

2 1 1.4529 1.5172 2 2 1.4204 1.4375

3 1 1.3859 1.4213 3 3 1.2651 1.3846

4 1 1.5217 1.4467 4 4 1.4294 1.6118

5 1 1.3865 1.4098 5 5 1.3462 1.2923

6 1 1.2584 1.2494 6 6 1.2041 1.3246

7 1 1.3428 1.1095 7 7 1.2869 1.2630

8 1 1.2482 1.1643 8 8 1.1137 1.0871 54

These regression coefficients are given in Table 9, and the analysis of variance in Table 10. The analysis of variance indicated conclusively that the growth rates were not the same for the various diets.

Table 10 Analysis of variance of the growth rates with second generation diets. Growth time equals the entire growth period. Test based on two replications of approxi*

Source DP SS MS P f -M F-l# Treatments 14 .4151 .02965 6.3545* 2.43 3.56

Error 15 .0700 .0046

Total 29 .4851

Therefore, a test for a significant difference be­ tween the check mean and the means of the other individual treatments was performed by the method of L. S. D. The results of these tests are given in Table 11. When no difference between the check mean and the treatment mean was found, there was considered to be no inhibition of

growth. When a difference between these means occurred,

growth was considered to be inhibited. 55

Table 11 Effect of various test diets on second gener ation roaches whose parents were fed test diets 1-8.

First Generation Second Generation Effect on Reared on Diet No. Reared on Diet No. Growth Rate

1 (0)* 1 (ck.) no inhibition 2 (6) 1 no inhibition 3 (12) 1 inhibition 4 (25) 1 no inhibition 5 (50) 1 inhibition 6 (100) 1 inhibition 7 (200) 1 inhibition 8 (400) 1 inhibition

2 (6) 2 no inhibition 3 12) 3 no inhibition 4 (25) 4 no inhibition 5 (50) 5 inhibition 6 (100) 6 inhibition 7 (200) 7 inhibition 8 (400) 8 inhibition

( )« Refers to ppm. of penicillin in particular diet.

Time to nAdult Stage”

Figure 14 represents, graphically, the time in weeks in which the cockroaches reached the ”adult stage” when reared on the various diets. Increase in time occurred chiefly if the parents of the young received 50 or more ppm. of penicillin, regardless of whether the young were reared on the check diet or the parental diet.

Mortality

The mortality in the second generation cockroaches, reared on the various diets, is given in Table 12 and

Figure 15. (continued on page 60) Weeks 15 10 ', Figure 1.4 Slashed bars represent cockroaches reared reared cockroaches represent bars Slashed 1.4 Figure on the diets of their respective, parents. Solid bars bars Solid respective, their parents. of diets the on represent cockroaches reared on the control diet. control the on reared cockroaches represent TIME IN WHICH SECOND GENERATION ROACHES GENERATION SECOND WHICH IN TIME REACHED THE ADULT STAGE, WHEN REARED WHEN STAGE, ADULT THE REACHED ON THE CONTROL AND PARENTAL DIETS PARENTAL AND CONTROL THE ON 56 Levels

57

Table 12

Second Generation Mortality through Eight Weeks

Level Initial Total Number Alive % Mortality A BA B A B Avg. A/B

1 30 30 26 30 13.3 0.0 6.7 2 30 30 28 29 6.7 3.3 5.0 2' 30 30 28 15 6.7 10.0 8.3 3 30 30 29 30 3.3 0.0 1.7 3' 30 30 28 29 6.7 3.3 5.0 4 30 30 29 29 3.3 3.3 3.3 4* 28 30 27 29 3.6 3.3 3.5 5 30 30 28 29 6.7 3.3 5.0 5» 30 30 28 30 6.7 0.0 ■ 3.3 6 30 31 18 27 40.0 12.9 26.2 6 ‘ 30 26 23 26 23.3 0.0 12.7 7 21 30 11 24 47.6 20.0 31.4 7* 30 29 25 24 16.7 17.2 16.9 8 30 30 24 30 20.0 0.0 10.0 8* 30 30 26 19 13.3 36.7 21.7 9 35 - 28 - 20.0 - 20.0 9 1 30 - 25 - 16.7 - 16.7 10 24 - 17 - 29.2 - 29.2 10» 26 - 20 mm 23.1 - 23.1 11 32 - 29 - 9.3 - 9.3 11 * 27 mm 23 - 14.8 - 14.8 12 18 mm 5 - 72.2 - 72.2 12* 15 - 0 - 100.0 - 100.0 13 12 - 0 - 100.0 - 100.0 13* 10 - 3 - 70.0 - 70.0 14 15 - 12 - 20.0 - 20.0 14f 11 - 8 mm 27.3 — 27.3 1lu e: 15' _ «• — 16 ------16* mm

(T) Indicates use of parental diet. The check diet was used for the others. 58

Figure 15 Mortality in the various levels at eight weeks in the second generation. (Slashed bars represent cockroaches reared on the diets of their respective parents. Solid bars represent cockroaches reared on the control diet.) % Mortality 100 80 90 60 70 40 60 MORTALITY IK THE VARIOUS LEVELS AT LEVELS IKVARIOUS THE MORTALITY 8

EK, n. GENERATION 2nd. WEEKS, 60

In .this generation, mortality was somewhat higher for those roaches whose parents received 100-200 or more ppm. of penicillin.

These mortality data include all second generation cockroaches used in the dietary treatments.

Reproductive Capacity

Second generation cockroaches, reared on either con­ trol or parental diets, appeared to reproduce normally if their parents received less than 50 ppm. of penicillin in their diets.

Third generation young produced by cockroaches whose parents received 50-100 ppm. of penicillin, hatched later than normal. Also, fewer nymphs per capsule were produced.

Cockroaches whose parents received 100 or more ppm. of antibiotic produced fewer egg capsules and these were often deformed. This resulted regardless of whether the second generation young were reared on the control or on the parental diets.

Experiments in rearing the young from adults re­ ceiving more than 400 ppm. of penicillin were rather

limited and therefore inconclusive. In general, only

enough insects were available to set up one replication.

However, sometimes, not even the necessary 30 were availa­ ble. On the average, these cockroaches reached the adult

stage in about thirteen to sixteen weeks, regardless of 61 whether they were reared on the control or parental diet.

Thl3 was the case with 50 young from adults reared on diet

10 (1560 ppm. of penicillin). The 50 young U3ed for the test, however, were the total from fifteen egg capsules.

Since a female cockroach normally produces 30-45 young per egg capsule, this sample was not considered adequate to reach any conclusions concerning the young which were produced. DISCUSSION AND CONCLUSIONS

The addition of penicillin to the dog food diet of the

German cockroach inhibited the growth rate if present In high enough concentration. Thus, the growth rate was either normal or inhibited, depending upon the total quantity of antibiotic in the diet.

In the first generation, test diet growth rates ap­ peared to be equivalent to check diet growth rates if the diets did not contain more than 400 ppm. of penicillin.

At 780 ppm. or more, however, Inhibition wa3 significant and became increasingly more severe up to 100,000 ppm. On the other hand, the offspring of the cockroaches which re­ ceived as little as 50 ppm. exhibited reduced growth rates, and this resulted whether the young were reared on the control diet or on the parental diet. Also, the offspring from adults which received any of the diets containing 50 ppm. or more of the antibiotic appeared to grow at ap­ proximately the same rate whether reared on the diet of their respective parents or on the control diet. There­ fore, it is concluded that in the first generation reared on diets containing 50-400 ppm. of penicillin, some ir­ reparable damage occurred which was not evident until the second generation. Analogous situations have been re­ ported In other insects. Adverse effects of insufficient diets may manifest themselves much more in the second or

62 63 succeeding generations. An example of this is the work of

Reynolds (1945). Prom experiments with Tribolium de*» structor, he reported that the rate of development of off­ spring was related to the nutritional value of the food on which the parents were reared. Second generation larvae developed more quickly when their parents had been fed a wholemeal flour than when fed white flour which was very deficient in B vitamins. This occurred whether the off­ spring were fed on white or on wholemeal flour. In fact, the offspring developed more quickly on white flour if the parents were fed wholemeal than they did on wholemeal if the parents were fed white flour.

In first generation German cockroaches fed on the various diets, inhibition of growth was not measurable until after four weeks. This inhibitory trend did not be­ come noticeable until the fifth week, even in the higher dietary levels. This is important because the insects grew very rapidly during the fir3t four week period. They almost doubled their weights from the initial weighing to the first week and also from the first to the second week.

Since no inhibition was apparent during these first weeks, the effect or effects of penicillin were latent or non- existant at this time. The penicillin could have inhibited the further accumulation or elaboration of some essential substance present in the nymphs at the time of hatching. 64

If this could occur, inhibition of growth would not be apparent until the supply of the unknown substance, present in the nymphs at hatching, was exhausted. This supposition

is somewhat substantiated by the fact that inhibition was evident earlier in the second generation, where it was first observed between the second and fourth week.

It is possible that inhibition, particularly at higher levels, could have arisen through reduction of the intra­ cellular bacteroida of the cockroaches. Brooks and

Richards (1955) found that 1,000 ppm. of aureomycin did not eliminate the bacteroids from the cockroaches. Higher levels eliminated the bacteroids, but also resulted in excessive mortality in three or four months time. However,

since no such excessive mortality occurred in first gener­ ation cockroaches fed diets containing as much as 100,000

ppm. of penicillin, it is possible that this antibiotic did not affect the bacteroids. The possibility al30 exists that penicillin is much less toxic to cockroaches than is aureomycin, and the excessive mortality associated with high concentration of aureomycin was due to its direct toxic action on the insect.

First generation cockroaches receiving less than 780

ppm. of penicillin reached the "adult stage" at approxi­ mately the same time. This was well correlated with the

fact that the growth rates were statistically the same. 65

In the levels containing 780 ppm. or more, however, there was an inverse relationship between time to "adult stage" and growth rate.

The cockroaches on all diets in both generations molted to the true adult stage at approximately the same weight. Thus smaller adults did not result from higher penicillin concentrations. It merely took them a longer time to achieve the normal size and weight for the molt to the adult stage.

Anorexia did not appear to be a problem in the various dietary levels with the possible exception of diet 16

(100,000 ppm. of penicillin). If the phenomenon actually occurred, it was not evident until after the fourth week, because, even on diet 16, growth was almost as great as the check up to that point. The cockroaches did not appear to be repelled by the penicillin in any way.

The reviews of Jukes and Williams (1953), Stokstad

(1954), and Jukes (1955) contain numerous references per­ taining to the acceleration of growth in vertebrate ani­ mals, Incited by the addition of small quantities of anti­ biotics in the food. In general, the antibiotics were added at about 10 ppm., and the extent to which it is ab­

sorbed by the animals at this low level is questionable.

Most evidence to date indicates that the antibiotic exerts

its action primarily on the intestinal biota. The various 66 possible ways in which an antibiotic may favorably affect the intestinal biota, and therefore elicit increased growth, have been often listed. According to Jukes and

Williams (1953) they are as follows:

wl) The antibiotic may inhibit or destroy organ­ isms which produce subclinical infections, that is, they suppress organisms which pro­ duce toxic reactions and cause a slowing of growth of the host animal.

2) Antibiotics may produce an increase in the number or activity of organisms which synthe­ size certain known or unknown vitamins or growth factors which are eventually made available to the host.

3) Antibiotics inhibit organisms which compete with the host for available nutrients.M

No acceleration of growth was observed in this study.

Had the cockroaches been reared under unsanitary conditions, or on a diet deficient in protein or vitamins, the results might have been different. In animals reared in clean quarters or under aseptic conditions, growth increases usually do not occur with antibiotic fortified feeds.

(See Jukes, 1952, Luckey, 1952; and Speer, 1950.)

The first generation data showed no appreciable difference in mortality even at the highest levels of peni­ cillin concentration. The high mortality with diet 9

(780 ppm. of penicillin), however, cannot be satisfactorily explained. There is a good possibility that the insects became diseased early in the course of the test. Dis­ regarding diet 9 the mortality varied between 3.7 percent 67 and 11.9 percent, which is considered quite low in view

of the faot that the insects were handled and anesthetized once each week from the time of hatching to the Madult stagen. In general, second generation cockroaches ex­ hibited higher mortality than the first generation cock­ roaches, even if their parents received as little as 100-

200 ppm. of penicillin. On the other hand, Steinhaus and

Bell (1953) found that 20,000-35,000 ppm. of penicillin added to grain killed 100 percent of the two species of

stored grain beetles tested, over a period of 30-60 days.

In the first generation, reproduction was apparently reduced by all dietary levels containing 200 or more ppm. of the antibiotic. This was of interest since the growth rate was not inhibited at less than 780 ppm., and excessive mortality was not observed at 100,000 ppm. Since very few young were produced at the higher levels of penicillin concentration, and no egg capsules were formed at the highest level (100,000 ppm.), there is a good possibility

that the ovaries of the females were impaired from function­

ing normally. Brooks and Richards (1955) found 5,000 ppm.

of aureomycin and 20,000 ppm. of sodium sulfathiozole

produced this effect with the German cockroach in 90 days.

Glaser (1946) reported the same results with sodium sulfa­

thiozole and calcium and sodium penicillin on the American cockroach. 68

Although fewer young were produced by adults receiving high concentrations of penicillin, those which did appear did not manifest all the characteristics shown by the naposymbiotic” cockroaches which resulted from the work of

Brooks and Richards (1955). They reported these nymphs possess the following characteristics: 1) the nymphs were slightly smaller than normal and light gray in color rather than dark blackish brown; 2) the embryonic cuticle, which is normally shed at the time of hatching, was not completely cast, but remained attached to the anal cerci; 3) the nymphs were weak and feeble; 4) 3ome nymphs died immedi­ ately, while others lay on their backs for several days waving their antennae; and 5) the nymphs were almost completely unable to grow on the stock dog food diet. The controls molted approximately every ten days on the dog food diet, and reached the adult stage at about 60 days.

The young Maposymbiotic” nymphs had not molted once by the end of thirty days.

In this 3tudy, the offspring from adults receiving as much as 50,000 ppm. of penicillin did not show all of the above listed characteristics . The color appeared normal in about half of the insects and the embryonic cuticles were completely detached. In addition, those that lived were able to reach the adult molt in thirteen to fourteen weeks on the normal dog food diet. This was not even twice 69 the normal growth period. Antibiotics differ considerably in structure and mode of action. Therefore, it is quite likely that with dietary penicillin the bacterolds were not eliminated or only partially so, and that the effects of the penicillin were directly on the cockroaches.

In all respects the second generation cockroaches were much more adversely affected than the first. Nowadays, when there is a great need for chemicals which are selectively toxic to economic pests, it would seem of value to investi­ gate further, the mode of toxic action of antibiotics on insects. Of course, it is always possible that resistant

strains could arise. In addition, the effects observed in this study were brought about only by feeding the cock­ roaches the penicillin all their lives. These results, how­ ever, lend support to the increasing evidence with verte­ brate animals that excessive doses of a seemingly non-toxic

antibiotic over long periods of time might lead to drastic

results. Although no noticeable effect in first generation

cockroaches was produced by feeding the penicillin at dosage

levels up to 780 ppm., the second generation was affected

even if their parents received as little as 50 ppm. There-

the possibility of dramatic results does not seem too

remote. PART II: EFFECTS OF DIETARY PENICILLIN ON CONCENTRATION OF B COMPLEX VITAMINS IN THE GERMAN COCKROACH

LITERATURE REVIEW

Rosenberg (1945) defines vitamins as follows:

"Vitamins are organic compounds which are required for the normal growth and maintenance of life of animals, including man, who, as a rule, are unable to synthesize these compounds by anabolic processes that are independent of environment other than air, and which compounds are effective in small amounts , do not furnish energy and are not utilized as build­ ing units for the structure of the organism, but are essential for the transformation of energy and for the regulation of the metabolism of structural units*!*

At least some of the known vitamins are utilized by every animal investigated. In insects, the B complex vita­ mins are reported to be important, but insects apparently neither use nor synthesize vitamins A, E, or the sterols of

the vitamin D group. Cholesterol, however, appears to be

a vitamin for all insects (Roeder, 1953).

The vitamins studied in this work were riboflavin,

niacin, biotin, and folic acid.

Riboflavin is synonymous with vitamin G, vitamin B2,

and lactoflavin. It is a yellow-green, fluorescent, water

soluble vitamin, widely distributed in plant and animal

cells. Generally, it is found combined with phosphoric

acid in the form of nucleotides. It forms a mononucleotide

known as riboflavin-5-phosphate, and a dinucleotide of

riboflavin and adenosine. The nucleotides act as prosthetic

groups in several oxidizing enzymes which play active roles 70 71 in cell respiration. They are important in carbohydrate and amino acid metabolism, oxidation of xanthine, and other chemical processes of tissues.

Niacin is synonymous with nicotinic acid. The amide of niacin is also biologically active and is the form of niacin predominately found in animal tissues. Niacinamide forms part of the pyridine, nucleotides which act as de- hydrogenoses chiefly in the oxidation of carbohydrates and proteins. These nucleotides are diphosphopyridine-nucleo­ tide (DPN), also known as coenzyme I or cozymase I, and triphosphopyridine-nucleotide (TPN), also known as coenzyme

II or cozymase II. Coenzyme I is made up of adenine, two molecules of phosphoric acid, two molecules of ribose, and niacinamide. Coenzyme II differs in having three molecules of phosphoric acid.

Biotin is synonomous with vitamin H and coenzyme R.

The mode of action of biotin is not completely known.

Studies indicate that it functions directly in at least three different types of reactions; deamination reactions, decarboxylation reactions, and oleic acid synthesis (Fruton and Simmonds, 1953).

Folic acid is synonomous with vitamin Bc , folocin, and

Lactobacillus easel factor. Folic acid is converted by the liver Into folinic acid or citrovorum factor, or leucovorin

(so called because it is necessary for growth of Leuco- 72 nostoc citrovorum)♦ This factor forms part of a coenzyme active in formate (HCOOH) transfer, and is an important step in the synthesis of purines and thymine, and therefore, nucleic acids. Formate carbon also appears in serine, choline, and methionine. (Houssay, ei; al., 1955.)

Data presented in some recent papers indicate a vita­ min sparing action in low level antibiotic fortified diets.

However, at high therapeutic doses, there are indications of vitamin loss. A few typical examples are listed below.

Lih and Baumann (1951) found that penicillin, aureo- mycin, and streptomycin stimulated the growth of rats re­ ceiving limiting amounts of thiamine, riboflavin, or panto­ thenic acid. The antibiotics were most effective in diets which contained enough vitamins for half maximum growth.

The growth responses due to the antibiotics were approxi­ mately equal to those observed when, in the absence of antibiotic, the vitamin content of the diet was doubled.

The relative effectiveness of the antibiotic varied di­ rectly with the vitamin deficiency. Calet, et al.. (1953) reported that rats which were fed aureomycin, had a higher growth rate than did normally reared controls. The aureo­ mycin increased the hepatic content of niacin, riboflavin, pantothenic acid, and vitamin B^g. Guggenheim, ejb al. (1963) reported that oral administration of penicillin, chloro- tetracycline, streptomycin, or terramycin at 50 ppm. stimu­ 73

lated growth of rata fed diets low in riboflavin and panto­

thenic acid. A similar effect was observed following peni­

cillin and oxytetracycline administration when the diet was

low in thiamine. These antibiotics had no growth promoting

effect in vitamin supplemented diets. Growth stimulation usually was associated with a higher level of the vitamin

in the liver and an increased urinary secretion. Subcu­

taneous injection of the antibiotics in rat3 receiving

diets low in these vitamins had no effect on growth or on

the accumulation of the vitamins in the liver. It was con­

cluded that the sparing action was due mainly to an alter­

ation of the intestinal flora.

Working with rats, Di Raimondo, et al.(1952) reported

that prolonged administration of therapeutic levels of

terramycin, aureomycin, and chloramphenicol produced macro­

cytic nutritional anemia, markedly decreased urinary ex­

cretion of vitamin A, nicotinamide, folic acid, and other

members of the B complex. These authors (1952) found that

approximately the same results occurred in healthy human

subjects. The administration of the antibiotics to under­

nourished humans produced definite signs of hypovitaminosis.

All the preceeding papers have concerned the effects

of antibiotics on vitamins in vertebrate animals. No

similar works with insects have appeared. MATERIALS AND METHODS

General Methods of Assay

In 1919, Williams reported that yeast could be used for the determination of "vitamine”, but only in recent years have the nutritive requirements of microorganisms been well enough known to permit their use as practical methods for the assay of vitamins.

Microbiological assays present numerous advantages in space, labor, and materials over biological assays which employ animals as the test organism. They are often pre­ ferred to chemical methods, because they may be used before the chemical configuration of the vitamin being assayed is known, and, in addition, are often more convenient and economical than available chemical methods.

A wide variety of microorganisms have been used in the assay of vitamins, but the lactic acid bacteria are most commonly employed. In general, they closely approach the Ideal for microbiological assay work because: 1) they are easily cultured in limited amounts of air, as in small test tubes; 2) they are nonpathogenic; 3) their nu­ tritional requirements are well known; 4) strains have been discovered which utilize some B complex vitamins.

In addition, lactic acid is produced as a metabolite

In direct proportion to their total growth. The amount of lactic acid present, indicating the extent of growth, is

74 75 readily determined by titration. Their growth may also be measured by any suitable nephelometric method (turbidity).

Thus, microbiological assay methods for vitamins are based on the fact that certain microorganisms utilize spe­ cific vitamins in growth. By using a dietary medium com­ plete in all respects except for the vitamin being tested,

growth responses of the test microorganisms are compared quantitatively in known standard and unknown solutions.

Either the acid or the turbidity produced by the test microorganism may be measured to determine the rate or extent of growth and thereby the quantity of vitamin in the test solution.

General Procedure for the Microbiological Assay of a Vitamin, with Riboflavin as the Specific Example

The general procedure here described is chiefly from

"Methods of Vitamin Assay" (Anonomous, 1951). The specific modification in respect to riboflavin is an adaptation of

the original microbiological method of Snell and Strong

(1939).

Preparation of Sample

As previously stated in Materials and Methods,Part I,

young adult male and female cockroaches were collected from

the various tests and kept frozen at -15°C. until the time

of assay. In natural materials, riboflavin generally occurs

combined either with phosphoric acid or with phosphoric and 76 adenylic acid, both of which may be combined with specific proteins to form oxidative enzymes. Therefore, the sample must be treated with acid or enzymes to release all the vitamin in the free state. The vitamin is acid stable and quite soluble.

At the time of assay, a number of the roaches were weighed and placed with a definite quantity of 0.1 N HC1 in a small Potter-Elvehjem motor-driven glass homogenizer.

The sample was then homogenized, and the homogenate poured into a test tube. An additional definite quantity of HC1 was used to rinse the homogenizer and was added to the homogenate.

This homogenate was then autoclaved at fifteen pounds pressure (121°C.) for fifteen minutes. After this, the sample was cooled to room temperature and the pH was ad­ justed to 4.5 with 0.1 N NaOH. All pH adjustments were measured with a line powered Model H2 Beckman pH Meter.

Following the adjustment in pH, the sample was placed in a volumetric flask, made to volume with distilled water, and filtered through Whatman no* 40 filter paper. Filtration was performed at pH 4.5 because this range is generally effective in removing bacteriological growth stimulants such as fatty acids and phospholipids. A specific quantity of the filtrate was then adjusted to pH 6.8 with 0.1 N NaOH, poured into a volumetric flask and made to volume with 77 distilled water. This constituted the completed sample, and was stored in a refrigerator at 6°C. until used.

Because riboflavin is light labile, all the above operations were performed in a darkened room.

Preparation of Stock Cultures

Stock cultures of the several strains of bacterial- used were transferred in triplicate every fourteen days.

The culture medium was Bacto Micro Assay Culture Agar

(B-319)2. The bacteria were transferred this often to have active cultures for preparation of inoculum. In transferring the cultures, deep stabs into agar were made and incubated for 16-24 hours at 37°C. After incubation, the cultures were stored in a refrigerator at 6°C. under aseptic conditions until used for inoculum or again trans­ ferred.

Preparation of Standard Tubes

Duplicate sets of standard tubes to be used for com­ parison with the unknown samples were prepared by adding to the tubes 0.0 (uninoculated blank), 0.0 (inoculated blank), 0.25, 0.50, 0.75, 1.0, 1.5, 2.0, and 3.0 ml. of a riboflavin working standard solution. This standard

^ Stock cultures were obtained from the American Type Culture Collection, 2029 M Street, N. W., Washington 6, D.C. D All dehydrated culture media used in this work were pro­ duced by Difco Laboratories Inc., Detroit 1, Michigan. 78

contained 0.1 microgram (meg.) of riboflavin per ml., and

was prepared by dilution of a stock solution containing 25 meg. of riboflavin per ml. The stock solution was stored

under toluene in a dark brown bottle in a refrigerator*

Sufficient distilled water was pipetted into each tube

to bring the volume up to 5.0 ml.

Then, into each of the tubes was pipetted 5.0 ml. of

the basal riboflavin assay medium (Bacto Riboflavin Assay

Medium (B325)). This assay medium contained all nutrients

except riboflavin necessary for the growth of Lactobacillus

casei ATCC 7469, the microorganism used as an assay agent.

The tubes were plugged with non-absorbent cotton and

placed in a test tube rack.

Preparation of Assay Tubes

Duplicate sets of assay tubes of the unknown samples

were prepared by adding three levels (1.0-3.0 ml.) of the

sample extract.

Sufficient distilled water was pipetted into the tubes

to bring the volume up to 5.0 ml.

To each tube was added 5.0 ml. of the assay medium.

The tubes were then plugged with non-absorbent cotton

and placed in wire test tube racks.

■Sterilization...... ■■ i ■ ■■ « The tubes were sterilized by autoclaving at fifteen

pounds pressure for ten minutes. They were then allowed to stand at room temperature -until there was no question that the temperatures of all were uniform. In these experiments, this was a minimum period of three hours. If this pre­ caution were not observed, the warmest tubes, after inocu­ lation, would exhibit a higher rate of growth and produce erroneous results.

Preparation of Inoculum

On the day prior to use, cells of L. oasei ATCO 7469 were transferred by sterilized needle to a tube containing

10 ml. of sterile inoculum broth (Bacto Micro Inoculum

Broth (B320)).

The Inoculated tube was then incubated for twenty hours at 37°G.

This culture was then centrifuged under aseptic con­ ditions in a Sorvall Angle head centrifuge until the cells were well layered In the bottom and side of the tube.

The supernatant liquid was discarded and the cells resuspended In 10 ml. of sterile isotonic (0.9$ w./v.)

sodium chloride solution. This procedure was used to re­ move riboflavin from the cells in the culture broth. The cell suspension was then further diluted 1:20 with isotonic

sodium chloride. The resultant suspension was the inoculum

as used. All of these procedures were performed aseptical- 80

Inoculation and Incubation

A sterile two ml. hypodermic syringe fitted with a number 27 needle was filled with the bacterial suspension.

The syringe was supported by a Burette clamp, clamped to a ring stand. The plug of each tube was then carefully re­ moved, one drop of the Inoculum introduced, and the plug replaced. When all tubes had been inoculated, the racks containing the tubes were placed in an incubator oven set at 37°C., and left undisturbed for twenty hours.

Upon completion of this incubation period, the racks were placed in a refrigerator (6°G.) to stop growth of the microorganism and were left there until the readings to determine amounts of growth were made.

Determination of Amount of Growth in the Sample

In determining the quantity of riboflavin or other vitamin in the sample, the amount of growth of the assay organisms in tubes containing a definite quantity of the sample extract was employed. The reduction in trans­ mittance of light, which varied in proportion to the numbers of organisms produced in any test sample, was used to measure the growth of the microorganisms. All transmittance readings were made with a Klett Summerson

Photoelectric Colorimeter fitted with a red filter with maximum transmission in the range of 640-700 millimicrons.

The actual percent transmittance was not calculated because 81

the colorimeter readings vary inversely with log percent

transmittance. For example, the uninoculated blanks were used to set the machine at 100 percent transmittance (or

zero optical density), which gave a colorimeter reading of 0 .0 . After the colorimeter was 3et at 0.0 with the uninocu­

lated blanks, readings were made on the remaining tubes.

This was the process involved in the reading of a tube: 1)

the contents of the test tube were agitated to suspend the

organisms uniformly; 2) the suspension wa3 poured back and

forth, twice, into a clean colorimeter tube to further in­

sure a uniform suspension; 3) the colorimeter tube with

its contents was placed in the machine, and the reading

recorded.

Construction of a Standard Curve with Known Solutions

After all readings were recorded, those of the known

solutions were used to construct a standard curve. This

was done by plotting amount of growth, as measured by the

readings, against the known number of micrograms of the

riboflavin per tube. An example of a standard curve is

shown in Figure 16.

Calculation of the Quantity of Vitamin in the Sample

By interpolation from this standard curve, the amount

of vitamin in the various levels of test solution was then

determined. The amount of vitamin per ml. of the sample 82

160

140 hO

S 1 2 0 cd (1)

* 100 0) +3 © S 80 O r - t O o 60

40

20

025 . 05 .075 .1 rig -275 Micrograms Riboflavin

Figure 16 Standard riboflavin curve. extract was then calculated. For example, if the tube con­ tained 2.0 ml. of the sample extract, the vitamin value for

1.0 ml. would be one-half of the total value. Thus, all values were calculated to the quantity of vitamin present in 1.0 ml. of sample. If three sets of these values differed by not more than fifteen percent of one another, the assay was considered valid.

The vitamin content of the test sample of roaches was 83

then calculated on the basis of a one gram sample by the

following formula: meg. /gram sample avg. meg./ml. sample X(vol.) ribo- / (wet wt.) » extract y dil’n flavin wt. of sample (gm.) A. factor

Niacin

In natural products, the majority of the niacinamide

is chemically bound to proteins. It may be liberated by

hydrolysis with strong acid or alkali (Melnick, 1942), or

by enzymatic treatment (Cheldelin and Williams, 1942). The

method of acid hydrolysis was used in these experiments.

The sample was homogenized with 10.0 ml. of 1.0 N

sulfuric acid, and the mixture autoclaved at fifteen pounds

pressure for 30 minutes.

After cooling, the pH was adjusted to 6.8 with 1.0 N

sodium hydroxide, diluted to the desired volume, and

filtered through Whatman no. 40 filter paper.

Lactobacillus arablnosus 17-5 ATCC 8014 was used as

the test organism.

All other methods were chiefly the same as those for

riboflavin.

Biotin

In natural materials, biotin is apparently rigidly

bound to other compounds, presumably proteins, but it may

be liberated by treatment with strong acid. 84

The samples were homogenized with 6*0 N sulfuric acid and autoclaved at fifteen pounds for 30 minutes.

After cooling, the homogenate was diluted volumetri- cally and filtered through Whatman no. 40 filter paper.

A definite portion of the filtrate was then neutral­

ized to pH 6.8 with 20 percent sodium hydroxide, and again diluted to a definite volume.

Lactobacillus arabinosus 17-5 ATGC 8014 was used as

the test organism.

All other procedures were chiefly the same as those for riboflavin.

Folic Acid (Pteroylglutamic Acid)

Successive molecules of glutamic acid may be attached

to the first glutamic acid radical in peptide linkage in natural materials. These compounds are called conjugates.

The organisms used in microbiological assays utilize these conjugates only to a small extent. However, they are uti­

lized to a considerable extent in some other animals. An

enzyme present in hog kidney, chick pancreas, and some other animal tissues splits the conjugates, liberating the vita­ min (Mims and Laskowski, 1945).

The samples were homogenized with 3.0 ml. of 0.2 N

phosphate buffer (2.723 gra. of monobasic potassium phos­

phate and 0.560 gm. of sodium hydroxide dissolved in dis­

tilled water and diluted to 100 ml.). 85

This homogenate was then heated for five minutes in a water bath at 100°C.

After cooling, 6.0 mg. of Difco Bacto Chicken Pancreas extract was added to each homogenate and the homogenates were incubated under toluene at 37°C. for 24 hours.

The homogenates were then heated in a boiling water bath for five minutes, diluted to a definite volume, and filtered through Whatman no. 40 filter paper.

Streptococcus faecalis ATCC 8043 was used as the test organism.

The other procedures were chiefly those used for ribo­ flavin.

Insects Used

Not many insects were available for the vitamin assay studies since it was necessary to save ample breeding stock for the second generation dietary studies. However, it was desired to make preliminary studies of the effects of the various levels of penicillin on several vitamins because it was not known if the antibiotic would produce a general or no effect on all, or if it would be limited in it3 action to one or more. Therefore, the limited samples of cock­ roaches available were used to run preliminary assays on four vitamins instead of a complete, replicated study of

one.

Tables 13-16 list the numbers and weights of the cock- roaches used for the vitamin tests. The insects used for comparative purposes were obtained from the culture kept at the University. Table 13

Weights and numbers of cockroaches used for riboflavin determinations

Males Female s Dietary Humber Wt. Avg. Wt. Number Wt. Avg. Wt". Levels Roaches Mg. Mg./Roach Roaches .. Mg. Mg./Roach

1 2 89*0 44.5 2 128.0 64.0 2 2 102.0 51.0 2 150.0 75.0 3 2 90.0 45.0 2 140.0 70.0 4 2 96.0 48.0 2 200.0 100.0 5 2 112.0 56.0 2 195.0 97.5 6 2 83.0 41.5 2 123.0 61.5 7 2 101.0 50.5 2 123.0 61.5 8 2 102.0 51.0 2 174.0 87.0 9 2 84.0 42.0 2 160.0 80.0 10 2 90.0 45.0 2 153.0 76.5 11 2 98.0 49.0 2 166.0 83.0 12 2 98.0 49.0 2 114.0 57.0 13 2 91.0 45.5 2 155.0 77.5 14 2 91.0 45.5 2 168.0 84.0 15 2 102.0 51.0 2 191.0 95.5 16 2 89.0 44.5 2 155.0 77.5 Table 14

Weights and numbers of cockroaches used for niacin determinations

Males Females Dietary Number Wt. Avg. Wt. Number l7t. Avg. Wt. Levels Roaches .Mg. Mg./Roach Roaches Mg. Mg./Roach

1 2 93.0 46.5 2 125.0 62.5 2 2 92.0 46.0 2 178.0 89.0 3 2 94.0 47.0 2 180.0 90.0 4 2 92.0 46.0 2 187.0 93.5 5 1 38.0 38.0 2 141.0 70.5 6 2 109.0 54.5 2 160.0 80.0 7 3 149.0 49.6 2 130.0 65.0 8 2 88.0 44.0 2 140.0 70.0 9 3 154.0 51.3 2 156.0 78.0 10 1 45.0 45.0 2 154.0 77.0 11 3 131.0 43.6 2 100.0 50.0 12 3 141.0 48.0 2 137.0 68.5 13 3 137.0 45.6 2 127.0 63.5 14 3 143.0 47.6 2 149.0 74.5 15 3 133.0 44.3 2 152.0 76.0 16 2 71.0 35.5 2 145.0 72.5 Table 15

Weights and numbers of cockroaches used for biotin determinations

Males______Females etary Number Wt. Avg. Wt. Number wt. Avg. Wt. vela Roaches . .Mg. Mg./Roach Roaches Mg. Mg./Roach

1 2 100.0 50.0 2 160.0 80.0 2 2 101.0 50.5 2 144.0 72.0 3 2 88.0 44.0 2 200.0 100.0 4 2 101.0 50.5 2 142.0 76.0 5 2 91.0 45.5 2 118.0 59.0 6 2 91.0 45.5 2 180*0 90.0 7 2 92.0 46.0 2 163.0 81.5 8 2 90.0 45.0 2 148.0 74.0 9 2 88.0 44.0 2 126.0 63.0 10 2 94.0 47.0 2 146.0 73.0 11 2 90.0 45.0 2 152.0 76.0 12 3 148.0 49.3 2 118.0 59.0 13 2 110.0 55.0 2 155.0 77.5 14 3 135.0 45.0 2 140.0 70.0 15 2 108.0 54.0 2 125.0 62.5 16 1 42.0 42.0 2 116.0 58.0 Table 16

Weights and numbers of cockroaches used for folic acid determinations

Males Female s Dietary Number Wt. Avg. Wt. Number Wt. Avg. Wt. Levels Roaches Mg. . Mg./Roach Roaches . . M r .. .. .Mg./Roach

1 2 101.0 50*5 2 138.0 69.0 2 2 85.0 42.5 2 201.0 100.5 3 2 96.0 48.0 2 183.0 91.5 4 3 152.0 50.6 2 215.0 107.5 5 2 87.0 43.5 2 122.0 61.0 6 2 103*0 51.5 2 155.0 77.5 7 2 92.0 46.0 2 191.0 95.5 8 3 137.0 45.6 2 146.0 73*0 9 2 117.0 58.5 2 179.0 89.5 10 2 85.0 42.5 2 138.0 69*0 11 2 106.0 53.0 2 143.0 71.5 12 4 213.0 53.2 2 158.0 79.0 13 3 143.0 47.6 3: 180*0 60.0 14 4 180.0 45*0 2 132.0 66.0 15 3 148.0 49.3 2 163.0 81.5 16 2 97.0 48.5 2 105*0 52*5 EXPERIMENTS AND RESULTS

The results of the assays for the four vitamins are given In Table 17. These results are represented graphi­ cally In Figures 17-21.

The data indicate reduction of vitamin content in both male and female cockroaches with respect to folic acid and riboflavin, but probably not with biotin and niacin.

The apparent reduction in riboflavin content appeared to occur if the diet contained 780 or more ppm. of peni­ cillin. This reduction, however, did not appear to be more drastic at 100,000 ppm. than at 780 ppm.

There appeared to be reduced quantities of folic acid in both male and female cockroaches if the diet contained

1,560 or more ppm. of the antibiotic.

No such reductions were observed with biotin and niacin. In fact, several of the levels contained somewhat' more of these vitamins than the check.

91 Table 17

Total quantity of four vitamins in the test cockroaches (Values calculated on net weight)

Riboflavin______Niacin_____ Biotin . Folic Acid Diet Me g. /g. Mcg./g. Mmcg./g.* Mmcg./g.* Males Females Males Females Males Females Males Females

1 9.8 9.7 33.0 37.0 100.0 100.0 420.0 635.0 2 6.8 9.5 41.0 39.0 99.0 93.0 510.0 850,0 3 10.0 8.8 32.0 31.0 126.0 99.0 345.0 730.0 4 8.0 9.6 33.0 27.0 116.0 100.0 355.0 715.0 5 7.3 10.3 42.0 34.0 113.0 148.0 320.0 450.0 6 7.3 10.0 34.0 34.0 114.0 108.0 330.0 620.0 7 8.5 8.0 33.0 33.0 109.0 118.0 595.0 675.0 8 6.0 8.5 34.0 32.0 116.0 101.0 410.0 575.0 9 3.7 6.3 32.0 31.0 110.0 145.0 390.0 480.0 10 3.7 5.5 32.0 29.0 104.0 143.0 55.0 130.0 11 3.8 6.3 29.0 31.0 121.0 132.0 110.0 250.0 12 3.7 5.3 23.0 29.0 75.0 173.0 98.0 340.0 13 4.2 5.2 29.0 27.0 90.0 151.0 70.0 70.0 14 3.7 6.0 33.0 28.0 99.0 109.0 175.0 190.0 15 3.6 5.5 30.0 30.0 77.0 122.0 80.0 50.0 16 4.5 6.8 39.0 35.0 99.0 119.0 90.0 83*0

^ Millimicrograms/ gram 93 QUANTITY OF RIBOFLAVIN PRESENT IN FEMALE ROACHES REARED ON THE VARIOUS DIETS IS

10

8

6

4

2

01 2 3 4 5 G 7 E 9 K) 11 ].g 13 14 1 5 16 c k. Levels

12 QUANTITY RIBOFLAVIN - MALES

10

8

6

4

2

Levels Figure 17 Le ve1s Le Levels 94 Figure 18 ON THE VARIOUS DIETS QUANTITY NIACIN - MALES IN IN FEMALE ROACHES REARED QUANTITY OF NIACIN PRESENT ck • ck ck. 0 1 2 3 4 5 6 7 8 9 10 11 32 1 13 4 15 16 50 40 anssTj, jo mBjg /•3pK anssjj, 95

QUANTITY OP BIOTIN PRESENT

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 ck. Levels

200 o

95u 150 ci>

U) 100 o a so

0 1 2 3 4 5 8 9 10 11 12 13 14 15 16 ck. Levels Figure 19 96

QUANTITY OP FOLIC ACID PRESENT IN HALE ROACHES REARED ON THE VARIOUS DIETS

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Levels Figure 20 M. Meg. / Gram of Tisane 100 200 -300 900 1 3 5 7 9 0 1 2 3 4 5 16 15 14 13 12 11 10 9 d 7 G 5 4 3 2 1 0 clc QUANTITY OF FOLIC ACID PRESENT PRESENT ACID FOLIC OF QUANTITY IN FEMALE ROACHES REARED REARED ROACHES FEMALE IN N H VROS DIETS VARIOUS THE ON iue 21 Figure 97 Levels DISCUSSION AND CONCLUSIONS

Noland (1949) reported that the omission of nicotinic acid from a synthetic diet used in rearing German cock­ roaches, resulted in extremely slow growth, and death be­ fore the adult stage. In similar tests, the omission of riboflavin resulted in retarded growth and some mortality.

However, the omission of biotin usually had no adverse effect upon either growth rate or survival. The omission of folic acid produced no abnormal effects.

Since many insects cannot live and grow on a diet which lacks any of the vitamins listed above plus other major B vitamins, It may be possible that folic acid and biotin are produced by intracellular or extracellular bacteria within the German cockroach, and in a form availa­ ble to the insect. Noland (1949) reported that the German cockroach accumulated more riboflavin than was present in the diet used, and that some accumulation would occur even if the diet were essentially devoid of the material. From this, it was concluded that the vitamin resulted from bac­ terial synthesis in the gut. Synthetic diets employed for rearing the German cockroach aseptlcally contain most of

the known B vitamins, including biotin and folic acid.

The riboflavin content of both male and female cock­

roaches receiving 780 or more ppm. of penicillin was de­

finitely lower than the check in the cockroaches tested.

98 99

The total quantity of riboflavin per gram was approxi­ mately the same as reported by Noland (1949) in cock­

roaches fed a riboflavin-free diet* Since some of the

penicillin-fed cockroaches had inhibited growth, it is

therefore quite possible that the penicillin prevented the

accumulation of this material. It Is not too likely that

the effect was due to destruction of riboflavin-producing

intestinal bacteria because they would have probably been

killed by leas than 780 ppm. of penicillin unless they were

highly resistant to the material.

Preliminary tests with untreated, id eat "normal”,

cockroaches reared on the dog food diet indicated that

riboflavin content Increased from approximately four micro­

grams per gram of whole cockroach in the first instar

nymphs to nine to twelve micrograms per gram in young adult

males and females. Approximately one month after reaching

the adult stage, and after reproduction, the females con­

tained only as much or less of this material than when they

reached the adult stage. The males, on the other hand,

contained about twice as much as when they reached the

adult stage. Loss of riboflavin in these normal females

probably was a result of egg production. Therefore, It Is

highly probable that an overall reduction of riboflavin

content in penicillin-fed roaches could have caused inhi­

bition of reproduction. However, the reduction in ribofla- 100 vln content was not in evidence if the diet did not contain

780 or more ppm. of penicillin. Since reproduction was in­ hibited at 200 ppm., some other factor must have been re­ sponsible for inhibited reproduction in levels containing

200-780 ppm. penicillin.

The apparent partial elimination of folic acid at high dietary penicillin concentrations undoubtedly could play a role in the inhibited growth of the cockroaches. It is likely that the vitamin i3 supplied by intestinal micro­ organisms because cockroaches can live on a diet devoid of the material. But here again, lower concentrations of peni­ cillin than those which resulted in elimination of some of

the vitamin, probably would have killed the bacteria, unless

they were in some way protected from contact with the full

penicillin dosage as by partial hydrolysis of the anti­ biotic .

In these studies, niacin and biotin appeared to be un­

affected in both male and female cockroaches. Actually,

some inhibition of these materials may have occurred at

the higher levels of penicillin concentration because it

took the cockroaches a longer time to attain the adult

stage, and therefore, they had a longer time to accumulate

the vitamin. In normal cockroaches, concentrations of both

of these vitamins increased up through the adult stage, but

losses occurred with age in the females. 101

It is very possible that the reduction of folic acid and riboflavin did produce some effect on the growth of the insects. However, this does not indicate why the second generation cockroaches had inhibited growth rates when their parents received as little as 50 ppm. of peni­ cillin.

Noland (1949) developed a synthetic diet on which

German cockroaches grew as rapidly or more rapidly than on a dog food diet. The offspring from adults reared on this diet were, however, abnormal. The egg capsules of the adults were often small, shriveled, and otherwise deformed, and only a small portion of the eggs hatched. Often only two or three live nynphs would result from an egg capsule.

These young required about twice as long to reach the adult stage as did the roaches reared on the dog food diet.

These were precisely the symptoms shown by the young of cockroaches fed diets containing 200 or more ppm. of peni­ cillin. On the other hand, the ’’aposymbioticM (without intracellular bacteroids) cockroaches which Brooks and

Richards (1955) obtained by feeding aureomycin to first generation cockroaches were scarcely able to grow on dog food diets at all. From this it appears that, at fairly low concentrations, penicillin might have inhibited the absorption or caused the destruction of some unknown nutri­ ent present in the natural dog food, but not in the syn­ 102

thetic diet. However, in addition, vitamin metabolism was

probably somewhat disturbed in the higher dietary levels of

penicillin.

Plippin (1955) states that the mode of action of peni­

cillin is not yet clearly defined, but apparently its maxi­ mum action is produced against rapidly multiplying bacteria:

that is, against actively multiplying bacteria it is bao-

tericidalj against bacteria which are not actively di­

viding, its action is bacteristatic in nature. In actively

dividint bacteria, some evidence indicates that penicillin

interferes with bacterial synthesis of proteins from amino

acids by blocking the process at a point which results in

abnormal accumulation of peptides. Since the cockroaches

received the penicillin all of their lives, the possibility

exists that this effect on dividing cells could have been

the same for the cockroach as well as the susceptible bac­

teria, particularly at higher levels of penicillin concen­

tration. Therefore, a reduction in the rate of growth in

the cockroaches fed high levels of penicillin could have

been a result of the action of penicillin on certain vita­

mins or on the protein syntheses in cell division.

In conclusion, the results of this investigation indi­

cate that penicillin, fed daily to German cockroaches all

of their lives prevents accumulation of two of the four

vitamins tested, particularly at high levels of penicillin 103 concentration. This vitamin reduction could produce an adverse effect on the cockroaches, but in all probability this is only one of several effects incited by the anti­ biotic. Since a quantity of penicillin much smaller in amount than that which affected accumulation of folic acid and riboflavin affected reproduction, and an even smaller amount affected growth in the second generation, a multi­ effect hypothesis appears tenable. SUMMARY

1.) Some effects of the oral administration of penicillin

on the German cockroach, Blattella germanica (Linn.), (Or-

thoptera: Blattidae), were studied. The antibiotic was added to a dog food diet at fifteen different levels of concentration ranging from six to 100,000 ppm. Starting with six ppm., each succeeding dietary level increased

geometrically by a factor of two. There was also a check

diet without penicillin. The insects were fed the various

diets all their lives and through two successive gener­ ations.

2.) The effects studied were: (1) the growth rate; (2)

time to the adult stage; (3) mortality; (4) reproductive

capacity; and (5) these same effects in the second gener­

ation. Total vitamin content of folic acid, biotin, ribo­

flavin, and niacin in young males and females at each die­

tary level in the first generation was also determined.

Microbiological assays were employed for the determination

of the vitamins.

3.) Test growth rates in the first generation appeared to

be equivalent to the check if the particular diet did not

contain more than 400 ppm. of penicillin. At 780 ppm. or

more, inhibition of growth was significant and became in­

creasingly more severe up to 100,000 ppm. In the second 104 105 generation, the offspring of the cockroaches which received as little as 50 ppm. exhibited reduced growth rates, re­ gardless of whether they were reared on the control or parental diet.

4.) First generation cockroaches receiving diets contain­ ing less than 780 ppm. of penicillin reached the adult stage in approximately the same length of time* In levels containing 780 or more ppm., however, there was an inverse relationship between time to adult stage and growth rate.

Offspring from first generation adults receiving 50 or more ppm. of penicillin took longer to reach the adult stage than did the checks*

5.) The first generation test cockroaches showed no ap­ preciable mortality over the checks, even at the highest dietary levels of penicillin concentration. Second gener­ ation cockroaches exhibited higher mortality than the checks if their parents received as little as 100-200 ppm. of penicillin.

6.) Reproductive capacity was apparently reduced in all dietary levels containing 200 or more ppm. of penicillin.

No eggs were formed by the cockroaches receiving the test diet containing 100,000 ppm. of penicillin. Offspring of adults receiving diets containing 50 or more ppm. of peni- 106 clllln exhibited reduced reproductive capacity.

7.) In all respects, the second generation cockroaches were much more adversely affected than the first, probably because of multiple and latent effects of the penicillin.

8.) Both male and female cockroaches receiving diets containing 780 or more ppm. of penicillin, had a lower content of riboflavin than the check. Both male and female cockroaches receiving the diets containing 1,560 or more ppm. of penicillin, had a lower content of folic acid than the check. The penicillin probably produced loss or prevented accumulation of these two vitamins. The niacin and biotin contents appeared to be approximately equiva­ lent at all the dietary levels used, in both males and females. REFERENCES

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I, James N. Liles , was born in Akron, Ohio, April 25,

1930. I received my primary and secondary school education in the public schools of Cuyahoga Palls, Ohio. My under­ graduate training was obtained at Miami University, Ohio, from which I received the degree Bachelor of Arts in 1951.

I entered the Ohio State University in the summer of 1952, and received the Master of Science degree in 1953, special­ izing in Insect Physiology. During the work on the Masterb degree I held the position of Research Assistant, first, at the Ohio Agricultural Experiment Station in Wooster, Ohio, and later In the Zoology and Entomology Department of the

Ohio State University. In the summer of 1953, I received a University Fellowship under the direction of Dr. R.H.

Davidson and Dr. P.W. Pisk, which I have held to the present.

I am a member of the Society of the Sigma Xi, Gamma.

Sigma Delta, Ohio Academy of Science, and the Entomological

Society of America.

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