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1964 Ecological and Nutritional Studies on Some Predators in Cotton Fields in Louisiana, With Special Reference to Coleomegilla Maculata, De Geer. Yousef Hanna Atallah Louisiana State University and Agricultural & Mechanical College
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Recommended Citation Atallah, Yousef Hanna, "Ecological and Nutritional Studies on Some Predators in Cotton Fields in Louisiana, With Special Reference to Coleomegilla Maculata, De Geer." (1964). LSU Historical Dissertations and Theses. 932. https://digitalcommons.lsu.edu/gradschool_disstheses/932
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A T A L L A H , Yousef Hanna, 1936— ECOLOGICAL AND NUTRITIONAL STUDIES ON SOME PREDATORS DM COTTON FIELDS IN IOUISLANA, WITH SPECIAL REFERENCE IX) C O L E O M E G I L L A M A C U L A T A DE GEER.
Lxiuisiana State University, Ph.D., 1964 Zoology University Microfilms, Inc., Ann Arbor, Michigan ECOLOGICAL AND NUTRITIONAL STUDIES ON SOME PREDATORS IN COTTON FIELDS IN LOUISIANA, WITH SPECIAL REFERENCE TO COLEOMEGILLA MACULATA DE GEER
A Dissertation
Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy
in
The Department of Entomology
by Yousef Hanna Atallah B .Sc., Ain Shams University, 1956 M .Sc., Ain Shams University, 1959 ACKNOWLEDGMENT
The writer wishes to express his sincere appreciation and gratitude to Professor L. D. Newsom for his interest, guidance, stimulating suggestions and making facilities available during the course of this work and in the preparation of the manuscript.
Appreciation is extended to Dr. J. H. Roberts for his assistance in photography and to Dr. H. B. Boudreaux and Dr. J. B. Sinclair for serving on the graduate committee.
He also wishes to express his appreciation to
Dr. N. W. Earle and Dr. J. B. Graves for their constant help during the course of these studies.
The writer is also grateful to Mrs. J. B. Chapin *
for help with identification of the coccinellids. TABLE OF CONTENTS
Page
ACKNOWLEDGMENT i i
LIST OF T A B L E S ...... v
LIST OF FIGURES...... viii
ABSTRACT...... xii
PART I
INTRODUCTION ...... 2
REVIEW OF LITERATURE...... 4
MATERIALS AND METHODS...... 12
RESULTS...... 14
Experiment Number 1, Hill Farm, 1962 ...... 14 Experiment Number 2, Perkins Road Farm, IS62 . . 20 Experiment Number 3, Hill Farm, 1963 ...... 29 Experiment Number 4, Perkins Road Farm, 1963 . . 36 Experiment Number 5, Perkins Road Farm, 1963 . . 45 Discussion of Results...... 46 •
CONCLUSIONS...... 51
PART II
INTRODUCTION ...... 54
REVIEW OF LITERATURE 5 5
BIOLOGY AND FEEDING HABITS OF FOUR COCCINELLID PREDATORS IN LOUISIANA ...... 62
Materials and Methods...... 62
ii i Page
R e s u l t s ...... 64
NUTRITIONAL STUDIES ON COLEOMEGILLA MACULATA DEG. 69
Materials and Methods ...... 69 D i e t s ...... 79
PART III
INTRODUCTION...... 115
REVIEW OF LITERATURE...... 116
MATERIALS AND METHODS ...... 118
RESULTS ...... 119
PART IV
INTRODUCTION...... 12 2
REVIEW OF LITERATURE...... 124
MATERIALS AND METHODS ...... 130
RESULTS ...... 137
Toxicity of* DDT to Coleomeqilla maculata. . . . 137 Toxicity of endrin to Coleomeqilla maculata . . 154 Toxicity of toxaphene to Coleomeqilla maculata. 162 Toxicity of toxaphene-DDT mixture to Coleomeqilla maculata...... 171
SUMMARY ...... 178
C ONC LU S I O N S...... 181
BIBLIOGRAPHY...... 182
VITA...... 198
iv LIST OF TABLES
TABLE Page
1 The numbers of squares and bolls damaged by the bollworm in Experiment Number 4. Total of squares and bolls on 15 plants counted July 9, August 1, 9, 16 and 29, Perkins Road Farm, 1963 ...... 39
2 Total numbers of prey and predators in all check plots, 1962-1963 ...... 49
3 Total numbers of prey and predators in all treated plots, 1962-1963 ...... 50
4 Development of different species of coccinellids on an aphid diet in the laboratory, Baton Rouge, Louisiana, July 15 to September 17, 1962...... 65
5 Time in hours required for development of each larval instar of Coleomeqilla maculata. 67
6 Number of aphids consumed per adult per day for different species of coccinellids in the laboratory, Baton Rouge, Louisiana,July,1963 68
7 Development of different stages of Coleomeqi 1 la maculata on Diet I ...... 83
8 Development of different stages of Coleomeqilla maculata on Diet I I ...... 86
9 Development of different stages of Coleomeqilla maculata on Diet III...... 89
10 Development of different stages of Coleomeqilla maculata on Diet I V ...... 91
11 Development of different stages of Coleomeqilla maculata on Diet V ...... 93
v Page
12 Development of different stages of Coleomeqilla maculata on Diet V I ...... 94
13 Development of different stages of Coleomeqilla maculata on Diet VII...... 96
14 Development of different stages of Coleomeqilla maculata on Diet V I I I ...... 98
15 Development of different stages of Coleomeqilla maculata on Diet X I ...... 101
16 Development of Coleomeqilla maculata on Diet XIII...... 105
17 Development of Coleomeqilla maculata on Diet X I V ...... 108
18 Development of Coleomeqilla maculata on Diet X V I ...... Ill
19 Effect of different population levels on egg production in Coleomeqilla maculata .... 120
20 Toxicity of DDT to diapausing and active adults of Coleomeqilla maculata collected at Boyce, Louisiana, February, 1963 ...... 138
21 The comparative effect of DDT on active and diapausing Coleomeqilla maculata. Beetles held at 80°F. and observed at the end of 72 h o u r s ...... 142
22 The effect of 40 micrograms of DDT per beetle on the reproductive and survival potentials of Coleomeqilla maculata .... 147
23 Toxicity of DDT to active adults of Coleomeqilla maculata. Baton Rouge, Louisiana, March, 1963 ...... 151
vi TABLE Page
24 Toxicity of endrin to diapausing and active adults of Coleomeqilla maculata collected at Boyce, Louisiana, February, 1963...... 155
25 The comparative effect of endrin on active and diapausing Coleomeqilla maculata. Beetles held at 80°F. and observed at the end of 72 hours...... 159
26 The effect of sublethal doses of endrin on the reproductive and survival potentials of Coleomeqilla maculata ...... 163
27 Toxicity of toxaphene tp diapausing and active adults of Coleomeqilla maculata collected at Boyce, Louisiana, February, 1963 165
28 The comparative effect of toxaphene on active and diapausing Coleomeqilla maculata. Beetles held at 80°F. and observed at the end of 72 hours...... 169
29 Toxicity of toxaphene-DDT mixture to active adults of Coleomeqilla maculata collected at Boyce, Louisiana, January, 1963 ...... 173
30 The effect of toxaphene-DDT mixture on active Coleomeqilla maculata. Beetles held at 80°F. and observed at the end of 7 2 h o u r s ...... 176
31 The effect of a "sublethal” dose of toxaphene-DDT mixture on the reproduction and survival potentials of Coleomeqilla maculata. 179
vii LIST OF FIGURES
Paye
1 Number of predators and prey in Check plot I, Hill Farm, 1962. The ordinate Y indicates the number of individuals...... 16
2 Number of predators and prey in Check plot II, Hill Farm, 1962. The ordinate Y indicates the number of individuals .... 18
3 Number of predators and prey in Treated plot I, Hill Farm, 1962. The ordinate Y indicates the number of individuals .... 21
4 Number of predators and prey in Treated plot II, Hill Farm, 1962. The ordinate Y indicates the number of individuals .... 22
5 Number of predators and prey in Check plot I, Perkins Road Farm, 1962. The ordinate Y indicates the number of individuals- .... 25
6 Number of predators and prey in Check plot II, Perkins Road Farm, 1962. The ordinate Y indicates the number of individuals .... 27
7 Number of predators and prey in Treated plot I, Perkins Road Farm, 1962. The ordinate Y indicates the number of individuals . . . 30
8 Number of predators and prey in Treated plot II, Perkins Road Farm, 1962. The ordinate Y indicates the number of individuals .... 31
9 Number of predators and prey in Check plot T, Hill Farm, 1963. The ordinate Y indicates the number of individuals . . . 33
Number of predators and prey in Check plot II, Hill Farm, 1963. The ordinate Y indicates the number of individuals l'. 35
viii FIGURE Page
11 Number of predators and prey in Treated plot I, Hill Farm 1963. The ordinate Y indicates the number of individuals...... 37
12 Number of predators and prey in Treated plot II, Hill Farm, 1963. The ordinate Y indicates the number of individuals...... 38
13 Number of predators and prey in Check plot I, Perkins Road Farm, 1963. The ordinate Y indicates the number of individuals .... 41
14 Number of predators and prey in Check plot II, Perkins Road Farm, 1963. The ordinate Y indicates the number of individuals .... 42
15 Number of predators and prey in Treated plot I, Perkins Road Farm, 1963. The ordinate Y indicates the number of individuals .... <*3
16 Number of predators and prey in Treated plot II, Perkins Road Farm, 1963. The ordinate Y indicates the number of individuals .... 44
17 Number of predators and prey in Experiment Number 5, Perkins Road Farm, 1963. The ordinate Y indicates the number of individuals...... 47
18 Adults held in a one pint carton with one vial of food and one vial of w a t e r ...... 71
19 A batch,of eggs laid on the side of a feeding tube. Magnification X 25...... 72
20 A batch of eggs laid on the artificial diet. Magnification X 2 5 ...... 73
21 Larvae growing in the feeding tubes on Diet X V I ...... 75
ix FIGURE Page
I 22 Adults feeding on Diet XVI in a one-half gallon carton ...... 76
23 A mating couple which were held in one of the oviposition cages and fed on Diet XVI plus vitamin E. Magnification X 14 .. . . 77
24 011a abdominalis feeding on Diet XVI . . . 113
25 Log time-probit lines for DDT-treated C. maculata (diapausing) collected at Boyce, Louisiana, February, 1963 ...... 139
26 Log time-probit lines for DDT-treated C. maculata (active) collected at Boyce, Louisiana, February 1963 141
27 Log dose-probit lines for DDT-treated C. maculata based on 7 2 hour observations, Boyce, Louisiana, February, 1963...... 144
28 Log time-probit lines for DDT-treated C. maculata (active) collected at Baton Rouge, Louisiana, and reared in the laboratory for 8 generations ...... 152
29 Log dose-probit line for DDT-treated C. maculata (active) collected at Baton Rouge, Louisiana,and reared in the laboratory for 8 generations ...... 153
30 Log time-probit lines for erdrin treated C. maculata (diapausing) collected at Boyce, Louisiana, February, 1963 ...... 156 * 31 Log time-probit lines for endrin treated C. maculata (active) collected at Boyce, Louisiana, February, 1963 ...... 159
32 Log dose-probit lines for endrin treated C. maculata based on 7 2-hour observations. Boyce, Louisiana, February, 1963...... 160
x FIGURE Page
33 Log time-probit lines Cor toxaphene treated C. maculata (diapausing) collected at Boyce, Louisiana, February, 1963 166
34 Log time-probit lines for toxaphene treated C. maculata (active) collected at Boyce, Louisiana, February, 1963 ...... 167
35 Log dose-probit lines for toxaphene treated C. maculata based on 72-hour observations. Boyce, Louisiana, February, 1963 ...... 170
36 Log time-probit lines for active C. maculata treated with toxaphene-DDT mixture, collected at Boyce, Louisiana, February, 1963...... 174
37 Log dose-probit line for active C. maculata treated with toxaphene-DDT mixture; based on 72-hour observations. Boyce, Louisiana, 1963 ...... 177
x 1 ABSTRACT
Population dynamics of Orius ins id iosus, Hippodamia convergens, Coleomeqilla maculata, Cycloneda spp., Scymnus
loewii, Scymnus terminatus, Stethorus sp. and Chrysopa spp. were studied in relation to some cotton pests in East Baton
Rouge Parish. These predators were found to be of significant
importance in preying upon Aphis gossypii Glover, Heliothis
spp. and Tetranychus spp. There was a strong correlation
between predator and prey populations, suggesting the presence
of a balance between the two populations. Disruption of the
balance resulted from the use of toxaphene-DDT-spray; this
treatment almost eliminated the Orius population. It caused
lacewing and coccinellid populations to oscillate at lower
general equilibrium positions. However, the coccinellid
population recovered much sooner than the lacewing population
after the treatment was stopped. In the treated plots the
aphid and spider mite populations developed high levels.
Although there was a correlation between predator and prey
populations in treated plots, it was not as strong as in the
untreated plots. The application of the insecticide obviously
changed the environment for all the organisms in the treated plots and favored some over the others.
Under field conditions the coccinellids showed the
following decreasing order of resistance to toxaphene-DDT spray: Hippodamia convergens, Coleomeqilla maculata, Scymnus terminatus, Cycloneda spp., Scymnus loewii and Stethorus sp.
An artificial diet and a laboratory rearing technique were developed for C^. maculata. Although reasonably satisfactory
in the laboratory, its use in the field failed to increase the population density of C. maculata. Crowding severely depressed
fecundity. The effects of competition were greater between
females than between males and females.
The effects of various insecticides on C^. maculata were found to be as follows: DDT had no effect on longevity,
increased the reproductive potential but lowered the survival
potential of the F^. It was found in the eggs and excreted
in the feces of females treated topically. Low levels of
DDT-resistance were found in one strain.
Endrin decreased longevity but had no effect on
oviposition or survival potential of .
Toxaphene decreased longevity slightly and prevented
oviposition,
Toxaphene-DDT mixture decreased longevity slightly,
x i i i lowered the reproductive potential and had no effect on survival potential of F^.
A log time-probit line technique was devised as a means of expressing the effect of a poison on longevity of an organism under laboratory conditions. This technique is discussed.
xiv PART I
The effect of toxaphene-DDT on population dynamics of some cotton insects and spiders in Louisiana INTRODUCTION
The most injurious pests of cotton in Louisiana are the boll weevil, Anthonomus grandis Boheman and the bollworm,
Heliothis zea (Boddie). The cotton aphid, Aphis gossypii
Glover, tobacco budworm, Heliothis virescens (F.), cotton fleahopper, Psallus seriatus (Reuter), cotton leafworm
Alabama arqillacea (Hubner), cabbage looper, Trichoplusia ni (Hubne’') , lygus bugs, Lygus lineolaris (Palisot de
Beauvois), and/or spider mites, Tetranychus spp. are of less importance. A complex of predators is more or less effective for suppressing populations of all of these pests except the boll weevil. However, when insecticides are applied for boll weevil control predator populations are depressed and biological control of such pests as the bollworm, cotton aphid, and spider mites becomes ineffective.
The major objectives of this study were to determine the effects of insecticide application on populations of predators and to explore the possibility of using artificial diets as a means of manipulating predator populations. The predators studied were: Orius insidiosus (Say), Nabis spp.,
Geocoris punctipes (Say), G. uliqinosus, Say, Podisus sp., 3
Scymnus loewii Mu Is., Scymnus terminatus Say, Coleomeg ilia maculata De G., Hippodamia converqens Guerin-Menevi1le,
Cycloneda sanquinea (L.), C. munda (Say), Stethorus spp.,
Cicindela spp., Chrysopa spp., and spiders. The prey studied
were: Heliothis spp. (H. zea (Boddie) and H. virescens
(Fabricius)), Aphis gossypii Glover, Tetranychus telarius (L.)
and Lygus spp.
Coleomeqilla maculata was chosen as the species to
use in studying artificial diets.
The effect of crowding on oviposition in this species
was also studied. In addition the effects of some insecticides
such as DDT, endrin, and toxaphene on Coleomegi11a maculata
were studied, since predators are always subjected to
pesticide applications under field conditions.
p REVIEW OF LITERATURE
For the sake of simplicity and understanding this review will be discussed in the following manner: the dynamics of insect populations; the natural enemies; and
the effect of insecticides on them with special reference to cotton insects.
Factors which affect insect populations have been
reviewed by Thompson (1956), Andrewartha and Birch (1954),
Milne (1957), De Bach (1956), Solomon (1949), and others.
All agree that predators are important in regulating popula
tions .
Thompson (1928) emphasized the importance of predators
in controlling aphid populations in central Florida.
« Thompson (1929) stressed the importance of micro-organisms,
entomophagous insects, and predators other than insects in
biological control. In the same paper he wrote that the
idea that predators are less reliable than parasites was
due chiefly to the belief that they are less specific;
there was little difference in specifity of parasites and
predators; and that the efficiency of either one depended
on the case.
4 Fleschner (1950) studied the searching capacities of the larvae of three predators of the citrus red mite in the laboratory and found that there was at least some similarity between prey and predator concerning phototropic and/or geotropic responses. He thought that searching was random and stimulation to feed was by contact.
Huffaker and Kennett (1956) concluded that the success of a predator depends on the close correlation of predator and prey biology, and whether the predators can survive for a significant period in the absence of the prey or at lower host density.
Burke and Dial (1956) stated that the larvae of the three species of Chrysopa they studied are voracious feeders on the cotton aphid, and that all three species occur in
aphid infested cotton fields.
Dean (1957) found that Stethorus punctum (Lee.) was the most important predator of Oligonychus pratensis
(Banks) which attacks maize and sorghum in the Lower Rio
Grande Valley in Texas.
Conrad (1959) found that the spotted lady beetle,
Coleomeqilla maculata (De Geer) was an important predator
of European corn borer eggs. 6
Simpson and Burkhardt (1960) evaluated some predators of Therioaphis maculata (Buckton) . They listed families of predators according to their relative importance as:
Coccinellidae, Chrysopidae, Anthocoridae, Nabidae and Syrphidae.
Hagen (1962) published an excellent review on the biology and
ecology of predaceous Coccinellidae.
Several of the studies reviewed emphasized the adverse
effect of insecticides on entomophagous insects. Among them
are those by Hauge and Peterson (1933), Ewing and Ivy (1943),
De Bach (1947), Newsom and Smith (1949), De Bach and Bartlett
(1951), Bartlett and Ortega (1952), Campbell and Hutchins
(1952), Ahmed, et al. (1954), Gaines (1954), Bartlett (1958)
and Pickett, et a3_. (1958). Ripper (1959) reviewed the
effect of pesticides on balance of arthropod populations.
He stated that DDT and parathion appear to be the insecticides
which most adversely affect predaceous arthropods.
Methods to study the efficiency of natural enemies
have not been standardized. The only method is published
by De Bach (1946) , De Bach, et al. (1949) , De Bach, et al.
(1951) and De Bach (1955). Even it is applicable only for a
limited number of entomophagous species, usually parasites
of scale insects on citrus.
Ewing and Ivy (1943) stated that applications of heavy dosages of calcium arsenate control the bollworm, but when the dosage is too light or when applications are not made at the proper time, cotton dusted with calcium arsenate becomes more heavily infested with bollworms than does undusted cotton. The increase in bollworm infestation is
frequently associated with increased aphid populations after
the plants have been dusted with arsenicals, and this may be due partly to the attraction of bollworm moths to aphid honey dew for food. Twelve species of predaceous insects
found on cotton readily fed and survived on bollworm eggs
Predators are listed below with the number of eggs consumed
per day in a laboratory experiment: Chrysopa rufilabris
Burm. larvae (27.4); Hippodamia converqens Guer. adult
(24.5); Coleomeqilla fuscilabris (Muls.) adult (22.1);
Hippodamia converqens Guer. larvae (14.1); CoHops balteatus
Lee. larvae (11.0), adult (8.8); Podisus maculiventris (Say)
adult (5.3); Zelus socius Uhl. adult (3.3); Z. renardii Kol.
nymph (2.8); Orius insidiosus (Say) nymph (2.8), adult (2.3);
Geocoris punctipes (Say) adult (2.7); Scymnus cpeperus Muls.
adult (2.1); Zelus renardii Kol. adult (1.8); and Melanophthalma
distinguenda (Com.) adult (1.7). Laboratory and field-cage
tests showed that when aphids were abundant the predators
fed on fewer bollworm eggs. 8
Newsom and Smith (1949) stated that the importance of natural enemies in the control of cotton pests had not been well studied, however, organic insecticide treatments proved to increase aphid and bollworm infestations, suggesting
that these materials were interfering with natural control normally achieved by insect enemies of this pest. All
insecticides studied reduced the populations of beneficial
species. The new organic insecticides were more detrimental
to two predators, Geocoris punctipes (Say) and Orius insidiosus
(Say), than was calcium arsenate-nicotine.
Heliothis virescens (Fab.) was the most destructive
pest of cotton in different valleys of the Central Coast of
Peru (1937-1950). Working on this problem, Wille (1951)
found that this pest was controlled by several natural enemies.
He recommended increasing the numbers of natural enemies at
the beginning of the season by planting corn early within
the rows of cotton fields as one means of controlling this
pest.
Campbell and Hutchins (1952) stated that coccine J.lids
were consistently observed feeding upon aphids and spider
mites in cotton fields. On one occasion a coccinellid larva
was seen eating into the dorsal portion of a Heliothis larva,
and Coleomeqilla was observed attacking eggs of the yellow 9 striped armyworm, Prodenia ornithogalli Guen., Geocoris punctipes was often observed with a mirid nymph impaled on its beak, whereas Nabis ferus (Linn.) seemed to prefer leaf- hoppers. On two occasions adult and nymphal Geocor is failed to pierce Heliothis eggs while in confinement in the insectary.
The eggs were probably those of H. zea, however, H. virescens was also present in the cotton fields. DDT caused lower mortalities in the predator population when used as a dust
than as a spray. In the laboratory DDT and toxaphene were more toxic to C. maculata than to H. converqens . All
insecticides used reduced the predator population. In
laboratory and field experiments hemipterous insects were more
seriously affected than coccinellids. H. converqens was the
species most tolerant to insecticides, 'but it was not abundant
after June. The peak in predator populations occurred in
% June and the lowest ebb in mid-July. During this ebb the
predators were very abundant on corn. This apparent buildup
on corn may explain why the predators were more numerous on
cotton adjacent to corn and suggests the possibility of
increasing populations of predators on cotton.
Brazzel et. al^. (3953) discussed the bollworm and the
tobacco budworm as cotton pests in Louisiana and Arkansas.
They found a high degree of natural control of eggs and 10 small larvae by a complex of predators consisting of lady beetles, the big eyed bug, Geocoris punctipes and the
insidious flower bug, Orius insidiosus. Use of insecticides
for other pests prior to deposition of bollworm eggs was
reported to be usually followed by deunaging infestations
unless control measures were applied. In 1952 there were
two periods of heavy egg deposition, in June (tobacco
budworm) and in late August (bollworm). The first was
completely eradicated by the predators. The August population
was also subject to heavy predation which was usually
sufficient to suppress infestations satisfactorily if
insecticides were not used.
Ahmed et a_l. (1954) studied the effect of feeding
some common predators of the cotton aphids on aphids poisoned
with systox. They found that the response varied from
highly susceptible to almost immune in different species of
predators and also in different stages.
Gaines (1954) stated that in the regular boll weevil
poisoning programs beneficial insects and spider populations
were practically eliminated after the second to the fourth
application; however, the lady beetles were slightly resistant,
also, to some extent the flower bugs, Or ius sp. Five species
of the carabid genus Lebia were reported by Whitcomb and Bell (1960) as predators of Heliothis zea eggs in cotton fields and some other crops in Arkansas. Bell and Whitcomb
(1961) stated that the dragonfly, Erythemis simplicicollis
(Say) is an important predator of the bollworm moth and several other noctuids. Bell and Whitcomb (1962) studied the efficiency of some egg predators of the bollworm under field conditions in Arkansas and estimated 60 to 95 per cent of the eggs could be consumed before hatching.
Dumas e_t a_l. (1962) found that the time of the day has a significant effect on surveys of predaceous insects in field crops. MATERIALS AND METHODS
For the purpose of this study five experiments were designed.
The first experiment was planted at Hill Farm, LSU, on June 1, 1962. On June 14, the field was divided into four plots. Each plot was 116 X 47.5 (12 rows) feet. The four plots were Check I, Check II, Treatment I and Treatment
II. Each plot had a buffer strip around it about 10 feet wide. The treatment used was one-half gallon per acre of a toxaphene-DDT emulsifiable concentrate mixture containing 4 pounds of toxaphene and 2 pounds of DDT per gallon in Xylene.
The treated plots were sprayed at 3-4 day intervals for a
total of 17 applications during the season. Counts were made within 24 hours after application. Sixteen counts were made during the season. For each count fifteen plants were chosen at random in each plot and direct counts of the numbers of squares, bolls, open bolls, squares damaged by bollworm, bolls damaged by bollworm, insects and spiders were
recorded on each plant. For aphid and mite counts, only the
number of individuals per five leaves on each of five plants were recorded in each count.
12 13
The second experiment was planted at Louisiana State
University Experimental Farm on Perkins Road. The experiment was divided into four plots in the same manner as the first experiment. Each plot was 10 rows wide and measured 116 X
39.5 feet. Treatments were carried out as in the first experiment. Seventeen applications were made. The technique of counting was the same in both experiments. Fifteen counts were made in this experiment.
In 1963 the two experiments of 1962 were repeated.
Each plot was 10 rows wide and measured 100 X 39.5 feet.
The third experiment at Hill Farm was planted 14 days later
than in 1962. Thirteen applications were made and 13 counts
were taken. The fourth experiment at Perkins Road Farm was
planted a month later than in 1962. Nine insecticidal
applications were made and eight counts were taken.
A fifth experiment was conducted in 1963 at Perkins
Road Farm. It received only four insecticidal applications
at the beginning of the season. The dosage rate of insecticide
was the same as in previous experiments. The checks for the
second experiment, 1963, were also used as checks for this
experiment. Eight counts were taken in this experiment. RESULTS
Results are summarized graphically and subjected to statistical analysis where appropriate.
Experiment Number 1, Hill Farm, 1962
Bollworm populations in check plot I damaged 8.5 per cent of the squares and 1.2 per cent of the bolls. Peak damage of squares occurred toward the middle of July, almost at the same time as the first bollworm population peak.
Most of the damage was done to the squares rather than the bolls since there were not as many bolls on the plant as there were squares. However, the bollworm population dropped to its lowest ebb toward the end of July and the beginning of August. This drop in the bollworm population was the result of biological control. Populations of the coccinellids coincided with those of the bollworm. The Qrius and Chrysopa populations started building up toward the end of July. It was obvious that there was not enough prey in the field for these predators during this period, so there was more stress on the bollworm population. However, toward the middle of
August, there was a buildup in the populations of aphids and 15 mites, which apparently released the pressure of the predators
on the bollworm eggs, resulting in an increase in the bollworm
population. The Qrius population did not respond to the
buildup of aphid, mite and bollworm populations, but the
coccinellid and Chrysopa populations showed increases at
this time.
The most important coccinellids based on numbers
present in the field during the season were: Hippodamia
converqens, Scymnus loewii, Coleomeqilla maculata, Scymnus
terminatus, Stethorus spp., Cycloneda sanguinea and C .
mund a.
Nabis populations were low at the beginning of the
season and built up toward the middle of August, almost at
the same time that the three prey populations (bollworm,
aphids and mites) built up their August peaks. Qeocoris
punctipes was more abundant than G. uliqinosus, and Podisus
sp. was present in low numbers. However, these three preda
tors were present in low numbers. There were no characteristic
peaks in spider populations. Data from the counts are illustrated
graphically in figure 1.
The populations in Check plot II were almost the same
as in Check plot I, except for the first peak in population
of bollworm eggs during mid-July which coincided with a 16
2200 2000
Qrius insidio3 us 1000 Coccinellids Chrysopa sp. Bollworm eggs Aphids Mi tes 500
400
300
200
100
0 16 19 2326 30 3 7 10 14 21 28 6 10 1618 22 31 June July August
Fig. 1. Number of predators and prey in Check plot I, Hill,Farm, 1962. The ordinate Y indicates the number of individuals. 17 similar peak, in population of Orius. Data from the counts are illustrated graphically in figure 2.
Bollworm populations in Treated Plot I deunaged
0.92 per cent of the squares and 0.52 per cent of the bolls.
The aphid population in this plot was 20 to 30 times greater than in the check plots. Soon after the second treatment the population reached a very high equilibrium position and oscillated around this point. Apparently none of the predators exerted any pressure on the aphid population because of the detrimental effect of the toxaphene-DDT sprays on predator populations. This insecticide seemed to have no toxic effect on the aphids. The mite population remained characteristically low during most of the season, except for a small peak toward the end of July, which soon dropped apparently because of competition with aphids. However, the’ mite population started building up toward the end of August when the aphid population dropped.
For the bollworm eggs there were two peaks, the first
and higher was during mid-July; the second was toward the middle of August. These two peaks coincided exactly with
those in the check plots, but the controlling factors differed.
In the treated plots the first peak was controlled by the
insecticide application. The second peak was controlled by 18
r y . Qrius insidiosus Coccinellids 500 Chrysopa sp. Bollworm eggs Aphids Mites
400
300
20 0
100
I I______1» » i r 1 . — ---1------16 19 23 26 30 3 7 10 14 21 28 6 10 1618 22 31 June July August Fig. 2. Number of predators and prey in Check plot II, Hill Farm, 1962. The ordinate Y indicates the number of individuals. 19
the insecticide applications, and also, there were almost no squares on the plant at this time and the bolls had hardened enough so that they were not suitable for the young larvae.
The Qrius population was almost eradicated from the plot.
The population of coccinellids was significantly
lower during the spraying period than that in the check plots.
When insecticide applications were discontinued, coccinellids built up tremendously high populations within 4 to 5 days,
resulting in a steep drop in the aphid population. The common
coccinellid predators present in the treated plot were:
Hippodamia convergens, Cycloneda spp., Scymnus terminatus,
Coleomegilla maculata, Stethorus spp. and £>. loewii, arranged
according to their population density in the plot. Cycloneda
spp. appear to te more resistant to toxaphene-DDT spray than
all other coccinellids with the exception of H. convergens.
Scymnus terminatus was more resistant to toxaphene-DDT than
S. loewii.
Chrysopa populations seemed to be the least affected
by the insecticide applications.
Nabis spp., Geocoris spp., and Podisus maculiventris
(Say) populations were almost eradicated from the plot.
Data from the counts are illustrated graphically in 20 figure 3.
Populations in Treated plot II were essentially the same as Treated plot I with the following exceptions:
1) The competition between aphids and mites was not
as pronounced.
2) The coccinellid population peaks after discontinuing
the insecticidal applications, came a week later
and were not as high.
So, in both check and treated plots the bollworm population was controlled; however, the reasons differed.
As a result of killing the natural enemies with toxaphene-
DDT, the populations of aphids and mites built up. Toxaphene-
DDT depressed Qrius populations significantly.
The bollworm damaged significantly more squares and bolls in the check than in the treated plots, 8.596 and 1.2% and 0.92% and 0.52%, respectively.
Data from the counts are illustrated graphically in figure 4.
Experiment Number 2, Perkins Road Farm, 1962
Bollworm populations in Check plot I damaged 4 per cent of the squares and 0.6 per cent of the bolls. The predator populations were at their peak during mid-July and 21
r Y Qrius insidiosua 10000 ' Coccinellids Chrysopa sp. Bollworm eggs 5000 - Aphids Mites
1000 500 f-
400
300
200
100
1619 2326 30 3 7 10 14 21 28 6 10 16 18 31 June July August
Pig. 3. Number of predators and prey in Treated plot I, Hill Farm, 1962. The ordinate Y indicates the number of individuals. 22
10000
Qrius insidiosus Coccinellids Chrysopa sp. 5000 Bollworm eggs Aphids Mites
1000 500
400
300
200
100
‘ "■ ^' *, _ , V ^ — - 0 ■ ^ JJ— a cr- ...... 1619 2326 30 3 7 10 14 21 28 6 10 16 18 22 31 June July August
Fig. 4. Number of predators and prey iji Treated plot II, Hill Farm, 1962. The ordinate Y indicates the number of individuals. 23 coincided with the peak square production. Bollworm infestation remained low in this plot. At the same time, the populations of the other prey were low, so the predator pressure was almost totally on the bollworm population. So, the bollworm population was depressed to the extent that there was no apparent peak in population of bollworm eggs during the season.
The predator populations reached their peak toward the middle of July and started dropping by the beginning of August, in spite of the fact that the mite populations were at their peak at the same time and the aphid population was building up. So, it looked as if the predator populations were not affected by build-up of non-reciprocating mite and aphid populations. However, the coccinellid population built up toward the middle of August and this buildup brought about a sudden drop in the aphid population.
The relative importance of different coccinellid species based on their numbers in the field during the season was Coleomeqilla maculata, Scymnus terminatus, Stethorus sp.,
Scymnus loewii, Hippodamia convergens and Cycloneda sanguinea.
Cycloneda munda was not present.
By comparing the relative importance (based on numbers) of different coccinellid species at the L.S.U. Hill
Farm and Perkins Road Farm, it is obvious that the most 24 important predator at one location might be of no importance in the other location. For example, H. convergens was much more abundant at the L.S.U. Hill Farm location than at the
Perkins Road Farm. The two locations are about 4 miles apart.
The Nabi3 population was very low with a peak at the end of July coinciding with peaks in populations of Geocoris punctipes, Podisus maculventris and spiders.
So the predators have their population peaks from the middle to the end of July, then they reach their lowest ebb by the beginning of August. This is at least partially responsible for the buildup of the populations of mites and aphids toward the beginning of August.
Data from the counts are illustrated graphically in figure 5.
Bollworm populations in Check plot II damaged 2.9 per cent of the squares and 1.1 per cent of the bolls.
The populations in Check plot II were essentially
the same as in Check plot I, except the peak of spider mite population was greater and the peak of aphid population came
toward the end of August when the mite population began declining. This emphasizes the importance of competition between aphids and mites.
Data from the counts are illustrated graphically in 25
700 r Y Qrius insidiosus Cocc inellids 600 Chrysopa sp. Bollworm eggs Aphids Mites
500
400
300
200
100
16 20 2426 303 7 10 17 24 8 14 21 1 June July August Sep
Fig. 5. Number of predators and prey in Check plot I, Perkins Road Farm, 1962. The ordinate Y indicates the number of individuals. 26 figure 6.
Bollworm populations in Treated plot I damaged 0.62 per cent of the squares and 0.14 per cent of the bolls.
The characteristic buildup of the aphid population in treated plots began after the first application. This shows the efficiency of the predators in keeping the aphid population under control. As soon as the predators were removed from the environment, the aphid population built up to tremendous numbers. Aphid populations remained high till the end of
August, when they dropped sharply as a result of leaf burning
(insecticide toxicity to the plants). Also, the cotton plant had almost finished fruiting at this time and was becoming senescent.
Mite populations remained characteristically low except for some oscillations. These low population densities were probably caused by interspecific competition with aphids.
There was a very slight peak at the end of July in the bollworm population, and another one toward the middle of August. Neither was of significant effect. Qrius populations were almost eradicated. The coccinellid populations were significantly lower them in the checks, with only one
small peak toward the end of July which dropped by the middle of August. This small peak corresponded to the highest aphid 27
950
900 Qrius insidiosus Coccinellids Chrysopa sp. Bollworm eggs 700 Aphids Mites
500
400
300
200
100
■ \ - . _
A 16 202426 303 7 10 17 24 31 148 21 1 June July August Sept.
Fig. 6. Number of predators and prey in Check plot II, Ferkins Road Farm, 1962. The Ordinate Y indicates the number of individuals. 28 population during the season. This conclusion is based on
the observation that there was no coccinellid peak in the check plots at this time. The relative importance of different coccinellid species based on their population density in this plot was: Scymnus terminatus, Coleomegilla maculata and
Scymnus loewii. The other coccinellids were not recorded
from the treated plot; however, they were present in only
low densities in the checks. Scymnus terminatus was more
resistant to toxaphene-DDT spray under field conditions than
all other coccinellids studied. The Chrysopa population was
not affected by the insecticide application, but this
population alone could not control the aphid population. The
average number of aphids one chrysopa larva could consume
in one hour was 32 + 21 under field conditions. This
observation was made on leaves practically covered with
aphids, so there was no searching time required for the
larvae to find the prey. Out of five larvae, each watched
for one hour, only two aphids fell from one's mouth. When
the larva moved among the aphids, they started leaving the
place (alarm substance or mechanical stimulation?). The
other entomophagous insects were almost eradicated in the
treated plot except for Cicindela spp.
The spider populations were significantly lower in 29 the treated plots than in the checks— Check I,_ 340; Check II,
360; Treatment I, 37; Treatment II, 33.
Data from the counts are illustrated graphically in
figure 7.
Populations in Treated plot II were essentially the
same as Treated plot I. Data from the counts are illustrated graphically in figure 8
Experiment Number 3, Hill Farm, 1963
Bollworm populations in Check plot I deunaged 3.93
per cent of the squares and 5.86 per cent of the bolls.
Maximum production of squares occurred toward the end of
July. This period coincided with the peak population of the
bollworm. However, the plant recovered from this infestation
because the bollworm population dropped to almost zero in a
week (the same as during 1962, Fig. 1). The Qrius population
was quite low during the whole season. It never built up to
a high level. Chrysopa populations did not build up to
appreciable levels till the last half of August, which was
too late for any significant effect on pest populations.
Coccinellids remained at high populations during the
whole season.
The aphid population oscillated around a low general 30
5000 Qrius insidiosus Coccinellids Chrysopa sp. 4000 Bollworm eggs Aphids Mites
3000
2000
1000
200
100
16 202426 30 3 7 10 17 24 31 8 14 21 1 June July August Sept.
Fig. 7. Number of predators and prey in Treated plot I, Perkins Road Farm, 1962. The ordinate Y indicates the number of individuals. 31
10500 10000 ....Qrius insidiosus Coccinellids v -•-Chrysopa sp. Bollworm eggs — Aphids 8000 o -Mites
6000
4000
2000
1000
200 L
100 .A
■ Fig. 8. Number of predators and prey in Treated plot XI, Perkins Road Farm, 1962. The ordinate Y indicates the number of individuals. 32 equilibrium position. The mite population had two peaks, the first during the last 10 days in July and the next during the second week in August. Data given in figure 9 shows that the bollwojrm and aphid populations were kept under control by the action of predators. The coccinellids present were not so effective on the mite population; however, when Chrysopa attained a high population density the mite population dropped. The coccinellids present according to their numbers in the plot were: H. convergens, Scymnus loewii, C. maculata, S_. terminatus and Cycloneda spp- Geocoris punctipes, G. uliqinosus and Podisus maculiventris were present at low population densities, however, populations of all three species increased slightly during the first week of August. Data from the counts are illustrated graphically in figure 9. Populations in Check plot II were essentially the same as in Check plot I except for: 1. A lower mite population, which built up toward the end of August. 2. A Chrysopa population which built up to a distinct peak toward the middle of August and started to 33 7 30 700 Qrius insidiosus Coccinellids Chrysopa sp. Bollworm eggs 600 Aphids Mites 500 400 300 200 100 23 27 2 6 10 15 19 26 2 9 17 24 June July August Sept. Fig. 9. Number of predators and prey in Check plot I, Hill Farm, 1963. The ordinate Y indicates the number of individualc. 34 > drop by the end of August, thus allowing the spider mite population to increase. Data from the counts are illustrated graphically in figure 10. Populations in both treated plots I and II were essentially the same. No bollworm eggs were observed in either plot during the whole season. However, one bollworm larva was found in Treated plot I on August 17, and two bollworm larvae were found in Treated plot II on August 2. Percentage squares and bolls damaged by bollworms were 0.55 and 0.9 3 in the former and 0.009 and 0.54 in the latter. The Or_ius population was almost eradicated. The aphid populations reached their peak at the end of July, dropped and increased again to a small peak at the middle of August. The coccinellid populations had two peaks corresponding to the aphid populations. The commonest lady beetles present were: H. convergens, C. maculata, Cycloneda spp., Scymnus terminatus and J5. loewii. There was no difference between the two Scymnus populations. Apparently the predators present did not affect spider mite populations. The Chrysopa populations had two peaks almost at the same time as the coccinellid population peaks occurred. 35 700 f y t .... Orius insidiosus Coccinellids ■- Chrysopa sp. 600 Bollworm eggs Aphids ►— Mites 500 400 300 200 100 23 27 2 6 10 15 19 26 2 9 17 24 2 June July August Sept. Fig. 10. Number of predators and prey in Check plot II, Hill Farm, 1963. The ordinate Y indicates the number of individuals. 36 In Treated plot I during the second peak of predators, Chrysopa was dominant, while in Treated plot II coccinellids were dominant. A significantly lower spider population was found in the treated than in the check plots. By comparing populations of the different species of coccinellids in the check with those in the treated plots it was obvious that they differed in their susceptibility to toxaphene-DDT spray. They could be arranged in descending order of their resistance as follows: H. convergens, C. maculata, Cycloneda spp., Scymnus terminatus and S. loewii. Data from the counts carried out in Treated plot I are illustrated graphically in figure 11, those from Treated plot II in figure 12. Experiment Number 4, Perkins Road Farm, 1963 * This experiment was planted a month later than the second experiment in 1962. The bollworm infestation was higher than in any of the previous experiments. The number of squares and bolls damaged by the bollworm is shown in Table 1. The bollworm damage reduced the crop tremendously. ■i The insecticide application brought the bollworm population under control, however, there was still some damage in 1300 Orius insidiosus 1200 Coccinellids Chrysopa sp. / Aphids / ■Mites / 1000 800 600 400 250 200 100 ~ — 23 27 2 6 10 15 19 26 2 9 24 2 June July ‘ August Sept. Fig. 11. Number of predators and prey in Treated plot I, Hill Farm, 1963. The ordinate Y indicates the number of individuals. 38 3000 Orius insidiosus Coccinellids Chrysopa sp. Aphids Mites , 2000 1000 500 40 300- 200 100 23 27 2 6 10 15 19 26 2 9 17 24 2 June July August Sept. Fig. 12. Number of predators and pray in Treated plot II, Hill Farm, 1963. The ordinate Y indicates the number of individuals. Table 1. The numbers of squares and bolls damaged by the bollworm in Experiment Number 4. Total of squares and bolls on 15 plants counted July 9, August 1, 9, 16 and 29, Perkins l*oad Farm, 1963. Number of Number of Per cent Per cent Number of Number of squares bolls damaged damaged Plot squares bolls damaqed damaqed squares bolls Check I 753 208 66 3 8.76 1.44 Check II 931 151 94 16 10.09 10.6 Treatment I 685 204 3 0 0.44 0.0 Treatment II 763 196 15 9 1.97 4.59 u> Treated plot II . Data from this experiment are graphically illustrated in figures 13, 14, 15 and 16. The populations of mites, Chrysopa and Orius were negligible. This afforded an excellent opportunity to study the ability of coccinellids to control populations of the cotton aphid without interference from other species. The coccinellid population was the only factor in the environment which kept the aphid population under control. In the check plots the coccinellid and the aphid populations fluctuated together, almost at the same time and in the same direction. In the treated plots where the coccinellid population was kept under control, the aphid population built up to a very high level. Intraspecific competition appeared to be the only factor which imposed limits upon the increase in aphid populations in the treated plots. The coccinellids present in the checks were: H. converqens, Scymnus loewii, £>. terminatus, Cycloneda spp. , and C. maculata in decreasing importance according to numbers present. In the treated plots the coccinellids present were: C. maculata, H. converqens and Cycloneda spp. £». terminatus and £>. loewii occurred in almost equal numbers. 41 7 50 Orlus insidiosus Coccinellids Bollworm eggs Aphids 500 375 250 200 150 100 50 18 22 29 1 9 16 24 29 July August Fig. 13. Number of predators and prey in Check plot I, Perkins Road Farm, 1963. The ordinate Y indicates the number of individuals. 42 500 Orius insidiosus Y Coccinellids Bollworm larvae Aphids 400 300 200 150 100 50 22 29 1 918 16 24 29 July Augvst Fig. 14. Number of predators and prey in Check plot II, Perkins Road Farm, 1963. The ordinate Y indicates the number of individuals. 43 5 300 5000 Coccinellids Aphids Mites 4000 3000 2000 1000 500 100 50 18 22 29 1 9 16 24 29 July August Fig. 15. Number of predators and prey in Treated plot I, Perkins Road Farm, 1963. The ordinate Y indicates the number of individuals. 44 6500 Coccinellids Chrysopa sp. Bollworm larvae 6000 Aphids 500 400 300 150 100 50 A. r 18 22 29 1 9 16 24 29 July August Fig. 16. Number of predators and prey in Treated plot II, Perkins Road Farm, 1963. The ordinate Y indicates the number of individuals. 45 Experiment Number 5, Perkins Road Farm, 1963 This experiment was carried out in the same locality as the fourth experiment and may be compared with that experiment. The idea for the experiment was to see if very high populations of predators could be built up in a plot 140 X 40 feet in size. Two-thirds of the plot was treated four times at 4-day intervals at the beginning of the season with one-half gallon per acre of toxaphene-DDT spray. The remaining one- third was left as a buffer zone. The insecticide treatment reduced the population of aphid predators and did not affect the aphids. The result was a high population density of aphids. When insecticide applications were discontinued the coccinellids built up a high population which soon brought the aphid population under control. Observations at night indicated that there were more bollworm moths flying over the treated than the check plot. Apparently they were attracted to the honeydew produced by the aphids, since some of the moths were seen feeding on it. A heavy bollworm population soon developed in the treated plot. It looked for a while that since there was a high population of coccinellids which had controlled the aphid population that predator activity would be directed toward the increasing bollworm population. However, this was not the case. When the coccinellids achieved the adult stage they started dispersing from the plot without laying eggs. The result was a buildup in the bollworm population and severe deunage to the squares and bolls. On August 29, 1963, the damage was 38% in the squares and 56.4% in the bolls. This damage was significantly higher than any bollworm damage in the four previous experiments. It could be concluded from this experiment that predator population manipulation under field conditions should be attempted with extreme care. Since coccinellid populations generally prefer aphids to bollworms and mites, apparently a complex of prey and predator should be present in the cotton fields in order to keep the bollworm population under control. Data from this experiment are graphically illustrated in f igure 17 . * Discussion of Results It is obvious that there are many factors in the environment of each organism and the factors are not separable in their effects on the population. So, an attempt was made to compare population deviations of prey and predators in treated and check plots as a means of determining total effects 47 1200 Coccinel1 ids 1100 Bollworm larvae Aphids 9000 700 500 300 125 100 50 18 22 29 1 9 16 24 29 July August Fig- 17. Number of predators and prey in Experiment Number 5, Perkins Road Farm, 1963. The ordinate Y indicates the number of individuals. 48 of the populations upon one another. Correlation of the two populations is of interest, not because one population is to be predicted from the other, but to know if there is a relationship between the two populations. Data from the check plots are summarized in Table 2 and show that there is a correlation between the two populations unless a one-in-ten chance has occurred in the sampling. In statistics 1% or 5% levels are usually used in testing the null hypothesis. The correlation between prey and predator populations was not significant at these levels. However, it should be emphasized that in studying population dynamics the statistic is very small in comparison to the parameter. Population dynamics is a relatively new field of study and the sampling techniques and interpretation of data are still under development. However, each number represented in the table is the sum of 13 counts made during the season at 4 to 7-day intervals. This should eliminate much of the chance. The ratio of prey to predator in these experiments ranged from 1.0 to 5.2 and averaged 2.5. Data from the treated plots are summarized in Table 3. The ratio of prey to predator in these experiments ranged from 7.3 to 106.6 and averaged 5 3.2. Table 2. Total numbers of prey and predators in all check plots, 1962-1963. ______Hill Farm Experiments Perkins Road Farm Experiments Checks 1,1962 11,1963 1,1963 11,1963 1,1962 11,1962 1,1963 11,1963 Prey X] 4959 2381 2384 1323 1648 .4219 1796 1282 Predators x2 2262 2268 456 536 1505 1549 537 548 n=8; r^O.65934; t=2.14813; degrees of freedom=n-2. Table 3. Total numbers of prey and predators in all treated plots, 1962-1963. Hill Farm Experiments Perkins Road Farm Experiments Treatments I,1962 II,1962 1,1963 II,1963 1,1962 11,1962 1,1963 II,1963 Prey x] 20512 24863 6010 11129 21685 30761 8693 7672 Predator X 2 2820 3161 98 210 755 661 84 7 2 n=8: r=0.5417; t=1.5786; degrees of freedom=n-2. CONCLUSIONS The experiments carried out in cotton fields in Louisiana proved that the predators play an important part, if not the most important, in controlling some pest populations when the other factors in the environment are ideal for development of these pests. For example in the fourth experiment it was evident that coccinellids can control aphid populations. When a selective insecticide is used that depresses predator populations a new environment is created which allows for rapid increases in populations of phytophagous animals such as the cotton aphid and spider mites. Hippodamia converqens , Scymnus terminatus , C. macu.l at a and Cycloneda spp. were the most resistant coccinellids to toxaphene-DDT spray of the species studied. Chrysopa populations were also resistant to the insecticide application. Once an insecticide treatment is used the balance is disturbed and a new balance is achieved in the new environment. Obviously the limited size of the plots in these experiments resulted in much more movement between plots of both prey and predator than would be the case in entire fields. 51 52 It is considered to be highly significant that the complex of predators which occurs in Louisiana cotton fields exerted almost as much control on the bollworm populations which developed as applications of toxaphene-DDT, one of the most effective insecticides available for control of Heliothis spp. Another highly significant point was the tremendous increase in aphid and spider mite populations which occurred in the treated plots as a result of the disruptive effects of applications of toxaphene-DDT mixture upon the complex of predators which normally keep these pests under control in untreated cotton fields. PART II Nutritional Studies on Coleomegilla maculata De G. INTRODUCTION Insect nutrition is becoming an increasingly active field of study in Entomology. Information on the food requirements and nutrition of insects is being accumulated rapidly as entomologists recognize that the injury done by insects or the benefits gained from them is the result of their efforts to obtain food. The aim of this study was to develop an artificial diet and an effective rearing technique for Coleomegilla maculata De G. so the insect could be reared continuously under laboratory conditions for ecological, physiological and toxicological studies. The value of mass culture and liberation of indigenous entomophagous insects for control of insect pests is still a debatable point. However, there seems to be a need for re-evaluation of the possibilities of controlling pests by this technique. Before trying to develop an artificial diet for this lady beetle, it was thought to be desirable to study the biology and feeding habits of some coccinellid predators so familiarity with this group of insects could be achieved. 54 REVIEW OF LITERATURE Forbes (1883) in his paper on insectivorous Coleoptera found that the guts of Megilla maculata (=Colemeqilla maculata) were full of pollens, some specimens also contained fungus spores. Aphids constituted only 10 per cent of their food. He also found that Coccinella movemnotata Herbst contained 90 per cent spores of fungi, and 10 per cent aphids. The gut of Hippodamia converqens Guerin-Meneville contained about 7 5 per cent fungus spores with the rest mites, aphids and a little pollen. Observations made by Clausen (1915) on Hippodamia converqens Guer., H. ambigua Lee., Coccinella californica Mann., Coccinella trifasciata Linn., Olla oculata Fabr., 0. abdominalis Say, Cycloneda sanquinea Linn., and Adalia bipunctata Linn, gave the following results: Between emergence and mating, 1.6-2.7 days; from mating to oviposition, 8.6-11.9 days; oviposition period, 28.2-48.1 days; larval development, 13-23 days; pupal stage, 3.5-8 days; number of eggs per female, 200-500; number of aphids consumed during larval development, 216-475; number of aphids consumed per day per adult, 15.6-56.1. Brues' (1946) idea about the origin of predaceous 55 56 insects was that early insects were vegetarian, which started feeding on plants and biting or actually feeding on other 'animals when opportunity presented itself, e.g. a phytophagus katydid, Conocephalus sp. Some coccinellids were found to feed on a mixed diet, since fungus spores and pollen were found with insect remains in the gut. One of the most abundant coccinellids, Coleomegilla maculata DeGeer was found to subsist in major part on spores and pollen; eggs and larvae of beetles were also found in its gut. Brues concluded that this mixed diet represents apparently a transition stage, similar to that which occurred at the time predatory insects first arose from phytophagus ones. At the Ninth International Congress of Entomology Szumkowski (1951) reported on some of hxs experiments with coccinellids particularly Coleomegilla maculata, stating that this insect ate eggs and larvae of Alabama sp., Feltia sp., Prodenia sp. and Laphygma sp. This species showed no preference for aphids except when the moth eggs had a protective covering of hair scales, e.g. as is the case with Laphygma sp. Cycloneda sanguinea preferred aphids to moth eggs. His field observations indicated that Coleomegilia and Cycloneda were effective egg predators of Laphygma 57 fruqiperda and Alabama argillacea. Trying to rear C. maculata on an artificial diet, he found that the larvae could not develop on a meat diet alone. Adding vitamin C gave no results. Adding "Multivitamin Roche" resulted in 3896 development, 86% development with liver plus "Multivitamin Roche"; 93% development with liver plus vitamin C. Feeding on fresh liver alone 38% of the larvae developed into adults. His data were published in more detail later (Szumkowski, 1961). However, several nutritional studies by other authors indicated that vitamin C (ascorbic acid) is not required by insects (Lipke and Fraenkel, 1956). It is added to the bollworm diet (Berger, 1963). Thompson (1951) disagreed with the idea that predators are general feeders. He stated that even in predaceous insects which are mobile and free living there is a high degree of specificity in host relations. He stated that predators might be less specific than parasites but in reality their behavior was just like their morphological characteristics in being specific and adaptive. Putman (1958) stated that Coleomegilla maculata legni (Timb.)' fed readily on Tetranychus telarius (L.) and the larvae developed normally on the same prey. This species also readily attacked Panonychus (=Metatetranychus) ulmi (Koch), the eggs 58 of Grapholitha molesta (Busck), and to a lesser extent, the crawlers of Pulvinaria vistis (L.). The food preferences of the larvae were generally similar to those of adults of the same species. All coccinellids concerned fed primarily on aphids, except Coleomegilla (=Ceratomegilla), which was more or less phytophagous. Smirnoff (1958) was the first to rear coccinellids on an artificial media. The diet was prepared by dissolving 1.3 g m . of agar, 16 gm. of sucrose and 6 g m . of honey in 100 gm. of hot water, then the mixture was cooled to 35-38°C. Separately, 4.5 gm. of royal jelly was added to 20 ml. of the original mixture, and stirred until a homogenous white emulsion was obtained. The two were combined and 0.5 gm. of alfalfa flour yeast and 2 gm. of pulverized dry insects which are natural prey of the species to be reared were added. The mixture was stirred vigorously and cooled to 5°C for storing. This diet was excellent for adults, but for larvae of some species it had to be supplemented with three parts beef jelly and one part royal jelly. Smirnoff listed several species of coccinellids which have been reared on this diet, however, Coleomegilla, Hippodamia and 011a were not among the genera mentioned. He did not develop a complete rearing technique for any of the species 58 of Grapho 1 itha molesta (Busck.) , and to a lesser extent, the crawlers of Pulvinaria vistis (L.) . The food preferences of the larvae were generally similar to those of adults of the same species. All coccinellids concerned fed primarily on aphids, except Coleomegilla (-Ceratomeqilla), which was more or less phytophagous. Smirnoff (1958) was the first to rear coccinellids on an artificial media. The diet was prepared by dissolving' 1.3 gm. of agar, 16 gm. of sucrose and 6 gm. of honey in 100 gm. of hot water, then the mixture was cooled to 35-38°C. Separately, 4.5 gm. of royal jelly was added to 20 ml. of the original mixture, and stirred until a homogenous white emulsion was obtained. The two were combined and 0.5 gm. of alfalfa flour yeast and 2 gm. of pulverized dry insects which are natural prey of the species to be reared were added. The mixture was stirred vigorously and cooled to 5°C for storing. This diet was excellent for adults, but for larvae of some species it had to be supplemented with three parts beef jelly and one part royal jelly. Smirnoff listed several species of coccinellids which have been reared on this diet, however, Coleomegilla, Hippodamia and Qlla were not among the genera mentioned. He did not develop a complete rearing technique for any of the species 59 he worked with. Smith (1960) found corn pollen in the gut of field collected larvae of C. maculata and other coccinellid species. During 1961 he stated that Coleomegilla maculata leqni Timberlake fed on various pollens developed from first instar larvae to the ad’ilt stage, whereas Cycloneda sanquinea and Coccinella trifasciata failed to mature on any pollen. Smith also found that C. maculata could be reared from first instar larvae to adult on the powdered stages of Rhopalosiphum maidis (Fitch), and also on yeast with relatively small mortality. Dry powdered food composed mainly of 3rewer's yeast {Mead Johnson and Co.), and water were also given separately. From this literature survey it was obvious that no standard method had been developed for rearing any of the coccinellid predators on an artificial diet. Palmer (1914) Nielson and Henderson (1959) and Nielson (1960) studied the biology and feeding habits of some coccinellids. Their estimated daily consumption of the spotted alfalfa aphid by Hippodamia converqens adults is quite different from one paper to another. In order to obtain additional information which might be helpful in developing a rearing technique for C. maculata, a number of additional articles about insect nutrition were reviewed. Most of these papers dealt with phytophagous 60 species. One paper dealt with a parasite, House (1958), but none dealt with predators. The extensive reviews published by Wigglesworth (1950), Trager (1953) and Lipke and Fraenkel (1956) were of great benefit. The insects which have been extensively studied for determining their nutritional requirements are: Drosophila melanoqaster Meigen, some flour beetles and moths, some stored grain insects and the German roach, Blatella germanica (L.). Generalizing about the food requirements of insects is difficult and often leads to wrong conclusions. The following generalizations are based on results obtained ♦ from thefew insects which have been studied. Further investigations on other insects might lead to different conelus ions: 1) Insects require in their food, adequate quantities of water, which is specific for each species. 2) Sources of energy differ greatly among insects. 3) Insects require the same ten essential amino acids classified as essential for the rat. These are arginine, lysine, leucine, isoleucine, tryptophan, histidine,phenylalanine, methionine, valine and threonine. 4) A nutritional requirement for vitamins other 61 than those of the B-group has never been substantiated. Synthesis of vitamin A from carotinoids never occurs in insects. Carotinoids are present in insects as pigments. 5) Insects require a dietary source of sterols. 6) Certain minerals and sulphur are required by most insects. 7) The fertility of an adult insect depends in whole or in part upcn the adequacy of its nutrition during the preimaginal stages. 8) The presence of micro-organisms in the gut of most insects, and the mycetomes complicate the study of their nutritional requirements. House (1955) stated that insects, possibly more than any other class of animals, are unusually diverse in feeding habits. The dietary requirements of all insects studied are found among about 30 nutrients and that no important differences having taxonomic significance have been brought to light. The requirements of insects studied have dealt only with one generation. Reliable determination of requirements must involve rearing the insect through several generations to detect symptoms of long-term deficiencies. BIOLOGY AND FEEDING HABITS OF FOUR COCCINELLID PREDATORS IN LOUISIANA Material and Methods Hippodamia converqens Guerin-Meneville, Coleomeqilla maculata De G. , Cycloneda munda (Say) , and Cycloneda sanquinea CL.) were collected from the field at Baton Rouge. They were reared in the laboratory at 80-90°F., 65 to 85% relative humidity, under continuous flourescent light provided by 400 watt, Cool White light giving 70 foot candles, fed on Aphis qossypii Glover, Rhopalosiphum pseudobrassicae (Davis), and R. maidis (Fitch). The adult beetles were sexed, then couples were placed in half or one pint white ice cream cartons covered with a Petri dish. The aphids were offered to the beetles on cotton leaves. The females usually oviposited on these leaves. The eggs were collected and allowed to hatch. Immediately after hatching five of the first instar larvae were placed in each of a one-half or one pint ice cream carton with a Petri dish cover and fed aphids until they pupated. When the adults emerged they were sexed and kept in couples in the cartons. Rearing the larvae of the four species was tried 62 63 first on a fresh milk diet. Drops of milk on a wax paper were placed in the rearing box.' Different instars were tested. The larvae survived for about 4-7 days depending on the instar, then died without any development. The living larvae continued normal growth if transferred to an aphid diet. So, neither the newly hatched larvae nor the larvae in later instars could develop on a fresh milk diet. A diet of either one of the three species of aphids used * was successful for all instars of the four species. RESULTS Cycloneda munda and C. sanquinea Both species are difficult to rear in captivity. Development under the previously described laboratory conditions is shown in Table 4. Hippodamia converqens Guerin-Meneville When held in one pint ice cream cartons in the laboratory the adults of this species were restless. Adults collected from cotton fields in Baton Rouge and vicinity on July 21, 1962, fed on an excess amount of aphids, started ovipositing July 24, 1962. If eggs were left with the adults they were eaten. At a light intensity of 5 foot candles the adults refused to lay eggs. Data on six individuals are given in Table 4. Coleomegilla maculata Adults were collected from cotton fields in Baton Rouge and vicinity July 10, 1962. The larvae did not survive on pollen of cotton although Smith (1961) found that C. maculata legni could 64 Table 4. Development of different species of coccinellids on an aphid diet in the laboratory, Baton Rouge, Louisiana, July 15 to September 17, 1962. Number of Days Pre- Preovi- Total No. of Incubation Larval Pupal mating position life Species observ. period______stages stage______period______period_____ cycle M. s .d. M. s .d M. s .d. M. s.d. M. s.d. M. s.d o o Cycloneda spp. 5 2.0 * 6.4 1.5 3.0 1.0 12 1.6 16.2 2.8 27.6 2.4 o o H. convergens 6 2.0 • 9.2 0.7 3.3 0.8 3.3 0.5 4.8 1.0 19. 3 2.0 C. maculata 6 2.0 0.0 8.7 1.4 3.0 0.0 7.5 1.4 10.5 1.0 24.2 2.1 M. =mean s.d.=standard deviation of the mean O' ui 66 develop on various pollens from first instar larvae to adult stage. Larvae were placed singly in half-pint ice cream cartons and provided with fresh ripened stamens of cotton which gave an excess of pollen. The experiment was replicated five times with five larvae per replicate. All larvae (25) died 4 days later. Adult females were provided with an excess number of cotton aphids. They consumed 5-11 of these per hour with an average of 7 aphids. If eggs were left with the adults they were eaten. Development under laboratory conditions is shown in Table 4. The time in hours required for development of each larval instar is shown in Table 5 Per cent survival was 75-90. The number of aphids consumed per adult per day is shown in Table 6 . Cannibalism is severe in the four larval instars of the four species. Although cannibalism among adults was observed in the laboratory, it is probably of relatively little, if any, significance. 67 Table 5. Time in hours required for development of each larval instar of Coleomeqilla maculata. ______Instars______Replicates First Second Third Fourth I 49 30 48 142 II 48 27 52 145 III 55 38 47 118 IV 52 48 51 98 V 51 45 50 1 0 0 VI 59 51 48 95 VII 48 49 47 ■ 93 VIII 72 71 48 76 IX 48 47 49 57 X 48 46 49 94 XI 47 49 48 8 6 XII 48 48 48 72 XIII 69 48 48 77 The means were: first instar 51.8+7; second instar 45.9 +10.6; third instar 48.7+1.5 and fourth instar 96.4+25.8. 68 Table 6. Number of aphids consumed per adult per day for different species of coccinellids in the laboratory, Baton Rouge, Louisiana, July, 1963. Species Number of Observations No. of Aphids Mean s.d. Cycloneda sp. 5 122 69 H. converqens 6 147 53 C. maculata 7 168 117 s.d .=standard deviation NUTRITIONAL STUDIES ON COLEOMEGILLA MACULATA DE G. Materials and Methods Coleomegilla maculata adults were collected from the field at Baton Rouge and vicinity during July, 1962. They were kept in the laboratory and fed living cotton aphids, since there was no adequate artificial diet. Since Szumkowski (1958) had reared a race of this beetle on fresh liver successfully, it was chosen as the basis for experimental diets. Taking into consideration the chemical analysis of liver and using the techniques which have been used successfully in developing artificial diets for the bollworm (Berger, 1963) and the boll weevil (Earle et^ al^. 1959) , seventeen experimental diets were formulated by addition or elimination of one material or compound at a time. All equipment used was washed thoroughly in tap water and distilled water, then dried at room temperature and held in an oven at 175°C. for at least 24 hours. Different sizes of Petri dishes, vials and ice cream cartons were tried for rearing the larvae and adults. Vials 1 . 6 cm. in diameter and 6 . 0 cm. high were found to be convenient for rearing and handling the larvae. 69 70 After preparing each diet, it was poured, while still hot, into the vials to a depth of 1-1.5 cm. Also a few Petri dishes 5 cm. in diameter were filled with the diet. The vials and Petri dishes were kept in the refrigerator at a temperature of about 35°F. until they were used. The rearing temperature was 80°F. +_ 1°F., and 25% relative humidity. A 50-watt incandescent bulb provided a continuous light source. The adults were placed in a one pint carton with one vial of food and another vial full of distilled water plugged with absorbent cotton for supplying moisture and for the adults to drink. The vial containing the water was found not to be necessary all the time depending upon the humidity in the rearing room. For purposes of holding the adults, up to 2 0 were kept in one box, to feed and mate. The females rarely oviposited under these conditions although it was observed once during the course of this study, Fig. 18. For oviposition a pair or a mated female was placed in each box. The females laid their eggs mostly on the sides of the feeding tube (Fig. 19) or on the media (Fig. 20). If there was not enough food or if the food decayed, the adults ate the eggs. The females laid eggs in batchs of 5-25. The eggs were removed from oviposition cages at 2 -hour periods, 71 Fig. 18. Adults held in a one pint carton with one vial of food and one vial of water. 72 Fig. 19. A batch of eggs laid on the side of a feeding tube. Magnification X 25. 73 / 4 Fig. 20. A batch of eggs laid on the artificial diet. Magnification X 25. 74 8 a.m. till 12 p.m., and held until they hatched. Newly emerged larvae were placed singly in feeding vials, plugged with non-absorbent cotton. The vials were then held together with a rubber band and placed upside down (Fig. 21) in the rearing room. Placing the tubes upside down reduced the contamination and decay of the diet which was a problem, and also reduced the rate of dessication of the diet. Sometimes it was necessary to change the feeding tube once or’twice because the medium decayed or dried out. This was usually required for the diets upon which larvae developed slowly. Trials were made to rear two or more larvae in one tube, but one always fed on the other. After the larvae completed their development they pupated in the vials, where they were left until they emerged as adults. Adults of the same age were kept together in boxes. For the purpose of keeping large numbers (over 100) half-gallon cartons were used, with a 5 cm. in diameter Petri dish full of food and a water tube placed on the bottom of the box (Fig. 22), a glass cover was used to cover the top of the box. The food and water were changed each 4-7 days. Under these conditions they fed and mated but did not lay eggs. Figure 23 shows a mating couple in one of the Fig, 21. Larvae growing in the feeding tubes on Diet XVI. Fig. 22. Adults feeding on Diet XVI in a one-half gallon carton. 77 Fig. 23. A mating couple which were held in one of the oviposition cages and fed on Diet XVI plus vitamin E. Magnification X 14. oviposition boxes feeding on Diet XVI plus vitamin DIETS The technique for preparing the diets was as follows 1. Half the amount of water required was placed in the Waring blender with all the ingredients except the agar and mixed thoroughly at medium speed for 3-4 minutes. 2. The other half was heated to boiling, then the agar was carefully dissolved in it, using an electric mixer. The agar solution was then added to the mixture in the blender, which was operated at high speed for 1 *5 -2 minutes. 3. The diet was poured while still hot into the containers vrhere it solidified. Diet I Ingredients Grams Distilled water 84 ml Casein 15 4M Potassium hydroxide 0.5 ml Wesson's salt 1.0 Sucrose 3.5 Choline chloride 0.1 Vitamin suspension 1.0 ml Ascorbic acid 0.4 79 80 Diet I (continued) Ingredients Grams Cholesterol 0.1 Inositol 0.1 Butterfat 0.2 Methyl p-hydroxybenzoate 0.15 Aureomyc in 0.03 Agar 2.5 Each ingredient contributed to the diet as follows: 1. Distilled water: To provide moisture and to give the diet a texture making it easier for the insect to eat. 2. Casein: Vitamin free; Nutritional Biochemicals Corp. product, "Devitarninized" by reprecipitation and exhaustive extractions with hot alcohol to remove both fat and water-soluble vitamins. Used in the diet for supplying amino acids. 3. Four Molar KOH: For adjusting the pH of the diet to about 6.5. 4. Wesson's salt: Nutritional Biochemical Corp. product. Formulated per the Wesson modification of Osborne and Mendle (salt mixture). Science 75: 339 (1932). Used in the diet for supplying the salt requirements. 81 5. Sucrose: Mallinckrodt Chemical Works product, analytical reagent. For supplying part of the carbohydrate requirement. 6 . Choline chloride: Mann Research Laboratory product. Used in the diet because it has proved to be required by some insects. Its exact function is not yet known, however, it is believed to play a great part in the cholinesterase system. 7. Vitamin suspension: Prepared in the following manner (Berger, 1963): Distilled water 100 ml Niacinamide hydrochloride 100 mg Calcium pantothenate (Dextroryatory) 100 mg Riboflavin 50 mg Thiamine hydrochloride 2 5 mg Pyridoxine hydrochloride 25 mg Folic acid (crystalline) 25 mg Biotin, crystalline 2.0 mg Vitamin 0-2 ml 9. Ascorbic acid (Vitamin C): General Biochemicals product. Nutritional studies under aseptic conditions (no extracellular microorganisms present in the diet or the gut, but there might be some intracellular, e.g. mycetomes) proved 82 that vitamin C is not required by some insects. However, it is used in the bollworm diet to increase reproduction. As mentioned in the review of literature, this compound is used because it is present in a relatively high concentration in the liver. 9. Cholesterol: Fisher Scientific Co. product (melting point, 148.2-149.2°C.; C 2 7 H 0 5 OH, molecular weight 386.665). Used because it has been proved to be required by some insects. 10. Inositol: Nutritional Biochemical Corp. product. Added because it is thought to be required by some insects. 11. Butter: Extracted from fresh milk. Added to provide the fatty acids. 12. Methyl p-hydroxybenzoate: HOC6 H4 COOCH3 ; M.W. 152.15; Eastman Organic Chemicals product. Added to inhibit the growth of microorganisms. 13. Aureomycin; Chlortetracycline HCl. Lederle Laboratories Division product. Added to inhibit bacterial growth in the diet. 14. Agar: Difco Laboratories product. Added to solidify the diet. The results of rearing individual larvae in single tubes on this diet appear in Table 7. Table 7. Development of different stages of Coleoneqilla maculata on Diet I. Humber developing to 2nd 3rd 4th Per cent of Replicates Initial Humber instar instar instar Pupa Adult development 1 10 7 0 0 0 0 0 2 10 8 2 0 0 0 0 3 S 4 0 0 0 0 0 4 10 5 0 0 0 0 0 5 10 8 1 0 0 0 0 6 10 8 3 1 0 0 0 7 10 6 0 0 0 0 0 8 12 10 1 0 0 0 0 9 10 6 0 0 0 0 0 10 8 7 0 0 0 0 0 oo U> 04 The individuals reaching the third instar were undersize and less tanned than normal. The larvae were very slow in development. Most deaths occurred either immediately before or during molting. Some larvae were reared in groups of 5 in 10 cm. Petri dishes. At the end of the first instar they became inactive, then after a day or two became cannibalistic. An average of 2 out of 5 survived to the 2nd instar. Usually one of the larvae fed on the other, then died in the third or fourth instar. In one case the larva pupated and emerged to an adult with a deformed tarsus. This adult died after one day. Adults obtained from larvae reared on liver and aphids then transferred immediately after emergence to Diet I, fed and survived for 45 days without laying eggs. Mating seldom happened. After 45 days the experiment was stopped. When 4 adults in one pint boxes were put together cammibalism frequently happened, especially when the adults were of different ages, e.g. 3 adults 2 days old fed on one adult one day old. Diet I was not successful. 85 Diet II Diet Illwas similar to Diet I except for: a. five grams of casein were used instead of 15; b. two hundred grams of fresh pork liver were added. Larvae were placed in individual tubes. The tubes were changed every 4-5 days because of contamination with microorganisms. The results of using Diet II appear in Table 8 . The larvae and adults were of normal size. The a larvae were slightly less tanned than normal. The adults were normally tanned. The tendency for cannibalism during P the larval stages was less than in Diet I. It seems that the death of some larvae was due to the decay of the diet. The larvae completed development in 12 days on an average compared to 10 days on the cotton aphid. Adults reared on this diet mated rarely. In most, cases the female rejected 4 the male and sometimes tried to bite him. Even when the- adults (regardless of sex) were feeding on the diet beside each other and one of them moved its leg near the other beetles, they would bite this moving leg. The females did not oviposit until their abdomens became distended with eggs, and it seemed they were forced to oviposit. In contrast the females fed on an aphid diet laid eggs before the abdomen was distended with eggs. The percentage of egg Table 8. Development of different stages of Coleomeqilla maculata on Diet II. Number developing to 2nd 3rd 4th Per cent of Replicates Initial No. instar instar instar Pupa Adults development 1 10 10 8 8 8 8 80 2 10 10 9 7 7 7 70 i 3 1 0 9 9 9 9 9 90 4 1 0 9 9 8 8 8 80 5 1 0 10 1 0 9 9 9 90 03 ON 87 hatch varied from 50 to 100. It seems that this depended on fertilization. The average hatch was 80%. Diet II was considered to be successful. Diet III Ingredients Grams Water 140.0 ml Casein 5.0 Soybean hydrolyzate (Enzymatic) 15.0 4M KOH 0.5 ml Wesson's salt 1.4 Sucrose 4.0 Choline chloride 0.15 Vitamin solution 1.6 ml Ascorbic acid 0.5 Cholesterol 0.1 Inositol 0.1 Butterfat 2.0 Methyl p-hyrodxybenzoate 0.1 Aureomycin 0.05 Agar 3.0 In this diet 15.0 grams of soybean hydrolyzate (enzyma tic) were substituted1for the fresh pork liver of Diet II 88 hoping that it would provide some of the essential amino acids deficient in casein. The soybean hydrolyzate was a Nutritional Biochemical product. There was no other difference between Diets II and III except for a very slight change in the ratios of the ingredients. The results of rearing C. maculata on Diet III appear in Table 9. High mortality during larval development was observed. Most of the fourth instar larvae did not pupate, however, when they did pupate a high mortality was again observed during the pupal development. The adults emerging were very weak and undersized. They died within one or two days after emergence. One adult had a deformed tarsus. No adults survived for more than two days regardless of the diet given to them. When the 4th instar larvae were fed on Diet IV, undersized adults were produced which survived. Diet IV This diet had the same constituents as Diet III except for: a. ten grams of casein were used instead of 5 grams; b. ten grams of soybean hydrolyzate were used instead of 15 grams; c. twenty-five grams of chicken liver were added. Decay was a problem. Results of rearing single Table 9. Development of different stages of Coleomeqilla maculata on Diet III. Number developing to 2nd 3rd 4th Per cent of Replicates Initial Number instar instar instar Pupa Adult development 1 10 5 5 4 2 1 10 2 10 7 6 5 3 0 0 3 10 1 0 0 0 0 0 4 10 4 3 3 1 1 10 5 1 0 3 3 2 1 0 0 6 10 5 4 3 0 0 0 oo 90 larvae in tubes on this diet appear in Table 10. This diet was successful. Adults were normal in size and behavior. Copulation readily occurred. Egg laying was average compared with individuals fed on aphid diets. Diet V , Ingredients Grains Water 140 ml Casein, enzymatic hydrolyzate 10 Soybean hydrolyzate 10 4M KOH 0.5 ml Wesson's salt 1.4 Sucrose 3 Choline chloride 0.15 Vitamin solution 1.5 ml Ascorbic acid 0.5 Cholesterol 0.1 Inositol 0.1 Butterfat 2.0 Glycogen 4.0 Methyl p-hydroxybenzoate 0.10 Aureomycin 0.05 Table 10. Development of different stages of Coleomeqilla maculata on Diet IV. Number developing to 2nd 3rd 4th Per cent of Replicates Initial Number instar instar instar Pupa Adult development 1 10 8 8 8 8 8 80 2 5 5 5 5 5 5 100 3 14 12 12 11 11 11 78.6 4 8 7 7 7 7 7 87.5 5 10 TO 10 10 10 10 100 6 10 8 8 8 8 8 80 7 13 11 11 10 10 10 76.9 92 Diet V (continued) Ingredients Grains Agar 3.00 The difference between Diets IV and V was: a. 4 grams of glycogen were substituted for the 25 grains of fresh chicken liver,- b. casein enzymatic hydrolyzate was substi tuted for casein; c. three grains of sucrose were used instead of four. Results of rearing single larvae in tubes on Diet V appear in Table 11. Diet V was not successful, however, it was better than Diet III. Diet VI Diet VI was similar to Diet V except for: a. fifteen grains of casein were used instead of 10 grams of casein enzymatic hydrolyzate; b. five grams of soybean hydrolyzate were used instead of 10 grams; c. one gram of yeast extractive was added. Ibis extractive is a product of Difco Laboratories. Data from rearing single larvae in tubes on this diet appear in Table 12. Except for high mortality during the first instar this diet appeared to be better than any previous diet without Table 11. Development of different stages of Coleomegilla maculata on Diet V. 2nd 3rd 4th Per cent of Replicates Initial Number instar instar instar Pupa Adult development 1 10 5 5 4 4 1 10 2 * 11 8 7 5 3 2 18.2 3 8 1 0 0 ‘ 0 0 0 4 7 1 0 0 0 0 0 5 10 2 1 0 0 0 0 6 4 0 0 0 0 0 0 VO Ul Table 12. Development of different stages of Coleomegilla maculata on Diet VI. Number developing to 2nd 3rd 4th Per cent of Replicates Initial Number instar instar instar Pupa *Adult development 1 7 2 2 2 2 1 14.3 2 ’ 10 4 3 3 3 3 30 3 7 4 4 4 4 4 57.1 4 10 5 5 5 5 5 50 5 9 3 2 2 2 2 2 2 . 2 6 5 3 1 1 1 1 20 v£> 95 fresh liver. Improvement may have been due to adding the yeast extract and lowering the amount of soybean hydrolyzate which proved to have a toxic effect at high concentrations (data from Diet IX). Diet VII Similar to Diet VI except for the addition of 25 grams of fresh chicken liver. This diet was originally designed to increasethe experimental population since the culture was being lost at this point. Data from Diet VII appear in Table 13. This diet was successful for larvae and adults. Larvae fed and developed normally on it. Adults had an * average size, copulation readily occurred and females laid eggs. The egg color was normal in comparison to those from females fed on aphids. The percentage of hatch averaged 83. Diet VIII Ingredients Grams Distilled water 140 ml Casein 10 Soybean hydrolyzate 10 4M KOH 0.5 ml Table 13. Development of different stages of Coleomeqilla maculate on Diet VII. Humber developing to * 2nd 3rd 4 th Per cent of Replicates Initial Humber instar instar instar Pupa Adult development 1 10 10 10 10 10 10 100 2 15 14 14 14 14 14 93.3 3 10 9 9 9 9 9 90 4 8 8 8 7 7 7 87.5 5 9 9 9 9 9 8 88.9 6 8 8 8 8 7 7 87.5 v£> O' 97 Diet VIII (continued) Ingredients Grains Wesson's salt 1.4 Sucrose 3 Choline chloride 0.15 Vitamin solution 1.6 ml Ascorbic acid 0.1 Cholesterol 0.1 Inositol 0.1 Butterfat ' 2.0 Glycogen 4.0 Yeast extract 1.0 Sorbic acid 0.1 Methyl p-hydroxybenzoate 0.1 Aureomycin 0.05 Agar 3.00 Diet VIII was similar to Diet V except for the addition of: a. one gram yeast extract; b. one tenth gram sorbic acid for inhibition of microorganisms. Sorbic acid is distributed by General Biochemicals, M.W. 112.1. Data from rearing single larvae in tubes on Diet VIII appear in Table 14. From the previous data it was conlcuded that soybean Table 14. Development of different stages of Coleomegilla maculata on Diet VIII. Number developing to 2nd 3rd 4 th Per cent of Replicates Initial Number instar instar instar Pupae Adult development \ 10 10 1 0 0 0 0 2 10 10 0 0 0 0 0 3 12 12 0 0 0 0 0 4 10 8 1 0 0 0 0 5 10 7 1 0 0 0 0 VO CD 99 hydrolyzate in the concentration used had a toxic effect on the larvae. Adults fed on this diet laid few eggs. Diet VIII E Similar to Diet VIII except for the addition of 0.2 ml vitamin E. There was no effect on the larvae as a result of adding vitamin E to Diet VIII. Host of the larvae died during the third instar. Those surviving died in the fourth instar. However, the adults fed on this diet, copulated readily and laid eggs readily. Diet IX This diet was similar to Diet VI except 0.1 gram sorbic acid was added and 0 . 1 gram ascorbic acid instead of the 0.5 gram in Diet VI. It was similar to Diet VI for larval development. The adults fed, copulation rarely occurred, and females laid few eggs. Diet IX E This diet was similar to Diet IX except for the addition of 0.2 gram vitamin E. There was no effect on larval growth. Adults fed and copulated more readily than 100 in Diet IX. Diet X Resembled Diet IX except for the use of 160 ml of distilled water instead of 140 ml, 4 grams of agar instead of 3, and 0.5 gram of ascorbic acid instead of 0.1 gram. This diet resembled Diet VI except for more distilled water, agar and the addition of 0 . 1 gram sorbic acid. It proved to be quite suitable for adults. Feeding, mating and egg laying were better than on any other diet not containing liver. Larvae developed on this diet until the third instar then most of them died. About 5% of the larvae continued development to form undersized adults. Diet XI Resembled Diet X except for substitution of 5 grams of liver fraction 2 for 5 grams of the casein. Liver fraction 2 is a product of Nutritional Bio- chemicals Corp., a dry powder containing the water insoluble fraction of liver. Data from rearing single larvae in tubes on Diet XI appear in Table 15. This diet was quite successful for larvae and Table .15. Development of different stages of Coleomeqilla maculata on Diet XI. Number developing to 2nd 3rd 4 th Per cent of Replicates Initial Number instar instar instar Pupa Adult development 1 10 10 9 9 9 8 80 2 12 10 6 6 6 6 50 3 4 1 1 1 1 1 25 4 5 1 1 1 1 1 20 5 8 7 5 5 4 3 37.5 101 102 adults, however, Diet VII which contained fresh chicken liver was better. Obviously something was still lacking in this diet. Diet XII Ingredients Grains Distilled water 150 ml Sucrose 3 Wheat germ 5 Vitamin solution 3 Butterfat 3 Glycogen 4 Liver extract concentrate 15 Pollen (bee collected) 5 Cottonseed oil 3 Sorbic acid 0.1 Methyl p-hydroxybenzoate 0.1 Aureomycin 0.05 Agar 5 In this diet the following ingredients were eliminated: casein, potassium hydroxide, Wesson's salt, choline chloride, ascorbic acid, soybean hydrolyzate, cholesterol, inositol, yeast extractive and liver fraction 2. The following was 103 added: wheat germ, liver extract 1 :2 0 , pollen and cotton seed oil. The amounts of the following ingredients were increased: vitamin solution, butterfat and agar. So, 13 ingredients were used in this diet instead of 19 ingredients in Diet XI. Wheat germ is a product of Kretschmer Wheat Germ Corp. Liver extract concentrate is a product of Nutritional Biochemicals Corp., a powdered 1:20 concentrate of liver containing appreciable quantities of the identified water soluable liver and B complex factors. Pollen was collected by honeybees. This diet allowed about 10% of the larvae to complete development. The adults did not feed well on it. Diet XIII Ingredients Grams Distilled water 150 ml Casein 7 Wesson's salt 1.25 Sucrose 3 Wheat germ 4 Vitamin solution 3 ml Ascorbic acid 0.4 104 Diet XIII (continued) Ingredients Grams Soybean hydrolyzate 3.0 Butterfat 2.5 Glycogen 2.1 Liver fraction L 3.0 Pollen balls 3.0 Corn oil 2.5 Sorbic acid 0.1 Methyl p-hydroxybenzoate 0.1 Aureomycin 0.06 Agar 5.00 Liver fraction L is a product of Nutritional Bio chemicals Corp., a solubilized dry concentrate containing the alcohol insoluble fraction of liver. Starting from this diet the data for development of larvae in different instars were discontinued since there was no considerable advantage of taking such data. Instead, data were taken for number of larvae, number of adults and time required for development only. Data on Diet XIII appear in Table 16. Adults fed, copulated and the females laid eggs. 105 Table 16. Development of Coleomeqilla maculata on Diet XIII. Period from egg No. of No. of Per cent of to adult No. larvae adults______development______Days______ 1 20 10 50 35-50 2 20 8 40 32-48 3 20 12 60 30-42 4 24 16 66.7 28-40 5 11 5 45.5 24-41 6 11 4 36.4 27-43 7 40 12 30 22-45 8 28 13 46.6 24-40 9 3 1 33.3 42 10 20 7 35 23-29 11 12 6 50 24-36 106 It was a fairly successful diet. A modification of this diet resulted in Diet XIV. Diet XIV Ingredients Grams Distilled water 200 ml Casein 6 Soybean hydrolyzate 3 Liver extract concentrate 3 Wheat germ 4 Butterfat 2 Glycogen 2 Sucrose 2 Dextrose 1 Corn oil 2 ml Brewer's yeast 1.0 Wesson's salt 1.25 Vitamin solution 2.0 ml Ascorbic acid 0.4 Sorbic acid 0.1 Methyl p-hydroxybenzoate 0.2 Aureomycin 0.06 * Agar 4.0 107 Data from rearing single larvae in tubes on Diet XIV appear in Table 17. The adults fed on this diet, copulated and laid eggs. However, the color of the eggs was abnormal in comparison to the color of the eggs laid by females fed on an aphid diet. The eggs were yellow instead of orange. Diet XV This was similar to Diet XIV except for substituting 2 chicken eggs for the one gram of Brewer’s yeast and using Liver fraction "L" instead of Liver extract concentrate. There was no significant difference between Diet XIV and Diet XV, however, microbial growth was more of a problem in Diet XV than it was in Diet XIV. Diet XVI Ingredients Grams Distilled water 200 Casein 6 Soybean hydrolyzate 3 Liver fraction "L" 3 Brewer's yeast 1 Cotton leaf extract (carotinoids) 50 leaves 108 Table 17. Development of C. maculata on Diet XIV. Period from egg No. of No. of Per cent of to adult No. larvae adults_____ development______Days______ 1 4 2 50 30 2 10 5 50 23-32 3 12 5 41.7 27-35 4 6 3 50 25-51 5 11 1 9 37 6 14 7 50 27-38 7 5 1 20 33 8 13 6 46.2 30-35 9 7 1 14 37 109 Diet XVI (continued) Ingredients Grams Glycogen 2 Butterfat 2 Corn oil 3 * Wheat germ 4 Vitamin solution _ 3 Sucrose 3 Dextrose 3 Ascorbic acid 0.5 Wesson's salt 1.25 Sorbic acid 0.2 Methyl p-hydroxybenzoate 0.2 Aureomycin 0.06 Agar 4.0 The cotton leaf extract (carotinoids) was made by a procedure described by Paech and Tracey (1955). 1. Leaves ground in a large blender and extracted 3 times with acetone. 2. Acetone extract diluted with 2 volumes of water. 3. This mixture was extracted several times with ethyl ether. Reduce ethyl ether extract to dryness (in a rotary evaporator under vacuum). 110 4. Sufficient ethanol (but not less than 10 ml) was added to the dry residue to dissolve it completely and then 60% (W/V) aqueous potash (1 ml/ 1 0 ml ethanol) was added with shaking. This mixture is covered with nitrogen (to prevent oxidation) and left for 12-16 hours (preferably over night) in the dark at room temperature. The solution is then diluted with 3-4 times its volume of water and extracted with an equal volume of peroxide-free ethyl ether. The extractions were continued until all the pigment is extracted. The combined ether extracts were washed with about one-half their volume of warm water; this was repeated until all the soaps were washed from the ether layer, b. The ether extract was impregnated on casein, dried under vacuum until the odor of the ether disappeared. The mixture was frozen until it was used. Data from rearing single larvae in single tubes on Diet XVI appear in Table 18. This diet was very successful for larval development. The period of development was not so variable as was the case with some other diets. However, changing the feeding Ill Table 18. Development of Coleomeqilla maculata on Diet XVI. Period from egg N o . of N o . of Per cent of to adult No. larvae adults development Days Q 1 7 87.5 21-28 2 5 4 80 21-37 3 2 2 1 0 0 24-25 4 8 8 100 21-26 5 2 2 1 0 0 29-30 6 2 1 50 30 7 12 11 91.7 28-31 112 tubes for larvae once or twice during their development was necessary. The adults reared on this diet fed, copulated and the females oviposited. Copulation could be increased by adding 0.2 ml vitamin E. Since this diet was a successful one it was used for rearing the beetles under laboratory conditions. Eight generations were reared on'this diet without observed loss in the viability of the culture. This diet was tried under field conditions in cages but it failed. Earwigs, some carabids and elaterids fed on the diet in the field. In all toxicological and ecological studies carried * on in the laboratory Coleomeqilla maculata was fed on this diet. It failed to support Coccinella novemnotata Herbst, Cycloneda spp. or Hippodamia convergens. 011a abdominalis (Say) fed on the diet as adults, but neither copulation nor laying eggs was observed (Fig. 24). This point needs further investigation. During the course of rearing C. maculata in the laboratory partial incompatibility was observed in some pairs which apparently resulted in a low percentage of egg viability. This could usually be corrected by changing the male. The causes for this are not known. 113 Fig. 24. Olla abdominalis feeding on Diet XVI. PART III The effect of crowding on egg production by Coleomeqilla maculata INTRODUCTION In order to obtain maximum egg production per female, an experiment was conducted to determine the effect of crowding on egg deposition by C. maculata, Many experiments have been done to study the effect of crowding on the fecundity of several insects under laboratory conditions. Different designs have been followed. The conclusions reached have differed from one experiment to another, mostly because.the workers tried to apply their conclusions to "naturally" occurring populations. 115 REVIEW OF LITERATURE Most of the experiments on populations have been done with insects under laboratory conditions. Some of the work has been done on the effect of density of the experimental population upon egg laying. One of the earliest workers was Pearl (1932) who found that as the population density of Drosophila melonoqaster in a limited or closed universe increases, the rate of egg production per female per day decreases. The effect of population density upon egg laying is immediate. Pearl also stated that this effect is probably a result of collision or interference action of the flies upon each other which alters the normal physiological equilibrium and processes of the individual particularly with reference to three major functions: a. food intake, b. energy output in muscular activity, c. oviposition. Later on several biologists worked on the same problem and arrived at the same conclusion, that the production of eggs or young per female decreased with increasing density. Among such workers are Kluijver (1951) who worked with wild populations of the great tit, Parus major L. (after Odum, 1962); Frank (1952) who worked on laboratory cultures of 116 117 Daphnia pulicaria (Forbes); Watt (1955) on Tribolinm confusum Jacquelin du Val; and Nicholson (1957) who stated that in cultures of Lucilia cuprina Wied. governed by larval competition only, a large number of flies laid so many eggs that larval over-crowding prevented any offspring from reaching maturity, whereas in those which were governed by adult competition alone, adult crowding prevented any eggs being produced. Nicholson also observed that the small flies laid fewer eggs. MATERIALS AND METHODS Adults were confined in one pint ice cream cartons with "ample" food and one water tube (See Part II, rearing technique). Seven different treatments were carried out: 1. One mated female and one feeding tube. 2. Two mated females and one feeding tube. 3. One male and one female and one feeding tube. 4. Two males and two females and two feeding tubes. 5. Three males and three females and two feeding tubes. 6 . Five males and five females and two feeding tubes. 7. Five mated females and two feeding tubes. A water tube was supplied to all treatments. The food was changed each 36 hours. The water tube was changed each 4 days.Eggs were collected at 12 hour periods (9 a.m. and 9 p.m.). The experiment was carried out from June 5 until July 11, 1963. 118 RESULTS Summarized data for egg production under different population levels within a standardized space,are shown in Table 19. Obviously intraspecific competition occurred. Even one female in a one pint box laid more eggs than two females together. A female with a male in one oviposition cage laid more eggs than the average laid per female if two of them were together. So it seems that not only the number of individuals present in a space but also the sex of these individuals affects oviposition. No eggs were obtained when more than 2 males and 2 females were held in these cages. It may be concluded that in a rearing program for this insect maximum egg production should be obtained by •confining mated females singly in the oviposition cages. • Satisfactory egg production was also obtained by caging one male and one female together. The last method was used in the rearing technique carried out in this work. 119 Table 19. Effect of different population levels on egg production in C. maculata. Reps. II______III______IV______Mean______s.d. Tr. No. B E_____ B______E B E B E B______E____ B______E 1- 10 41 4 21 3 19 2 8 4.75 22.25 3.6 13.7 2 3 13 4 12 5 27 3 18 3.75 17.5 0.9 6.9 3 2 17 . 4 16 1 3 4 23 2.75 14.75 1.5 8.4 4 2 8 2 7 2 8 0 0 1.5 5.75 1.0 3.86 5 0 0 0 0 0 0 0 0 0 0 0 0 6 0 0 0 0 0 0 0 0 0 0 0 0 7 0 0 0 0 0 0 0 0 0 0 0 0 Reps.=Replicates; Tr.-Treatment. For description of the treatments see "Materials and Methods.“ B=number of egg batches E=total number of eggs PART IV The effect of DDT, toxaphene and endrin on the reproductive and survival potentials of Coleomegilla maculata De Geer INTRODUCTION Few studies have been reported on the effect of insecticides on entomophagous insects. So, the toxicity of DDT, toxaphene, endrin and toxaphene-DDT mixture to active and diapausing adults of the spotted lady beetle, Coleomegilla maculata were studied. During the course of these studies it was found that the effect of the previously mentioned insecticides on the reproductive and survival potential of this beetle should be determined. This point has not been investigated before. An interesting paper which deals partially with this subject was published during the course of this study. Pickett and Patterson (1963) studied the effect of arsenates on the fecundity of some diptera; four species of adult Diptera, Rhagoletis pomonella (Walsh), Drosophila melanogaster Mg ., Drosophila hydei Sturt., and Musca domestica L., showed varying degrees of reduced fecundity when sublethal doses of arsenic were included in their food. Egg production was greatly reduced when the newly emerged adults were fed sublethal doses of arsenates. The ovaries were examined under a stereoscopic microscope about 3 weeks after emergence. An orchard test confirmed the laboratory results. Arsenates may influence the fecundity of many species, or other orders of insects or even other classe of animals. REVIEW OF LITERATURE Many papers have been published on the toxicity of a certain pesticide to several pests, or on the toxicity of several pesticides to a certain pest. There are also some published papers on the LD50 of several pesticides mostly on mammals and arthropods. Few papers have been published on the effect of sublethal doses of a chemical on an insect. This review will pertain only to the papers published on the effect of sublethal doses. Also there will be a short review on detection and estimation of chlorinated pesticides in vege tables and some animal products, since such a review was necessary as a basis for developing a rapid method for the estimation of DDT in eggs, adults and feces of Coleomegi11a maculata. Friederichs and Steiner (1930) stated that the older larvae of the pine moth, Bupalus piniarius L., are not always killed by arsenical dusts. Both poisoned and unpoisoned larvae reached the pupal stage, but three times as many of the latter produced adults. The pupae of poisoned larvae were noticeably smaller, the fertility of females from them 124 125 being much impaired. Breeding experiments with eggs collected in stands dusted in the previous year showed that the resultant larvae produced 3.4% of pupae, as compared with 19% in the non-poisoned groups. Nenyokov and Tareeva (19 31) concluded that incomplete poisoning affects the metabolic process in insects and would probably reduce their reproductive power. This conclusion is in agreement with data obtained during a campaign against the cedar moth, Dendrolimus sibiricus Tshtv., when calcium arsenite was used. Owing to unfavorable weather conditions some of the larvae were incompletely poisoned and pupated; some of the pupae, however, did not give rise to adults and a considerable percentage of moths that emerged had under developed wings and either did not oviposit or only laid about 30% the usual number of eggs. Pielou and Glasser (1952) in selecting for DDT resistance in Macrocentrus ancylivorus Rohw, found that after a month of selection, the resistance of the females became 4.35 times, and that of the males 3.7 2 times, as great as that of the original stock. Hoskins and Gordon (1956) published an excellent review on arthropod resistance to chemicals. One of the points they discussed was "The biochemical basis of insecticide 126 resistance." They suggested that insecticides kill by interfering with some "sensitive mechanism" essential to life. The action of the insecticide on the sensitive mechanism may be countered in two ways: a. by addition of a "protective mechanism" which somehow prevents the interaction of insecticide with the sensitive mechanism or b. by changing or replacing the sensitive mechanism by some "insensitive mechanism" not affected by the insecticides. Two of the protective mechanisms discussed and of .interest to this work are: 1. Detoxification mechanisms, e.g. an enzyme system that can quickly destroy the insecticide. The detoxification of DDT in the housefly and some other insects to DDE is an example. 2. Storage mechanisms such as the storage of insecticides in regions of the insect body where it has no harmful effect. It is well known that the highly lipoid-soluble insecticides such as DDT are accumulated in the fat of rats and other mammals. Mobilization of the fat reserves during starvation releases this stored DDT, which then causes the characteristic symptoms of DDT poisoning. An analogous situation may occur in the larvae of DDT susceptible houseflies reared 127 on media containing DDT. The larvae contain large quantities of unchanged DDT (presumably stored in the fat reserves), which is released during pupation and kills the adult before it can emerge or after ward. Adult resistant flies can contain relatively large quantities of unchanged DDT or lindane. King (1958) found that different strains of Drosophila melanogaster differ in response to selection for resistance. All evidence points toward a polygenic system of resistance. In one line out of four there were sex linked resistance factors, but thi-s line carried autosomal factors as well. The crosses between lines had an LD50 intermediate between the parental values and was always close to the geometric mean showing an absence of dominance and generally additive effects on a logarithmic scale. Beard (1960) stated that insecticides applied to populations should be considered as ecological factors affecting insect numbers. He found that DDT treated house fly populations showed a modified reproductive biology disadvantageous to the flies. The various population patterns testify that different insecticides differ in their long range effects and their influence is not limited to their lethal action in merely removing a portion of the flies. 128 Borkovec (1962) in a study on chemosterilants, stated that most chemosterilants have some degree of species specificity. He suggested a relationship between chemo- sterilants and so called antitumor compounds used in cancer chemotherapy, such as alkylating agents, antimetabolites, radiomimetic compounds and mitotic poisons. Hays and Cochran (1964) found that certain mammalian hormones and related compounds lowered the normal fertility rate or sterilized adults of the plum curculio, Conotrachelus nenuphar (Herbst) when administered by different methods. The effect of those compounds was on both sexes. In larval treatments, only the adults of those larvae treated with Enovid or progesterone-diethylstilbestrol produced no off spring. No deviation from normal mating behavior was observed. A high mortality rate occurred in some treatments. Bartlett (1963) studied the contact toxicity of some pesticide residues to hymenopterous parasites and coccinellid predators. He stated that the effect of each pesticide upon most kinds of adult parasitic Hymenoptera may be . anticipated with a high degree of reliability while the effect upon predatory coccinellids is much less predictable. Bartlett listed some materials as broadly toxic to most of the entomophagous species. DDT and endrin were among those 129 materials. For adults of Hippodamia converqens, DDT had a low toxicity, toxaphene a medium toxicity and endrin a high toxicity. For interpretation of the toxicity data the methods of Abbott (1925) and Finney (1952) were used. For the determination of DDT in Coleomegilla maculata adults, eggs and feces a method was developed from the methods suggested by Mills (1961), Johnson (1962), Onley and Mills (1962), Mills, Onley and Gaither (1963), and Blinn and Gunther (1963). These papers dealt with the detection and estimation of chlorinated pesticides in milk, milk products, chicken eggs and some vegetables. MATERIALS AND METHODS The chemicals used were: DDT: 1,1,1-trichloro- 2,2-bis (£-chloropheny1) ethane; endrin: 1,2,3,4,10,10- hexachloro-6 ,7-epoxy-l,4 ,4 a,5,6 ,7,8 ,8 a-octahydro-l,4-endo- endo-5,8 -dimethanonaphthalene; toxaphene: chlorinated camphene containing 67-69% chlorine. Four different treatments were tested on Coleomeqilla maculata. The treatments were DDT, toxaphene, endrin and toxaphene-DDT 2:1 mixture. The first three materials were tested on diapausing and active beetles. Toxaphene-DDT was tested only on active beetles. Beetles for most of the tests were collected at Boyce, Louisiana, from their diapausing quarters, mostly under loose bark and trash near the stems of large pecan trees growing in cotton fields. The beetles were in aggregations of hundreds and sometimes thousands. Tne diapausing beetles were brought to the laboratory and divided into two groups. Group one was left in diapause, and held in a 60°F. room in 1/2 gallon cartons with some trash and bark. In group two the beetles were separated from the trash, placed in 1/2 gallon cartons with food and water tubes 131 and held at 80° F. under a 16-hour photoperiod provided by a mercury vapour lamp. This treatment was designed to break diapause. Mating was the category used to decide that the beetles had become active. In some of the treatments laboratory reared beetles, from parents collected at Baton Rouge, Louisiana, were used. For each treatment information about the location where the beetles were collected and whether they were laboratory reared or field collected will be indicated. In all tests acetone was used as a solvent. The dosage used was applied topically at the point where the posterior margin of the pronotum meets with the midline of the elytra. One microliter of acetone solution containing the desired amount of insecticide was used per beetle. The ^treated beetles were then placed in one pint cartons, 10 i beetles in each carton with a food tube and a water tube as previously described in Part II, Fig. 1. Counts for live and dead beetles were made at 0.0, 1.0, 2.0, 3.0, 5.0 and 7.0 days. Abbott's formula was used for computing the per cent killed by the treatment in each count. The fourth count (72 hours) was used for computing the log dose-probit line for each material (Finney, 1952). For each dosage, time versus mortality was drawn in a log 132 time-probit line. This line is a theoretical line, however, it demonstrated the relative effect of the dosage on longevity. All the probit analyses were programmed at the University computer center. Biotic potential is the ability of the population to reproduce and survive, i.e., natality versus mortality. Some writers mix the term biotic potential with reproductive potential and consider them synonymous, but there is a difference. Biotic potential can be divided into two categories: a. Reproductive potential. b. Survival potential. Reproductive potential is the natality rate of the organism. Survival potential is the ability of the organism to live under different environments, i.e., the ability of the organism to withstand environmental resistance. The survivors of sublethal treatments were kept and the effect of the treatment on their biotic potential was studied. They were placed in pairs in cartons containing food and water tubes. The number of eggs produced by each female was recorded until the female died. This was the way the reproductive potential was measured. The effect of the 133 treatment on survival potential of the progeny of the treated adults was measured as follows: the larvae were reared in single tubes using the rearing technique described above. The number of larvae reaching the adult stage was recorded. Since the treatments, especially DDT, characteristically affected the biotic potential of the insect, estimation of DDT in and "on" the treated beetles and their eggs was essential. A rapid method for estimation of DDT was developed. Reagents; a. Acetonitrile technical grade. Purified by the following procedure: to 1 gallon add 1 ml 8596 H 3PO , 30 grams P2O 5 an<3 boiling chips and distill " f at 81-82°C. Do not exceed 82°C. b. Anhydrous Na2S0^, analytical reagent. c. Distilled water. d. Hexane, Chromatoquality Reagent 99+mol%. Matheson Coleman and Bell. e. Saturated solution of NaCl, analytical reagent. f. Acetone, analytical reagent. Apparatus: a. Graduate cylinders, 50 ml, Pyrex. b. Fritted glass funnels, Buchner type, 30 ml, Pyrex. c. Separatory funnels, pear shaped, 125 ml, Pyrex. 134 d. Pesticide Residue Analyzer (Gas chromatograph), G C - 1607; Microtek, Baton Rouge. e. Waring Blender, 50 ml. f. Test tubes, 30 ml, Pyrex. g. Rubber stoppers to fit the test tubes. h. Pressure pump. All glassware was washed thoroughly in soap and water then washed twice in tap water, twice in distilled water and rinsed with acetone analytical reagent grade, then placed in an oven for at least 24 hours at 190-200°C. Method: 1. To 20 beetles (.24409+0.00005 grams) or at least 40 eggs (0.00256+0.0002 grams) add 15 ml acetonitrile. Place in the Waring Blender. Mix at low speed for 2 minutes, then at high speed for 1 minute. Filter under pressure through a fritted glass funnel into a 50 ml graduate cylinder, wash the Waring Blender jar twice, each time with 3-5 ml acetonitrile and filter in the same funnel into the same graduate cylinder. Record volumes of the acetonitrile used and the volume of the filtrate. 2. Transfer measured filtrate to a 125 ml separatory funnel. Carefully measure 10 ml hexane in the same graduate cylinder used for the filtrate. Pour the hexane into the 135 separatory funnel and shake vigorously for 3-4 minutes. Add 1 ml saturated Na Cl and 60 ml distilled water, and mix gently but thoroughly. Allow to separate, discard aqueous layer and wash twice successively, each with 10 ml distilled water. Discard washings, transfer the solvent layer to a 50 ml graduate cylinder and record volume. 3. Transfer the solvent to a test tube, add 1.5- 2.00 grams anhydrous Na2S0 ^ and shake vigorously. Then the solvent should be ready for chromatography. Calculations: Sample represented by the solvent=S X(F/T )X(y^). S=grams of sample used, or number of beetles or eggs used. F=volume of filtrate. T=total volume of acetonitrile used. H=volume of solvent (Hexane), from Step 3. Gas Chromotography: Inject a known volume of the solvent (usually 1 to 9 microliters) in the pesticide residue analyzer. During analysis the gas chromatograph was adjusted o o o on: inlet, 210 C ; oven, 180 C.; detector, 190 C.; attenuation, 8x1; the flow of gas, 200 ml/min., 30 volt pulse. Under these conditions the DDT started appearing on the chart 136 after 8 minutes and ended at 11 minutes. If the solvent has a high concentration of DDT (more than 4 nanograms of DDT per microliter), appropriate dilution should be made to 1 to 2 nanograms of DDT per microliter. The previously mentioned method was also used for estimation of DDT in the feces of the beetle. « RESULTS Toxicity of DDT to Coleomeqilla maculata Data obtained from treating diapausing and active adults topically with DDT are summarized in Table 20. Effect of DDT on longevity Log time-probit lines for treatments at 10, 30 and 60 micrograms per beetle in diapause are shown in figure 26. For the 20 and 40 microgram treatments, the line was almost horizontal, so the points were out of the range of the scale used on the graph. The log time-probit line is completely theoretical, however, it gives a good indication of the effect of a certain treatment on longevity of the adults, a point which has not been reported before. The slope of the line actually indicates the effect of the insecticide on the longevity of the beetle. The steeper the slope, the stronger the effect of the insecticide. The homogeneity or heterogeneity of the population might have some effect on the slope, however, it should be nullified since beetles under comparison are drawn from the same experimental population. From figure 25: 0 10 = 0.233 137 Table 20. Toxicity of DDT to diapausing and active adults of Coleomeqilla maculata collected at Boyce, Louisiana, February, 1963. Microqrams of toxicant per beetle Days after 10 20 30 40 60 Check Treatment Dead Total Dead Total Dead Total Dead Total Dead Total Dead Total Diapausinq 1 6 60 14 60 13 52 21 60 29 60 0 60 2 11 60 14 60 13 52 21 60 29 60 0 60 3 11 60 14 60 13 52 21 60 29 60 0 60 5 14 60 14 60 14 52 21 60 31 60 0 60 7 14 60 15 6'. 16 52 23 60 33 60 0 60 Active 1 3 60 7 60 12 60 15 60 24 60 0 60 2 9 60 14 60 14 60 20 60 29 60 0 60 3 9 60 16 60 14 60 21 60 30 60 0 60 5 9 60 17 60 21 60 27 60 34 60 0 60 7 11 60 17 60 22 60 32 60 27 60 0 60 OJ 00 Per cent mortality 95 98 40 80 90 60 50 70 20 i. 5 Lg iepoi lns o D-rae . auaa (diapausing) maculata C. forDDT-treated lines time-probit Log 25.Fig. 0.01 0.020.03 olce a oc, oiin, eray 1963. February, Louisiana, Boyce, at collected boss 30micrograms perbeetle 60micrograms perbeetle 10 micrograms perbeetle Time in days logarithmicon scale Probits 140 0 30 — 0.073 0 60 = 0.147 0 45 3 Average slope for the three lines = — =0.151. Since the slope is relatively small, the DDT treatments have almost no effect on the longevity of the survivors. The log time-probit line for active beetles is shown in figure 26. The slopes of the lines for different treatments are: 0 10 = 0.25 0 20 = 0.25 © 30 = 0.25 0 40 = 0.32 0 60 = 0.24 DDT tended to affect longevity more in active than in diapausing beetles but the effect, though consistent, was not significant. The effect was not consistent in the case of the diapausing beetles. Toxicity of DDT to diapausing and active beetles The summarized data for tests carried out to determine the toxicity of DDT to diapausing and active beetles are given in Table 21. The data are from three tests carried out January 5, February 13 and February 20, 1963. The beetles Per cent mortality . . 0.3 0.10.2 40 80 10 50 90 60 70 95 i. 6 Lg ieroi lns o DTtetd . auaa (active) collected C. maculata for DDT-treated lines time-rpobit Log 26.Fig. 20 30 5 - - 6 /beetle 60 — . . 40 tBye Lusaa Fbur, 1963. February, Louisiana, Boyce,at 20 10 /beetle Z Z1 1 /beetle /beetle TimeTime in days logarithmicon scale ? 3 ? l 10 20 20 Probits Table 21. The comparative effect of DDT on active and diapausing C. maculata. Beetles held at 80°F. and observed at the end of 72 hours. Dose in micrograms ______Diapause______Active per beetle Dead Total % mortality Dead Total % mortality Check, 0 0 80 0.00 0 80 00.0 10 13 80 16.25 10 80 12.50 20 18 80 22.50 18 80 22.50 30 18 80 22.50 17 80 21.25 40 30 80 37 .50 24 80 30.00 60 39 80 48.75 40 80 50.00 142 were collected at Boyce, Louisiana, and held at 80°F. The data are based on observations at the end of 72 hours. Log dose-probit lines for diapausing and active beetles are illustrated in figure 27. The was 79.1 micrograms per active beetle, which is 6482.3 milligrams per kilogram. For diapausing beetles the bD^Q was * 3 micrograms per beetle, which is 6328.6 milligrams per kilogram. The difference in toxicity of DDT to diapausing and active beetles is not significant. The slopes of the two lines indicate that there was great heterogeneity in resistance to DDT in the experimental population which was slightly greater in the diapausing than in the active beetles. The average weight of the active beetles based on a 100 beetle sample from both sexes was 0 . 0 1 2 2 0 2 gram, while that of diapausing beetles was 0.012207 gram. The effect of DDT on the reproductive and survival potentials Adult males and females'which survived the 40 microgram DDT treatment (LD^) were kept together, whether diapausing or active in half gallon ice cream cartons with appropriate food and water. The untreated animals were handled similarly. Whenever a male and female populated, they were Per cent mortality i. 7 ID oepoi lns o D-rae . auaabsdo 7 hour 72 on based maculata C. IxDg forDDT-treated lines dose-probit 27. Fig. 40 60 20 80 70 90 2 bevtos Bye Lusaa Fbur, 1963. February, Louisiana, Boyce, observations, 3 5 4 Diapause Active 10 Dosage logarithmicon scale 0 0 0 50 40 30 20 0 200 100 Probits 145 isolated in a one pint ice cream carton with a food tube and a water tube which provided conditions appropriate for laying eggs . Within 10 days 20 pairs were isolated from the check, while in the survivors of the treatment, no copulation took place. This was probably due to the effect of the insecticide. So, copulation was designated as the point of recovery from treatment. However, the treated beetles did not feed on the diet for two days after treatment, most of them started feeding on the third day. The adults recovered, copulated and started laying eggs 16 days after treatment. A record of the number of egg batches and the number of egqs per batch was kept for each couple in the treatment and the check. The egg record was carried on till all the females died (February, March, April, May, June and July). The eggs were collected at two-hour intervals from 7 a.m. till 11 p.m., except for a few times during the five month period. . However, most of the eggs were laid during May and June. The eggs were held till they hatched, then the first instar larvae were reared individually using the rearing technique previously described in Part II. The number of larvae which pupated and the number of adults which emerged 146 were recorded. From this record the effect of DDT on the F^ of treated beetles was studied. F^ had never been subjected to DDT. So, this study covered the effect of DDT on the reproductive potential (number of eggs) as well as the survival potential of F^ (number of larvae which completed development). The summarized data obtained are given in Table 22. In the 6 treated couples one female did not lay any eggs. Whether this was due to the treatment or to chance is not known. As a result of this case the variations were very high in the statistical analysis and none of the differences were significant at the 0.05 level. There was 6 6 .8% more eggs laid by the treated than by untreated females and the number of larvae was significantly higher in the treatments than in the check unless a one-in- ten chance occurred in sampling. For treated beetles the percentage of F^ larvae which pupated was 20.9, while it was 52.7 in the check. As in the previous case the number of pupae in the treatment dropped and the t test indicates no significant difference between the two groups. The percentage of pupae which gave rise to adults was 96.3 in the checks and 89.2 in the treatment. Table 22. The effect of 40 micrograms of DDT per beetle on the reproductive and survival potentials of Coleomeqilla maculata. No. o f N o . o f No . o f No. o f N o . o f Batches______eggs______larvae______pupae______adults_____ Category couples Tot. P.C. Tot.______P.C._Tot. P.C.___ Tot. P.C._____Tot. P.C. Check 6 30 5.0+2.6 211 35.2+15.7 148 24.7+7.6 81 13.5+3.4 78 13.0+3.2 Treatment 6 37 6 .2+3.5 352 58.7+31.3 277 46.2+24.6 65 10.8+6.1 58 9.7+4.8 Per cent development: Check, 52.7: Treatment, 20.9. Tot.=total; P.C.^per couple 148 The differences found between the DDT-treated beetles and the check is due to either a genetic difference obtained as a result of selection by DDT or to the presence of DDT or its metabolites in the beetles and their progeny. In either case the inducing factor was DDT. So, DDT causes disturbances in the reproductive and survival potential of this insect. Determination of DDT in treated beetles and their progeny The beetles treated with DDT had a higher reproductive potential than the checks, and the of those beetles had a lower survival potential. There was a probability that these effects might be due to the presence of DDT in the F^ of the treated beetles. The method previously described was used to determine DDT in the adults, eggs and feces of the beetles. Beetles collected in February, 1964, at Boyce, Louisiana, were treated with 40 micrograms of DDT per beetle and then held in oviposition cages. The eggs were collected in acetonitrile and held in a freezer at 0 + 3°F. till analyzed. Samples of twenty beetles each were collected at 0, 0.5, 1.5, 2.5, 3.5, 5.5, 8.5, 12.5, 17.5, 24.5 and 30.5 days . DDT was qualitatively determined and found to be 149 present in all the specimens collected from the treated beetles, while there was no DDT in the checks, DDT was also found to be present in the feces of the treated beetles. During the analysis an unknown compound appeared on the chromatograph at 4-6 minute intervals. It was present in the adults and eggs of both treated and untreated beetles. Since the checks were field collected beetles this point was considered to be of special importance and is still under investigation. The presence of DDT in the eggs of the treated beetles was of special interest since it might be responsible for the lower survival potential of the F^ . The amount of DDT in the eggs laid after a.5 days after treatment was roughly estimated to be 15 nanograms of DDT per egg (108.7 milligrams per kilogram). Such amounts might be responsible for the high mortality in - In the eggs of the check beetles there was not any detectable amount of DDT by the methods used. Toxicity of DDT to Coleomeqilla maculata collected at Baton Rouge, Louisiana In July, 1962, 20 adults (males and females) were collected on the Louisiana State University Experimental Farm on Perkins Road. The adults were brought to the laboratory and reared on aphids and artificial diets. In March, 1963, there was enough progeny from these beetles (7th and 8 th generations) to run the toxicity test on them. The treated beetles and checks were held at 80CF. for observation. Summarized data obtained from this test are given in Table 23. A point which deserves attention is that at the beginning of the rearing program for these beetles there was a high mortality rate in F^ and progeny. The slope of the log time-probit line in figure 28 indicates that the effect of DDT on the longevity of beetles of the Baton Rouge strain is greater than the effect on the Boyce strain. The log dose-probit line for this experiment is shown in figure 29. The slope of the line indicates more homogeneity in response to DDT treatment in the Baton Rouge than in the Boyce population. The LD5 0 was 13.7 micrograms per beetle (1124.5 milligrams per kilogram), which is significantly less than that for the Boyce population. This suggests that this insect is not "naturally resistant" to DDT, but that DDT resistance has developed in the Boyce strain. 151 Table 23. Toxicity of DDT to active adults of C. maculata Baton Rouge, Louisiana, March, 1963. Micrograms of DDT per beetle______Days after ____ 20______40______60______Check treatment Dead Total Dead Total Dead Total Dead Total 0 0 30 0 30 0 30 0 30 1 18 30 17 30 30 30 0 30 2 19 30 18 30 30 30 0 30 3 2 1 30 23 30 30 30 0 30 5 26 30 27 30 30 30 0 30 7 26 30 27 30 30 30 0 30 14 29 30 29 30 30 30 0 30 Per cent mortality i. 8 Lg iepoi lns o DTtetdC mclt (cie collected (active) maculata C. DDT-treated for lines time-probit Log 28.Fig. 40 80 10 50 60 70 90 95 30 .2 0.03 0.02 eeain. cu generations. tBtn og, oiin, n rae n h lbrtr for laboratory the reared in and Rouge, Louisiana, Baton at - /* 0 4 beetle / - 20 0.05 / P ie ndy n oaihi scale logarithmic on indays Time beetle 0.1 . 0.3 0.2 0.5 1 2 8 i 3 Probi ts Probi -1 Per cent mortality i. 9 Lgds-rbt ie o DTtetdC mclt (cie collected (active) maculata C. for DDT-treated line dose-probit Log 29.Fig. 40 20 10 30 50 60 70 80 55 SO generations. tBtn og, oiin, n rae i te aoaoy for laboratory the in reared and Louisiana, Rouge, Baton at 1 2 oaeo lgrtmc scale logarithmic on Dosage 4 5 4 3 10 20 0 0 50 60 30 8 90 7 Probits u> U 1 154 Toxicity of Endrin to Coleomeg illa maculata Summarized data obtained from treating diapausing and active adults with endrin topically is shown in Table 24. Experimental animals were held at 80°F. and observed at 1, 2, 3, 5 and 7-day intervals. Effect of endrin on adult longevity The log time-probit line concept was previously discussed in this work. The effect of endrin on the longevity of diapausing beetles is illustrated in figure 30. Slope values for different treatments are: 9 1 = 0.30 9 2 = 0.66 9 4 = 0.63 9 6 = 1.01 9 8 = 1.29 At a very low dosage (LDq 4 ) endrin had no effect on longevity of the beetles which survived the treatment. However, when the dosage was LD5 (2 micrograms of endrin per beetle) an effect on longevity became obvious. Beyond this point the higher the dosage, the greater the effect of endrin on adult longevity. Table 24. Toxicity of endrin to diapausing and active adults of Coleoipeqill^ maculata collected at Boyce, Louisiana, February, 1963. Micrograms per beetle Days after 1 2 4 6 8 Check treatment Dead Total Dead Total Dead Total Dead Total Dead Total Dead Total Diapause 0 0 60 0 60 0 60 0 60 0 60 0 60 1 1 60 0 60 3 60 10 60 15 60 0 60 2 1 60 3 60 12 60 24 60 31 60 0 60 3 1 60 3 60 14 60 34 60 45 60 0 60 5 2 60 6 60 22 60 45 60 59 60 0 60 7 4 60 11 60 28 60 51 60 59 60 0 60 Active 0 0 30 0 30 0 30 0 30 0 30 0 30 1 ^ 0 30 1 30 1 30 3 30 2 30 0 30 2 0 30 2 30 3 30 9 30 8 30 0 30 3 0 30 5 30 9 30 15 30 2 0 30 0 30 5 1 30 11 30 15 .30 23 30 30 30 0 30 7 3 30 16 30 2 0 30 27 30 30 30 0 30 U1 Ln Per cent mortality i. 0 Lg iepoi lns o edi tetdC mclt (diapausing), maculata C. treated endrin for lines time-probit Log 30.Fig. 40 20 30 60 10 80 70 -0 90 95 5 olce a Bye Lusaa Fbur, 1963. February, Louisiana, Boyce, at collected / / 4 1 2 3 4 50 40 30 20 10 5 4 * / ie ndy n oaihi scale logarithmic on indays Time y / / ' A / y / y - 4 ------ y . 6 8.P 1 ^ / beetle ^ / F 2 Z / beetle beetle f1 / 1 / beetle beetle / 100 200 A ■ ■A 7 Probits The log time-probit lines for active beetles are shown in figure 31. The slopes of different lines are: © 1 = 1.71 © 2 = 0.96 © 4 = 1.08 0 6 = 1.16 0 8 = 2.00 The slopes of the lines indicate that any of the endrin treatments used will affect the longevity of the active beetles. Although endrin affects the longevity of both diapausing and active beetles, the effect is greater on active beetles. Toxicity of endrin to diapausing and active beetles Summarized data for tests carried out to determine the toxicity of endrin to diapausing and active beetles are given in Table 25. The data are from two tests carried out February 13 and February 20, 1963. The beetles were collected at Boyce, Louisiana and held at 80°F. The data are based on observations at the end of 72 hours. The log dose-probit lines for diapausing and active beetles are illustrated in figure 32. The LD^q was 5.8 micrograms per active beetle (471.8 milligrams per kilogram) Per cent mortality i. 31 Fig. 10 40 20 30 50 60 eo 95 70 90 5 , -4 -1 8 6 2 tBye Lusaa Fbur, 1963. February, Louisiana, Boyce,at o tm-rbt ie fr nrn rae . auaa (active) maculaca C. treated endrin for lines time-probit Log P / P / P )* / f f / beetle / / beetle beetle beetle beetle ie ndy n oaihi scale logarithmic on in days Time 4 5 4 3 i . * it 10 0 2 0 50 40 90 :o3lected Probits U 00 1 Table 25. The comparative effect of endrin on active and diapausing C. maculata. Beetles held at 80°F. and observed at the end of 72 hours. Treatmenti micrograms per beetle in one Diapause______Active microliter acetone Dead Total % mortality Dead Total % mortality Check, 0 0 1 0 0 0 0 . 0 0 0 30 0 0 . 0 0 1 1 60 1.67 0 30 0 0 . 0 0 2 3 60 5.00 5 30 16.67 4 14 60 23.00 9 30 30.00 6 35 60 56.67 15 30 50.00 8 45 60 75 .00 2 0 30 66.67 159 Per cent mortality i. 2 Lgds-rbt ie fr nrn rae . auaabsdo 7 hour 72 on based maculata £. treated endrin for lines dose-probit Log 32.Fig. 20 40 0 1 30 50 60 80 95 . bevtos Bye Lusaa Fbur, 1963. February, Louisiana, Boyce, observations. Active Diapausing oaeo lgrtmc scale logarithmic on Dosage 20 Probits o 161 and 5.6 micrograros per diapausing beetle (454.9 milligrams per kilogram). The slope of the line indicates a more or less homogeneous response to endrin in both active and diapausing animals. The effect of endrin on the reproductive and survival potentials Adult males and females which survived the 2 and 4 microgram endrin treatments whether diapausing (LD^ and LD^q) or active (LD^q and LD 3 3 ) were held together in half-gallon ice cream cartons with appropriate food and water. The same was done for the checks. Whenever a male and female copulated, they were isolated in a one pint ice cream carton with a food tube and a water tube which provided appropriate conditions for laying eggs. The treated adults started mating 6 days after treat ment. Six pairs from both the check and treatment were held in oviposition cages. A record for the number of egg batches and the number of eggs per batch was kept for each couple. The eggs were collected at two-hour intervals from 7 a.m. until 11 p.m., except for a few times. Records were continued until all the females died. There are three factors which 162 should be kept in mind that might have interfered with the number of eggs laid by the treated females while not influencing the checks: a. the effect of the presence of endrin or its metabolites in the female's body; b. selection might have happened since not all individuals could survive the "sublethal" dose; c. as previously discussed endrin has an effect on the longevity of the beet’es, and such effect might have reduced the number of eggs laid by each female- In any case endrin was the inducing agent. The eggs were held till they hatched, then the first instar larvae were reared individually. The number of larvae which pupated and the number of adults which emerged were recorded. Since the F^ was never subjected to endrin, this study covered the effect of endrin on the reproductive potential (number of eggs) as well as the survival potential of (number of larvae which completed development). The summarized data appear in Table 26. There was no significant difference between endrin treated and untreated beetles. Toxicity of Toxaphene to Coleomeqilla maculata The summarized data obtained from treating diapausing and active adult3 with toxaphene topically are given in Table 26. The effect of sublethal doses of endrin on the reproductive and survival potentials of C. maculata. No. of couples No. of Batches No. of Eggs No. of Larvae No. of Pupae No. of Adults T. P,C. s.d. T- P.C. s.d. T. P.O. s.d. T. P.C. s.d. T. P.C. s.d. Check 6 30 5 2.6 211 35.2 15.7 148 24.7 7.7 81 13.5 3.5 78 13 3.2 Treated 6 27 4.5 1.6 187 31.2 6 . 6 131 21.8 4.9 87 14.5 2.9 85 14.2 2.9 % of development of larvae: Pupa, Check, 54.7; Adult, Check, 52.7. % of development of larvae: Pupa, Tr., 66.4; Adult, Tr., 64.9. Tr.=treatment; T.=total; P„C.=per couple; s.d.*standard deviation. 164 Table 27. The data arc summarized from two tests carried out February 13 and February 20, 1963. The beetles were collected at Boyce, Louisiana, and held at 80°F. The data are based on observations at the end of 72 hours. Effect of toxaphene on adult longevity The effect of toxaphene on the longevity of diapausing beetles is shown by the log time-probit lines, figure 33. Slope values for different* treatments were: 9 10 = 0.728 9 20 = 0.521 9 30 = 0.589 9 40 = 0.403 9 60 = 0.707 All dosages used affected the longevity of the dia pausing beetles. It seems that regardless of the amount of toxaphene {between 1 0 and 60 micrograms per beetle) the effect on longevity was nearly the same. The effect of different doses of toxaphene on the longevity of active beetles is illustrated in figure 34. The slopes of the lines of the different treatments were: 9 10 - 0.626 9 20 = 0.673 Table 27. Toxicity of toxaphene to diapausing and active adults of Coleomegilla maculata collected at Boyce, Louisiana, February, 1963. ______Microqrams per beetle______Days after ____ 10______20______30______40______60______Check satment Dead Total Dead Total Dead Total Dead Total Dead Total Dead Toti Diapause 0 0 60 0 60 0 60 ' 0 60 0 60 C 60 1 0 60 5 60 6 60 11 60 15 60 0 60 2 2 60 10 60 14 60 19 60 20 60 0 60 3 5 60 13 60 20 60 23 60 31 60 0 60 5 7 60 19 60 29 60 28 60 45 60 0 60 7 12 60 25 60 34 60 30 60 46 60 0 60 0 0 60 0 60 0 60 0 60 0 60 0 60 1 3 60 13 60 22 60 19 60 11 40 0 60 2 9 60 24 60 35 60 39 60 21 40 0 60 3 12 60 28 60 40 60 47 60 27 40 0 60 5 18 60 37 60 50 60 58 60 38 40 0 60 7 26 60 41 60 55 60 60 60 39 40 0 60 Ln Per cent mortality i. 33 Fig. 40 0 1 20 30 50 60 70 80 90 95 5 - olce a oc, oiin, eray 1963. February, Louisiana, atBoyce, collected o tm-rbt ie fr oahn tetdC mclt (diapausing) maculata C. treated toxaphene for lines time-probit Log 5 0 0 0 0 50 40 30 20 10 5 4 ie ndy n oaihi scale logarithmic on indays Time 40 60 0 2 10 30 F F F F F i i / / / / / i a - * * - a beetle beetle beetle beetle beetle Jk 100 200 Probits & Per cent mortality i. 4 Lg iepoi lns o txpee rae . auaa (active) maculata C. treated toxaphene for lines time-probit Log 34.Fig. 40 60. 10 20 50 80 90 95 70 . 03 . 0.5 0.4 0.3 0.2 mh -- 30 . 40 ^ / beetle ... - b 60 ^ / beetle olce a oc, oiin, eray 1963. February, Louisiana, Boyce, at collected 0 2 0 1 P I f2 1 / beetle / beetle / beetle Time in days on logrithmic scale logrithmic on in days Time -r' 2 r 10 20 obits >0 - j 16b © 30 = 0.741 © 40 - 1.207 © 60 = 1 . 129 The higher the dose the greater the effect on longevity of active adults. This was not the case with diapausing beetles on which all doses used affected longevity. Toxicity of toxaphene to diapausing and active beetles The summarized data for tests carried out to determine the toxicity of toxaphene to diapausing and active adults of Coleomegilla maculata are summarized in Table 28. The data are from two tests carried out February 13 and February 20, 1963. The beetles were collected at Boyce, Louisiana, and held at 80°F. The data are based on observations at the end of 72 hours. The log dose-probit lines for diapausing and active beetles are illustrated in figure 35. The LD^q was 44.2 micrograms per active beetle (3618.9 milligrams per kilogram) and 21.8 micrograms per diapausing beetle (1789.1 milligrams per kilogram). These differences were statistically significant at the 0.05 level. So, the diapausing beetles withstand higher doses of toxaphene than the active beetles. The slopes of the lines indicate Table 28. The comparative effect of toxaphene on active and diapausing C. maculata. Beetles held at 80°F. and observed at the end of 72 hours. Treatment micrograms Diapause Active per beetle Dead Total % mortality Dead Total % mortality Check, 0 0 80 0 . 0 0 0 60 0 0 . 0 0 1 0 6 80 7.5 12 60 2 0 . 0 0 20 23 80 28.75 28 60 46.67 30 28 80 35.00 40 60 66.67 40 43 90 53.75 47 60 78. 33 60 47 80 58.75 45 60 75.00 169 rig. 35. Log dose-probit lines for toxaphene treated treated toxaphene for lines dose-probit Log 35.rig. Per cent mortality 40 20 10 50 60 . 70 80 . 90 95 3 5 0 0 0 0 50 40 30 20 10 5 4 3 2 X bevtos Bye Lusaa Fbur, 1963 February, Louisiana, Boyce, observations. Active - - Diapausing -- X X X i.l I oaeo lgrtmc scale logarithmic on Dosage XX X X X C. 0 0 1 auaabsd n 7 on based maculata 2 hour 210 X 0 0 3 0 0 4 7 Probits 171 a more or less heterogeneous population from the standpoint of resistance to toxaphene. This indicates that the population may be in the process of developing resistance to toxaphene. The effect of toxaphene on the reproductive and survival potentials Adult males and females which survived any of the treatments ( 10, 20, 30, 40 and 60 micrograms of toxaphene per beetle) were held in their appropriate group in half gallon ice cream cartons with appropriate food and water. The beetles started to copulate 15 days after treatment. Each copulating pair was isolated in a one pint ice cream carton with a food tube and a water tube which furnished appropriate conditions for laying eggs. Twenty pairs were isolated, each in the oviposition.cage. None of the females laid eggs. Egg laying was normal in untreated beetles. Toxicity of Toxaphene-DDT Mixture to Coleomegilla maculata Toxicity of toxaphene-DDT mixture used in the ratio 2 parts toxaphene to 1 part DDT to active adults of the spotted lady beetle was studied. A solution with a known concentration was first prepared for each of the insecticides. Appropriate amounts of the two solutions were mixed together so that each microliter of the resulting acetone solution contained the desired dose per beetle. The doses used were: 5:2.5, 10:5 and 20:10 micrograms per beetle. Beetles for the experiment were collected from a field at Boyce, Louisiana, January 1963 and held in the laboratory on an artificial diet until March 1963, when the experiment was carried out. The beetles were treated topically and held at 80°F. for observation. The summarized data obtained from this experiment are given in Table 29. Abbott's formula was used for adjustment of the number of dead beetles in the treatment. Effect of toxaphene-DDT mixture on adult longevity The effect of toxaphene-DDT mixture on the longevity of active beetles is shown in figure 36. The slopes of the lines were: 6 5:2.5 = 0.913 9 10:5 = 0.611 G 21:10 = 0.483 The mixture of toxaphene-DDT affected adult longevity. The highest dose killed the beetles so rapidly that the lower end of the line moved up, while the upper point did not move up as fast because the per cent mortality was already more J 7 Table 29. Toxicity of toxaphene-DDT mixture to active adults of C. maculata collected at Boyce, Louisiana, January, 1963. 5:2.5______10:5______20 : 10 Days after Micrograms per beetle______treatment Dead Total Dead Total Dead Total 0 0 30 0 30 0 30 1 4 27 17 27 24 27 2 7 27 19 27 24 27 3 7 27 19 27 24 27 5 17 26 23 26 25 26 7 2 2 26 25 26 26 26 14 23 25 25 25 26 26 Per cent mortality i. 6 Lg iepoi lns o atv C mclt tetdwt toxaphene- with treated maculata C. active for lines time-probit Log 36.Fig. 40 70 • 80 10 20 60 30 50 90 95 0.05 20:10 ^ / beetle / ^ 20:10 0 510: /*5:2.5 D itr cletd tBye Lusaa Fbur, 1963. February, Louisiana, Boyce, at collected DDT mixture 0.1 / P / beetle beetle 0.2 ie ndy o lgrtmc scale logarithmic on indays Time . 0.40.5 0.3 1 3 5 4 3 2 10 Probits 175 than 90 on the third day. This is the reason for the low slope of the line at a very high dose. Thus, the effect of an insecticide on the longevity of any insect should be better studied on low or moderate doses and not on high doses if the log time-probit line is to be used for interpreting the data. Toxicity of toxaphene-DDT mixture to the active adults The summarized data of tests carried out to determine the toxicity of toxaphene-DDT mixture to active adults are given in Table 30. The procedure followed was the same as that described for the active adults. Log dose-probit lines for active beetles treated with toxaphene-DDT mixture is illustrated in figure 37. The LD50 was 7.1-3.6 micrograms per active beetle {584.0 milligrams toxaphene and 292.0 milligrams of DDT in a mixture per kilo gram) . The indicates a synergistic action of the two insecticides against this insect. According to the formula of Sun (I960) the co-toxicity coefficient of the mixture^ 484.5, indicating a strong synergistic effect. The slope of the log dose-probit line indicates more or less homogeneity in the population in response to toxaphene-DDT mixture. 176 Table 30. The effect of toxaphene-DDT mixture on active C. maculata. Beetles held at 80°F. and observed at the end of 72 hours. Treatment Dead Total Check 3 30 5:2.5 jx / beetle 11 30 10 : 5 jx / beetle 22 30 20:10 n / beetle 28 30 Per cent mortality i. 7 Lg oepoi ln fr cie . auaa rae ih toxaphene- with treated maculata C. active for line dose-probit Log 37.Fig. 40 60 20 50 80. 30- 70 90 95 - D itr; ae n 2hu bevtos Bye Lusaa 1963 Louisiana, Boyce, observations. 72hour on based mixture; DDT X 4 oaeo lgrtmc scale logarithmic on Dosage X 5 X t * t - 1 10 A X 20 1 A1 21 5 7 Probits -> j 178 The 'effect of toxaphene-DDT mixture on the reproductive and survival potentials of Coleomegilla maculata Survivors from 5-2.5 toxaphene-DDT mixture treatment were held in half-gallon ice cream cartons and treated the same way as was done in the study of the effect of DDT on the reproductive and survival potentials. The summarized data appear in Table 31. There was no significant effect of toxaphene-DDT treatment on the reproductive potential of the treated females. However, the number of eggs laid by the treated females was 35% less than the checks. The toxaphene-DDT treatment reduced the survival potential of the and the progeny production of the treated beetles significantly at the 0 .01 level. Summary Effect on longevity DDT did not affect longevity but endrin, toxaphene and toxaphene-DDT mixture reduced the longevity of the treated beetles. Toxicity The Boyce strain was about 6 -fold more resistant to Table 31. The effect of a "sublethal" dose of toxaphene-DDT mixture on the reproduction and survival potentials of £, maculata. Tr.=treatment; T.=total; P.C.=per couple; s.d.=standard deviation. No. Of couples No. of Batches No . of Eqqs No. of Larvae No. of Pupae No. of Adults - T. P.C. s.d. T. PiC.-s.d. T. P.C. s.d. T. P.C. s.d. T. P.C. s.d. Check 6 30 5 2.6 211 35.2 15.7 148 24.7 7.7 81 13.5 3.5 78 13 3.2 Tr. 6 21 3.5 1.05 137 22.8 5.7 97 16.2 4.3 48 8.0 1.4 47 7.8 1.6 % development of larvae to pupa: Check, 54.7; Tr., 49.5 % development of larvae to adult: Check, 52.7: Tr., 48.5 179 180 DDT Baton Rouge strain. Active and diapausing beetles treated with DDT or endrin responded alike, however, active and diapausing beetles treated with toxaphene differed significantly in their response. Diapausing beetles could withstand higher doses of toxaphene than active beetles. There was a synergistic effect between toxaphene and DDT in their toxicity to this insect. Effect on Reproductive and Survival Potentials DDT increased or at least disturbed the reproductive potential of the treated females. DDT significantly lowered the survival potential of from treated pairs. Endrin had no effect on the reproductive and the survival potentials of the treated beetles. Toxaphene treated females did not lay eggs. Toxaphene-DDT mixture lowered the reproductive and survival potentials of the treated beetles. From the previous comparisons it is obvious that Coleomegilla maculata has developed resistance to DDT and is in the process of becoming almost "immune" to this compound. All evidence indicates that this insect is also in the process of developing resistance to toxaphene and endrin. CONCLUSIONS The action of natural enemies, especially a complex of predators, kept bollworm, Heliothis zea; tobacco budworm, H* virescens; cotton aphid, Aphis qossypii; and spider mite, Tetranychus spp. populations on untreated cotton at general equilibrium positions below the threshold for economic damage. The use of insecticides for boll weevil control disrupted the prey-predator balance. Manipulation of predator populations under field conditions may be possible. Coleomegilla maculata can be reared satisfactorily on an artificial diet under laboratory conditions. 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From September, 1959 to August, 1961, he worked as am instructor of Entomology at Shebin El Kom College of Ag riculture, during the same period he was studying for the Ph.D. degree in the Department of Entomology, College of Agriculture, Ain Shams University. He received a scholarship from the government of the United Arab Republic in September, 1961 to study insect ecology in the United States of America, He has attended Louisiana State University since September, 1961, except for the fall semester, 1963-64, which he spent at the University of California at Riverside, 198 for some special work in Insect Ecology. He is now a Candida e for the degree of Doctor Philosophy at Louisiana State University. EXAMINATION AND THESIS REPORT Candidate: Yousef Hanna Atallah Major Field: Entomology Title of Thesis: Ecological and nutritional studies on some predators in cotton fields in Louisiana, with special reference to Coleomegllla maculata De Geer. Approved: Major Professor and Chairman & D e a n of the G r a d u a t e School EXAMINING COMMITTEE: v m l y , Date of Examination: May 15, 1964