This dissertation has been microfilmed exactly as received 69-15,938
LYON, William Francis, 1937- BEHAVIORAL RESPONSE OF THE ADULT CEREAL LEAF BEETLE Oulema melanopus (Linnaeus), (Coleoptera, Chrysomelidae) TO VARIOUS CHEMICAL LURES.
The Ohio State University, P h .D ., 1969 Entomology
University Microfilms, Inc., Ann Arbor, Michigan BEHAVIORAL RESPONSE OF THE ADULT CEREAL LEAF BEETLE
Oulema melanopus (Linnaeus), (Coleoptera, Chrysomelidae)
TO VARIOUS CHEMICAL LURES
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By
William Francis Lyon, B.Sc., M.S.
f ********
The Ohio State University
1969
\ MA O -
Advisers Faculty of Entomology ACKNOWLEDGMENTS
The author is indebted to his co-advisers, Dr. Robert E.
Treece of Wooster, Ohio and Dr. Ralph H. Davidson of Columbus,
Ohio for their guidance, encouragement, and suggestions during
the course of these investigations.
Special appreciation is given to Dr. Frank W. Fisk for the
use of his laboratory and equipment, and for guidance in the
attraction studies; and to Mr. John McCabe, who provided host
oat plants for the beetle attraction tests.
Thanks are also extended to Dr. C. R. Weaver for suggesting
experimental designs and analyzing the data; to Dr. Roy W. Rings
for assigning the author a research assistantship; and to my wife,
Marjorie, for her patience during the many hours the author spent ,
away from home testing the attractants.
This research was supported primarily from Hatch 282 funds made available to the Ohio Agricultural Research and Development
Center, Wooster, Ohio. VITA
January 24, 1937 ...... Born - Marion, Ohio.
1959 ...... B.Sc. - The Ohio State University, Columbus, Ohio
1959-1961 ...... County Extension Agent, 4-H, Kenton, Ohio
1961-1962 ...... Teaching Assistant, The Ohio State University, Columbus, Ohio
1962 ...... M.Sc., The Ohio State University, Columbus, Ohio
1962-1964 ...... Survey Entomologist, Ohio Agricultural Research and Development Center, Wooster, Ohio
1964-1966 ...... Research Assistant, The Ohio State University, Columbus, Ohio
1966-1969 ...... Instructor, Extension Entomology, The Ohio State University, Columbus, Ohio
PUBLICATIONS
Lyon, W. F., and R.H. Davidson. 1965. The effect of humidity on the volatilization of certain insecticides. J. Econ. Entomol. 58:1037.
Lyon, W. F., and R.E. Treece. 1968. The cereal leaf beetle in Ohio. Cooperative Extension Service Leaflet L-150, 11-15. The Ohio State University, Columbus, Ohio. CONTENTS Page
ACKNOWLEDGMENTS ...... ii
VITA ...... iii
LIST OF TABLES ...... v
LIST OF FIGURES ...... viii
INTRODUCTION ...... 1
REVIEW OF LITERATURE ...... 7
MATERIALS AND METHODS ...... 13 Controlled air flow olfactometer ...... 13 Cage type olfactometer ...... 23 Test insects ...... 26 Outdoor insect c a g e ...... 30 USDA synthetic lures ...... 32 Plant extraction techniques ...... 35 Filter paper and agar substrates ...... 38 Field traps ...... 43 Field treatments ...... 43 Statistical analysis ...... 47
RESULTS - LABORATORY ...... 50 Olfactometer reliability ...... 50 USDA synthetic lures ...... 51 Live beetles, dead beetles, and beetle feces ...... 53 Host and non-host plants ...... 53 Oat host plant studies ...... 54 Light effects ...... 61 Filter paper and agar substrates ...... 64 Response between sexes ...... 66 Antennectomy studies ...... 68
RESULTS - FIELD ...... 71 Trap reliability ...... 71 USDA synthetic lures ...... 71 Oat host plant studies ...... 73
SUMMARY AND CONCLUSIONS ...... 80
APPENDIX...... 84
LITERATURE CITED ...... 91 iv LIST OF TABLES Number Page
1 Volatilization rates of various compounds in a controlled air flow olfactometer ...... 17
2 Test materials with treatment numbers illustrating the solvent or method used, plant condition, and type of storage in field investigations ...... 46
3 Response of 7/7/65 field collected unsexed adults, tested 7/10/65 in a cage type olfactometer, to succulent oat leaves ...... 50
4 Response of 7/7/65 field collected unsexed adults tested 7/10/65 in a controlled air flow olfactometer ...... 51
5 Response of field collected unsexed adults, tested in controlled air flow and cage type olfactometers, to USDA synthetic lures ...... 52
6 Response of 4/20/66 field collected unsexed adults, tested 4/25/66 in a cage type olfac tometer, to leaves of ginkgo, oats, red clover, wheat, alfalfa, and red kidney beans .... 54
7 Response of 5/22/66 outdoor cage unsexed adults, tested 5/25/66 in a cage type olfac tometer, to dark green, light green, and green-yellow oat leaves ...... 55
8 Response of 5/6/66 outdoor cage unsexed adults, tested 5/9/66 in a cage type olfac tometer to dried, succulent, and mature oat leaves ...... 56
9 Response of field collected unsexed spring and summer adults, tested in a cage type olfactometer, to various oat leaf conditions ..... 57
10 Response of 7/14/65 field collected unsexed adults, tested 9/17/65 in a cage type olfac tometer, to freeze dried oat extracts and distilled water ...... 57 v Number Page
11 Response of 7/15/65 field collected unsexed adults, tested 9/23/65 in a controlled air flow olfactometer, to freeze dried extracts of mature and succulent oats, and distilled water ...... 58
12 Response of field collected unsexed adults, tested in a cage type olfactometer, to various oat plant extracts obtained by different methods ...... 59
13 Response of field collected unsexed adults, tested in a cage type olfactometer, to various oat extracts ...... 59
14 Response of 7/12/68 field collected unsexed adults, tested 7/15/68 in a controlled air flow olfactometer, to oat extracts from various solvents and plant extraction methods .... 60
15 Response of 7/7/66 field collected unsexed adults to water and acetone extracts of succulent dark green oat leaves upon agar substrates and a check agar substrate ...... 66
16 Response of 4/20/66 field collected sexed adults, tested 5/25/66 in a cage type olfactometer, to dark green oat leaves ...... 68
17 Response of 7/4/68 field collected unsexed adults, tested 7/6/68 in a cage type olfactometer, to green oat leaves. Beetles were in various stages of antennectomy...... 69
18 Response of adult cereal leaf beetles to various USDA synthetic lures tested 6/2/66 in a wheat field near New Carlisle, Indiana ...... 72
19 Response of overwintering adult cereal leaf beetles to various oat plant treatments tested 5/26/67 in a winter wheat field near New Carlisle, Indiana ...... 74
20 Response of overwintering^ adult cereal leaf beetles to various oat plant treatments tested 5/27/67 in a winter wheat field near New Carlisle, Indiana ...... 75 vi Number Page
21 Response of new summer adult cereal leaf beetles to various oat plant treatments tested 7/12/67 in an oat field near New Carlisle, Indiana ...... 76
22 Response of new summer adult cereal leaf beetles to various oat plant treatments tested 7/13/67 in an oat field near New Carlisle, Indiana ...... 78
23 Analysis of variance of adult cereal leaf beetle response in 1967 field tests at New Carlisle, Indiana, to various treatments of oat plant extracts ...... 85
24 Analysis of variance of adult cereal leaf beetle response in 1967 field tests at New Carlisle, Indiana, to various treatments of oat plant extracts ...... 86
25 Mean response of adult cereal leaf beetles in 1967 field tests at New Carlisle, Indiana, to various treatments of oat plant extracts ...... 87
26 Mean response of adult cereal leaf beetles in 1967 field tests at New Carlisle, Indiana, to various treatments of oat plant extracts ...... 87
27 Mean response of adult cereal leaf beetles in 1967 field tests at New Carlisle, Indiana, to various treatments of oat plant extracts ...... 88
28 Analysis of variance of unsexed spring and summer adult cereal leaf beetle response to various oat plant conditions in the laboratory .... 89
29 Analysis of variance of unsexed spring and summer adult cereal leaf beetle response to various treatments of solvents in the laboratory ...... 89
30 Analysis of variance of unsexed spring and summer adult cereal leaf beetle response to various treatments of oat plant extraction methods in the laboratory ...... 90
vii LIST OF FIGURES Number Page
1 Severe damage to wheat plants at New Carlisle, Indiana ' ...... 3
2 Undamaged (top), and damaged (bottom), spring oats at Berrian County, Michigan...... 4
3 A controlled air flow olfactometer (initial design) ...... 15
4 Diagrammatic sketch of the olfactometer (top view) ...... 16
5 Air flow measurement with manometers ...... 19
6 The port end of the reaction chamber during a test ...... 20
7 Portable air pump aspirator ...... 22
8 Screened cage housing 2 port controlled air flow olfactometer ...... 24
9 Lighting over the olfactometer set - u p...... 25
10 Cage type olfactometer with Plexiglasv-4...... 27
11 Screen cage type olfactometer ...... 28
12 Outdoor cereal leaf beetle cage with zippered doorway ...... 29
13 Interior view of the outdoor cereal leaf beetle cage ...... 31
14 The freeze drying apparatus ...... 36
15 Lipid and aqueous portions ...... 39
16 Volume reduction with an air s t r e a m ...... 40
viii Number Page
17 Agar substrates in plastic refrigerator chambers ...... 42
18 A field trap ...... 44
19 Schematic field design of replicated and randomized treatments ...... 47
20 Collecting adult cereal leaf beetles from field traps ...... 48
21 Accumulative response in various time intervals of 5/10/68 field collected unsexed adults, tested 5/11/^68 in a cage type olfactometer, to different storage conditions of water solutions of green oat plant extracts ...... 62
22 Accumulative response in various time intervals of 7/3/68 field collected unsexed adults, tested 7/4/68 in a cage type olfactometer, to different storage conditions of water solutions of green oat plant extracts ...... 63
23 Accumulative response in various time intervals of 5/25/66 field collected unsexed adults, tested 5/27/66 in a controlled air flow olfactometer, to freeze dried oat extracts with and without lights ...... 65
24 Adult cereal leaf beetles during the act of copulation ...... 67
25 Antennae of adult cereal leaf beetle (greatly enlarged) ...... 70
ix INTRODUCTION
The cereal leaf beetle, Oulema melanopus (Linnaeus) has been a pest of small grains in Europe for many years (Hodson, 1929). Losses in Russia have been estimated to range from 25 to 50% of the crop during certain seasons. Attacks were so severe in Rumania in 1931 that crops had to be plowed under, while in Spain during 1938 the wheat crop was almost totally destroyed (Anonymous, 1964).
The first identification of this pest in the United States occurred in July of 1962 (Anonymous, 1962) from specimens obtained in
Berrien County, Michigan. At that time oats were being severely damaged and several farmers plowed this crop under and replanted to another crop. Without question the cereal leaf beetle had been present for several years prior to its discovery and identification.
(Unpublished reports of Michigan Department of Agriculture, 1962).
Oats in Ohio were found infested for the first time in north western Williams County in May of 1963 (Anonymous, 1963). During the following weeks, additional specimens were collected in Fulton,
Defiance, and Allen Counties. Five years later, in 1968, virtually all of Ohio was found to be infested. The known distribution of the beetle in North America now includes parts of Michigan, Indiana, Ohio,
Pennsylvania, Illinois, Kentucky, West Virginia, and Ontario, Canada
(Anonymous, 1968). 1 Host crops of the cereal leaf beetle in Europe are similar to
those in North America, and include oats, Avena sativa; wheat,
Triticum aestivum; barley, Hordeum vulgare; and rye, Secale cereale (Manolache, C., 1923) (Fig. 1). Host grasses both in
Europe and North America include wild oats, Avena fatua; quackgrass,
Agropyron repens; timothy, Phleum pratense; rye grass, Lolium sp.; orchardgrass, Dactylis glomerata; reed canarygrass, Phalaris arundinacea; and canary grass, Phalaris canarienses. Corn, Zea mays, is also an infrequent host (Castro, Ruppel, and Gomulinski, 1965).
In general, grasses in the family Gramineae are hosts to a varying degree.
The economic importance of the cereal leaf beetle is due to the
fact that both larvae and adults infest succulent young plants and _ chew out long strips of tissue between the leaf veins (Fig. 2). These
seedlings may die or become stunted in growth as a result of heavy
feeding damage. Research has demonstrated that if an average of one
larva completes its development per stem, oat yields will be reduced
2.24 bushels per acre (Treece, 1967). With Ohio leading the United
States in 1968 with an estimated average yield of 68 bushels per acre (387> increase in oat acreage over 1967), the cereal leaf beetle could be a definite threat to the farm economy (Anonymous, 1968).
Although insecticides such as carbaryl, malathion, and azinphos- methyl have been found effective as control chemicals against the cereal
leaf beetle (Wilson, Ruppel, and Treece, 1965; Ruppel and Yun, 1965), it was felt that a better understanding of the behavior of the beetle Fig. 1.--Severe damage to wheat plants at New Carlisle, Indiana. 4
Pig. 2.--Undamaged (top) and damaged (bottom) spring oats at Berrien County, Michigan. 5 would be a prerequisite to improving the efficiency of chemical and
biological control programs.
Since it has been demonstrated with other host specific insects
that specificity is related to the presence or absence in plants of
certain chemical substances, such as glucosides, essential oils,
alkaloids, saponins, or tannins (Fraenkel, 1953), it seemed conceiv
able that there could be a chemical basis for host selection of the
cereal leaf beetle. This dissertation describes the investigation of
the effect of odors on the behavior of the adult cereal leaf beetle.
Adults were selected as the stage to be tested because of availability best locomotion response, and hardiness. Larvae were not bioassayed i because of poor locomotion responses and their failure to move from
plant to plant as readily as do adults.
Olfaction studies with the cereal leaf beetle have been in pro gress since autumn 1964 at The Ohio State University, Columbus, Ohio, and in New Carlisle, Indiana.
The overall objectives of these investigations have been to (1) construct an efficient cereal leaf beetle olfactometer, (2) screen
USDA synthetic lures, (3) confirm or refute the attractiveness of certain known host plants, (4) prepare and test various plant ex tracts, (5) observe behavioral patterns in beetles particularly attraction or arrestant responses, and (6) confirm the results of laboratory investigations with field tests.
Since chemical attractants and arrestants, in combination with chemosterilants, insecticides, and traps, have proved to be of value f in the control of certain insect pests, it seemed that the response of the cereal leaf beetle to a certain group of plants could be utilized as a control measure. An attractant in traps would lure
the beetles and these could be killed or sterilized, or an arrestant scattered in the environment might confuse the beetles
to the extent that they would be unable to make a mass attack on a specific crop (Wood, 1961). REVIEW OF LITERATURE
Since Vershaffelt (1910) first reported that sinigrin, present
in cruciferous plants, governed feeding by the larvae of cabbage
butterflies, Pieris rapae L. and P. brassicae L., the literature has
contained conflicting theories concerning insect host specificity and
odors. Fraenkel (1953) argues that host specificity is determined by
the presence or absence in plants of odd chemical substances such as
glucosides, essential oils, alkaloids, saponins, or tannins. He
indicates that the chemical composition of leaves, as far as it is of
importance in the nutrition of insects, seems to be very similar in
different plants and therefore specificity of host plants for their
insects cannot be explained by the chemical differences in food
substances as protein, carbohydrates, fats, minerals, sterols, or
vitamins. He feels that it is the "odd substances" with no nutritional
value which act as chemical stimuli, and therefore determine the
specificity of a food plant.
Thorsteinson (1960) states that the effect of host plant odors
is negligible beyond a few meters and host selection beyond that distance is probably random. He feels that there are other factors
in food plant selection besides plant odors such as vision, phototaxis, geotaxis, and hygrotaxis. Also, food plant selection by insects is believed determined by perception and plant availability. Dethier
(1953) proposed orientation to feeding, biting response, and continued 8
feeding in host selection. Callahan (1965) believes that insects can
detect different wavelengths in the infrared spectrum emitted from
other insects, animals, and plants. He feels that odors are rela
tively unimportant in host specificity.
Von Frisch (1937) believed that both odor and sight served to
guide an insect to a substance. He devised an experiment to determine
whether color or scent attracted bees. A number of bees were trained
to fly to a yellow box possessing rose scent. Later two boxes were
used; a yellow box without rose scent and a white box with rose scent.
The bees flew towards the yellow box but when very near it veered away
and went to the white box with the rose scent. He concluded that bees
recognized flowers from a distance by their color but when near the
flowers the sense of smell predominated.
Moncrieff (1967) indicates that smell is a primitive sense which
emerged long before vision. Even the simplest of animals, such as
Protozoa, have some sort of chemical sensitivity. As the scale of
animal life rises, this sensitivity differentiates into taste and
smell. The ewe recognizes its own lamb by sniffing. Hounds follow
the scent of a fox or raccoon over the ground. This ability to smell
serves the needs of the organism particularly in nutrition and repro
duction. If these two needs are not met, the species will disappear.
Fifty years ago, Mclndoo (1919) gathered evidence to indicate
that specific plant odors had a definite role in host plant selection by insects. He confirmed that plants emit odors, and insects are 9
guided by these odors. The effective odor emitted by a plant may be
a combination of all volatile constituents or one odor may be so
strong that it masks others. Moncrieff (1967) indicates that a
variety of odors are emitted from roots, stems, leaves, and flowers
of plants. He states that 738 different kinds of plant leaves have
been examined and about half of them were characterized by the fresh
green odor of a,b-hexylene aldehyde, a compound partly responsible
for the flavor of tea. He further points out that the perfumes of
fragrant flowers are usually complex and not due to one chemical
constituent, but to the combined effects of many. Only occasionally
one individual odorant is almost entirely responsible for the flower
perfume such as methyl anthranilate in orange blossom perfume.
Beroza and Green (1963) state that attractants are either natural or synthetic in origin. Natural sources would include the
particular test insect, host animal, or host plant, whereas synthetic
sources would be man made.
Dethier (1968) states that no group of animals has smell or taste
so diversified and sharply delineated as insects. An insect may be so associated with a particular plant that it is named for its food host.
Examples are the potato beetle, hog louse, tobacco hornworm, and alfalfa weevil. 10
In olfaction studies, entomologists must also consider other
factors which induce insects to feed. Many compounds are reported as active phagostimulants (Davis, 1968). Carbohydrates are present in all plants, and sucrose has been frequently recognized as a feeding stimulant (Ito, 1960; Davis, 1961; Augustine et al., 1964; and Ridgway et al., 1966). Fructose, raffinose, arabinose, rhamnose, galactose, maltose, and melizitose also have some influence in elicit ing feeding responses (Ito, 1960; Davis, 1961), but glucose has very slight or no influence. Yamamoto and.Fraenkel (1960), Thorsteinson and Nayar (1963), and Heron (1965) report that glucosides are phago- stimulatory in low concentrations and inhibitory in higher concentra tions. Beck (1956) discovered that larvae of the European corn borer fed selectively on a diet of tissue containing the highest concentra tion of sugars. He considered this fact very important in host plant selection.
Mittler and Dadd (1964) and Robbins et al. (1965) used proteins with a phosphate buffer to elicit feeding. Gluten was moderately effective in eliciting feeding response in the confused flour beetle.
Davis (1965) reported that the feeding response of certain insects is intensified by combining amino acids with sucrose.
Lipids have also been associated with insect phagostimulation.
Wheat germ oil and soybean oil enhance the feeding activity of locusts and silkworms (Ito, 1961). Stride (1965) prepared an oil from fresh leaves of Solanum campylacanthum which contained phagostimulants for
Epilachna fulvosignata. B-Sitosterol is a phagostimulant for the 11 silkworm (Hamamura et al., 1961; and Nayar and Fraenkel, 1962).
Phospholipids are effective stimulants to grasshoppers (Thorsteinson
and Nayar, 1963).
Thorsteinson (1960), and Fraenkel and Gunn (1940), and Dethier
(1947) stated that there is no reason not to consider hygroreception
as a type of chemoreception.
Maxwell et al. (1963) discovered a powerful arrestant and feeding
stimulant for boll weevils, Anthonomus grandis Boheman, to exist in water extracts of all cotton plant parts and square components investi
gated. Wood et al. (1961) found that moisture played an important role
in response of Scolvtus multistriatus (Marsham). Dethier (1968) points out that flies can distinguish between water, sugar, and salt. Davis
(1961) found that cold and hot water extracts of flour made from germinated rye seed elicited a biting or feeding response from the
larvae of the prairie grain wireworm. On the other hand, ethanol and petroleum either extracts and the residue from extractions did not.
It is now generally known that the principal olfactory sites in insects are the antennae (Schoonhoven and Dethier, 1966). They also
indicate that other appendages on the head, especially the maxillary and labial palpi bear olfactory organs. The basic question always arises: what makes a substance odorous and detectable by insects while other substances are not? Mbncrieff (1967) gives three pre requisites for a substance to be odorous and detected by insects:
(1) It must be volatile so that molecules can be lost to the atmosphere and detected in the olfactory sensitive region. (2) The odorant must 12 be capable of being adsorbed on the sensitive surface of the olfac tory epithelium. (3) The substance must be one which is not already present on the olfactory epithelium, so that when it arrives a change causes sensation.
In summary, over the past years the study of insect olfaction and the influence of "odors" in host plant selection has been a subject of much entomological work. Although much information has been discovered, Thorsteinson (1960) states that the extraordinary variety of insect-plant relationships is likely to be based on more diverse mechanisms than can be foreseen at the present time. MATERIALS AND METHODS
Controlled air flow olfactometer
If insect responses to stimuli are purely mechanistic, results obtained from a device to measure reaction to specific stimuli are dependable only when all stimuli but one remain constant. This requirement is nearly impossible to achieve. The complexity of a device such as an olfactometer would depend on the extent to which variables are to be controlled. Variables involved would be quality and intensity of light, temperature, humidity, air flow, and the stimulus in question.
The degree of reliability of an olfactometer would be reflected by the intensity and consistency of response. The devise should provide a means of observing the insect response, have the criteria to measure the particular response, and have a means to know the concentration of the stimuli. With these goals in mind, much time and labor were involved in designing, constructing, and calibrating an olfactometer useful for the cereal leaf beetle, Oulema melanopus (L.)
The instrument employed was modified from one described by
Dethier and Yost (1952). It was constructed in the shape of a truncate sector of a circle with vation of the test insects and to allow for introduction and removal of insects as desired. One-fourth inch plywood boards were used on 13 14 the sides and the bottom of the olfactometer with the overall dimensions as 3 feet long, 4 1/2 inches across the truncate end,
14 inches across the circle end, and 3 1/2 inches deep. A three port olfactometer was used initially (Fig. 3). However, after several preliminary tests, a two port olfactometer was found to be more effec tive in the attraction studies (Fig. 6).
The two port olfactometer principle of operation was simple.
The air source was split into three streams each of which was metered.
These streams ultimately entered into a reaction chamber through two ports. Stream c. passed directly to the port which was to serve as the control. Streams a and b ended at the test port. Of the two streams entering the test port, a was passed through a bottle contain ing the compound on test and into a second air mixing bottle where the remaining stream b entered to dilute the desired concentration before the material entered the final test port. In other words, odor con centration was regulated by varying the ratio of the rates of flow of streams a and b. Stream a passed through the saturator containing the test compound in a liquid state (Fig. 4). The test compound in the liquid state was placed in a 5 ml beaker containing a cotton dental roll wick. The liquid evaporated from the wick and was carried by the air stream to the test port. The concentration was ascertained by measure ment of the weight loss of the liquid after a measured amount of air had passed within a given period of time. Fig. 3.--A controlled air flow olfactometer (initial design). air flow 16
' KEY
1. Air purifier and flow equalizer 2. Flow meter 3. Saturator 4. Mixing bottle 5. Fluorescent light 6 . Control or test air streams port 7. Reaction chamber 8 . Stockinette material 9. Screened cage 10. Exhaust port 11. Light bulb
i nr
air flow 11 Fig. 4.--Diagrammatic sketch of the olfactometer (top view). 17
After several preliminary tests, a standard "odor concentration"
was established for each of the materials tested. The standard was
based on a total air flow rate of 60 liters per hour with stream a
comprising one part and stream b comprising nine parts of the 60 L/hr.
Volatility rates in milligrams/liter and millimoles/liter were calcu
lated after an hour's test period. The compound volatilization rates
are in descending order.
TABLE 1
VOLATILIZATION RATES OF VARIOUS COMPOUNDS IN A CONTROLLED AIR FLOW OLFACTOMETER
Hourly Hourly Hourly Compound Formula Mol. Wt. Wt. Loss Mg/L Loss mM/L Loss
Chloroform CHCI3 119.4 .6420 10.70 .0895
Acetone c2h 60 58.1 .5620 9.36 .1611
Hexane G6h 14 86.2 .5120 8.53 .0989
Petroleum Ether — -- .4962 8.27 —
Methyl Alcohol ch40 32.0 .4204 7.00 .2187
95% Ethyl Alcohol 46.1 .4080 6.80 C2H6° .1475 Hot Water Oat Extract —— .0706 1.17 --
Cold Water Oat Extract — -- .0700 1.16 --
Freeze Dried Extract —— .0690 1.15 --
Water h 2o 18.0 .0673 1.12 .0622 Benzene FPn 78.1 .0476 .79 .0101 18
The system of switching hoses at the two ports permitted
directing the control stream or the test stream to either the right
or left port. In all tests the rate of air flow through the control
port was made equal to the total air flow through the test port (both
streams a and b combined). The air flow rate was measured by manometers with a range of 0.36 to 3.6 liters of air per minute at the
standard conditions of 14.7 psi absolute pressure and with a tempera
ture of 20 C. Sixty liters per hour of air flow were used in testing
all synthetic lures and plant extracts (Fig. 5). ‘The laboratory
compressed air which carried the odors into the test chamber was first passed through a Koby "Junior K i n g " ® a i r purifier and flow equalizer to remove impurities.
The reaction chamber was constructed so the two air streams, control and test, passed through it with little or no mixing. This was verified by blowing cigar smoke into the reaction chamber and observing the pattern of smoke movement. A vacuum was employed at the exhaust port permitting the exhaust of odors at a slightly greater rate than that when they entered the reaction chamber. In this manner turbu lence and dead air space areas were reduced to a minimum.
Insects to be tested were placed in the truncate (square) end of the reaction chamber and allowed to make free movements to the control or test ports. A single two-foot General Electric Cool White fluores cent tube (15 watts) yielded light through the ports and attracted beetles to the circular (port) end of the reaction chamber during the test (Fig. 6). In a control experiment, when pure air passed through 19 Fig. 6 .--The port end of the reaction chamber during a test. 21 both ports, the beetles distributed themselves equally on the screen
of the ports. When odor replaced the air at one port, the distribu
tion of beetles shifted so that a greater number congregated at the
test port if the odor was attractive and a lesser number if it w a s
repellent. After the 30-minute test period, the fluorescent light was turned off and the beetles were removed from the reaction chamber by means of a 250 ml flask connected by TygonGD tubing to a portable
vacuum pump. This device worked efficiently in serving as an
aspirator for gathering the beetles. Beetles could also be rapidly
counted when using this device (Fig. 7). In testing it was felt that
the control port took care of the light bias and any right or left bias was corrected by alternating the test and control ports. Beetles
were presented with three choices, namely, the control port, the test
port, or some neutral area in the test chamber. The response criteria
involved the positive movement of the beetles to the ports with a given
number of beetles counted on the port screen. Each test required 30 minutes. Generally 90-95% of the beetles would move towards the ports but not completely locate on the screens.
Beetles apparently became fatigued if used over and over again in
consecutive 30-minute tests, and the results were not consistent in
repeat tests with the same test sample. Therefore, beetles used more
than once responded more consistently if an hour or more elapsed before
retesting.
Another method of testing materials in the controlled air flow
olfactometer involved using a piece of cotton wick held upright by a Fig. 7.— Portable air pump aspirator pin stuck into a cork. The cotton wick was impregnated with a synthetic lure or plant extract and placed near the port (Fig. 3).
An air flow directed over the impregnated cotton wicks carried the odor down the chamber to the test beetles. Criteria used for posi- tive response involved the presence of the test beetles within a
11/2 inch radius from the center of the wick position at the end of a 30-minute test period.
It was necessary to construct a screened cage 30"x46"x48M in order to house the "controlled air-flow olfactometer" during a test.
This screened cage was used to prevent escape of flying beetles into the room, which occasionally occurred when raising and closing the
chamber. Also, at the time that the tests were initiated Ohio had strict quarantine laws since the beetle was not yet in Franklin County. This large cage contained a 30-inch piece of stockinette material attached to a rectangular frame on one side of the cage in order that insects and materials could be inserted or removed with ease (Fig. 8). Over the head of this screened cage was placed a pair of six foot General Electric Cool White'^(&) fluorescent tubes (40 watts each) to insure uniform light intensity at all areas within the olfactometer (Fig. 9).
Cage type olfactometer
The second type of olfactometer used functioned as a "non- conttolled air flow olfactometer." The test chamber involved a 15-inch cubed box with the top, bottom, and one side made facilitate easy observation. Different synthetic lures or plant Fig. 8.— Screened cage housing 2 port controlled air flow olfactometer.
26 extracts were placed at three corners with the fourth corner as the control or check Beetles were introduced into the center of the floor of the cage and allowed to move freely into any one of the four corners. This test chamber contained an 8" x 8" door to intro duce or remove beetles and materials. Small circular air vents were present in the PlexiglasO^ side with a ventilation screen in the ply- wood side to permit aeration (Fig. 10).
Also used as a "non-controlled air flow olfactomer" was a cubicle type screened cage. Each cage was assembled into a 1 foot cube, using
1-foot-square alumir urn window screen panels on back, sides and top and sheet aluminum on tte bottom. A 30-inch piece of stockinette material was attached to a rectangular frame on the cage front permitting easy introduction or removal of insects and materials. The stockinette material was long enough to permit it being tied into a knot (Fig. 11).
After each test, caies were washed with hot, soapy water, rinsed with cool water, and dri^d.
Test insects
During the investigation virtually all beetles used in tests were field collected. 0 /erwintering spring adults were collected in early
April and new summe r adults were collected in early July. Beetles were then stored in pint size ice cream cartons at about 40 F* hundred beetles were placed in each carton with a moistened paper towel,
Cartons were then inspected periodically to make certain that the beetles had adequate moisture on the paper towels. Usually, when beetles were held for 2 1/2 to 3 months, about 80-90% mortality occurred. Fig. 10.--Cage type olfactometer with Plexiglass Fig. 11.— Screen cage type olfactometer. 29
Fig. 12.--Outdoor cereal leaf beetle cage with zippered doorway. 30
Beetles tested 1-4 days after field collection responded best to materials in the olfactometer. Beetles kept 5-6 weeks in cold storage gave decreased response to materials in the olfactometer.
Test beetles were removed from the refrigerator at least 24 hours before testing and were given 5% sucrose solution absorbed on cellu- cotton. This time allowed beetles to adequately warm up to room
temperature and to obtain liquid food and water prior to actual olfac
tion tests.
During testing great variations in response could usually be attributed to improper insect pre-conditioning or poor light distri bution. Experience gained during the testing program indicated that cereal leaf beetle adults were very phototactic and controlled light
ing was essential in laboratory tests.
Outdoor insect cage
In order to facilitate the availability of cereal leaf beetle adults for testing purposes throughout the year, an outdoor insect
t cage obtained from the Ohio Agricultural Research and Development
Center was utilized. The overall cage dimensions were 9 lxl5'x6l.
Three by 6-foot screened panels were bolted together to form the cage.
Also, a hinged door 2 1 wide x 5 1 high, located near the center of one
side of the cage, provided easy access into or out of the structure.
It was necessary to install inside the doorway stockinette material
split vertically by a 60" zipper to prevent escape of flying beetles when one entered or departed from the cage. The zipper could be operated from inside or outside the cage (Fig. 12). 31
Fijf^ 13.--Interior view of the outdoor cereal leaf beetle cage. The soil within the cage was tilled, planted with oats, wheat and barley, straw-mulched, and watered regularly depending on weather conditions (Fig. 13). After plant growth occurred 10,000 summer adult cereal leaf beetles were introduced into the cage.
These beetles fed on the plants for about 2 to 3 weeks and then went into estivation for the remaining summer months. As autumn and winter approached, it was noticed that many of the beetles had entered into individual straws of the wheat stubble mulch. This behavior was found advantageous since merely collecting the wheat straws containing beetles provided the test insects. The straw was brought into the laboratory, placed on a table under a heat lamp, and beetles soon crawled out from the straws. Beetles were then quickly collected with an aspirator and stored in the refrigerator or used immediately in tests if needed.
USDA synthetic lures
It was felt that possibly certain synthetic lures would be attractive to the adult cereal leaf beetles. At this particular time
(in 1964) there existed an urgent need for an additional survey and detection tool to aid the existing cereal leaf beetle sampling techniques which was usually net sweepings. Consequently, 25 USDA synthetic lures were tested in the laboratory for attractant pro perties. Listed on the following sheet with their entomology numbers are the materials which were investigated. 33
Entomology Number Material
1170 Anisyl alcohol
30686 m-Dioxane, 5-ethyl-2-phenethyl- 4-propyl-
11010 Linoleic acid
673 1-Acetomaphthone
21040 Veratrole, 4-allyl- (or Methy- leugenol)
203 Citronellal
24118 Carbostyril, 1-methyl-
4466 2-furaldehyde
222 3-Buten-2-one, 4-(p-methoxyphenyl)- (Anisalacetone)
24225 Ether, butyl vinyl
24483 Nopinene
25001 Eugenol Terpenes (a complex mixture of terpenes having the general formula C^0**16^
25073 beta-Ionone
18142 Alpha-phellandrene
514 Safrole
11510 Fhenethyl alcohol, alpha-ethyl
23404 2-Butanone, 3-hydroxy-3-methyl-
31627 Butyric acid, 3-piperonyl-
15356 Isoeugenol
5514 Butyrophenone, 4-methoxy-
4094 Propriophenone, 4-methoxy- 34
Entomology Number Material
24486 p-Menthane
24594 alpha-Pinene
20991 Benzene, l-allyl-2-butoxy-
206 Geraniol
The USDA synthetic lure stock was diluted at the rate of 0.2 ml of lure per 2 ml of acetone as suggested by Michigan State
University personnel (personal correspondence). The cotton wick control was immersed in acetone and allowed to evaporate as with the given synthetic lure wick_jprior to testing. The completion .of the acetone evaporation could be detected by a smell test.
Listed below are six additional USDA synthetic lures which had been laboratory and field tested by Michigan State University personnel. These six materials showed enough beetle response to warrant further screening by Ohio personnel (Jantz, 1965).
Entomology Number Material
3294 Acetic acid, cyclohexyl ester
4162 Octanoic acid
11747 Unknown
24748 l,2,cyclohexonediol, l-methyl-4- isopropenyl-, 2 acetate
24766 Butyric acid, 2-ethyl-, ethyl ester
30443 Formic acid, decyl ester 35
Plant extraction techniques
The extraction of volatile compounds from oat foliage was made possible by using a "Virtis Freeze-Drying
combination with a "Cenco-Megavac Vacuum methyl cellusolve (ethylene glycol monomethyl ether) and dry ice
(chunks of solid carbon dioxide) in the condenser provided the necessary refrigeration to the vacuum chamber (Fig. 14).
Fresh green oat foliage was clipped by scissors and either
placed into a plastic freezer bag and stored in the deep freeze
until use, or was used immediately after clipping. Oat foliage
was placed in the freeze-drying flask and infrared heating or a hot
water bath outside the flask greatly aided in the rapid plant
dehydration. As the foliage dehydrated under vacuum, volatiles
would move as vapors to the cold surface of the central well,
then condense and immediately freeze. After each plant extraction
the vacuum ports of the freeze-drying apparatus were tightly
stoppered, the central well emptied of the methyl cellusolve-dry
ice solvent, and the mixture of water vapor and volatile fraction
frozen on the outer surface of the well was allowed to thaw and
collect in the bottom flask. Then the collected freeze-dried
samples of volatiles were immediately used in bioassay. The
dehydration of a 40 g oat foliage sample to 10 g occurred in about Fig. 14.— The freeze drying apparatus. o\ 37
2-2 1/2 hours yielding approximately 25 ml of water vapor plus volatile fractions. No explanation is known for the fact that some of the extract (approximately 5 ml) was lost in the process.
Cold water extraction consisted of soaking oat leaves for 24 to 48 hours in a stoppered Erlenmeyer flask. After extraction, the leaves were removed and stripped of the water and the plant residue was discarded.
Hot water extraction involved blanching the oat leaves in boiling distilled water for three minutes to denature destructive enzymes and then quick freezing to cause as much cellular disruption as possible. After thawing, the material was macerated with all the blanching water as the extracting solvent. The plant residue was then discarded. Both cold and hot water extracts were held in the refrigerator before testing.
A few tests were made using beetle feces, dead beetles, and live beetles as lures. Feces and dead beetles were macerated and mixed with distilled water before testing. Live beetles were wrapped in cheese cloth prior to testing.
Ten gram samples of fresh green oat foliage were plunged into a
"Waring" blendor containing 125 ml of 80% ethanol. This rapid proces
sing of the leaves was necessary in order to inactivate enzymatic
reactions, especially the hydrolysis of sucrose. Samples were blended
for 5 minutes after which the macerated foliage and alcohol were poured
into a flask with an additional 25 ml of alcohol used to rinse the 38
"Waring" blendor. This flask was allowed to stand with occasional shaking for a period of about 12 hours. Next the material was filtered using a Buchner funnel which was also rinsed with an additional 25 ml of 80% ethanol.
The filtrate was poured into a separatory funnel with 125 ml of chloroform (Fig. 15). After the layers separated, the aqueous portion contained the amino acids and sugars, while the chloroform portion contained the lipids. The total volume in both portions was reduced by a slow stream of air without the use of heat (Fig. 16).
After the alcohol and chloroform solvents had been removed, the residue was brought to 5 ml in volume with distilled water and then used in bioassay.
Filter paper and agar substrates
Augustine et al. (1964) treated filter paper with an extract from bean plant leaves. Mexican bean beetle biting marks on the filter paper indicated the presence of a short range attractant in the volatile constituents and arrestants from the sugars of the host plant extract.
This filter paper method was employed with the cereal leaf beetle. However, very few feeding marks were observed on the treated filter paper saturated with the oat volatile and a 2% sucrose
solution. Feeding mark examination on the filter paper was made using a binocular microscope. Apparently mandibular action failed to leave distinct feeding prints on the filter paper as was so obvious with the Fig. 15.--Lipid and aqueous portions. Fig. 16.— Volume reduction with an air stream. 41
Mexican bean beetle. However, the cereal leaf beetles were attracted to the filter paper.
Consequently, agar powder prepared from Difco "Bacto-agar" was used as a substrate for the cereal leaf beetle adults in place of filter paper. A 2% agar solution in 100 ml of distilled water was prepared. Acetone and water extracts of oat plants were added on the cold agar surface immediately prior to testing.
Discs 2 mm in diameter were cut out of the cold agar sheet with a cork borer. Discs were cut approximately 0.2 mm thick to encourage beetles to attack the top surface of the agar disc and not the sides.
Disc thickness was accomplished by measuring the desired height of the liquid agar after pouring into a flat bottom glass dish.
There were four agar discs per petri dish and four petri dishes per plastic refrigerator box. Petri dishes were individually covered with screened ice cream carton lids. Four clear plastic refrigerator boxes contained the 16 petri dishes and 64 agar discs. These plastic chambers were suspended on rods above a 2-foot General Electric Cool
White fluorescent light (Fig. 17). The light was placed under the chambers, the beetles remained at the bottom of the petri dish and responded to the agar disc surface as desired. Each plastic refrig erator box contained a short glass nipple through the lower left and upper right corners at the ends permitting thorough air ventilation and diffusion. The humidifier consisted of a 1 1/2 gallon bottle 1/3
filled with water and fitted with a fidtted glass aerator placed inside near the bottom. A second hole was drilled in the bottle neck with a
"Y" tube inserted to serve as the humidity outlet. Tygon tubing Fig. 17.--Agar substrates in plastic refrigerator chambers. 43 connected the outlet to the four chambers. The rate of laboratory compressed air bubbled through the bottle determined the percent humidity in the testing chambers. Tests were conducted at 80% RH and a room temperature of 72 F. Tiny 3x3" Taylor "humidiguides" were used to measure the humidity and temperature.
Field traps
The field trap used was obtained from the USDA Methods Improve ments Laboratory at.Niles, Michigan. The trap consisted of a canary yellow painted masonite sheet measuring 5 inches wide by 9 inches long with a 1 1/2 inch diameter hole located 1/3 the distance down from the end and in the center of each sheet. A hairpin stapled slightly above the hole to which a cotton wick was attached served as the attractant carrier. A piece of galvanized tin roofing was bent over the upper 1/3 of the trap, protecting the lure-impregnated cotton wick from rainfall
(Fig. 18). "Stickem" (polymerized, isobutene, and butane 97%) was spread on both sides of the lower 2/3 of the masonite sheet with a putty knife. Then the entire trap was wire suspended 2 feet above the host field plant foliage by use of a steel rod bent in an inverted "L" shape. All traps were placed at 25 ft intervals from one another within the test field and suspended 2 ft above the plant foliage.
Field treatments
It was discovered that certain oat plant extracts displayed some attractiveness or arrestiveness to adult cereal leaf beetles under 44
Fig. 18.— A field trap. 45 laboratory tests. Consequently, effort was made to confirm the results of laboratory investigations with field tests.
Oat extracts derived from solvents of 957. alcohol, chloroform, water, and freeze drying processes were both refrigerated and frozen prior to field tests. Extracts were prepared from both green and dried plant foliage in the Insect Physiology laboratory at The Ohio
State University and transported to New Carlisle, Indiana, for field tests. The extracts stored in vials were packed in 4 to 5 pounds of dry ice within a styrofoam ice chest and taken to the field in frozen condition. The refrigerated samples were maintained at refrigerator temperatures until the time of testing.
Listed are the test materials with designated treatment numbers illustrating the solvent or method used, plant condition, and type of storage (Table 2).
Fresh test materials were put out at the beginning of each test day and trapped adult beetle counts were recorded before the end of the test day (Fig. 20). The 20 treatments were replicated four times and randomized within the replicates (Fig. 19). Spring adults were trapped the last weeks of May and summer adults trapped during the first weeks of July. 46
TABLE 2
TEST MATERIALS WITH TREATMENT NUMBERS ILLUSTRATING THE SOLVENT OR METHOD USED, PLANT CONDITION, AND TYPE OF STORAGE IN FIELD INVESTIGATIONS
Treatment Solvent or Plant Type of Number Method Used Condition Storage
1 95% alcohol Green Frozen 2 95% alcohol Green Refrigerated 3 Water Green Frozen 4 Water Green Refrigerated
5 Chloroform Green Frozen 6 Chloroform Green Refrigerated 7 95% alcohol Dried Frozen 8 95% alcohol Dried Refrigerated
9 Water Dried Frozen 10 Water Dried Refrigerated 11 Chloroform Dried Frozen 12 Chloroform Dried Refrigerated
13 Freeze Dried Green Frozen 14 Freeze Dried Green Refrigerated 15 Freeze Dried Dried Frozen 16 Freeze Dried Dried Refrigerated
17 95% alcohol 18 Water 19 Chloroform 20 Check 47
Listed is the schematic field design.
WEST
Black-Top Road
13 8 17 11 19 14 ' 20 16 1 16 2 10 20 9 9 2 15 7 13 6 8 4 18 17 4 11 15 1 16 12 13 5 Dirt 10 6 8 12 18 2 15 1 (NORTH Lane 2 19 4 9 5 7 19 10 17 9 20 3 13 11 8 3 5 18 7 14 10 1 12 11 14 3 5 16 6 17 6 9 12 20 19 18 15 3 14 4
A B C D
Fig. 19.— Schematic field design of replicated and randomized treatments.
Statistical analysis
These data were analyzed at the Statistics Laboratory of the Ohio
Agricultural Research and Development Center at Wooster, Ohio.
Laboratory data from 1965 and 1966 were analyzed by using the normal
curve concerning proportions of beetles responding. In tests where
these laboratory treatments were unbalanced, the necessary statistical
adjustments were incorporated before final computations. If the normal
curve approximation was suitable it was so stated as "OK." If the normal curve could not be used, it was stated "NCNA" meaning normal
curve not applicable. The insufficient sample size due to inavail
ability of test beetles and the low percentage of beetles responding 48.
Fig. 20.— Collecting adult cereal leaf beetles from field traps. 49 in these tests resulted in the need for certain statistical correction factors.
Field data in 1967 were analyzed by an analysis of variance to test for differences among the means. Specific mean differences were determined by calculating a least significant difference (LSD) accord ing to the method of Steel and Torrie (1960). Both the 1967 and 1968 data came from tests where the sample size was adequate and the number of beetles responding was sufficient for satisfactory statistical analysis.
In interpreting the results of these analyses, it was important to learn any significant differences which existed among the treatments and to observe any consistent treatment patterns which might exist over a period of time both in the laboratory and in the field investi gations .
Listed in the Appendix are tables of statistical analyses from
1967 and 1968 data. RESULTS - LABORATORY
Olfactometer reliability
Since the degree of reliability of any olfactometer is reflected by the intensity and consistency of response, it was first necessary to evaluate the effectiveness of two types of olfactometers in tests with the adult cereal leaf beetle.
During preliminary testing, many tests were conducted where the number of insects per test and the number of test replications were unequal in size. Consequently the use of statistical correction factors was necessary before evaluating data. The proportions of beetles responding were listed as "OK" or "NCNA" as explained in the Materials and Methods Section.
TABLE 3
RESPONSE OF 7/7/65 FIELD COLLECTED UNSEXED ADULTS, TESTED 7/10/65 IN A CAGE TYPE OLFACTOMETER, TO SUCCULENT OAT LEAVES
Test Repli- Number Number of Percent Confidence Normal Materials cations of Test Beetles Beetle Limits Curve ______Insects Responding Response 95%______
Oat leaves 4 1390 753 54.1 51.5-56.7 OK Control 4 4170 104 2.4 2.0-2.9 OK
50 51
As shown in Table 3, adult beetles responded quite significantly
to oat leaves in preference to the control in the cage type olfacto
meter.
TABLE 4
RESPONSE OF 7/7/65 FIELD COLLECTED UNSEXED ADULTS, TESTED 7/10/65, IN A CONTROLLED AIR FLOW OLFACTOMETER
Test Repli Number Number of Percent Confidence Normal Materials cations of Test Beetles Beetle Limits Curve Insects Responding Response 95% - - Oat leaves 2 450 144 32.0 27.6-36.3 OK Control 2 900 108 12.0 9.8-14.1 OK
Likewise as shown in Table 4, adult beetles responded signifi
cantly to oat leaves in preference to the control in the controlled
air flow olfactometer. After observing subsequent beetle preference
response to the known host plant oats in both types of olfactometers,
attention was next turned to testing USDA synthetic lures.
USDA synthetic lures
During this particular testing period, beetle availability was
limited and some tests were conducted where the number of insects per
test and number of test replications were unequal in size. Likewise,
the use of statistical correction factors was necessary before
evaluating data. Only the percent of the proportions of beetles
responding to a test material and its suitability to the normal curve
is given in Table 5. 52 TABLE 5
RESPONSE OF FIELD COLLECTED UNSEXED ADULTS, TESTED IN CONTROLLED AIR FLOW AND CAGE TYPE OL FACTOMETERS, TO USDA SYNTHETIC LURES
Test Percent Beetle Suitability of Normal Materials3, Response Curve (Proportions of Beetles Responding)
1170 4.8 NCNA 30686 4.9 NCNA 11010 1.9 NCNA 673 5.3 NCNA 21040 9.2 OK
203 10.2 OK 24118 5.8 NCNA 4466 5.9 NCNA 222 4.0 NCNA 24225 4.2 NCNA
24483 1.1 NCNA 25001 4.7 NCNA 25073 3.6 NCNA 18142 2.6 NCNA 514 5.8 NCNA
11510 5.9 NCNA 23404 6.2 NCNA 31627 2.9 NCNA 15356 3.8 NCNA 5514 4.5 NCNA
4094 8.2 NCNA 24486 6.3 NCNA 24594 4.2 NCNA 20991 3.7 NCNA 206 7.6 NCNA
Control 6.7 NCNA
See pages 33 and 34 for names of test materials. 53
The screening of 25 USDA synthetic lures resulted in no particular lure appearing outstanding. Only citronellal and methy- leugenol gave any significant results in attracting adult cereal leaf beetles. Many of the lures attracted fewer beetles than the control, indicating a possible repellent action. However, it is quite possible that lower concentrations of these synthetic lures might have proven attractive rather than repellent to the beetles. Byrne et al. (1966) reported that alfalfa produced a volatile substance which elicited an olfactory response by the alfalfa weevil. A 0.0257o dilution of the substance elicited a maximum weevil response with the undiluted form causing repellency.
Live beetles, dead beetles, and beetle feces
Subsequent preliminary tests involved exposing test beetles to other live beetles, dead beetles, and their feces. Test insects were
field collected and unsexed with the controlled air flow olfactometer used. Best response occurred when the beetle feces were presented as an attractant followed by live beetles, dead beetles, and the check.
However, beetle response was very low in all treatments. Thus, further
tests of this type were abandoned in order to examine host and non host plant preference.
Host and non-host plants
Tests were conducted to determine whether cereal leaf beetle adults could be attracted to legumes as well as to grasses in the
olfactometer. Treatments involved the fresh succulent plant leaves of the particular plant in question. 54
TABLE 6
RESPONSE OF 4/20/66 FIELD COLLECTED UNSEXED ADULTS, TESTED 4/25/66 IN A CAGE TYPE OLFACTOMETER, TO LEAVES OF GINKGO, OATS, RED CLOVER, WHEAT, ALFALFA, AND RED KIDNEY BEANS
Test Repli Number Number of Percent Confidence Normal Materials cations of Test Beetles Beetle Limits Curve Insect-s Responding Response 95%
Ginkgo leaves 4 200 3 1.5 0.1- 3.1 NCNA Oat leaves 4 200 96 48.0 41.0-54.9 OK Red Clover leaves 4 200 17 8.5 4.6-12.3 NCNA Check 4 200 12 6.0 2.7- 9.2 NCNA Wheat leaves 4 200 62 31.0 24.0-37.4 OK Alfalfa leaves 4 200 16 8.0 4.2-11.7 NCNA Red Kidney bean leaves 4 200 8 4.0 1 .2- 6.8 NCNA Check 4 200 10 5.0 1.9- 8.0 NCNA
Higher beetle response occurred at the oat and wheat leaves than
any of the other treatments (Table 6). Beetles would randomly wander
to leaves such as ginkgo, but would fail to remain on or near this
leaf. Also, red clover, alfalfa, and red kidney bean leaves did not
reflect feeding marks. Oat and wheat leaves were severely fed upon.
Oat host plant studies
The next test involved the response to different stages of plant
growth. Oat plants 14 days old (4-5 inches tall, deep green color);
21 days old (7-8 inches tall, light green color), and 28 days old 55
(9-10 inches tall, greenish yellow color) were tested for
attraction properties.
TABLE 7
RESPONSE OF 5/22/66 OUTDOOR CAGE UNSEXED ADULTS, TESTED 5/25/66 IN CAGE TYPE OLFACTOMETER, TO DARK GREEN, LIGHT GREEN, AND GREEN'-YELLOW OAT LEAVES
Test Repli- Number Number of Percent Confidence Normal Materials cations of Test Beetles Beetle Limits Curve Insects Responding Response 95%
Dark green leaves 4 200 55 27.5 21.3-33.6 OK Light green leaves 4 200 34 17.0 11.7-22.2 OK Green yellow leaves 4 200 22 11.0 6.6-15.3 OK
Check 4 200 7 3.5 0.9- 6.0 NCNA
Adults were attracted to all stages of plant maturity (Table 7).
However, the most favorable response was recorded on the deep green color youngest plants. Severe feeding damage resulted on the deep green and light green plants with less feeding damage on the greenish yellow plants. The younger plant leaves were more palatable and attractive to the adult beetles during the test.
It was assumed that if a volatile substance was responsible for olfactory attraction, the volatile could be removed by drying the plant leaves. Consequently, 14-day-old oat leaves were dried by placing the blades between sheets of blotting paper for 30 days. 56
TABLE 8
RESPONSE OF 5/6/66 OUTDOOR CAGE UNSEXED ADULTS, TESTED 5/9/66 IN A CAGE TYPE OLFACTOMETER, TO DRIED, SUCCULENT, AND MATURE OAT LEAVES
Test Repli Number Number of Percent Confidence Normal Materials cations of Test Beetles Beetle Limits Curve Insects Responding Response 95%
Dried oat leaves 4 100 5 5.0 0.7- 9.2 NCNA Succulent oat leaves 4 100 36 36.0 26.5-45.4 OK Mature oat leaves 4 100 20 20.0 12.1-27.8 OK
Check 4 100 6 6.0 1.3-10.6 NCNA
Succulent- oat leaves were definitely favored by the adult
beetles (Table 8). Beetles moved to the succulent oat leaves and
usually began feeding immediately on the blades. Mature oat leaves
were the second choice of the beetles with the dried and check treat ments attracting less insects. Several of the beetles crawled to the
olfactometer top or sides and could not be counted as a response.
Additional plant condition tests were conducted to determine
whether spring and summer adults preferred the same plant conditions
(Table 9, Statistical Analysis in Appendix Table 28).
Green oat leaves were preferred over the other treatments by both the spring and summer adults. Beetle response was practically
immediate and feeding occurred shortly thereafter. Yellow plants were
the second choice and brown plants the third choice. Severe feeding occurred on the green oat leaves. It did seem that the summer adults 57 were more active than the spring adults in overall response. However there was no statistically significant difference in response between spring and summer adults.
TABLE 9
RESPONSE OF FIELD COLLECTED UNSEXED SPRING AND SUMMER ADULTS, TESTED IN A CAGE TYPE OLFACTOMETER, TO VARIOUS OAT LEAF CONDITIONS
Type Number of Number of Percent Treatment Response After Adults Test Replica 30 Minutes Insects tions Fresh Green Fresh Green Dried Yellow Check
Spring 100 4 36.2 10.7 16.7 5.2 Summer 100 4 55.7 9.2 15.0 3.0
Freeze dried extracts collected from fourteen-day-old succulent oat plants were offered to beetles in a cage type olfactometer.
TABLE 10
RESPONSE OF 7/14/65 FIELD COLLECTED UNSEXED ADULTS, TESTED 9/17/65 IN A CAGE TYPE OLFACTOMETER, TO FREEZE DRIED OAT EXTRACTS AND DISTILLED WATER
Test Repli- Number Number of Percent Confidence Normal Materials cations of Test Beetles Beetle Limits Curve Insects Responding Response 957.
Freeze dried oat extract 4 60 18 30 18.4-41.5 OK Distilled water 4 60 0 0 0-0 NCNA
Although the number of test insects was relatively low (Table 10),
these beetles immediately responsed and moved upon the filter paper 58 impregnated with freeze dried oat extract. Beetles lowered both their head and antennae when in contact with this filter paper. In contrast, beetles moved on and off the filter paper impregnated with distilled water.
Next, freeze dried extracts of 14-day-old succulent oat plants and 28-day-old mature oat plants were compared with distilled water.
TABLE 11
RESPONSE OF 7/14/65 FIELD COLLECTED UNSEXED ADULTS, TESTED 9/23/65 IN A CONTROLLED AIR FLOW OLFACTOMETER, TO FREEZE DRIED EXTRACTS OF MATURE AND SUCCULENT OATS, AND DISTILLED WATER
Test Repli- Number Number of Percent Confidence Normal Materials cations of Test Beetles Beetle Limits Curve Insects Responding Response 95%
Freeze dried succulent oat volatile 2 380 136 35.7 30.9-40.6 OK Distilled water 2 380 60 15.7 12.1-19.4 OK
Freeze dried mature oat volatile 2 90 22 24.4 15.5-33.3 OK Distilled water 2 90 4 4.4 0.1-8.7 NCNA
Oat volatiles were more attractive than the distilled water in these tests. The freeze dried succulent oat volatiles were preferred over freeze dried mature oat volatiles as shown in Table 11.
Further testing involved comparing the response of both spring and summer adults in a cage type olfactometer with freeze dried, cold water, and hot water oat extracts with check. 59
TABLE 12
RESPONSE OF FIELD COLLECTED UNSEXED ADULTS, TESTED IN A CAGE TYPE OLFACTOMETER, TO VARIOUS OAT PLANT EXTRACTS OBTAINED BY DIFFERENT METHODS
Type Number of Number of Percent Treatment Response Adults Test Repli After 30 Minutes Insects cations Freeze Cold Hot dried water water Check
Spring 100 4 17.2 16.5 18.7 5.0 Summer 100 4 20.2 25.0 25.0 4.2
Both spring and summer adults responded to freeze dried, cold water, and hot water extracts in preference to the check (Table 12,
Statistical Analysis in Appendix Table 30). It was surprising to ob serve that the hot water extract seemed to be more preferred by the beetles. Summer adults responded significantly greater than spring adults and the oat extracts were significantly preferred over the check.
Tests were next set up to compare spring and summer adult response in a cage type olfactometer with oat extracts from water, chloroform, and alcohol.
TABLE 13
RESPONSE OF FIELD COLLECTED UNSEXED ADULTS, TESTED IN A CAGE TYPE OLFACTOMETER, TO VARIOUS OAT EXTRACTS
Type Number of Number of Percent Treatment Response After Adults Test Replica 30 Minutes Insects tions Water CHCI3 Alcohol Check Spring 100 4 19.0 2.0 4.5 17.2 Summer 100 4 33.0 2.0 5.7 10.5 60
Beetle response was greatest toward the water oat extract and
check treatments (Table 13, Statistical Analysis in Appendix Table
29). A low response occurred with the chloroform and alcohol oat
extracts. There was no significant difference among spring and summer
adult response to the treatments. However, water and check response
were significantly greater than the chloroform and alcohol response.
The controlled air flow olfactometer was used to test summer adult
preference to oat extracts of various solvents using different extrac
tion methods.
TABLE 14
RESPONSE OF 7/12/68 FIELD COLLECTED UNSEXED ADULTS, TESTED 7/15/68 IN A CONTROLLED AIR FLOW OLFACTOMETER, TO OAT EXTRACTS FROM THE VARIOUS SOLVENTS AND PLANT EXTRACTION METHODS ) Solvent Choice Number of Beetle Response Percent and Port Test After Total Location Insects 30 Minutes Response
Water oat extract 30 22 73 Check 6 20 Freeze dried extract 30 18 60 Check 8 27 Water 30 10 33 Check 8 27 Benzene 30 9 30 Check 8 27 Methyl alcohol 30 7 23 Check - 13 95% ethyl alcohol 30 6 20 Check 3 10 Hexane 30 5 16 Check 4 13 Acetone 30 5 16 Check 4 13 CHC13 30 3 10 Check 2 7 Pet. ether 30 3 10 Check 5 16 61 Summer adults responded more positively toward the water oat ex tract and freeze dried extract than to any other treatment (Table 14).
Descending order of preference is as follows: water, freeze dried, wateraalone, benzene, methyl alcohol, 95% ethyl alcohol, hexane, ace tone, chloroform, and petroleum ether. However the checks gave a relatively high response.
Water extracts from green oat plants were frozen (0 F.), refrig erated (40 F.), and held at room temperature (72 F.) before testing beetle response. Particular interest involved the effect of storage on the response of both spring and summer adults. Effort was made to determine the effects of storage on the length of time that beetles
could continue to be attracted.
Spring adults responded greatest during the first 10 minutes to room temperature, refrigerated, and frozen oat extracts (Fig. 21).
The initial response was to room temperature, refrigerated, check, and frozen extracts. However, the check preference decreased after
the initial response and leveled off over the 120 minutes. The other
treatments decreased in attractiveness after the first 10 minutes.
The active"fraction" appears to be most attractive at room temperatures.
Cooler temperatures appear to reduce attraction initially until the
extract warms up.
Initially, summer adults responded similarly to the spring adults
(Fig. 22). Peak response was reached at 5 minutes and decreased thereafter.
Light effects
During early preliminary laboratory tests it was discovered that
summer adult cereal leaf beetles were phototactic. Unequal lighting 62
100
frozen ______refrigerated room temperature ------check
at 15 m _ , g 14
S 13
60 120 Minutes
Fig. 21.--Accumulative response in various time intervals of 5/10/68 field collected unsexed adults, tested 5/11/68 in a cage type olfactometer, to different storage conditions of water solutions of green oat plant extracts. i. 22.“-AccumulativeFig.response in various timeintervals of Percent Total Beetle Response 17 18 - 20 19 ■ 15 16 1 1 13 . 14 - 10 12 IOC 4-1 8 9 4 r 5 6 7. 21 3. 1
- -
-
extracts. conditionsof watersolutions greenof oat plant 7/3/68 fieldcollected unsexed adults, tested 7/4/68 ina cagetype olfactometer, todifferentstorage 1 3 60 30 10 5 Minutes \ check — refrigerated «— — ______t— 120 r0omtemperature frozen
63
64 in the room as a result of huge windows caused beetles to orient towards the light regardless of the attractant on test. Therefore, tests were set up to observe the effects of light on spring adult response in a controlled air flow olfactometer with freeze dried oat extract treatments. Counts were made at 1, 2, 5, 10, 15, and 20 minutes.
Results in Figure 23 provided evidence that spring adults are phototactic as was observed with summer adults. A significant response occurred when the lights were present in the olfactometer. Unfortun ately, response to treatments was rather low. However, beetles had to travel 3 feet to this type olfactometer in contrast to 1 foot in the cage type olfactometer to give a recorded response. Most of the beetles moved only a few inches or not at all in total darkness.
Filter paper and agar substrates
Discovering from the previous laboratory tests that freeze dried and water extracts of oat foliage yielded positive olfactory response from beetles, it was decided to treat the surface of agar substrates in an attempt to record any feeding marks that might occur (Table 15).
By placing the lights under the petri dishes, responses were confined to the bottom of the dishes and on the agar. The agar treated with oat extracts attracted more beetles than the check. Beetles rapidly flexed their antennae and performed strange foot movements when
r moving toward and at the oat extract treatments. Feeding marks on the agar were difficult to separate from clay or foot marks. Few beetles appeared to be biting the agar. Percent Total Beetle Response 100 Fig. 23.--Accumulativeresponse in various timeintervals of 10 11 12 13L 15. 16 17- 20 - . - 5/27/66in acontrolled flowair olfactometer, to 5/25/66 fieldcollected unsexed adults, tested freeze driedextractsoat withand lights. without (Normalcurve95% - OK or NCNA) Minutes without lightswithout ih lightswith
66
TABLE 15
RESPONSE OF 7/7/66 FIELD COLLECTED UNSEXED ADULTS TO WATER AND ACETONE EXTRACTS OF SUCCULENT DARK GREEN OAT LEAVES UPON AGAR SUBSTRATES AND A CHECK AGAR SUBSTRATE
Test Repli Number Number of “ Percent Confidence Normal Materials cations of Test Beetles Beetle Limits Curve Insects Responding Response 95%
Oat water extract 4 160 53 33.1 25.8-40.4 OK Oat acetone extract 4 160 42 26.2 19.4-33.0 OK Check 8 320 14 4.3 2 .1- 6.6 NCNA
Response between sexes
The next question arose as to whether plant preference differed
between the two sexes. Field collected overwintering spring adults
were often 60-70% females in a population. Therefore, beetles had to
be sexed and tested as a 50:50 ratio to determine difference between
response of males and females.
The best method of sexing the beetles involved separation during
the act of copulation (Figure 24), which shows the male uppermost.
In addition (Myser and Schultz, 1967) discovered a method for sexing
the beetles based on morphological characters. They found that the
males were flat or concave in the intercoxal process of the first
abdominal sternite, whereas the females were humped or convex at this
process. This technique of sexing assisted in the tests. However,
it was still time consuming, since one needed a binocular scope to
individually sex each beetle. 67
Fig. 24.--Adult cereal leaf beetles during the act of copulation. 68
TABLE 16
RESPONSE OF 4/20/66 FIELD COLLECTED SEXED ADULTS,TESTED 4/24/66 IN A CAGE TYPE OLFACTOMETER, TO DARK GREEN OAT LEAVES
Test Repli Number Number of Percent Confidence Normal Materials cations of Test Beetles Beetle Limits Curve Insects Responding Response 95%
Dark green oat leaves 8 2408$'s 39 16.2 11.5-20.9 OK Check 8 24(®'s 5 2.0 0.2-3.8 NCNA Dark green oat leaves 8 240^f,g 29 12.0 7.9-16.2 OK Check 8 2 4 0 # t s 17 7.0 3.8-10.3 NCNA
There was no significant difference in response to test materials between the two sexes (Table 16). Males showed a trend to be more exploratory than females.
Antennectomy studies
The antennae of adult cereal leaf beetles are composed of 12 segments. The first segment is small and when in a natural position is somewhat hidden by the second segment (Fig. 25). The second segment is thick and globular. All successive segments are almost equal, cylindrical, and provided with hairs. Observations were made on response of beetles having both antennae intact, the distal 1/2 of both antennae removed, and both antennae completely removed.
However, it may be quite possible that the distal 5 to 7 segments were removed rather than the distal 6 segments. Segments were rapidly removed with a single edge razor blade. 69
TABLE 17
RESPONSE OF 7/4/68 FIELD COLLECTED UNSEXED ADULTS,TESTED 7/6/68 IN A CAGE TYPE OLFACTOMETER, TO GREEN OAT LEAVES. BEETLES WERE IN VARIOUS STAGES OF ANTENNECTOMY
Insect Number of Number Beetles Percent Total Treatment Test Responding After Response Insects 30 Minutes
Both antennae removed 25 4 16 Distal 1/2 of both antennae removed 25 10 40 No antennae removed 25 18 72
It was interesting to observe that the beetles with approxi mately 1/2 of the distal part of both antennae removed oriented surprisingly well to the succulent green oat plant leaves (Table
17). Orientation decreased when both antennae were removed. Fig. 25.--Antennae of adult cereal leaf beetle (greatly enlarged), a.— X360; b.— X970. RESULTS - FIELD
Trap reliability
Various kinds of field traps were tried by Indiana and Michigan personnel shortly after the entry of the cereal leaf beetle. These early traps were often ineffective and caught only a few beetles. It was not until 1965, that "canary yellow" was found to be the most attractive color for adult cereal leaf beetles (Jantz, 1965). Utiliz ing this color plus "stickem," the field trap as described in the
Materials and Methods Section was built by the Methods Improvement
Station of the Plant Pest Control Division at Niles, Michigan. After development, this trap was used by Michigan and Ohio personnel as the
field trap in attractant and arrestant studies. During the 1967 season of Ohio field tests, statistical analysis revealed a mean per trap per day of 3.5 beetles from the check and 8.2 beetles from an oat plant extract (Table 27). However, daily catches often would range
from 0 to 30 beetles per trap depending on the weather conditions.
USDA synthetic lures
The 25 USDA synthetic lures previously tested in the laboratory were field tested. None of the lures attracted more beetles than the
control or checks. No further effort was spent on field screening
these lures.
71 72
Attention was next turned to testing six additional USDA synthetic lures. These six USDA lures gave the best beetle response out of 431 lures field tested by Michigan personnel in 1965 (Jantz,
1965).
TABLE 18
RESPONSE OF ADULT CEREAL LEAF BEETLES TO VARIOUS USDA SYNTHETIC LURES TESTED 6/2/66 IN A WHEAT FIELD NEAR NEW CARLISLE,INDIANA
Test Mean Number of Materials3, Replications Beetles Trapped
3294 2 12.0 4162 2 12.0 11747 2 11.5 24748 2 10.5 24766 2 10.5 30443 2 12.0 Control 2 8.5 Check 2 9.0
cL See page 34 for names of test materials.
As shown in Table 18, the number of beetles trapped were essentially the same among the six USDA candidate lures. However, all lure catches were higher than the control and check treatments which does support the Michigan findings. At that time, I did not feel that these six lures showed enough promise for further replica tions. Effort was shifted to testing various plant extracts which had looked good in previous laboratory investigations. 73
Oat host plant studies
It was discovered in laboratory tests that plant condition, method of extraction, and method of storage were all influential in determining oat plant preference. Consequently, the final step involved confirming the results of laboratory investigations with field tests.
Tests were conducted during the spring and summer months of 1967 at New Carlisle, Indiana, in wheat and oat fields.
Treatments were applied at 10:00 AM and adult counts taken at
11:00 AM and 4:00 PM (See Table 19). The temperature was 70 F. and the wheat plants were 18 inches high.
A few adult beetles were caught one hour after treatments were
•employed. Most accumulation of adults occurred up until 4:00 PM.
More beetles were trapped than expected in spite of a strong wind from the southeast at 15 to 20 miles per hour. The beetles were not removed from the board until the final 4:00 PM reading. A common putty knife worked well when removing the beetles from the sticky board traps. It was noted that attraction was slightly greater toward the green oat extract water solvent and freeze dried process than the other treatments. 74
TABLE 19
RESPONSE OF OVERWINTERING ADULT CEREAL LEAF BEETLES TO VARIOUS OAT PLANT TREATMENTS TESTED 5/26/67 IN A WINTER WHEAT FIELD NEAR NEW CARLISLE, INDIANA
Treatment Replicate Counts at: Accumula Number3, A B C D 11 :00 AM 4:00 PM tive Total Trap Board Side Count East West East West
1 4 6 2 4 2 5 4 12 16 2 3 5 4 2 1 6 5 9 14 3 8 7 5 1 0 11 3 18 21 4 3 2 5 2 0 6 2 10 12 5 2 3 4 0 4 0 4 5 9
6 3 1 1 1 0 2 0 6 6 7 2 3 3 1 1 3 2 7 9 8 3 2 2 2 0 2 1 8 9 9 9 6 3 4 1 12 4 18 22 10 5 4 4 3 1 10 1 15 16
11 2 1 2 0 0 1 0 5 5 12 3 1 1 2 2 1 4 3 7 13 8 4 3 4 2 8 7 12 19 14 7 8 3 6 2 10 3 21 24 15 6 6 2 3 1 10 2 15 17
16 4 4 5 3 1 11 2 14 16 17 3 3 4 1 1 8 1 10 11 18 4 3 2 4 0 5 2 11 13 19 0 0 0 0 0 0 0 0 0 20 4 3 1 5 1 5 4 9 13
See page 46 for treatment number identification. 75
TABLE 20
RESPONSE OF OVERWINTERING ADULT CEREAL LEAF BEETLES TO VARIOUS OAT PLANT TREATMENTS TESTED 5/27/67 IN A WINTER WHEAT FIELD NEAR NEW CARLISLE, INDIANA
Treatment Replicate Counts at: Accumula Number3- A B C D 11:00 AM 4:00 PM tive Total Trap Board Side Count East West East West
1 2 0 2 2 0 3 2 4 6 2 2 3 4 1 1 5 4 6 10 3 9 4 2 2 0 10 1 16 17 4 6 2 2 0 1 6 3 7 10 5 2 3 4 2 2 3 3 8 11
6 1 2 2 1 1 3 2 4 6 7 2 0 1 2 2 0 2 3 5 8 2 1 3 0 2 1 2 4 6 9 6 4 2 1 3 8 4 9 13 10 7 3 3 4 4 12 5 12 17
11 1 1 0 3 2 3 2 3 5 12 2 3 1 1 1 1 3 4 7 13 7 2 1 3 3 5 6 7 13 14 6 3 3 4 2 8 2 14 16 15 7 4 2 2 2 9 3 12 15
16 5 3 4 3 3 10 4 11 15 17 2 1 0 3 2 3 2 4 6 18 3 3 2 2 3 6 3 7 10 19 1 1 0 2 1 2 2 2 4 20 2 2 1 2 1 4 2 5 7
aSee page 46 for treatment number identification.
The total beetle counts were slightly less than the preceding
day probably due to cooler temperatures at approximately 65 F. or
less. New treatments had been put out in the morning at approxi
mately 10:00 AM. A refrigerator in the Purdue Mobile Trailer kept
the samples either frozen or refrigerated as desired. The water 76 solvent seemed to attract slightly more beetles than the other treatments. Alcohol appeared undesirable in beetle attraction.
Beetles were trapped primarily on the west side of the trap board.
After testing spring adults in wheat fields, the same treat ments were used for summer adult response in oat fields.
TABLE 21
RESPONSE OF NEW SUMMER ADULT CEREAL LEAF BEETLES TO VARIOUS OAT PLANT TREATMENTS TESTED 7/12/67 IN AN OAT FIELD NEAR NEW CARLISLE, INDIANA
Treatment Replicate Counts at: Accumu Number3 A B C D 11:00 AM 4:00 PM lative Trap Board Side Total North South North South Count
1 4 5 2 2 8 1 11 2 13 2 4 6 5 1 9 0 15 1 16 3 8 8 4 3 8 2 20 3 23 4 12 9 9 2 7 2 28 4 32 5 2 3 2 0 1 0 6 1 7
6 3 1 4 0 1 0 5 3 8 7 2 3 3 2 3 0 8 2 10 8 3 1 4 1 2 1 5 4 9 9 10 6 4 5 12 2 20 5 25 10 7 5 5 2 8 0 18 1 19
11 3 2 2 0 0 0 3 4 7 12 2 2 1 0 0 0 1 4 5 13 9 7 5 2 12 3 18 5 23 14 9 11 4 3 10 2 24 3 27 15 6 9 2 4 10 0 20 1 21
16 7 7 5 2 8 0 18 3 21 17 3 4 3 2 5 0 10 2 12 18 6 6 5 4 11 1 18 3 21 19 2 3 2 3 1 0 5 5 10 20 4 2 2 2 2 0 6 4 10 cl See page 46 for treatment number identification. 77
The wind came from the northwest at about 20 miles per hour.
Most of the adults were trapped on the north side of the board.
The response data received from these treatments were analyzed at the statistics laboratory at Wooster, Ohio. As shown in the
Appendix Table 23, the differences among the type plant condition and solvents (methods of extraction) were significant, whereas dif ferences among the types storage were nonsignificant. The fresh succulent plants were significantly better than dried brown plants and the water solution and freeze dried extracts were significantly better than chloroform and 95% alcohol extracts. However, there was no significant difference between frozen and refrigerated stored extracts.
As indicated in the Appendix Table 24, the plant-storage combi nation was significant with the green refrigerated extract better than the green frozen extract. The interaction of solvent times plant-storage combination was not significant.
The LSD's as shown in the Appendix Tables 25 and 26, indicate that the green plant was significantly better than the dried plant in beetle preference. Also, the refrigerated water extract was best since it gave the highest mean of trapped beetles. The chloroform - extract attracted significantly fewer beetles when stored by refrigeration than when frozen. 78
TABLE 22
RESPONSE OF NEW SUMMER ADULT CEREAL LEAF BEETLES TO VARIOUS OAT PLANT. TREATMENTS TESTED 7/13/67 IN AN OAT FIELD NEAR NEW CARLISLE,. INDIANA
Treatment Replicate Counts at: Accumu- Numbera A B C -D - 11:00 AM 4 : 0 0 P M " lative _ Trap Board Side Total North South North South Couni
1 6 3 3 2 5 2 8 6 14 2 5 4 3 4 8 0 12 4 16 3 12 8 9 2 12 1 28 3 31 4 10 9 6 6 14 2 26 5 31 5 4 . 5 6 6 10 0 20 1 21
6 3 1 3 3 3 0 7 3 10 7 2 3 3 0 6 0 8 0 8 8 5 6 3 2 6 0 14 2 16 9 8 6 4 1 11 0 19 0 19 10 11 7 2 2 7 1 15 7 22
11 7 2 2 2 5 0 10 3 13 12 2 4 1 1 4 0 8 0 8 13 6 5 3 3 7 1 14 3 17 14 9 8 5 3 12 0 20 4 24 15 9 11 2 4 11 1 25 1 26
16 7 5 4 3 5 2 12 7 19 17 6 1 1 0 4 1 7 1 8 18 9 5 7 7 10 1 23 5 28 19 4 1 0 0 2 0 4 1 5 20 3 3 2 2 5 0 8 2 10
0 See page 46 for treatment number identification. 79
After study of the beetle means of the individual treatments, it was interesting to discover that water alone yielded 6.93 beetles in contrast to alcohol alone yielding 4.24 beetles and chloroform alone yielding 2.11 beetles (Table 27).
It appeared that the water could be considered an attractant or arrestant, whereas the alcohol and chloroform were repellent to cereal leaf beetle adults. SUMMARY AND CONCLUSIONS
Field collected unsexed adult cereal leaf beetles responded significantly in olfactometers when oat leaves as lures were com pared with controls. Of the twenty-five USDA synthetic lures screened in the laboratory for attractant or arrestant properties, only citronellal and methyleugenol elicited enough beetle response to warrant further testing.
Subsequent preliminary laboratory tests indicated the following order of attractiveness: beetle feces > live beetles > dead beetles > control. However, most treatments usually had a very low overall beetle response resulting in the normal curve not being applicable from a statistical standpoint.
Host preference tests indicated that adult cereal leaf beetles have a restricted host range. Beetles consistently responded to oat and wheat leaves over red clover, ginkgo, alfalfa, and red kidney bean leaves. No legumes were found attractive.
The host plant condition was very important in laboratory tests.
Succulent green leaves were always preferred over mature yellow leaves and the control by both spring and summer adults. Also, freeze dried oat extracts were significantly preferred over distilled water by both spring and summer adults. 81
Various oat plant extracts were evaluated in the controlled air flow olfactometer. Order of beetle preference was oat extracts from water > freeze dried > water alone > benzene > methyl alcohol> 957o ethyl alcohol > hexane > acetone > chloroform > petroleum ether.
Usually, the greater the volatility of the solvent, the less the attractancy in laboratory olfactometer tests.
Highest beetle response usually occurred during the first 5-10 minutes of an olfactometer test. Freeze dried volatiles were pre ferred to distilled water extracts initially, but lost attractiveness after 10-15 minutes. The "active fraction or fractions" are appar ently short-lived because of high volatility. Beetles responded slightly to agar substrates treated with water extracts of oats but
failed to leave distinct feeding marks in the agar. Beetles did
display antennal and foot movements on oat extract agar substrates.
Beetles are phototactic. Light was found to be a definite
auxiliary stimulus in laboratory olfactometer tests. Response of male and female beetles was similar. Also, antennae studies revealed
surprisingly good response to oat treatments when the distal halves
of both antennae were removed. Orientation decreased greatly when
both antennae were removed.
Field investigations revealed that extracts of fresh succulent
oat plants were significantly more attractive than dried brown oat
plants, and that water plus freeze dried extractions were significantly
better than chloroform and 95% alcohol extractions. There was no
significant difference between frozen and refrigerated storage of 82 extracts. No explanation is known for the fact that the refrigerated extract of green oats was more attractive than was the frozen extract of green oa ts . The most attractive test material was the refrigerated water extract of fresh oat plants.
These results indicate that water alone could be a possible
"arrestant" and in combination with certain plant substances a pos sible "attractant" for adult cereal leaf beetles. It is well known that under certain weather conditions plants transpire greatly. There undoubtedly exists in nature a water gradient with molecules moving from a higher concentration to a lower concentration. The water molecules could possibly carry the plant arrestant or attractant which the beetles would detect through their highly refined receptor system. Beetles would then orient toward the source and move to the water-odor origin.
The following hypothesis is proposed:
1. The volatility of a given plant substance could be influenced by its reaction with water. Water could serve as a catalyst in the decomposition of a plant substance resulting in greater volatility.
Water is the best solvent known because more substances dissolve in
it than in any other solvent. Certain substances dissolved in water
could readily ionize because water molecules by virtue of their own dipolar charged nature could act as small magnets attracting the
ions of the plant substance, thereby weakening the attraction in the material itself. 2. Water molecules could act as a carrier of "odor" molecules.
A moving humid air flow would hydrotransport the plant odor molecules away from the plant. Water does tend to form hydrogen bonds which link relatively negative atoms like oxygen, nitrogen, fluorine, or chlorine present in one molecule, to the relatively positive hydrogen atoms in another.
Consideration must be given to temperature, humidity, wind velocity, light, insect stage, plant condition, and other factors.
Then after recognizing that various stimuli do exist; farmers can forecast the stage of crops most susceptible to attack, survey teams can more accurately detect this pest in uninfested regions, and entomologists can best wage the war of pest destruction. APPEN DIX
84 APPENDIX
TABLE 23
ANALYSIS OF VARIANCE OF ADULT CEREAL LEAF BEETLE RESPONSE IN 1967 FIELD TESTS AT NEW CARLISLE, INDIANA, TO VARIOUS TREATMENTS OF OAT PLANT EXTRACTS
Source DF MS F
Replication 3 225.71 33.313 Solvent 3 325.67 48.067** Plant 1 71.31 10.525** Storage 1 .01 .001 ns
Solvent x Plant 3 13.67 Solvent x Storage 3 19.03 2.809* Plant x Storage 1 .88 Time 7 1742.32
Mean 1 5630.70 Error 345 6.77 Total 368
**Significant at the .01 level of probability
^Significant at the .05 level of probability
ns Nonsignificant 86
TABLE 24
ANALYSIS OF VARIANCE OF ADULT CEREAL LEAF BEETLE RESPONSE IN 1967 FIELD TESTS AT NEW CARLISLE, INDIANA, TO VARIOUS TREATMENTS OF OAT PLANT EXTRACTS
Source DF MS F
Replication 3 216.48 31.530 Solvent 3 410.21 59.746** Plant-Storage Combination 4 20.28 2.954*
Solvent x Plant-Storage Combination 11 11.00 1.603 ns Time 7 2080.20 Mean 1 6598.21
Error 408 6.86 Total 437
“Significant at the .01 level of probability
Significant at the .05 level of probability
ns Nonsignificant 8.7
TABLE 25
MEAN RESPONSE OF ADULT CEREAL LEAF BEETLES IN 1967 FIELD TESTS AT NEW CARLISLE, INDIANA, TO VARIOUS TREATMENTS OF OAT PLANT EXTRACTS
Plant Condition Mean Collection3
Green 5.57 Dried 4.69
^ S D (.05) = .53
TABLE 26
MEAN RESPONSE OF ADULT CEREAL LEAF BEETLES IN 1967 FIELD TESTS AT NEW CARLISLE, INDIANA, TO VARIOUS TREATMENTS OF OAT PLANT EXTRACTS
Storage3,
Solvent Refrigerator Freezer
Alcohol ' 4.15 3.82 Water 7.63 6.58 Chloroform 2.58 3.67 Freeze Dry 6.15 6.47
aLSD (.05) = .74 88
TABLE 27
MEAN RESPONSE OF ADULT CEREAL LEAF BEETLES IN 1967 FIELD TESTS AT NEW CARLISLE, INDIANA, TO VARIOUS TREATMENTS OF OAT PLANT EXTRACTS
Treatment3, Mean Number Response
4 _ 8.24 10 7.19 18 6.93 13 6.80
3 6.76 9 6.59 15 6.33 16 6.33
14 6.15 2 5.41 1 4.76 17 4.24
5 4.15 20 3.47 11 3.37 7 3.06
8 3.06 6 2.85 12 2.50 19 2.11
See page 46 for treatment identification. TABLE 28
ANALYSIS OF VARIANCE OF UNSEXED SPRING AND SUMMER ADULT CEREAL LEAF BEETLE RESPONSE TO VARIOUS OAT PLANT CONDITIONS IN THE LABORATORY
Source DF MS F
Replications 3 33.66 A Treatments9 1 98.00 3.814 ns B Treatments*3 3 2776.08 108.058** AB Interaction 3 227.75 8.865** Error 21 25.69 Total 31
aSpring and Summer Adults Fresh green plants, green dried plants, fresh yellow plants, and check •jf Significant at the .01 level of probability nsNonsignificant
TABLE 29
ANALYSIS OF VARIANCE OF UNSEXED SPRING AND SUMMER ADULT CEREAL LEAF BEETLE RESPONSE TO VARIOUS TREATMENTS OF SOLVENTS IN THE LABORATORY
Source DFMSF
Replications 3 52.75 A Treatments3 1 36.12 2.346 ns B Treatments*3 3 924.08 60.033** AB Interaction 3 150.04 9.747** Error 21 15.39 Total 31
aSpring and Summer Adults ^Water, Chloroform, Alcohol, and Check •Jf Significant at the .01 level of probability nsNonsignificant 90
TABLE 30
ANALYSIS OP VARIANCE OF UNSEXED SPRING AND SUMMER ADULT CEREAL LEAF BEETLE RESPONSE TO VARIOUS TREATMENTS OF OAT PLANT EXTRACTION METHODS IN THE LABORATORY
Source DF MS F
Replications 3 18.75 A Treatments3 1 - 144.50 10.847** B Treatments*3 3 514.75 38.640** AB Interaction 3 32.41 2.433 ns Error 21 13.32 Total 31
aSpring and Summer Adults
^Freeze dried, cold water, hot water, and check
•jf |||j Significant at the .01 level of probability
ns • . Nonsignificant LITERATURE CITED
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