smote:- OF ms mxum. EEHAVIOI or m: ADULT CEREAL LEAF BEETLE, 90mm. MELANOPgs (L) 118 009 THS The“: for {In Dagn- of M. 5. WCHEGAN STATE UNIVERSITY L. Gualberto Merino M. 1966 H9515, Airgun . Shfgan State ‘ mvctsiry ABSTRACT STUDIES OF THE SEXUAL BEHAVIOR OF THE ADULT CEREAL LEAF BEETLE, Oulema melanopus (L.) by L. Gualberto Merino M. The cereal leaf beetle, Oulema melanopus (L.) (Coleoptera, Chrysomelidae), is an imported pest that has threatened the produc- tion of cereal grains in the midwestern region of the United States of America. Considerable effort has been directed to obtaining in- formation regarding the biology of this insect that may either improve control measures or lead to more desirable methods of control. A laboratory research project was conducted to investigate the adult sexual behavior of this beetle. In general, the work was carried out in two steps. The first consisted of careful daily obser- vations of individuals in populations of 20 insects confined in small cages over barley seedlings. The second consisted of daily observa- tions of two groups of insects, one composed of isolated females captured in their first or one of their early copulations and the other of isolated pairs captured in their first or early copulation. The adults feed parallel to the veins of the leaf from either the dorsal or ventral surface of the leaf. Their heads may be oriented either upward or downward while feeding. They usually feed through and consume a narrow strip of the leaf, although less commonly one of L. Gualberto Merino M. the cuticle or surfaces may be left intact. Females while in copula- tion appear to feed more voraciously. The rate of feeding is about two millimeters of strip length per minute. Courtship prior to mating has not been established but several manifestations on the part of the male have been observed after mounting. The time of actual copulation ranges from 4 to 121 minutes. There is frequently a time lapse between the end of copulation and separation of male and female. Mounting without copulation and in- terruptions of intercourse are common. Definite homosexual behaviour in the male of this species was observed during this study. Prior to egg deposition the female orients herself longitu- dinally, parallel to the leaf veins, and near the center of the leaf. The eggs are laid parallel to the leaf veins and near the center of the leaf. The average interval between orientation of the female and oviposition is 2.3 minutes. Oviposition usually takes place after mating. Seventy-eight percent of the eggs are laid on the upper leaf surface. One or two eggs are laid at one time. One egg was deposited in 78% of the observations and two eggs in the remaining cases. Females that were isolated from males after a single mating laid as many eggs as females that were continuously caged with males. Only 45% of the eggs laid by the isolated females hatched while 62% of the eggs laid by the females that were caged with males were fertile. STUDIES OF THE SEXUAL BEHAVIOR OF THE ADULT CEREAL LEAF BEETLE, Oulema melanopus (L.) BY L. Gualberto Merino M. A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Entomology 1966 ACKNOWLEDGEMENTS The author is deeply grateful to Dr. Robert F. Ruppel for his foresighted conception of this fundamental study. Sincere apprecia- tion is extended to Dr. Gordon Guyer, Chairman of the Department of Entomology, and to Drs. Roland Fischer, Roger Hoopingarner and Mr. Richard Connin for their critical suggestions and contributions to the manuscript. The author is indebted to Messrs. Melvin Gomulinski and David Cobb for their help in conducting this work. Special appreciation is extended to the Entomology Research Division of United States Department of Agriculture for providing important re- sources during the active research period. ii TABLE OF CONTENTS INTRODUCTION . . . . . . . MATERIALS AND PROCEDURE RESULTS OF OBSERVATIONS Feeding Habits . . Mating Behavior . . . . . . Homosexual Behavior of the Males Oviposition Behavior . . . Influence of Multiple Mating on Egg and Fertilization . . . DISCUSSION . . LITERATURE CITED . . . . . . iii l4 16 21 23 25 LIST OF TABLES Table Page 1. The Rates of Feeding by the Adult Cereal Leaf Beetle . . . . . . . . . . . . . . . . . . . . . . . . 7 2. Times of Pre-copulation Mounting, Actual Copulation and Mounting without Copulation of Male Cereal Leaf Beetles O I O O O O O O O O O O O O O O O O I I O 1]- 3. Grouping of Eggs of the Cereal Leaf Beetles on the Upper and Lower Surfaces of the Leaves of "Hudson" Barley Seedlings . . . . . . . . . . . . . . . . . . . 19 4. Grouping of Eggs of the Cereal Leaf Beetles on Leaves of ”Hudson" Barley Seedlings . . . . . . . . . . 20 5. Number and Fertility of Eggs Produced by Female Cereal Leaf Beetles Following Single and Multiple Matings . . . . . . . . . . . . . . . . . . . 22 iv INTRODUCTION The cereal leaf beetle, Oulema melanopus (L.), long known as a pest of small grains in Europe is now threatening crops in the New World. Farmers near Galien, Michigan, reported spraying with in- secticides for control of the pest in 1959, but its presence was not officially recorded until June, 1962 (Castro, 1964 and Castro _£l_l., 1965). Like many other introduced species in different parts of the world, the cereal leaf beetle has thrived in its new range and is damaging cereals in Michigan, Indiana, and Ohio. The only reliable method now known for the control of this pest is the use of insec- ticides (Ruppel _£__l., 1964). Cognizant of limitations on the use of chemicals for insect control, e.g. populations resistant to the insecticide and undesirable side-effects, entomologists are seeking to find better means for insect control. There are a few examples of successful control based on the behavior and responses of the pest species. The control of the screw worm fly, Cochliomyia hominivorax (Coq.), by means of radiation-induced male sterilization, a technique developed with exact knowledge of the sexual behavior of this species, is an outstanding example (Metcalf and Flint, 1962). Utilization of such information emphasizes the necessity of having a thorough know- ledge of the pest in order to plan satisfactory control measures. Despite the fact that insects have been known to-inflict dis- astrous effects on man's economy since ancient times, detailed studies 1 2 of the behavior of insect pests are extremely scattered. This is true of even the more familiar pests where the life cycle and responses to toxicants are commonly fully documented, but notes on behavior of the Species are commonly restricted to a few casual observations and often nothing is mentioned of the other factors that must affect the exist- ence of a living organism. The research reported herein was undertaken to provide knowledge of some of the behavioral characteristics of the cereal leaf beetle which might lead to the improvement of its control. The life cycle, biology, and habits of this pest in the New World are described by Castro (1964), and his paper should be consulted for a general view of the pest. MATERIALS AND PROCEDURE The research discussed herein consists of two sets of observa- tions on the adults of Oulema melanopus (L.): (1) Its different be- havior manifestations, and (2) the influence of multiple mating on egg production and fertilization. The insects used for the behavior study were collected in the field near Galien, Michigan on September 25, 1964 and stored in a refrigerator at 430 F until December 23, 1964. The adults were re- moved from storage and placed in cages in lots of 20 each. The cages consisted of seven-inch glass lantern chimneys placed over 4.5 inch diameter flower pots containing young "Hudson" barley seedlings. The mouth of the lantern globes were capped with fine nylon screen and bases sealed to the soil surface by liberally sprinkling white quartz sand over the soil surface. In order to facilitate observa- tions, seedling plants were removed as necessary. Fresh plants were provided by removing the insects from the old cage with an aspirator and placing them in a new cage. A temperature of approximately 80° F and relative humidity of 40% were maintained throughout the study. Light was from 350 foot candles of white daylight florescent tubes. A photoperiod of 16 hours (7 a.m. to 11 p.m.) was maintained from December 23 until January 14, at which time it was changed to 24 hours of light. This change was made in accordance with the general rearing practices used for the pest. Careful observations were made and notes 3 4 were taken daily on those manifestations currently under observation. There were a total of 104 hours of close and accurate observations taken on 49 days with an average of 2.1 hours per day. The maximum and minimum times of continuous observation were 3.3 and 0.3 hours, reSpectively. Observations were not taken December 25, 27, and January 3, 4, 8, and 27. The study of the influence of copulation on egg production and fertility, was conducted from July 15 through August 24, 1965 in the Entomology Laboratory of Michigan State University using adults reared in the laboratory. The adults had been placed in storage at 380 F on May 17, 1965. Two hundred seventy adults were removed from storage on July 15, 1965, and placed in a 16" x 16" x 16" plexiglass cage containing seedlings of "Hudson" winter barley. The experiment was conducted at a temperature of 780 - 80° F under a 24-hour photo- period. Adults in the large cage were observed and isolated into two groups. One group consisted of 20 pairs of adults observed copulating and subsequently isolated and maintained together in a glass chimney cage. The second consisted of 21 individually isolated females which were captured while copulating and maintained singly in glass chimney cages. Copulation was considered to occur if the aedaegus was observed within the female. The cages were periodically observed for feeding, mating, and oviposition. The number of eggs in each cage was recorded and the female or pair moved to new cages when fresh barley was needed. The eggs were held until hatching was completed and the number of larvae counted. RESULTS OF OBSERVATIONS Feeding Habits During the first three hours after removal from storage all of the adults remained motionless on the sand. At the end of 24 hours no feeding had taken place and three adults were found on the walls of the cage, four remained on the sand, and the remainder were at about mid-height on the seedlings. Three days later five adults were found on leaves near the screen cap where the leaf tips had been bent by the screen. There was very little feeding on the leaves except at the bending leaf tips, where considerable feeding had occurred. Thus, at first it appeared that adults preferred the tips of the leaves. However, later observations of active adults indicated a positive phototropism and the resultant feeding may be the first indication of this manifestation. One adult was observed at the tip of a leaf with its head bent toward the surface and moving the mandibles, creating the impression of feeding. Examination of the resultant damage re- vealed only five uneven scratches on the upper cuticle of the leaf with the lower cuticle intact. When feeding, the insect orientates itself longitudinally parallel to the leaf, and a narrow strip of leaf tissue is removed as the head is moved rhythmically from side to side and the insect moves forward. The insect feeds indifferently upwardly or downwardly on either the upper or underside of the leaf. The forelegs are 5 6 directed obliquely ahead, making an angle of about forty-five degrees to the longitudinal axis of the body, while the adult is actually feeding. Usually both sides of the leaf are eaten but infrequently one of the cuticulary layers is left. Occasionally feeding may take place on the stem. Even when food is abundant adults will feed so close to eggs on the leaves that the eggs appear to have been deposited on the edge of the feeding scar. Females that have males mounted on them have been observed to feed more voraciously regardless of whether copula- tion is taking place or not. Some of the females were disturbed when carrying the male, as evidenced by sporadic and scattered feeding. Feeding by two insects was interrupted for 8'to 35 seconds by noise, voices, and by striking the glass chimney cage with a finger on several occasions. Interruptions in feeding by the female during mating were observed on some occasions at the time the copulatory fluid became visible. The length of uninterrupted feeding varies, but ranges from 8 to 20 milimeters. The consumption of food for a given time varies considerably. One female during and after copulation was observed to consume a 70 mm. strip of leaf tissue in 128 minutes with some inter- ruptions. Other females were observed to eat 40 mm. in 12 minutes, 30 mm. in 33 minutes, and 40 mm. in 45 minutes with some interruptions, while copulating. It appears that as the adults grow older, food consumption is reduced. On February 8, 129 mm. of leaf tissue were consumed by three adults in 20 hours. No feeding by these adults occurred on February 12, during a three-hour observation period, and only 58 mm. were consumed 7 in 19 hours on February 13. An estimate of the rate of feeding in a lot of eleven adults observed at random on eight occasions under a 24 hour photoperiod is provided in Table 1. TABLE l.--The rates of feeding by the adult cereal leaf beetle f J Length of Feeding Continuous Time Rate of feeding Sex Stripe (mm.) of feeding (min.) (mm./min.) Not determined 8 4 2.0 Copulating female 10 5 2.0 Female 10 3 3.3 Not determined 15 8 1.9 Not determined 15 8 1.9 Not determined 15 5 3.0 Copulating female 15 9 1.7 COpulating female 19 10 1.9 Copulating female 20 12 1.7 Female 20 10 2.0 Not determined 20 14 1.4 Less common observations which express the range of variability in feeding habits include: the occurrence of damage to plant tissues which appear as scratchings; feeding on the central unfolded tender leaf; and feeding at the extreme tips of leaves. Mating Behavior There is an apparent 1:1 sex ratio in the cereal leaf beetle 8 (Yun, unpublished). Sex differentiation of the active adults remains a serious problem for researchers working with this species. It is impossible to identify living males and females with certainty unless the pair have actually been seen copulating. The observed homo- sexuality in this Species makes the identification of adults by sex difficult. With some certainty, a male can be identified on the basis of behavior which may take the form of agile acrobatics, short flights, a rapid climb of the plants to the leaf tips or walls of the cage, and a drop to the surface of the cage. Females were recognized only late in the course of this study on the basis of poor body condition or the posterior dorsal segment appearing sticky with copulatory fluid left by the male. Generally a female in poor condition had her elytra separated along the posterior medial dorsal line and loosely attached to the thorax with the abdomen appearing to be somewhat bent downward. While considerable effort was made to determine a courtship pattern no manifestation of its existence was detected. In general males mount females slowly or quickly, either from the front or rear. Thus it appears that a regular courtship pattern does not exist. On several occasions males were observed to mount dead adults and in one instance the aedeagus was inserted into the posterior abdominal seg- ment of a dead female and remained long enough to give the impression that copulation did in fact take place. While no courtship prior to mounting has been seen, the male was frequently observed to turn on the mounted female and stroke the posterior abdominal dorsum of the female. The first observed copulation was on the fifth day after removal of the adults from the 43° F storage and the last mating 9 was observed 29 days later although observations of earlier copulations have been recorded (Castro, 1964 and Castro _£,_1., 1965). During mating the female is grasped by the male by stretching his prothoracic legs to the dorsal-lateral edges of the confluence of the head and pronotum of the female. The head of the male rests near the junction of the femur and tibia and is directed straight ahead. The head of the male during copulation is about 3 mm. above the dorsum of the female. This position is necessitated by the need to deflect the abdominal end of the male during mating. Once engaged in copulation the insects are difficult to separate, even when disturbed. Female behavior during mating is varied and includes the brushing of her antennae with the forelegs and grasping or stroking of the sides of the abdomen of the male with either mid- or hind legs. Frequently the females separate or attempt to dislodge the male by pushing one of the mid- or hind legs of the male with her fore-,mid- or hind legs. Generally the attempt is not successful or results in a momentary separation of the male's leg from her body. The female has also been observed to scrape the femora and tarsi of the mid- and hind legs from the same side together. On numerous occasions females have been observed feeding and walking while mating is taking place. The appearance of a conspicuous drop of copulatory fluid is characteristic of mating and the droplet may acquire considerable size. The time interval between the insertion of the aedeagus and appearance of the droplet in 13 copulations observed over a period of eleven days averaged 16.2 minutes and ranged from 8 to 58 minutes. Mounting in this species does not mean actual mating will occur. Several mountings may be made by the male without copulation or there 10 may be considerable delay before copulation occurs. The observed time of c0pulation varies from as short as 4, to as long as 121 minutes, and averaged 42 minutes (Table 2). When c0pulation terminates the male releases the female and passes over the female's head. As a consequence of this habit the dorsum of the abdomen of the female becomes smeared with fluid if the droplet is large enough. Post mating behavior apparently follows no set pattern although the female frequently moves away from the male. A common phenomena is the mounting of a second male of couples in copulation or on other males engaged in homosexual play. Whatever the stimulus the second male assumes the position of copulation on the principal male. The second male may shift his body to one side of the couple and bring his head near that of the female. On other occasions the second male may assume the mating position, evert the aedaegus, and hook it in the underside of the principal male close to the apices of the elytra. On still other occasions the second male may remain atop the mating couple, but in a rear-facing position. Cases of tertiary mounting of adults were observed only twice. Sec- ondary males have been observed to mount and dismount frequently for short periods of time. A few cases were observed in which a preference for a partic- ular female by a male was exhibited. This statement is based on observations of mating male adults leaving one female, mounting a second female and then returning to the first mate. Mating is not restricted to monogamous couples. At least two matings of the same female with different males were seen on the same day and one male ll as mesa .m spmsunmm HNH on on mm an am On mm om mm as Nu ma momfi .m sumsunmm me am m on 0 cm 0 mm a ma HN om mm ma ma mm mm mesa .H sumsunmm Hm ma o mm ma on «m NH m mm mm mm ma NH ed on 0H mm mm 0H ma ma OH mu 0H 0H q NH 0H mm mm ma m NH mm «N mm «H m NH m dN mu qH m 0H OCH qm «q mH m 0H m «N mm Ha m ma m mm HH 0H HH ma N mm On a m «H 5 mm ma m N mH ma HN Nu mesa .H sumscmm OH mH m Hm mm Hm m 0H mm om no Hm m m OH NH mm om w m 0H 0H mm mm HH m 0H mood .m stanzas Ho coma .wm “mesmoma a mama .H stanzas mouscwz muma mmuscwz mama mmuscfiz mama aw meH ca mEHH :fl mEHH cowumfismou usozuHB use :oHumHDQOU cowumfismoo mam wcflucsoz :H mafia Hmsuo< mo oEHH waflucdoz cmmSDwn meH mmaummn mama Hmoumo mama mo coflumHsdoo usonufiz mewucsoe paw coHumfisaoo HmSuom .wcfiucooe cowumasaou-mua mo mmEHH--.N mandH 12 was observed to repeat copulation with a different mate 45 minutes after the first. A number of events related to mating were observed during this study which, while they were observed infrequently, appeared to be sufficiently systematic and organized to be considered behavioristic. Most of these events were observed and recorded only once in the course of the study but behavior, or actions similar to those mentioned, were also observed in large numbers of adults not under direct study. 1. After mating females may take a position such that the abdomens of the pair are opposed and separated by a short distance and remain in this position five to ten minutes. The distance between abdomens was estimated at 10 mm. In one case observed the female then proceeded to face the motionless male who after five minutes twice mounted the female. 2. An "anger" reaction may have been observed when a mating male, disturbed by an annoying male, dismounted and stepped onto the secondary adult from the front, resulting in both adults falling to the bottom of the cage. 3. Odoriferous attractive substances may be secreted in the mating process as evidenced by an awareness and interest by both the male and female adults in the anal portions of both sexes. Often mounting and copulation may follow. 4. Antennal contact between sexes appears to occur fairly com— monly but with no consistent behavior afterwards. Male and female adults were observed brushing or cleaning their antennae while mating. 5. A running, chasing, and excited behavior among adults occurs but it is difficult to establish a direct relationship between 13 behavior prior to and mating itself. 6. Copulation may be interrupted for no apparent reason for short time followed by re-mating. 7. While mating some males were observed to rub their hind legs together. 8. During the copulatory process the aedaegus remains in- serted in the female abdomen. However, on numerous occasions it is visible while copulation is not taking place. A male has been ob- served with aedaegus visible mounted atop the female for 46 minutes, and except for five brief periods of trial insertion no copulation occurred. On the sixth insertion copulation took place for six minutes and afterwards antennal contact occurred by the female with another male. 9. A self-induced separation, was observed after 48 minutes of copulation. The male then returned to mate. Ejaculation took place on the elytra of the female and this beetle was observed for several days. While this female was not observed continuously, the following unusual events occurred during a three-day observation period. The female was in the normal position with a male adult for five minutes. The male then attempted a lateral copulation four suc- cessive times without success. This male afterwards copulated suc- cessfully with another female. On the second day the female approached a male who mounted, reversed his position twice, attempted to copulate laterally and failed. Soon afterward the female laid an egg. Later in the day the female was mounted twice without mating. The third day the female was observed to present the abdomen to a male who l4 walked toward but did not mate with this female. Two questions arise from this behavior: (1) Are attempted matings stimuli for egg laying? and (2) Did the failure to mate indicate a repellency or physical barrier due to the dry copulatory fluid? 10. Midway through the study one male was observed to extrude and retract the aedaegus repeatedly. Near the end of the observations, when only one female and three males survived, a male was observed to excrete a small white pearly drop of fluid while eating. Later in the study, with only two males surviving, one of the two adults was ob- served to extrude and retract his aedaegus 60 and 100 times in two successive days, respectively, while clinging transversely to the axis of the leaf edge. Movement was accompanied by a curving down of the abdominal tip and use of the hind-legs to rub the tip of the abdomen. Homosexual Behavior of the Males The author is convinced that homosexuality exists among the males of this species. The earliest manifestations of homosexuality or lack of sex differentiation was observed when one male briefly mounted a second that had just ended copulation. The second male had everted his aedaegus twice and was rubbing his posterior legs to- gether when mounted twice, briefly, by the other male. Numerous observations were recorded where mutual mounting be- tween males occurred. Characteristically, males exchange partners and roles frequently. The maximum observed period of homosexual play was 58 minutes. Activity was generally continued about 15 minutes and roles may be exchanged, or remounting may occur. The maximum and 15 minimum recorded time for the mounting of one male atop another was 38 and 4 minutes, reSpectively. Behavior appears to be similar to normal male-female relation- ships in that the homosexual males will exhibit the restless chasing and circling of the glass chimney cage prior to mounting. It is note- worthy that on one occasion a male who had demonstrated homosexual activity by mounting a male, mounted the above mentioned female with copulatory fluid on her elytra six minutes later. He performed a series of rotations with his body on the female while bringing his head to several peripheral sites on the body of the female, but no mating occurred. The occurrence of three homosexual males was observed most fre- quently. Generally a mating pair of males appeared to attract a third male. Apparently the third male signals his intent by mounting the mating pair. He may dislodge the principal male and assume the mating position himself or dismount. In either case, the non-mating male circles the cage and mounts the pair at the end of one cycle. This circling and mounting continues until mating ceases at which time the secondary male may mount either of the mating males, but apparently preferring the pseudo-female. A change of roles often occurs afterward. Since the sex ratio was abnormal near the end of the observa- tions, with a high proportion of males to females in the cage, it was felt that a study of the effect of sex ratios on male homosexuality should be conducted. However, due to time limitations, only a situa- tion involving a complete absence of females was observed. 16 The excited behavior of the males was verified by this study. Behavior and mounting occurred as previously described. Furthermore, it was determined that the aedaegus is inserted in the anus of the pseudo-female for at least a short time (no longer than two minutes in the case observed). When withdrawn, the proctodaeum was protruded and appeared as a sclerotized slender tapering cylinder. Several males were observed both copulating and attempting to do so but in no case was there a droplet of copulatory fluid as observed in bisexual copula- tion. As many as four males were observed to be mounted atop a single individual, three facing in the opposite direction to the other two. Since this study was conducted under a 24-hour photoperiod it is dif- ficult to state affirmatively that homosexuality of males may also occur under a l6-hour photoperiod, although some affirmative evidence is available. Adults of both sexes removed from a l6-hour photoperiod and placed in a 24-hour photoperiod were grouped by sex and closely observed for 9 days. At the end of 30 hours short periods of mounting were observed among the males. At the end of three days one pair was observed mounting for 35 minutes and on the fourth day actual copula- tion occurred. Observations made thrice daily for nine days gave no indication of homosexuality in a lot of five egg-laying females. Oviposition Behavior Oviposition began eight days after removal of the adults from storage at 43° F, and continued for 36 days. In the egg-laying posi- tion the female orientates herself longitudinally to the leaf and parallel to the veins of the monocotyledonous leaf, generally l7 straddling the midvein. Her head may be either up or down the leaf. Normally she attempts to place the venter of the body on the surface of the leaf while the legs are somewhat extended. With very rare ex- ceptions, the eggs are deposited with their sides paralleling the veins of the leaf. Before oviposition the female protrudes and retracts the egg several times. On six different dates during the observation period the elapsed time between assumption of the egg-laying position and oviposition was: 3, 2, l, 2, 3, and 3 minutes. In 24 observed cases 18 ovipositions consisted of one egg and in six cases two eggs were laid. In the cases where two eggs were laid the female was observed to move slightly up or down the leaf to deposit the second egg. The case of three or more eggs laid in sequence by the same female was not observed. However, instances were observed where eggs were deposited between two previously laid eggs or adjacent to previously laid eggs giving the appearance of a perfect column of three eggs. While these circumstances could contribute to mis-interpretation as to the number of eggs deposited at one time, differences in colora- tion due to development of the egg make identification possible pro- vided that the eggs are not laid at approximately the same time. Hence, a continuity of eggs does not indicate that they were laid simultaneously by the same female. Two cases were observed in which the same female laid two eggs after a single copulation. In both cases the second egg was deposited on a leaf different from the first. The time lapse between oviposition of the first and second egg was 8 and 9 minutes, respectively. In another case, a second oviposition was observed 15 minutes after the 18 first with a period of copulation between. Twelve observations were made during the study to determine the elapsed time between the end of copulation and oviposition. The times recorded were: 4, 5, 7, 7, 8, 8, 10, ll, ll, 17, 50, and 66 minutes, with an average of 17 minutes. These data indicate that about 80% of egg laying occurs 4 to 17 minutes after copulation. On three occasions copulation was observed 15, 92 and 17 minutes after laying thus, a short cycle of copulation-oviposition-copulation apparently occurs. Observations were made to determine the number of eggs laid singly or in columns of two or more, the total number of eggs on the upper and lower surfaces of the leaves, and the occurrence of ovi- position a single or two adjacent eggs on the upper or lower surfaces of the leaves. The data were recorded when fresh plants were pro- vided for adult feeding. The results are presented in Tables 3 and 4. In terms of mean percentages, 77.7% of the eggs were laid on the upper surface of the leaves and 22.3% on the under surface. Eighty-three percent of the eggs laid in columns of two were laid on the upper surface of the leaves. The grouping of eggs is recorded as observed. Groups of two are assumed to have been laid simultaneously and groups of 3 or 4 are assumed to have been formed by later ovipositions. Eggs laid in groups of two occurred in 20.7% of the ovipositions, and 79.3% occurred singly. These data corroborate the actual observation of the 24 ovipositions cited previously. Noteworthy are the following miscellaneous observations: 1. On two occasions, one egg was found constricted, thus appearing to possess a waist. 19 TABLE 3.--Grouping of eggs of the cereal leaf beetles on the upper and lower surfaces of the leaves of "Hudson" barley seedlings No. of Single No. of Groups E883 of Two Eggs Upper Lower Upper Lower Date Surface Surface Surface Surface January 17, 1965 48 l8 l3 6 18 78 7 37 5 19 91 8 3O 1 20 49 9 11 0 21 48 10 21 l 22 42 29 14 7 24 79 19 26 0 26 62 26 26 5 28 47 21 14 10 30 49 24 7 5 31 17 9 3 1 February 2, 1965 10 2 3 0 4 3 2 0 O 6 3 l 0 O 20 .wwo mech a mo mmz GOHuHmoaH>o map 0655me mm3 uH ..EE N Sana whoa was wwwm cmmsumn GOHumumamm onu me 0.0 0.0 0.0 0.00H q 0 0.0 0.0 0.0 0.00H q a 0.0 o.o 0.8 0.8 m N 3338 0.0 0.0 m.mH n.00 m Hm 0.0 0.0 H.¢H a.mm 0 on 0.0 H.H w.mN H.mn w wN 0.0 N.H 0.mN n.Nm m 0N 0.0 0.0 0.HN 0.0m m 0N 0.0 H.N q.NN m.mn HH NN 0.0 ¢.N m.0N p.05 NH HN 0.0 m.N m.mH m.wm NH 0N 0.0 0.m H.MN a.mm NH 0H 0.0 o.H 0.Nm w.N0 NH wH H4 m.~ SAN 0.? .2 $3 .2 Dances noom mo mmsouo moufiH mo masonu mwmm 038 mmwwm mUH50< mama CH vcsom GH mason Ho museum CH mech mo wwwm ucmoumm wwwm unmoumm coHuHmomH>0 unmouom ucooumm umnssz mwaHHpmmm %mHumn :comvdm: mo mm>mmH co mmHummn mmmH Hmmuoo mcu mo wwwm mo wchdouuuL.¢ MHm¢H 21 2. One egg was deposited while the female was situated obliquely to the longitudinal axis of the leaf. Surprisingly, the egg was placed normally on the leaf. Throughout the observation period all eggs were deposited on the leaves with exception of two instances when eggs were deposited on the glass cage. Influence of Multiple Mating on Egg Production and Fertilization Only 23.8% of the females isolated from the males after copula- tion laid eggs, while 50% of the females that were caged with a male laid eggs. The isolated pairs averaged 11 matings in an average period of 24 days. The average number of eggs per ovipositing female was 138.0 for the isolated females and 97.2 for the females that were caged with a male. The percent eclosion was 62.3 for the eggs laid by the females that were caged with males, and only 45.2 for those laid by the isolated females. Both the number of females that laid eggs, and the number of eggs laid per female were less than observed in the mass rearing pro- gram for the beetle. It has been observed that egg production is reduced by frequent handling of the reproductive adults. This factor may account for the reduction of egg production in this test. The present test indicates that multiple mating results in a higher fertility of the eggs than do single matings of the cereal leaf beetle. The results of this test are summarized in Table 5. 22 TABLE 5.--Number and fertility of eggs produced by female cereal leaf beetles following single and multiple matings Percent Mean No. Percent Number Females of Eggs per Fertile of Matings Ovipositing Ovipositing Female Eggs Single 23.8 138.0 45.2 Multiple 50.0 97.2 62.3 DISCUSSION As in most preliminary research projects new avenues of explora- tion have unfolded, each of which must be further examined to fully understand the behavior of this insect. While only a few definitive conclusions can be stated at the present time the author feels that a systematic observation of cereal leaf beetle behavior will disclose facts which will assist in understanding the species. The origin of the abundant fluid which is usually secreted at mating deserves to be determined. It appears to be seminal fluid, but its origin may be different since there is some evidence that the seminal fluid is a white pearly liquid which is not nearly as abundant as the fluid observed after mating. If the seminal fluid is confirmed to be a white pearly liquid it suggests that the possible source of the mating fluid may be the female and may be a consequence of the voracious feeding of the female at mating. The mating fluid, if secreted by the female may be attractive to males but this is undetermined. In only one case was a male de- tected being attracted by the mating fluid. Thus, present evidence would tend to indicate it is not an attractant and only secreted by the female during copulation. Since males were observed to insert the aedaegus into the anus of other males the data of Table 2 may be inconclusive for normal bi- sexual copulation because one couple classified as male and female 23 24 could have been two males. The lower percentage of ovipositing females found in the isolated females and couples of male and female could also be at least partially a consequence of improper sex identification due to the homosexualism between males. A sure means of determining sex in living adults must be found as bisexual copulation can be verified only if the female is identifiable. At present the living female is identified on the basis of oviposition which would be inexact in the case of sterile females. Whether the homosexual behavior of the male is due to the lack of sex differentiation, the imbalance of sexes, or even a physiological consequence, theoretically this behavior could be of practical value in controlling the pest. If an accurate means of sexing can be devised and if it can be proven that the males that behave as females continue this behavior, then the appropriate release of these males would re- duce the probability of fertilization of the females. The success of this theoretical possibility would be enhanced if fertilization occurs in multiple matings. LITERATURE CITED Castro, T. R. 1964. Natural History of the Cereal Leaf Beetle Oulema melanopus (L.) and its behavior Under Controlled Environmental Conditions. Ph.D. Thesis, Michigan State University. Castro, T. R., Ruppel, R. F., and Gomulinski, M. S. History of the Cereal Leaf Beetle in Michigan. Expt. Sta. Quart. Bul. 47:623-653. 1965. Natural Mich. Agric. Metcalf, C. L. and Flint, W. P. 1962. Destructive and Useful Insects. McGraw Hill, N.Y. 1071 pp. Ruppel, R. F., Gomulinski, M. S., Cobb, D. L., Yun, Y. M., and Castro, T. R. 1964. Cereal Leaf Beetle. 47:259-270. Test of Insecticides to Control the Mich. Agric. Expt. Sta. Quart. Bul. Yun, Y. Mok. Unpublished data of Department of Entomology, Michigan State University. 25 Is I p ‘ u “can ‘33; 1|‘5M11511; E Ural VERS' Yi'BHL'k F '11 1111 11: 11111 1‘ 93 03146 3643