“WV- v. THE BIONOMICS 0F DIAPARSIS N. SP. (Hymenoptera: ‘ Ichneumonidae) A LARVAL PARASITOID OF THE CEREAL LEAF BEETLE, OULEMA MELANOPUS (L) (Coleoptera: ChrysomeI-idae) Dissertation, for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY DAVID I. MILLER 1977 LIBRARXa Michigan Stat: University “1. This is to certify that the thesis entitled THE BIONOMICS OF DIAPARSIS N.SP. (Hymen0ptera: Ichneumonidae) A LARVAL PARASITOID OF THE CEREAL LEAF BEETLE, OULEMA MELANOPUS (L.) (Coleoptera: Chrysomelidae) presented by David J. Miller has been accepted towards fulfillment of the requirements for PH . D . degree in ENTOMOLOGY flatten Major professor DateMlfllZ— 07639 ABSTRACT THE BIONOMICS OF DIAPARSIS N.SP. (Hymenoptera: Ichneumonidae) A LARVAL PARASITOID OF THE CEREAL LEAF BEETLE, OULEMA MELANOPUS (L.) (Coleoptera: Chrysomelidae) BY David J. Miller Laboratory and/or field investigations of the biology and ecology of Diaparsis n.sp. (newly established in the U.S.) and Diaparsis carinifer (Thomson) (not established in the U.S.), parasitoids of the cereal leaf beetle, were carried out in Michigan and Yugoslavia from 1973-1975. Distinct differences in oviposition behavior between the two species were found. Longevity, fecundity, development, and identification characters of the immature parasitoids were determined. Field investigations in Michigan showed overwinter survival of Diaparsis n.sp. to be good. Emergence began in late May and continued for two to three weeks. Synchrony with the host was relatively good and parasitism rates were as high as 40%. Although some multiparasitism occurred, Diaparsis n.sp. appeared to mesh well between generations of Tetrastichus julis (Walker), a eulophid parasitoid of the cereal leaf beetle. Some indication was found that the presence of wild flowers as a source of food for adult parasitoids may be important in maximizing parasitization. David J. Miller Field observations in Europe and the U.S. showed behavioral differences between European and U.S. populations of the cereal leaf beetle. The possible significance of this difference is discussed. A number of possible strategies for management of Diaparsis n.sp. in an agricultural situation are suggested and discussed. THE BIONOMICS OF DIAPARSIS N.SP. (Hymenoptera: Ichneumonidae) A LARVAL PARASITOID OF THE CEREAL LEAF BEETLE, OULEMA MELANOPUS (L.) (Coleoptera: Chrysomelidae) BY David J. Miller A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Entomology 1977 ACKNOWLEDGMENTS I would like to thank Dr. James Bath, Chairman, Department of Entomology and the U.S.D.A. for providing facilities and funds for this research project. Also, I would like to thank Thomas Burger and his staff at the U.S.D.A. CLB Parasite Rearing Laboratory at Niles, Michigan for providing laboratory and field space for the field research. In addition the excellent help of Gary Montgomery and Nancy Fisher in the field and laboratory research respectively was greatly appreciated. Thanks is also due to my Guidance Committee, Drs. Dean L. Haynes, Richard W. Merritt, Brian A. Croft, and Milo B. Tesar for their valuable suggestions and criticisms in reviewing this manuscript. Much help was also received from Mr. Kenneth Dimoff of the Entomology Department in proper data analysis for which I am very grateful. I would like to particularly thank Dr. Frederick W. Stehr, my advisor, for his suggestions, encouragement, and patience in the lengthy task of conducting research and developing it into an acceptable thesis. Typist Marilyn Feikema also deserves thanks for typing the rough drafts and special thanks goes to my wife and ii family Marian, Gina, Tim, and Joie without whose encouragement this thesis may never have been completed. iii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . . . INTRODUCTION . . . . . . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . . . . S T UD Y AREA 0 0 O O O O O O I O O O O O O O O TAXONOMY O O O O O O O O O O O O O O O O O O LABORATORY BIOLOGY . . . . . . . . . . . . . Oviposition Behavior . . . . . . . . . Prediapause Development . . . . . . . . Post-diapause Development . . .‘. . . . Longevity and Fecundity . . . . . . . Mating Behavior . . . . . . . . . . . . Identification of Immature Parasitoids FIELD BIOLOGY . . . . . . . . . . . . . . . Emergence . . . . . . . . . . . . . . . Parasitism Rates . . . . . . . . . . . Synchrony . . . . . . . . . . . . . . . Encapsulation, Superparasitism, and Multiparasitism . . . . . . . . . . . Adult Parasitoid Density . . . . . . . Overwinter Mortality . . . . . . . . . Flight Activity . . . . . . . . . . . . Adult Parasitoid Food . . . . . . . . . Interaction with Tetrastichus julis . . EUROPEAN WORK . . . . . . . . . . . . . . . Species Composition . . . . . . . . . . Flight Activity . . . . . . . . . . . . Emergence . . . . . . . . . . . . . . . Adult Density . . . . . . . . . . . . . Field Observation . . . . . . . . . . . Host Behavior . . . . . . . . . . . . . iv vi viii 11 18 18 26 33 39 45 46 49 49 70 81 81 96 101 104 113 119 124 125 126 130 132 134 138 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . 141 LITERATURE CITED . . . . . . . . . . . . . . . . . . . 150 APPENDICES . . . . . . . . . . . . . . . . . . . . . . 155 Table l. 10. 11. LIST OF TABLES Recent publications dealing with the cereal leaf beetle, Oulema melanopus . . . . . . Location of oviposition in Oulema melanopus larvae by Diaparsis n.sp. and Q. carinifer Time of oviposition in seconds of Diaparsis n.sp. and Q. carinifer from point of antennal contact until ovipositor withdrawal . . . . . . . . . . . . . . . . Fecundity of Diaparsis n.sp. and Q. carinifer determined from laboratory tests and dissections. . . . . . . . . . . . . . Emergence trap catch of Diaparsis n.sp. at Niles, Michigan (1973-1975) . . . . . . . Probit equations for cumulative percent Emergence of Diaparsis n.sp. plotted over D O O O O O O C O O I O O O O O O O O O 48 Emergence trap catch of Diaparsis n.sp. from tilled and untilled areas . . . . . . . . CLB larval densities, parasitized CLB larval densities, and Diaparsis n.sp. parasitism rates in three oat strips for three years at Niles, Michigan . . . . . . . . . . . . Summary of emergence of Diaparsis n.sp. at Niles, Michigan (1973-1975) . . . . . . . Cumulative percentages of cereal leaf beetle larvae and parasitized cereal leaf beetle larvae at Niles, Michigan (1973-1974) . . Cumulative percentages of cereal leaf beetle larvae and parasitized cereal leaf beetle larvae at Niles, Michigan (1975) . . . . . vi Page 23 24 44 55 57 67 76 77 82 83 Table Page 12. Encapsulation of Diaparsis n.sp. eggs and larvae occurring singly in the host . . . . . 85 l3. Encapsulation of smooth vs. knobbed Diaparsis n.sp. eggs occurring singly in the host . . . . . . . . . . . . . . . . . . . 86 l4. Encapsulation of Diaparsis n.sp. eggs and larvae with two parasitoids present . . . . . 87 15. Effect of superparasitism and multiparasitism on encapsulation rate of Diaparsis n.sp. . . . 89 16. Combinations of superparasitism and encapsulation of Diaparsis n.sp. (2 parasitoids only) . . . . . . . . . . . . . 92 l7. Multiparasitism of CLB by Diaparsis n.sp. and Tetrastichus julis and Lemophagus curtus at Niles, Michigan . . . . . . . . . . . . . . 93 18. Seasonal trends of multi- and superparasitism of CLB larvae by Diaparsis n.sp. at Niles, Michigan (1975) . . . . . . . . . . . . . . . 94 19. Density of adult Diaparsis n.sp. at Niles, Michigan determined with a DVac sampling machine . . . . . . . . . . . . . . . . . . . 98 20. Survival of Diaparsis n.sp. from cocoon formation to spring emergence at Niles, Michigan (1974) . . . . . . . . . . . . . . . 102 21. Flowers reported by Bjegovic as food plants for adult Diaparsis spp. (Listed in order of preference) . . . . . . . . . . . . . . . . 115 22. Flowers found at Niles, Michigan to serve as food plants for adult Diaparsis n.sp. . . . . 116 23. Numbers of adult Diaparsis spp. caught in sweepnet samples in cats at Zemun, Yugoslavia . . . . . . . . . . . . . . . . . . 133 24. Observations of searching and attack by Diaparsis spp. in the field at Zemun, Yugoslavia . . . . . . . . . . . . . . . . . . 135 25. CLB larval densities in oats and wheat at Zemun, Yugoslavia . . . . . . . . . . . . . . 139 vii Figure 1. 2a. 2b. 10. LIST OF FIGURES Diagram of the Niles insectary showing crop locations and topography . . . . . . . Scanning electron microscope photographs showing the difference in the occipital rim between Diaparsis n.3p. and Q. carinifer . . . . . . . . . . . . . . . Scanning electron microscope photographs showing the difference in the texture of the temples between Diaparsis n.sp. and Q. carinifer . . . . . . . . . . . . . . . Terminal abdominal segments and ovipositors of Diaparsis n.sp. and Q. carinifer (Note curvature of n.sp.) . . . . . . . . . . . . Duration and frequency of prediapause life stages of Diaparsis n.sp. under laboratory conditions 0 O O O O O O O O O O O O O O O Drawings showing relative size of early and late first instar Diaparsis n.sp. larvae . Diaparsis n.sp. cocoons . . . . . . . . . . . Post-diapause development of Diaparsis n.sp. All three specimens were out of cold temperatures for 14 days . . . . . . . . . Duration and frequency of several post- diapause development stages of Diaparsis n.sp. under laboratory conditions . . . . . Terminal segments of prepupal male and female larvae of Diaparsis n.sp. showing primordia of external sex characters . . . Chamber used for longevity and fecundity tests . . . . . . . . . . . . . . . . . . . viii Page 10 15 16 17 27 3O 31 35 36 38 40 Figure Page 11. Frequency distribution of longevity of Diaparsis n.sp. and D. carinifer under laboratory conditions . . . . . . . . . . . . 42 12. Drawings showing difference between mandibles of first instar larvae of Diaparsis n.sp. and 2. carinifer . . . . . . 48 13. Photographs showing (a) emergence trap set up in stubble field and (b) part of catch in the container at the top of the trap . . . 50 14. Diagram of Niles insectary showing layout of crops and location of emergence traps (1973-1975) x=emergence trap . . . . . . . . 51 15. Variance of degree days accumulated at various degree day bases at initial emergence date of Diaparsis n.sp. at ‘ Niles, Michigan (1973-1975) . . . . . . . . . 54 l6. Probit curves of emergence of male and female Diaparsis n.sp. at Niles, Michigan (1973) . . . . . . . . . . . . . . . 58 l7. Probit curves of emergence of male and female Diaparsis n.sp. at Niles, MiChigan (1974) o o o o o o o o o o o o o o o 59 18. Probit curves of emergence of male and female Diaparsis n.sp. at Niles, Michigan (1975) . . . . . . . . . . . . . . . 6O l9. Probit curves of emergence of Diaparsis n.sp. at Niles, Michigan (1973) . . . . . . . 6l 20. Probit curves of emergence of Diaparsis n.sp. at Niles, Michigan (1974) . . . . . . . 62 21. Probit curves of emergence of Diaparsis n.sp. at Niles, Michigan (1975) . . . . . . . 63 22. Probit curves of emergence of Diaparsis n.sp. at Niles, Michigan. Comparison 1973, 1974, and 1975 . . . . . . . . . . . . 64 23. Emergence curves of Diaparsis n.sp. from spring planted oats and from untilled stubble (1974-1975) . . . . . . . . . . . . . 68 ix Figure Page 24. Total CLB larval incidence and parasitized larval incidence for Oats-1 at Niles, Michigan (1973-1975) . . . . . . . . . . . . 72 25. Total CLB larval incidence and parasitized larval incidence for Oats-2 at Niles, Michigan (1973-1975) . . . . . . . . . . . . 73 26. Total CLB larval incidence and parasitized larval incidence for Oats-3 at Niles, Michigan (1973-1975) . . . . . . . . . . . . 74 27. Diagram of the sampling scheme for parasitism by Diaparsis n.sp. in Oats-2, 1975. Numbers 1-12 represent the sampling sites (ca. 20 yds. apart). Field pepper- grass was planted around sites four and ten . . . . . . . . . . . . . . . . . . . . . 78 28. Mean parasitism rates in Oats-2 at each sample site and results of analysis with Duncan's multiple range test. Points with the same letters are statistically similar . . . . . . . . . . . . . . . . . . . 80 29. Percent superparasitism of CLB larvae by Diaparsis n.sp. and percent multi- parasitism by Diaparsis n.sp. and T. julis or L. curtus at Niles, Michigan (1973-1975) . . . . . . . . . . . . . . . . . 90 30. Seasonal trend of superparasitism of CLB larvae by Diaparsis n.sp. and multi- parasitism by Diaparsis n.sp. and T. julis or L. curtus (1975) . . . . . . . . . . . . . 95 31. Type of malaise trap used to monitor flight activity of Diaparsis n.sp. . . . . . . . . . 105 32. Diagram of the Niles insectary showing location of malaise traps and crops in 1974 and 1975 . . . . . . . . . . . . . . . . 106 33. Cumulative percent malaise trap catch of Diaparsis n.sp. plotted over 0D48 (1974) . . 107 34. Cumulative percent malaise trap catch of Diaparsis n.sp. plotted over 0D48 (1975) . . 108 35. Total daily malaise trap catches of Diaparsis n.sp. over 0D48 (1974) . . . . . . 111 X Figure Page 36. Total daily malaise trap catches of Diaparsis n.sp. over 0D48 (1975) . . . . . . 112 37. Comparison of emergence curves of Diaparsis n.sp. and T. julis. Adapted from Gage, 1974 . . . . . . . . . . . . . . . . . . . . 120 38. Diagram of the study area in Yugoslavia and the location of the malaise traps . . . . . . 128 39. Regression line and equation with .95 confidence interval showing relation of percent males to total catch of Diaparsis n.sp. in malaise traps in Yugoslavia . . . . 129 40. Photographs showing Diaparsis n.sp. attacking an early instar larva (a) and the same larva left sticking on its back on the leaf (b) 137 xi INTRODUCTION The cereal leaf beetle, Oulema melanopus (L.), (Coleoptera: Chrysomelidae) is a native of Europe and Asia, ranging from Scandinavia to northern Africa and from the Atlantic coast and England to central Asia (Dysart, et. al., 1973). It is apparently an accidental introduction to the U.S. and was first identified in Berrien Co., Michigan in. 1962 (Castro, et. al., 1965). Over most of its native range it is not of pest status or only sporadically a pest with the exception of the Balkan states where it is more frequently economically damaging. However, farmers in the U.S. were Spraying for control of this pest as early as 1959 (Castro, et. al., 1965). In 1963 a biological control program was begun by the U.S. Department of Agriculture with the initiation of field surveys in Europe. These were conducted by the European Parasite Laboratory to determine the presence and abundance of the CLB (=Cerea1 Leaf Beetle) and its natural enemies in Europe. Contracts were also made with Michigan State University and Purdue University to study the pest in the U.S., and two PL-480 projects were funded in Europe, one in Poland and one in Yugoslavia. In 1966 the Cereal Leaf Beetle Parasite Rearing Station was established at Niles, Michigan for rearing and dissemination of CLB parasitoids in the U.S. (Dysart, et. al., 1973). The result of the European work was the introduction of five parasitoids of the CLB into the U.S. One of these, Anaphes flavipes (Foerster), was a mymarid egg parasitoid, and the other four were larval parasitoids, Lemophagus curtus Townes, Tetrastichus julis (Walker), Diaparsis carinifer (Thomson), and Diaparsis n.sp. They were first released in southern Michigan and northern Indiana (Annon, 1972) and except for Diaparsis carinifer, all have been recorded as established AStehr, (1970), Maltby, et. al., (1971), Stehr and Haynes, (1972), and Stehr, et. al., (1973)]. Little other than host and general life cycle was known about any of these parasitoids prior to their establishment. Since then, Gage (1974) has studied the biology and population dynamics of T. igTTg which was the first larval parasitoid to build up a considerable population. Diaparsis n.sp. populations subsequently began to increase also, which resulted in the decision to more thoroughly study the biology and bionomics of this parasitoid. This research was part of a much larger study of the cereal leaf beetle, its dynamics, its behavior, and its parasitoids. The overall objective of the total research effort was to develop a management program for this pest. The objective of this research was twofold; l). to elucidate characteristics of the biology and bionomics of Diaparsis spp., 2). to provide data useful to the CLB management program. The European work and much of the U.S. work was * supported by two cooperative agreements. Ent. Res. Div., Agr. Res. Serv., USDA cooperative agreements 12-14—100-10, 905 (33) and 12-14-1001—23. LITERATURE REVIEW Various authors (Hodson, 1929, Hilterhaus, 1965, Venturi, 1942) have described the biology and behavior of Oulema melanopus in Europe. The natural history in Michigan has been described by Castro, et. al., (1965). Since the cereal leaf beetle has become a serious pest of small grains in the U.S., considerable study has been directed toward elucidation of its behavior, biology, and population dynamics. Wellso, et. al., (1970) have published a major bibliography on the CLB. Since then a number of additional papers dealing with various aspects of CLB behavior, biology, and dynamics have been published. They are listed in Table 1. To simplify understanding the parasitoid behavior and relationship to the CLB, a review of the life cycle follows. The CLB is univoltine and the overwintering stage is the adult beetle. These emerge in the spring with peak abundance from mid-April to mid-May. Early populations are in wheat, but oats is attacked as soon as it is available. Immature stages consist of egg, four larval instars, prepupa and pupa. Pupation occurs in the soil and the adults emerge from mid-June to mid-July. Table 1. Recent publications dealing with the cereal leaf beetle, Oulema melan0pus. Topic Author Age specific mortality Helgesen and Haynes, 1972 Behavior and survival Casagrande, 1975 Host resistance Gallun, et. al., 1973 Host resistance Webster, et. al., 1973 Host resistance Casagrande and Haynes, 1976 Interactions with the host Gage, 1972 Interactions with the host Jackman, 1976 Laboratory oviposition studies Wellso, et. al., 1973 & 1975 Laboratory oviposition studies Wellso and Cress, 1973 Laboratory oviposition studies Wellso, 1976 Parasitoid relations Gage, 1974 Population management Haynes, 1973 Population management Tummala, et. al., 1975 Population monitoring Ruesink and Haynes, 1973 Population monitoring Fulton, 1975 Diaparsis Spp. are mentioned only briefly in the literature by European authors who were investigating the cereal leaf beetle (Venturi 1942, Hilterhaus 1965). Venturi (1942) reared a parasitoid he called Thersilochus moderator (L.) from Q. melanopus in Italy and gave a brief description of the life cycle and host preference. Knechtel and Manolache (1936) also reared a Thersilochus sp. from Q. melanopus in Rumania. Hilterhaus (1965) referred to a parasitoid reared from the same species by the name Thersilochus carinifer Thomson. A description of the occurance and activity of these parasitoids in EurOpe under the name 2. carinifer, was given by Dysart, et. a1. (1973). Also Bjegovié (1972, 1973) gave a similar report on these parasitoids in Yugoslavia, and Miczulski (1973) did the same for Poland. A recent publication referring to Diaparsis spp. (Montomery and DeWitt, 1975) deals with the taxonomic separation of the larvae of parasitoids attacking the CLB. Dysart, et a1. (1973) outlined the collection of CLB parasitoids in Europe and their subsequent shipment to the U.S. Releases have been made in Michigan, Indiana, Ohio, W. Virginia, Virginia, New York, and Pennsylvania. Through 1975, recoveries of Diaparsis n.sp. have been made in four counties in Michigan, three in Ohio, two in Indiana, and one each in W. Virginia, Pennsylvania, and New York. In several Michigan counties recoveries have been made at more than one site. The primary host of these two Diaparsis species is Oulema melanopus. However, in Poland Miczulski (1973) reported finding a single dead male Diaparsis carinifer in a pupal cell of Lema cyanella (L.). He also reported having two 2. carinifer emerge from host cells during the same season as they were collected. One of these emerged from g. melanopus and the other from Q. gallaeciana. At the time of European collection and subsequent release, it was not known that two species of Diaparsis were involved. Consequently all of these were released as Diaparsis carinifer and Stehr and Haynes (1972) reported the establishment of this species. Further work has shown that it was actually a new species, Diaparsis n.sp., which was established, and Q. carinifer has not been recovered as yet. STUDY AREA The research was primarily conducted in three locations from 1973-1975. The laboratory work was done at Michigan State University, and the field work was done near Niles, Michigan. Three months were spent in EurOpe in 1974, primarily in Yugoslavia, for the collection and rearing of parasitized CLB larvae in order to obtain material for laboratory work. Various other investigations including parasitoid flight activity. Parasitoid and host density, and observations on the behavior of parasitoid and host were also conducted. The work at Niles was done at the CLB field insectary in cooperation with the USDA APHIS CLB Parasite Rearing Station there. The field insectary is located west of Niles near Galien, Michigan and is managed for maximum production of CLB parasitoids. The management scheme is essentially the same as that outlined for the MSU, Gull Lake Research Station (Gage, 1974). The insectary is divided into halves, with one half tilled and planted to creps one year and the other used the following year. This leaves the crop stubble undisturbed for one full year to allow maximum parasitoid survival and emergence. In order to have an extended season for CLB oviposition and development, two crops are planted each year. The first is winter wheat which is planted in a single strip about five acres in size. The second crop is spring oats which is planted at three dates beginning as early as possible and continued at weekly intervals with each planting comprising about two acres. (Figure l). Hereafter the oat crOps will be refered to as Oats-l, Oats-2, or Oats-3 from the earliest to the latest respectively. Since it is believed that most emigration of parasitoids is along the north edge of the insectary, an additional strip of oats (trap crOp) is planted here to help retain the parasitoids in the insectary. The topography varies considerably with most of the area being rolling, well drained loamy soil. Approximately the southern one third, however, is flat and low lying with poorly drained muck soil. 10 .mnmmumomou can mGOwumooH mono mcw3osm humuommcfl mmaflz ecu mo Emummwa .H mucosa 88... «DO :3 .60 8%.. III """ é / I'll—.381!!! n «30 50:3 _ £00 N 500 n 3.5 no.0 a2... ~20 a2... oz TAXONOMY The parasitoids investigated were originally placed in the genus Thersilochus. However, in a revision of the European Tersilochinae, Horstman (1971) placed them in the genus Diaparsis. Initially the imported specimens were thought to all be 2. carinifer. Further examination by the USDA Systematics Entomology Laboratory in Washington, D. C., and consultation with Dr. Horstmann of the Institute far Angewandte Zoologie, Wfirzburg, Germany, revealed that two species of parasitoids were involved. One was Diaparsis carinifer (Thomson) and the other a new Species, referred to hereafter as Diaparsis n.sp. Also from these Specimens, R. W. Carlson was able to establish a tentative distribution of the two species in EurOpe. In a letter (1971) to Horstmann he indicated that the distribution of Diaparsis n.sp. appears to be limited to lower Austria, France and Yugoslavia. Q. carinifer, however, apparently occurs over all of Europe. Failure to recover Q. carinifer in the U.S. is noteworthy, particularly since it is probable that both species were released. According to reports from the USDA 11 12 (Anon, 1972) more than 60,000 Diaparsis spp. adults from Yugoslavia were released in the U.S. Since both species are known to occur in Yugoslavia, both species should have been included in the U.S. releases. Both species have typical ichneumonid, parasitoid life cycles consisting of egg, several instars, prepupa, pupa and adult. They are univoltine and overwinter as prepupae in cocoons within the CLB cells in the soil. The two species are nearly impossible to separate in the field except perhaps by oviposition behavior. In the laboratory, however, it is relatively easy to distinguish them since several characteristics are distinctly different. In an unpublished key for recognizing Diaparsis Spp. parasitic on Oulema melanopus, Carlson (1972) described the characters in the following couplet. "Lower portion of the occipital rim (the outer margin of the occiput, usually and inappropriately referred to as a carina) scarcely visible in lateral view. Temples polished and distinctly punctate, if somewhat shagreened in males, then still with a distinct luster. Abdominal tergites occasionally mostly furruginous, but often largely black (more so in males) Diaparsis n.sp." 13 "Lower portion of occipital rim easily visible in lateral view. Temples strongly shagreened, dull and only obscurely punctate. Abdominal tergites usually not extensively darkened except at the abdominal apex of some males. Diaparsis carinifer" In addition to the characters outlined above (Figures 2a and 2b) a consistent difference in the ovipositors was observed (Figure 3). The ovipositor of Diaparsis n.sp. is slender and definitely curved upward whereas 2. carinifer has a somewhat stouter ovipositor, slightly broader, and with considerably less curvature. This difference was found to be reliable for separation of live females of the two species in the laboratory. Since the ovipositor of Q. carinifer has less curvature, it projects slightly farther beyond the abdomen. Diaparsis n.sp. also tends to have more black coloration on the dorsal aspect of the abdomen than 2. carinifer. Since the laboratory work required identification of the parasitoids prior to their use, it was necessary to find a way to positively identify the live material. To do this the parasitoid and a small piece of cotton were placed in the lower half of a 5 cm plastic petri dish which was upside down on a note card. Since the parasitoid tended to walk upside down on the inside of the petri dish, it was carefully picked up and placed over the cotton. 14 This held the parasitoid tightly enough to allow examination of the key characters under a dissecting microscope. 15 Diaparsis carinifer 50X Figure 2a. Scanning electron microscope photographs showing the difference in the occipital rim between Diaparsis n.sp. and Q. carinifer. Figure 2b. 16 Diaparsis carinifer lOOX Scanning electron microscope photographs showing the difference in the texture of the temples between Diaparsis n.sp. and Diaparsis carinifer. 17 ““““‘-«\ .1 - ‘m‘fir‘“‘ Diaparsis n.sp. Diaparsis carinifer Figure 3. Terminal abdominal segments and ovipositors of Diaparsis n.sp. and 2. carinifer. (Note curvature of n.sp.) LABORATORY BIOLOGY Oviposition behavior Methods Oviposition behavior was observed in the laboratory by placing a single parasitoid in the lower half of a plastic 5 cm petri dish which was upside down on a note card under a dissecting microscope. Host larvae were introduced on leaves of greenhouse grown barley seedlings. A leaf with a larva on it was broken off and slipped under the edge of the petri dish. The larvae were of various sizes; however, mostly second and third instars were used and generally only one larva was presented at a time. To avoid disturbing the parasitoids a piece of heavy paper was taped around the dish to shield hand movements. Results and Discussion Observation of oviposition revealed a number of characteristics of Diaparsis spp. In the situation described above, the parasitoid appeared to walk randomly around the dish until it contacted the leaf. It then began to search along the leaf, tapping the surface constantly 18 19 with the antennae. If the leaf was contacted in the middle and the initial search for the host was in the wrong direction, the parasitoid turned and searched the entire length of the leaf until the host was located. If an area of host feeding was contacted, the parasitoid became more excited and investigated the area thoroughly with its antennae. It was found that the plant juices occuring where the leaf was broken off elicited a more intense searching behavior from the parasitoid. In several cases it elicited a brief oviposition response, with the ovipositor being exserted and a few probes being made. Stimulation to oviposit was received primarily from the host larval fecal coat. Contact with this caused the parasitoid to exsert the ovipositor and probe repeatedly for the host body even when the fecal coat was not on the larva. Several larvae with little or no fecal coat were presented to the parasitoids which were slow to accept them or rejected them altogether. In one case where Q. carinifer rejected such a host, some fecal material was added from another host and it was stung immediately. Most larvae were accepted immediately, regardless of size; however, some were refused by the parasitoid for no apparent reason. In one case six larvae were rejected by a Q. carinifer, but when they were cleaned of their fecal 20 coat and dabbed with fecal material from other larvae they were accepted immediately. Since Diaparsis spp. are solitary parasitoids, only one egg was normally deposited each time the ovipositor was inserted. In a few instances two eggs were deposited in the same puncture, and in several cases the eggs passed down the ovipositor simultaneously. Generally larvae were parasitized only once, but occasionally a female oviposited twice in one host. If the parasitoid left the leaf after oviposition, and subsequently relocated it, she did not search along it. In cases where two or more larvae were close together, normally only one was stung. Only the very tip of the ovipositor was inserted and this apparently stimulated release of the egg. Once this was accomplished, oviposition was completed regardless of circumstances unless the puncture was made for feeding purposes. During one oviposition attempt, a parasitoid accidentally placed its mesotarsus in the mandibles of the larva which immediately fastened on it. Following oviposition, the parasitoid struggled to free itself. In struggling it pushed with its ovipositor and accidentally punctured the host integument. Struggling ceased immediately and an egg was deposited. This occurred twice before she was able to free herself. Observation of oviposition led to the discovery that sometimes Diaparsis spp. feed on host larval fluid. This 21 was observed in Diaparsis n.sp. more frequently than in Q. carinifer. Parasitoids were observed to insert the ovipositor tip, generally into the abdomen, and wiggle it back and forth laterally until a drop of haemolymph oozed out. They then lowered their mouthparts to it and ingested for several minutes. This often occurred with one of the first larvae presented and was then followed by a period of oviposition. Several significant differences in oviposition behavior between the two species were noted. One of these was the location of oviposition. Diaparsis n.sp. nearly always oviposited in the cervical or gular region of the larva. This was generally accomplished by orienting parallel to the larva and probing along it with the ovipositor until the head capsule was located, whereupon oviposition took place immediately behind the head capsule. The role of the host head capsule became apparent when a parasitoid was observed probing normally along a host which had a head capsule and larval skin from the previous instar stuck to its side. The ovipositor happened to contact this and oviposition occurred immediately at this point. This was observed on two separate occasions. In contrast, 2. carinifer was much less specific about the location of oviposition. When the parasitoid located a larva it exserted the ovipositor and attacked rapidly with a violent, stabbing ovipositor motion. No particular location was selected for oviposition; it occurred where 22 the ovipositor first was inserted. Consequently it occurred most often in the abdominal region. In order to analyze this difference more closely, records were kept of the location of a number of ovipositions of each species. The host larvae were divided into three regions - cervical, thoracic, and abdominal. The results are shown in Table 2. The time required for oviposition also appeared to differ in the two species, so the ovipositions of a number of individuals of each species were timed with a stop watch to see how much difference existed. The timed period was from the time the parasitoid antennae contacted the host until the ovipositor was withdrawn (Table 3). The data resulting from this test were analyzed with a two-level nested analysis of variance for unequal sample sizes. (Sokal and Rohlf, 1969). This analysis tested for differences among wasps within a species as well as for a difference between the species. Since the wasps used did not all oviposit the same number of times it was necessary to use a test allowing for unequal sample sizes. 23 Table 2. Location of oviposition in Oulema melanopus larvae by Diaparsis n.sp. and Q. carinifer. Total Cervical Thoracic Abdominal Diaparsis n.sp. Number 185 155 25 5 Percent 84 14 2 Diaparsis carinifer Number 151 2 50 99 Percent l 33 66 «N mv SN mH 0N mN SH mH mH 0N mH mH SH mH 0H SH HN mH mH mN mH NN MH mH mH mm NN mN mN mN MN NN «N Hm mm 0N SH mH SH SH vH mH mH SH mm ON mN mm mH 0N mH SH vH mH mH mH SH mH mH mH ON NMQ‘lnkOl‘ m NH mH «H SH SH HH NH SH mH mH mH OH mH mH mH NH mH mH mH ON Sm mH NH mN H HmMHCHHmo mHmummoHa 24 NN HN Hm Nv HN mm mm mm mm mH mm mH om mm mN mH SH mm mN mH mN NS mH mN mH Hm SH Sm mN SH NN mH mm Sm mm Hm Nv Nm m «N om Hv HN mN oN m mm vm mm mm ow Sm Hm Nm mm ev Nm om Hm Hm S mN mN mN ¢m mN vm Hm 0N mN o ow mv av Sm Nv me ow mm vm mm ov em Sv mm mw vm mm vm Hm m ov Nm SN mm Hm Sm mm om NN om w Sm mN mv we SN om SN SN mN m mN mm mN ow Sm Nv SN vN om oN om om mN mN oN N mN Nm Hm mv om H .mm.c mHmummoHo m>HMH some you mEHB * muHmmnmm .Hm3ouonuH3 HouHmomH>o HHucs pomucoo Hoccmucm mo ucHom Eoum .mmm mHmHmQMHa mo mocoomm GH coHuHmomH>o mo mEHB .m OHQMB 25 The results of this analysis* indicate that a significant difference in oviposition time exists at the .01 level both within each species and between the species. The difference in ovipositor morphology between the two species (Figure 3) may be related to the variation in oviposition behavior discussed above, 2. carinifer being more adapted to thrusting quickly and Diaparsis n.sp. more adapted to careful selection of a particular location on the larva. Post oviposition behavior was the same in both species. When the ovipositor was withdrawn, the head was lowered toward the surface of the larva with the mandibles widely spread. It was held there briefly, sometimes just barely touching the fecal coat but usually slightly above it. Following this the parasitoid walked a short distance away and cleaned itself thoroughly. The host larva usually exhibited some reaction to the parasitoid. As soon as the antennae tapped the larva it raised its head from the leaf surface bending back considerably. This occurred more frequently in third and fourth larval instars than in first and second. Gage (1974) reported that the reaction of CLB larvae to approach Fr ANOVA Table Source of variation df SS MS F ** Among Species l 18,410.53 18,410.53 21.07** Among Wasps 14 6,484.87 463.21 3.92 Within Wasps 202 23,896.67 118.3 Total 217 48,792.07 26 by Tetrastichus julis is to raise the abdomen. This was never observed to occur with the contact of Diaparsis spp. In the field it was noted that this defense reaction resulted in the larva falling from the leaf surface. This occurred almost invariably in larger larvae - fourth instar and late third - and the result was avoidance of parasitization. Prediapause Development Methods To examine development of Diaparsis n.sp. from egg to prepupa, laboratory-parasitized host larvae were placed on barley seedlings grown in pots and held at room temperature. Dissections were then made at daily intervals and parasitoid eggs and larvae were preserved in 70% ETOH. Results and Discussion The results showed that development of prediapause Diaparsis n.sp. larvae was quite variable making it difficult to determine the duration of each developmental stage. Figure 4 shows an approximation of the frequency of several stages and their duration. Diaparsis spp. lay two types of eggs. One is a smooth, typical, ichneumonid egg and the other more common type has a lateral projection on one side which is referred to as a knob. Q. carinifer lays only knobbed eggs whereas Diaparsis n.sp. lays both types. In the latter species the 27 .mcoHuHccoo SHOHMHOQMH Moons .mm.c mHmnomMHo mo mommum omHH mmDMQMHcon mo mucosvoum can COHumuzn .v musmHm «Hon 8 n. o. m H H H H .I H _ H H H H H H H _ IH H H H _ meow o d 4 on m «u av 33.... I co. m... n.nv nu I, I on S nu 33%... .. co. N no N I on I oo. 28 knobs are at times only partially developed (Montgomery and DeWitt, 1975). The knob, which is flattened on tOp, apparently functions to attach the egg to host tissue. Most often this is the inside of the host integument, but occasionally it may attach to other tissues. The adhesive nature of this structure became evident in dissections of female parasitoids which had been preserved and dissected in alcohol. In dissection of eggs from the ovaries, it was found that lightly touching the flat side of the knob with the dissecting needle caused it to adhere firmly to the needle. The eggs hatch in from four to seven days with two changes becoming visible prior to hatching. The first change is the appearance of a highly visible, dense, very white spot in the middle of the egg, and the second change is the appearance of the larva within the egg. The larvae pass through several instars in their development. The first and last instars are recognizable by a sclerotized head capsule and well defined mouthparts, but the intermediate instars lack these making it very difficult to separate them. The changes in appearance are discussed by Montgomery and DeWitt (1975). One developmental characteristic of Diaparsis n.sp. was that it goes through only one instar before the host larva spins its cocoon. This became apparent when only 29 first instar parasitoid larvae were found in field dissections. Late first instars may reach considerable size before molting, whereas early first instars are relatively slender, the body being the same diameter as the head capsule (Figure 5). This characteristic, coupled with the fact that Diaparsis n.sp. females parasitize any instar host larva, seems to account for the variation in development of individual larvae. Since development is not completed until after the host spins its cocoon, an egg deposited in a first instar host obviously takes longer to develop to the prepupal stage than one which is deposited in a third or fourth instar. When the parasitoid reaches the prepupal stage, it exits from the host skin and spins its cocoon inside the host cocoon. This is a three-layered structure with the outer layer of fibrous brown silk, the middle layer a smooth, tough material, and the inner a thin, transparent membrane. A cream colored band encircles the middle of the center layer and is visible externally. One end of the outer layer has more extraneous silk and is fuzzy in appearance (Figure 6). Inside the cocoon the head of the larva is always oriented in this direction. The development of Q. carinifer is assumed to be the same as that of Diaparsis n.sp. The same study outlined above was also attempted with Q. carinifer, but only a few parasitoid larvae were obtained. Of 81 dissections of CLB 30 Early Late Figure 5. Drawings showing relative size of early and late first instar Diaparsis n.sp. larvae. 31 Figure 6. Diaparsis n.sp. cocoons. 32 larvae known to be stung by D. carinifer, only seven (8.6%) were found to contain parasitoids and five of these were encapsulated. The reason for the lack of parasitoids in hosts which were known to be stung by Q. carinifer is not clear but two hypotheses are suggested, both of which likely contribute to these findings. The first is that encapsulation may interfere with locating the parasitoids in dissections. As pointed out above, five of seven found were encapsulated which indicated that encapsulation was frequent. If the eggs were encapsulated while they are attached to the epidermis, it would have been easy to miss them in the dissections, particularly if they were melanized or reduced in size. Examination of cast host skins showed that some eggs were lost at the time of molting, remaining attached during the molting process. Whether or not this occurs depends on the stage of host development at the time of oviposition. If the egg is placed into the haemocoele of the host, as is normal, it would not be lost at molt since it would be inside the epidermis. However, if the host has secreted a new cuticle from which the old cuticle has separated, it may be that the ovipositor does not penetrate the epidermis. The result would be deposition of the egg between the old and new cuticle which is possible with either Diaparsis species since they normally insert only the very tip of the ovipositor. 33 The first point discussed above, encapsulation, probably is the main cause of unsuccessful parasitism by Q. carinifer and also probably accounts for the lack of establishment of this species in the U.S. Post-diapause Development Methods Post-diapause development of Diaparsis n.sp. was also studied in the laboratory using cocoons obtained by rearing field collected CLB larvae. Following emergence of adult beetles, the remaining cells were held at room temperature for 12 weeks and were then placed in 40°F for an additional 12 weeks. They were then held at room temperature for parasitoid development. During this entire time the cells were sprayed with water periodically to preventdehydration. Development was examined by dissection of cocoons at daily intervals after their removal from diapause temperatures. Results Development could essentially be divided into eight easily recognizable stages. These were 1) thoracic constriction, 2) appearance of eyespots, 3) voiding of the meconium, 4) pupation, 5) eyes dark, 6) thorax dark, 7) abdomen dark, and 8) adult emergence. These various stages grade into one another to some extent except for pupation and emergence which are more definite stages in development. 34 When the eyes first appear they are pale red, but they become progressively darker, and following pupation they become very deep red. The body of the parasitoid also goes through several color changes. The prepupal larvae are a grey color mottled with white from fat granules showing through the integument. As they near pupation they become cream colored. After pupation they remain this color until about the time the eyes darken. The rest of the body then becomes a semi-translucent white, which gradually darkens until it is the adult color. Figure 7 shows several of the stages of development. In addition to the development stages, Figure 7 also illustrates the variable development rate of Diaparsis n.sp. The three specimens shown were all subjected to the same conditions. Although the bulk of the population develops at the same rate, there is a great deal of individual variation. Records received from the Newark, Delaware USDA lab on emergence of European-collected Diaparsis n.sp. to be used in laboratory studies, also showed great variability. The earliest emergence was a male which emerged in 14 days and the latest was two females which emerged in 55 days. With such great variation, it is difficult to characterize a population and conclude that a given stage of development covers a definite period of time. Figure 8 shows an approximation of the duration and frequency of several of the stages discussed above. .mamo vH mom monoumuomsmu oHoo mo #50 ouo3 mowEHoomm owns» HHd .mm.c mHmummMHo mo undemoHo>mo mmommchIumom .S muomHm 36 .mcowuHocoo ShouMHOQMH noon: .mm.c mHmHmmmHo mo mommum ucosmon>oo mmDMQMHqumom Hmum>mm mo Socmsomum can coHumudo mace 0» mm ON a. o. n HHHHHHHH.HHHHHHHLHH4H_HHH_HH 332a IIIIIIIIIIIIIIII-I £2.»on T ildldl cozoaam 8:09.25 I—I-IqJII. .m whomwm O O 100 [mo—la .1 av .J - m .u nxu mw lin:u_mfl no me .IAun mw n Au 1 m 10 100 00. 37 In some of the dissections late in the development period, larvae were found which had apparently not undergone any development but were still viable. It has recently been found (Montgomery, pers. comm.) that some Diaparsis n.sp. go through two seasons of diapause prior to emergence. This apparently is the explanation for the lack of develOpment found in some of the dissections. For this reason the prepupal stage in Figure 8 is represented as open ended. It was also found that prepupal larvae can be sexed. This was reported by Wilson and Ridgeway (1975) for Campoletis sonorensis, an inchneumonid parasitoid of Heliothis virescens. In E. sonorensis the sex is apparent in the fourth instar, the female being distinguished by having several ventral ovoid depressions in segments 10, 11 and 12, and the male having only one in segment 12. However, in Diaparsis n.sp. sex differences cannot be distinguished prior to the prepupal stage at which time they appear in the terminal segments (Figure 9). The male structures are simply a pair of small round bodies. The female structures are rod-shaped, with four of them lying side-by-side, and with the anterior pair having the distal ends bent sharply laterally. These characters are most easily visible in preserved specimens and seem to be more 38 .mnouomumno xwm Hmcuouxo mo MHUHOEHHQ mcHsonm .mm.c mHmHMQMHo mo mm>HMH mHmEmm one ons Hmmsmmum mo mucmsomm HmcHEHoB mHmEom GHMZ .m musmHm 39 apparent in larvae preserved in FAA+ than in those preserved in 70% ethyl alcohol. Longevity and Fecundity Methods To examine longevity and fecundity a single female parasitoid and four host larvae on barley leaves were placed in small, covered, plastic dishes ca. 6 cm dia x 3 cm deep (Figure 10). A hole cut into the bottom of the dish and covered with fine mesh copper screen provided ventilation. A water drop was placed inside and honey was streaked on the screen. The dish was kept coverside down in a 16 hr. photoperiod. Larvae were changed every 24 hours except in several instances when this was not feasible and they were left for 48 hours. The larvae removed from the cages were either dissected immediately or preserved in FAA for later dissection. Since larval instars were estimated visually when they were placed in the cage, head capsules were measured at dissection to accurately determine the instars present. The number of eggs in each larva was counted, and the tests were continued until the parasitoids died. Both Diaparsis n.sp. and Q. carinifer were tested. +FAA is a larval preservative with the following composition: 50 parts H20, 47 parts 95% ETOH, 2 parts formalin, 1 part glacial acetic acid. 40 Figure 10. Chamber used for longevity and fecundity tests. 41 Results and Discussion Longevity Longevity of the parasitoids was calculated from the date of emergence until death. This was not the same as the number of days they were exposed to hosts since some parasitoids were held from one to several days before they were tested. The tests on 14 females of each species showed that the two species live nearly the same length of time in the lab. The figures for Diaparsis n.sp. longevity are i = 11.4 i 5.0 days, Range = 3-18 and for Q. carinifer i = 11.3 i 2.7 days, Range 5-15. Figure 11 shows a frequency distribution of this data. The field longevity of Diaparsis spp. may be longer. An approximation can be obtained by comparing the final emergence trap catch with the final malaise trap catch which will be discussed later. The last parasitoid caught in a malaise trap was 18 days after the last parasitoid emerged in an emergence trap. This, of course, can not be construed to be an accurate measure of longevity, however, it is probably considerably closer than the laboratory data. The physical constraints of a small unnatural environment and being handled daily may have affected the length of life in the laboratory. 42 .mcoHuHocoo SHODMHOQMH Hops: HoMHcHHmo .o ocm .mm.c meHMQMHo mo muH>wmcoH mo COHuanuumHo Socosoonm .HH mHDmHm no»... goo 0.20.9198 :o_mo S o n¢ n N _ I. IN I» I'm w n1 3:530 32303 a nu I, a .m. B. INW... I¢ dd... «.2365 43 Fecundity Fecundity of the two species was also obtained from these tests. Although the results obtained for 2. carinifer appeared to be reasonably accurate, those for Diaparsis n.sp. appeared much lower than they should have been. Consequently ten females of each species were dissected and the eggs in the ovaries were counted. These females had been fed water and honey but had not had access to hosts and only the apparently mature eggs in the ovaries were counted. The results are given in Table 4. In 2. carinifer the mean from the dissections compares favorably with that found in the test. The mean for Diaparsis n.sp., however, was much higher in the dissections than in the test and is likely much nearer to the true fecundity. The reason for the low numbers of eggs deposited by Diaparsis n.sp. in the lab test may be due to one or both of the following reasons. First, it may be more sensitive to the constraints of a small, unnatural environment which did not allow normal behavior. The second reason may be that it has a stronger defense against superparasitism than 2. carinifer. Any defense the latter may have against this apparently broke down completely since in the host dissections it was not uncommon to find more than 20 eggs in a single larva. One first instar contained 26 eggs and a third instar contained 31. In contrast, most parasitized 44 Table 4. Fecundity of Diaparsis n.sp. and Q. carinifer determined from laboratory tests and dissections. Q. carinifer Diaparsis n.sp. x eggs/female 131.1 29.9 Tests SD 76.8 25.3 ' Range 24-291 4-70 N l4 14 i eggs/female 143.8 185.0 Dissections SD 20.4 20.7 Range 119-186 131-202 N 10 10 45 larvae from the test with Diaparsis n.sp. contained only one or two eggs although one second instar contained 25. This, however, was an exception. MatingiBehavior Mating of Diaparsis spp. was observed a number of times in the laboratory. The specimens used for mating experiments were isolated prior to emergence so they were unmated prior to the observations. Both species were observed and no differences in behavior were noted. Apparently a pheromone is operative in mating of these parasitoids, but it did not appear to be a strong attractant. Males became excited only after contacting a female or walking across a spot where the female had been. If the female had left the area, the male continued to search but did not seem to be able to orient toward the female. When a male contacted a female he became obviously stimulated. Movement became more rapid, and the antennae were used to search for further contact with the female. The wings were held up over the back at an angle and buzzed in rapid, short bursts and the abdomen was lowered and curled forward. Coupling occured in a typical male-above position and lasted from 30-45 seconds. Once a male released the female he did not mate immediately again, but if other males were present, they often mated with the female immediately. 46 If the female was receptive, she mated readily; however, some were found to be unreceptive. These struggled and usually succeeded in dislodging the male by kicking with the hind legs. Often the females struggled initially but then submitted to mating. Since two closely related species were being studied, a number of attempts to cross mate them were made. Diaparsis n.sp. males and Q. carinifer females mated readily, but it is not known if these matings were successful since progeny were not obtained due to encapsulation which has been discussed earlier. The reciprocal cross, Diaparsis n.sp. females with 2. carinifer males, did not take place. The males attempted to mate repeatedly, but the females would not submit. The females kicked with their back legs and also exserted the ovipositor to prevent coupling by the males. This latter behavior was never observed except in this cross mating attempt. The same females, when placed with Diaparsis n.Sp. males, mated immediately. Identification of Immature Parasitoids One of the objectives of this research was to find a way to distinguish between the two species of immature parasitoids in order to be able to identify the species present in field dissections. Since both Diaparsis n.sp. and Q. carinifer remain as first instars until the host 47 spins its cocoon, this is the most important stage to differentiate. Therefore, first larval instars preserved in 70% alcohol, were cleared in 10% KOH for approximately one hour and then rinsed in distilled water and mounted in glycerin on micrOSCOpe slides for examination. A definitive character for their separation has not yet been found, however, it appears that the mandibles may supply this character. To examine the mandibles it is necessary to place pressure on the coverslip to help orient the mandibles in the proper plane, since they project somewhat ventrally from the head of the larva. The two types of mandibles observed in this study are shown in Figure 12. Since only five positively identified 2. carinifer were available, the conclusions presented here are only tentative. The mandibles of both species are sickle-shaped, and have a similar general appearance. However, one significant difference is the curvature on the inner aspect of the mandible. In Diaparsis n.sp. this is a sharp curve which forms a nearly parallel sided U. In 2. carinifer the inner curve is much more open with the sides divergent. 48 Diaparsis n.sp. Diaparsis carinifer Figure 12. Drawings showing difference between mandibles of first instar larvae of Diaparsis n.sp. and Q. carinifer. FIELD BIOLOGY Emergence Methods Emergence of overwintering Diaparsis n.sp. was monitored by the use of emergence traps (Gage, 1974) covering one yd2 at the base. (Figure 13). The bottom of the container at the t0p of the trap was filled with ethylene glycol which killed the trapped parasitoids and preserved them for removal and examination. The traps were set up at a density of three per crOp strip except in 1975 when 15 traps were placed in second oats and seven in each of the other strips (Figure 14). The 15 traps in second oats were placed adjacent to the k yd2 plots where samples for the overwinter study had been taken. Thus the same traps served for evaluating normal spring emergence and overwinter mortality. The extra four traps in each of the other strips were set up as a check on the trapping of previous years. However, no difference in the emergence curves for the three years was found which could be attributed to better data obtained from a greater trap density. 49 (b) Figure 13. Photographs showing (a) emergence trap set up in stubble field and (b) part of catch in the container at the top of the trap. 51 Stu bblo Gate 3 Gate 2 Date I Wheat Oote Stubble Stubble Date Date 2 Date I Wheat Stubble Date Date 3 Gate 2 Date I Wheat l975 Figure 14. Diagram of Niles insectary showing layout of crops and location of emergence traps (1973-1975). x = emergence trap. 52 The traps were checked daily over the entire period of emergence and one week beyond emergence of the last parasitoid. Each day the parasitoids were removed from the traps and taken to the laboratory for positive identification. To determine if Diaparsis n.sp. was able to survive and emerge from a field which was plowed and planted, a small section of trap crop stubble was plowed, tilled, and planted to oats in the spring of 1974 and 1975. A series of six emergence cages was placed in the plowed strip immediately after planting and six control traps were placed in the untilled stubble parallel to the first six. These were monitored daily with the emergence traps in the rest of the insectary. However, the data from these traps were not used in calculation of the emergence curves. Air temperatures were used in calculating the degree days for plotting the emergence curves. Although soil temperatures would be more accurate for predicting emergence, it was not feasible to record them. Since there is no weather station at Niles which records temperature data, this was taken from two other stations, Dowagiac, which is 14 miles NNE of Niles, and South Bend, Ind. which is eight miles south of Niles. The daily maximum and minimum temperatures from each station were averaged and the means were used as the maximum and minimum temperatures for Niles. 53 The degree days were calculated using the formula 0 max + min D = 2 - Base Temp. for all days when the minimum temperature was above the base temperature. If the minimum temperature was below the base, the Baskerville-Emin (1969) sine curve method was used. In a laboratory study Gage (1974) determined the developmental threshold temperature of Tetrastichus julis to be 50°F. Similar attempts to determine this temperature for Diaparsis n.sp. were unsuccessful. Consequently an alternate method was used to obtain an approximation. This. was a comparison of the variances of heat accumulation for different degree day bases at the time of initial emergence (Figure 15). The fluctuation in variance is probably due to the fact that only three years data were available for analysis. Although 49°F was the point of least variance, 480 was used in the analysis in order to facilitate comparison with Gage's (1974) work on T. iuTTs which uses 480 as a base temperature. Also the variances at the 48 and 49 degree day bases were not significantly different at the .05 level. Results and Discussion Emergence trap catches are summarized by crOp strip in Table 5. The data from this trapping program were analyzed using a two level analysis of variance for unequal 54 .HmSmHImSmHV cmmHnon .meHz om .mm.c mHmHmmmHo mo dump wocmmnmfim HmHuHcH um momma moo mooHHm> Hm omDMHseooom mSmo mmummo mo mUCMHHm> .mH ouomHm omom Soc 353 on 3 I no N... .n on 2. 2. Sc 2. av H H J H H H H H H H O O .. 8. e e e O O 4 1 C) (D 9 8 J C) 8 eauogmA 4 02. 1 00m 55 Table 5. Emergence trap catch of Diaparsis n.sp. at Niles, Michigan (1973-1975). Year Wheat Oats-l Oats—2 Oats-3 Trap CrOp 1973 Total 1 9 28 26 x/Trap .33 3.0 9.33 8.67 1974 Total 25 15 8 12 44 i/Trap 8.33 5.0 2.67 4.0 14.67 1975 Total 2 118 408 68 i/Trap .29 16.86 27.20 9.71 56 '1: sample sizes (Sokal and Rohlf, 1969). The results indicate that a significant difference in emergence at F.05 occurred between the years and also between the strips. Initially a three level analysis was used considering parasitoid sex as the third level. However no differences were found so this was combined with the error term to increase the degrees of freedom. The emergence curves shown in Figures 16-22 were fitted to the data using probit analysis. The probit equations for these curves are listed in Table 6. The curves in Figures 16-18 show that males emerge consistently earlier than females. Although considerable overlap occurs, a given percent emergence occurs from three to five days earlier in males than in females. Sex ratio for each year was analyzed using a Chi square analysis resulting in the values 1.503, .154, and 12.722 for 1973, 1974, and 1975 respectively. This indicates a significant difference in 1975 only, when the number of females was greatest. According to Flanders (1939, 1946) and Clausen (1939, 1940) a number of factors influence egg fertilization and thus sex ratio in parasitoids. These include host size, * ANOVA Table Sour: df SS MS Fs* Ya- Y Years 2 1,664.495 832.248 4.270*** Yb- Ya Strips 10 4,330.372 433.037 4.997 Y - Yb Error 113 9,793.11? 86.665 Y - Y Total 125 15,787.984 57 Table 6. Probit equations for cumulative gercent emergence of Diaparsis n.sp. plotted over D43. Curve Equation Chi sq. df 1973 Males Ep = -5.359 + .0192X 1.10972 14 1973 Females Ep = -3.002 + .0138X .50029 17 1974 Males Ep = -7.291 + .0231X .09615 13 1974 Females Ep = —5.124 + .0169X .21977 17 1975 Males Ep = -5.970 + .0216X 5.46107 16 1975 Females Ep = —6.795 + .0218x 1.55005 12 1973 Total Ep = -3.765 + .0158X .7128 18 1974 Total Ep = -4.006 + .0158x .3617 17 1975 Total Ep = -5.722 + .0202X 12.8316 16 58 mSmH .2”.onon .mudz E .mw.z 2355.“. Batu... ozm HE: mo 3sz55 .._o mu>m=u two”: .9 35¢ we mmmm mmmomo coo omS 8S 0mm coo 0mm cow omv _ . _ . l r _ . XS . _ . r . _ . _ I a Jmafi . t e... a. x n a x o x n a m momma... I x _ o 3:... 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Irffrrrrr SE 20 3g IOU enunreerE BEBCEMI EHEBGEMCE T T ff] C3 ‘3‘] fit I r 0 mSmH .zmoHrqu .monz Sm .mm.z memcmcHo mo muzmoxmzm mo m>m=u SHmomm .HN mmson wv mwcm wmmomo owo oom omS DOS com 0mm com owe 00v p H P r H S h h k b b 63 171 T T 1 09 1 T Tfifl 3888 92 SL 001 BONBOHBNE 1N3383d BAIIUWHNHQ CI "’1 (T) 5" Bl MIFES b. } EWEBSEHCE (E 580311 CHEAE .85 51' £187 LL] C 0 II CD [I] If.) C3 (11 H- (11. "\ I ‘1') <33 .11 J L‘ “’1 LB. C‘) —J - ’1 [351mm F) CIJ —( CU 64 com H .mSmH 02¢ .eSmH .mSmH mo zomHmmmzoo .zcoHIOHz .meHz kc .mm.z mHmmcmcHD mo mm>m30 meomm .NN mmonu mv mmmm MMMOMD own DOS 0mm Dom 0mm Dom omw 00¢ H H P H H H H H H H H H H H H I r0 I Z [S I v 8 1 ex. . Q.— 1 IO 6 I ow. - r [H 1'9 H 001 33N3083N3 1N3383d BAIIUWHNHJ HIIIIIIIIHIIIII 4 ,iI 4 ID 1’ . UJ F7 EHEZIOHX .mmbs Hm .rm.x mwmmmmgw -5 mmumH mHummn mmmH Hmmumo pmufluwmmumm paw mm>nma maummn mama Hmmumo mo mommucmoumm O>Humasesu .OH mHnt 83 OCH OCH mm m.hm v.mm 00H ooa 00H ooH MN H.hm m.o> m.mm o.mm v.mm m.mm ma m.mv N.Hv m.vm H.mm o.mm OH m.mm OH m.ma m.vH H.mm H.mh h.Nm m.mm NH «.mm m.mn m v.vm o.om o.vv m.Hm m H.0v m.mv m m.mm m.vv N OGSH m.mH H.mN o.HH m.mm mm m.o O.HH H.N m.m mm >82 mAU mHHU mAU mAU mAU mAU OUMD Umufiuwmmumm omNHuwmmumm omufluflmmnmm mnmumo mlmumo Humumo .AmOmHV cmmwnowz .mmHHz um mm>HMH waummn mama Hmwuwo pmufiuflmmumm can mm>HmH maumwn mama Hmmnoo mo mommucmoumm m>flumHsESU .HH OHQMB 84 Results and Discussion Tables 12 and 13 show the encapsulation rate of Diaparsis n.sp. eggs and larvae which occurred singly in host larvae. Encapsulation in this situation was lowest in 1974 and highest in 1973, and in all years larvae were encapsulated less than eggs. Overall encapsulation for the three years was 7.5% for eggs and 0.5% for larvae. The rate including both eggs and larvae occurring individually in the host was 1.8%. Smooth eggs are more frequently encapsulated than knobbed eggs, Table 13. Perhaps the knob interferes in some way with encapsulation. Encapsulation was higher when two parasitoids were found in the same host larva (Table 14), occurring more frequently in superparasitism than in multiparasitism with either Tetrastichus julis or Lemophagus curtus. In one case T. julis was found to be encapsulated, while in the other 54 cases of multiparasitism neither parasitoid was encapsulated. It is possible that some of these encapsulations occurred prior to deposition of the second egg. This could not be determined since these are field data and the time of oviposition was not known. However, since encapsulation was more frequent in hosts which were superparasitized than in hosts with a single parasitoid, it appears that super- parasitism triggers encapsulation of one parasitoid. 85 Table 12. Encapsulation of Diaparsis n.sp. eggs and larvae occuring singly in the host. Parasitoid stage 1973 1974 1975 Total Eggs Encaps. 2 l 17 20 Unencaps. 31 49 168 248 % Encaps. 6.0 2.0 9.2 7.5 Larvae Encaps. 2 3 l 6 Unencaps. 134 373 698 1205 % Encaps. 1.5 .8 .1 .5 Totals % Encaps. 2.4 .9 2.0 1.8 86 Table 13. Encapsulation of smooth vs. knobbed eggs occuring singly in the host. Egg Type No. Encaps. No. Unencaps. $ Encaps. Smooth 8 67 11 Knobbed 12 181 6 87 Table 14. Encapsulation of Diaparsis n.sp. eggs and larvae with two parasitoids present. Superparasitism - 152 Cases Both Encaps. 2 1.3% One Encaps. 85 55.9% None Encaps. 65 42.8% Multiparasitism - 62 Cases Diaparsis Encaps. 7 11.3% T. julis Encaps. l 1.6% T. curtus Encaps. 0 0% None Encaps. 54 87.1% 88 From the data it appears that supernumaries in multi- parasitism are more commonly eliminated by direct larval competition than by encapsulation. According to Askew (1971) this competition may be by fighting, with one larva physically destroying the other, or by destruction of excess larvae by toxins produced by one of the parasitoids, or by depletion of available 0 by one of the parasitoids. In 2 the latter case the excess larvae are destroyed by suffocation. According to Dysart, et. al., (1973) Diaparsis n.sp. most likely is the successful parasitoid in competition with T. Julis. In dissections of European material they observed the larvae of Diaparsis biting T. julis larvae, and in several instances healthy Diaparsis were found with dead or wounded T. julis. However, in cases where the host larvae contain mature T. julis larvae it may not be possible for Diaparsis to destroy them. The outcome of Diaparsis - T. curtus competition is presently unknown. The effects of super-and multiparasitism on encapsulation are shown in Table 15. In cases where Diaparsis n.sp. was found in various combinations with other Diaparsis n.sp., T. igTTg, or T. curtus eggs or larvae, 43.9% had at least one Diaparsis encapsulated. In contrast only 1.8% encapsulation occured in hosts containing one parasitoid. Figure 29 shows the percentages of superparasitism and multiparasitism in each year, 1973-1975. In 1975 89 Table 15. Effect of superparasitism and multiparasitism on encapsulation rate of Diaparsis n.sp. Cases of single parasitism 1479 85.7% Encaps. 26 1.8% Unencaps. 1453 98.2% Cases of super- or multiparasitism (l) 246 14.3% Encaps.(2) 108 43.9% Unencaps. 138 56.1% Total 1725 100% (1) Superparasitism of Diaparsis n.sp. and multiparasitism with either Tetrastichus julis or Lemophagus curtus. In several cases all 3 were present. (2) One Diaparsis encapsulated only. 9O .. H H d 1 1 d I975 vM0w0w0w0MOMOMOMOMOM00.00.0r0r00000M000r0M.x. 00.00000.....0.0.0.0. .00....0.0.........0..00......0...0...0000. ..0.0...0...0.0.0,00.00.00.00.00»... V V @ SUPERPARASITISM‘ MULTIPARASITISM % 4 Percent superparasitism of CLB larvae by Diaparsis n.sp. and percent multi- ddefiflw .§%.00 ..%§§§ 000009 I973 I61- I2)- _ p b p b m m m a s 2m.._._mo OmuuoHa .om.c mHmHmmmHQ mo nonmo mmnu mmHmamE ucwoumm w>wustEsU .mm wusmflm O¢Do 000.. 000.. 00m 00m 000 000 Oak 00h 000 000 Ono 000 00¢ _ H _ H _ H _ H _ _ _ .a . o 03 O l . .1 Om 3.9 m m w" 4% m I . w. I. Rya\ ILO¢_I. x. xx _\HO O aw .mlmlzl 0 m an .. x o\ 1 OO m \ .1 HO _x \ “u .. Q C _1I:IIW... . \. I an... \\ AlnI : I. oo ......o o m. .49.... .x... . I . . a . , . . O. I oo. 108 .Hmhmav wwoo Hm>o pmuuon .mm.c mammonao mo noumo mmnu wmflmame uswouwm w>HHMHSEsU .vm musmflm O¢O 0 ONO.— ONOJ ONO; Ohm ONO ONO ONO Obs. 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I IS HH «\H“ OH 1 3 thaom \ Hx /\ 1 OO o :hCOZIW. mm I.ON_HH I100. .I.OON .I OVN 113 weather factors discussed above, there are two possible hypotheses explaining the high peak with a subsequent drop. One of these is that it is a reflection of behavior. If the parasitoids spend a period of time feeding just after emergence, followed by a period of more intense searching activity, a result such as this may be observed. A second hypothesis is that this observation may be the result of increased host densities. Rogers and Hassell (1974) conclude that interference of parasitoids resulting from a high parasitoid density leads to emigration of the parasitoid from high density areas. This may have been the. case with Diaparsis n.sp. in 1975. The 139% increase in emergence in 1975 over 1974 could have led to frequent interference between the adults with a resultant dispersal giving rise to the high malaise trap catches early in the season. This may also help explain the lower rate of parasitism in 1975 as compared to 1974 in spite of the much higher emergence density. Adult Parasitoid Food Part of the overall biological control effort aimed at the CLB was to investigate the host-parasitoid relationships as they occur in areas where the CLB is native. In an unpublished summary of one of these studies (1972), Dr. Bjegovié, of the Institute for Plant Protection in Belgrade, Yugoslavia, reported that Diaparsis spp. do considerable feeding on various wild flowers in the field. 114 As far as is known this is the first report of this being observed for Diaparsis spp. although it is known for other species. The species which Bjegovic reported as food sources for adult Diaparsis spp. in Yugoslavia are listed in Table 21. In addition to the plants listed, I also observed fair numbers of Diaparsis spp. feeding on yarrow, Achillea millefolium, in Yugoslavia. The order of importance of the plants listed in‘ Table 21 was determined by Bjegovic by observations in the laboratory of relative numbers of parasitoids visiting each plant. Following the report of the findings in Yugoslavia, observations were made on flowers occuring in the Niles insectary. In 1974 Diaparsis n.sp. was found feeding on yellow rocket (Barbarea vulgaris) and field cress (Lepidium campestre). In 1975 a more thorough examination was made in this area and seven species were found with Diaparsis n.sp. feeding on them. These are listed in Table 22. In addition to these, several pots of greenhouse- reared Brassica kaber were set out in the field and several Diaparsis n.sp. were observed to visit and feed on this. Bjegovié (1973) attempted to test the effect of the absence of flowers in the field by monitoring parasitism in two wheat fields, one of which was treated with herbicides to destroy the flowering plants. Parasitism was monitored 115 Table 21. Flowers reported by Bjegovié as food plants for adult Diaparsis spp. (Listed in order of preference.) Stellaria media Vill. Chickweed Sinapsis alba L. White mustard Lepidium draba L. Hoary cress Capsella bursa-pastoris L. Shepherds purse *Lamimum amplexicaule L. Dead nettle *In the laboratory only. 116 Table 22. Flowers found at Niles, Michigan to serve as food plants for adult Diaparsis n.sp. Barbarea vulgaris (L.) Yellow rocket Lepidium campestre (L.) Field cress Erigeron annuus (L.) Daisy Fleabane Stellaria media L. Chickweed Achillea millefolium L. Yarrow Viburnum sp. Viburnum Rubus sp. Dewberry 117 by dissection of CLB larvae collected daily throughout the season and also by rearing CLB larvae. The results of this investigation revealed a 5.9% parasitism rate in the field treated with herbicides and 9.7% in the untreated field. Although these data show a higher parasitism rate with flowers present, it was felt that since the fields were only about 500 feet apart there may have been some fly-over from one field to the other, which may have influenced parasitism. Also, since Diaparsis spp. appears to be more abundant in oats, more definitive results could perhaps have been obtained by using oats rather than wheat. In the work at Niles, an attempt was also made to evaluate the effect of wild flowers on parasitism. The design of this test was outlined earlier in the discussion of parasitism rates. Initially it was not known that such a wide variety of flowers serve as a source of food for Diaparsis n.sp. Therefore, the proximity of various flowers to the test area could have affected the results and made them largely inconclusive. In addition to the feeding which was observed on flowers, it was found in the laboratory that Diaparsis n.sp. also feeds on host-larval fluid at times. This has been reported for other parasitoids (Flanders, 1942, Leius, 1961) and likely occurs in the field as well as in the laboratory. 118 Discussion Observations in both Yugoslavia and Niles suggest that flowers are an important requisite for Diaparsis n.sp. Both males and females feed heavily on those mentioned above. At Niles, males were found more readily on the flowers growing in the emergence area and females were found more readily on flowers in or adjacent to the area where hosts were located. In Yugoslavia where food plants were nearly all in or bordering the crop, both males and females were found on them. With regard to wild flowers and the effectiveness of Diaparsis n.sp., it appears reasonable to assume that there is a relationship. Such a relationship is indicated in the literature for other species. Wolcott (1942) attributes the successful introduction of a parasitoid of the mole cricket into Puerto Rico directly to the presence of two flowers on which the adult parasitoids feed. When these flowers were absent, establishment did not occur even though hosts were abundant. A more recent study of parasitism in unsprayed non- commercial orchards (Leius, 1967) showed an 18X increase in parasitism of tent caterpillar pupae (Malacosoma americanum (F.)) in orchards with a rich growth of flowers over orchards with few or no flowers. Parasitism of the codling moth (Carpocapsa pomonella L.) was 5x greater with flowers present. van Emden and Williams (1974) feel that areas of floral diversity outside the crop should be maintained 119 until we are certain that their beneficial capacities can be replaced by biological control or planned diversity. However, in the Diaparsis situation it appears that such diversity is a necessary component of biological control. Interaction with Tetrastichus julis In an effort to establish effective biological control of the CLB in the U.S., three larval parasitoids have been established. The dynamics of one of these, Tetrastichus julis, has been studied by Gage (1974). The dominant parasitoids in areas where all three have been established are T. julis and Diaparsis n.sp. Since these are both larval parasitoids, it is important to look at their interaction. The following discussion is not meant to be definitive, but several observations may be made concerning interactions in the field. The life cycles of these two parasitoids are essentially the same except that T. iEliE has a partial second generation. The emergence curves are compared in Figure 37 although the relationship between these curves may vary from year to year depending on weather factors. A general relationship is seen, however, in that first generation T. julis emerges earlier than Diaparsis n.sp. There is considerable overlap between these parasitoids during the first generation of T. julis which might lead to a great deal of interference between them, however, multiparasitism is lowest early in the season. 120 .vbma .Ommu ECHO OOn wmummod .mHHHHn. .M can .mmé mfimummHOHo mo merHHHo mosmmeEm mo somwummaoo OH.O. OON. oo: 000. com com 02. com 000 00¢ H 1 H 201 H H H H \ H H H 2.2 H 13:22.3 HHON ...: ..H. .9 .HH stage IvH H H H H H H H H Alcozotocoo S. H aza— ..H. .Om «HOOHH Lt 0N On OH. 00. 3008983743 % BAILVWONI'IO 121 (See Figure 30). This is likely related to several factors. From emergence trap catches and from monitoring parasitism rates at Niles, it is evident that the first generation of T. ngTs is quite small (although it may be much larger at other localities). Therefore interference with Diaparsis n.sp. in the early part of the season is minimal. Host densities are also generally high early in the season, which helps to avoid multiparasitism. According to Gage (1974) T. igTTg ends its first generation at about 850 0D So when Diaparsis n.sp. is 48' most active, T. julis is not present. Much more multiparasitism occurs late in the season as the second generation of T. ngis appears and host densities decrease, but even here it is not serious. The total multiparasitism involving T. jETT§_and Diaparsis n.sp. was only 36 (2.1%) in three years. The following question needs to be answered, "Do these two parasitoids negate each other in any way?" This cannot be definitively answered at this point, but it does not appear that they do. Since T. igTTs is bivoltine and Diaparsis n.sp. fits essentially between the two generations, effects on each other are minimal. The greatest effect of having Diaparsis n.sp. in the system from the viewpoint of management of the CLB if Diaparsis n.sp. is valued is that it narrows, if not closes, the biological window for insecticide application discussed and predicted by Gage (1974). Since Diaparsis 122 n.sp. is present and active until T. igTTg begins its second generation, there is virtually no time when it would be feasible to apply insecticides to the larval population without affecting some of the parasitoid populations. Although, from the observations above, it does not appear that Diaparsis n.sp. and T. julis have any great effect on each other, it seems that both have not attained high populations in the same area to date. In Michigan, both parasitoids were released at the MSU research farm at Gull Lake in Kalamazoo County and in the USDA insectary at Niles, in Berrien County. At both localities both parasitoids have been established, but at Gull Lake the dominant parasitoid has been T. juTTg and at Niles, only about 60 miles southwest, it has been Diaparsis n.sp. Also, in other insectaries established by the Niles laboratory in the Midwest and Northeast U.S. by sub- colonizations from the Niles insectary, a similar trend is developing (Burger, pers. comm.). In some of these T. julis is more abundant and in some Diaparsis n.sp. is more numerous. In addition to the above considerations, it appears that a similar situation exists in Europe. In their European work Dysart, et. al., (1973), give the percent parasitism in a number of localities over a seven year period (Table 7). In nearly all cases parasitism by one parasitoid is high and the other low or both are low. In 123 no cases were both high except in one locality in Sweden where parasitism by Diaparsis was 18.2% and by T. julis 27.2%. It would appear that there is some ecological factor operating which favors one over the other in some areas and vice versa in other areas. Any attempt at this point to define what this factor might be would be mere speculation. More study is needed to evaluate the relationship of these two parasitoids and their effect on each other in various ecological conditions and population densities, but it does tend to support the position of many species introductions vs. single species introductions. EUROPEAN WORK Part of the cooperative agreement with the United States Department of Agriculture under which this work was conducted, stipulated that an effort be made to find a means of separating the two species of parasitoids in the larval stage. This would make it possible to identify the species present in dissections of field-collected hosts and. avoid the problem of rearing them to the adult stage. To do this it was necessary to obtain parasitoids directly from Europe since only Diaparsis n.sp. is available in the U.S. Consequently, the 1974 field season, mid-April to mid-July, was spent in Europe, primarily for the purpose of collecting parasitoids, but also to make observations of the CLB and Diaparsis spp. in their native areas. According to communications from the USDA Parasite Laboratory in Sevres, France, the area around Belgrade has a consistently high population of CLB. Therefore, approximately two months were spent in Zemun at the Biological Control Division of the Institute for Plant Protection of Belgrade. Since a main objective in the European work was collection of material for laboratory use, considerable 124 125 time was spent rearing CLB larvae to obtain parasitoid cocoons. Using a method developed by Gruber, et. al. (1972), a total of 19,500 larvae was reared. The cocoons obtained from these rearings were held for emergence of CLB's and non-diapausing parasitoids. The remaining cells were then sent via the USDA Laboratory in France to the quarantine lab at Newark, Delaware where they were held for emergence and subsequent shipment to MSU. A few CLB cells were also collected in Central Denmark near Kolding which is where the CLB was reported as having the highest densities. In 1974, however, densities were very low and only 44 unemerged CLB cells were shipped to the U.S. Some of these contained Tetrastichus julis and some Lemgphagus curtus so the actual number of Diaparsis spp. obtained was rather low. The agricultural conditions and host behavior in Europe, were considerably different than expected, so not all the work could be carried out as planned. Some problems were also encountered due to an exceptionally rainy season, with the result that much of the information gathered is in the form of observational data rather than data subject to statistical analysis. Species Composition The exact geographical distribution of the two species of Diaparsis in Europe is not currently known; however, both species occur in Yugoslavia with Diaparsis n.sp. 126 predominating. This was apparent in several of the sampling procedures, one of which was malaise traps. From a total of 4487 Diaparsis spp. caught in these, 85% were Diaparsis n.sp. and 15% were 2. carinifer. These figures are only relative since the sampling schemes may be selective in some way due to differences in species behavior. Also the mortality in lab rearing and emergence may differ between the two species. It seems clear, however, that Diaparsis n.sp. is the dominant species in Yugoslavia. Although only a small number of larvae could be collected for rearing, the situation in Denmark appears to be different. All 15 adults received at MSU were found to be 2. carinifer. This of course is not a large enough sample to rule out the possibility of Diaparsis n.sp. being present, but it does seem to indicate that Q. carinifer is at least dominant in Denmark. Flight Activity Part of the work in Yugoslavia was a Diaparsis spp. population monitoring program done with two malaise traps designed the same as those used at Niles (Figure 31). The traps were set up on May 25 and were monitored daily for the remainder of the parasitoid season. Both traps were located between oat and wheat fields in narrow uncultivated areas. Trap 1, with the long axis north-south and thus Open to the east and west, was located at the 127 corner of a wheat field with a hedgerow containing trees and shrubbery behind and to one side of it. Trap 2 was in a more open area and was oriented in an east-west direction opening to the north and south (Figure 38). The total catch of Diaparsis was 4487 with the highest catch occuring on May 28 when 730 were caught in one 24-hr. period. The total catch for the season was lower in Trap 1 (657) than in Trap 2 (3830). In terms of sex ratio it was found that the catch of Diaparsis n.sp. is about equally divided with 47% of the total being males and 53% females, but in Q. carinifer only 11% were males and 89% females. Although the overall sex ratio was nearly 121 in Diaparsis n.sp. it appeared that males predominated early in the season. Consequently the catches of both species for the first 15 days of trap operation were analyzed to see if a relation existed between percent of males captured and the total catch. The initial test used was Kendall‘s tau which is a coefficient of rank correlation. The result of this showed no significance in correlation of percent males to total catch for Q. carinifer, but for Diaparsis n.sp. the correlation was significant at the .002 level. This was further analyzed using a least squares linear regression statistic by which the equation of the regression line (Figure 39) was determined to be Y=13.18+ 0.09lx with an R2 value of .5665. The T value (HO:B=O) for this regression was 4.122 which is significant at the .001. 128 SOUTH GEIABL ALFALFA H 005 TRAPI WHEAT WHEAT 2‘9 UNCULTIVATED I-T-I 3 I TRAPZl O I‘.‘ ‘2‘ g POTATOES 22: CORN 3 “1 OATS 5 '2 -’ In I 3 U 3 I Figure 38. Diagram of the study area in Yugoslavia and the location of the malaise trap. 129 .¢H>¢Hmoo:> 2H mmcmh mmH¢4¢z 2H .mm.z mHmzmmmHo mo zuhcu Amhoh ow mm4¢z Hzmummm mo onH¢4um oszozm 4c>¢mHzH wuzuonzou mm. :HHz onhmaam 02$ qu4 onmmumomz .mm .OHH .onHquH OO OO Oh OH on ON OH O- Prb - b rfiLhP—FhkPhr5rh—Pbb P—h pr: — n b j I Tfl T 92 1 I ' I I 001 SEWUH 1N33838 (7)7“ HI T T In: I\H T T IO 2 T H 10L HI_\ X T V. 1 flH‘HHIHHHIHHHH4AJ4HHHHHH311441H4411HHHk/MITIO 04 09 mm 0v om ow OH OI HonLmHoH LmHOH m OM>HOH w OM>HOH m mumo m mumo H mumo mnaH I .sOHz .mOHHz um mmHmH EmHuHmmHmm can mmHuHmaOU HO>HOH mHO "Hm.> xHUsOmmd 163 OH m.o m m.N m CH m.o O HO O.w H HHH5O. ON o.oH m OH o.v m om m.HN ON ov N.mH H HHH5O. om H.¢H H OHH5O. ow 0.0N vN Nm v.ON ON mu m.mm ON om m.ON oN om m.mN «N we m.O¢ ON Nm m.mv OH mN m.mN 0N wm o.mm ON we N.mN MH ow m.ON mH om O.mm OH Nm m.ON 0H mN m.OH mH mm m.mN mH mm m.m w mN m.m oH mash vm o.OH oH OCSb Om m.o m mash .mumm um\ .mumm um\ .mumm OM\ w OM>HOH w OM>HOH w OM>HOH m mpmo N mumo thH I .SUHS HmOHHZ ”Hum mOUMH EmHHHmMHMQ UCM wmflflflmfimfl HMNKHMH mHHU .Hh.> OHOOOOOO 164 .mmusmHm Hmsuom swap HO30H OH OmuHsmOH O>mn NOE mHmEmm GONon mo GOHHOOmmHU OHOHt m.v MN «H o.o mN «NH O.NN mH om v.mH mH v.O mN N.MN OH O.ov «H m H.OH ON mm m.ON NH Nm m.wv NH O o.wv mN m.Nv m vv m.H¢ m NN m.mm mH mv N.mm m «m m.mm m ON o.mm OH om m.¢m N mash Om o.vm N OGSb m.H¢ OH NN O.om mN mH m.om mN OH 0.00 OH when OH 0.0m OH HO: O O.NO ON Om: .mnmm um\ .mumm HM\ .mumm um\ w OM>HOH m OM>HOH w OM>HOH m mumo N mumo H mumo mOmH 1 .HOHE .mOHHz um mmumu EmHuHmmHmm UGO mmHuHmsmp Hm>HmH mHU “Ho.> xwuswmmd 165 Appendix VI: Data from soil samples and spring emergence for analysis of overwinter mortality of Diaparsis n.sp. at Niles, Mich. 1974. Fall Samples Spring Emergence Total Catch Sample # # Alive # Dead Total 2 l 29 l 30 23 2 6 2 8 6 3 ll 3 14 2.5 4 19 6 25 11.5 5 7 4 11 25 6 15 0 15 30 7 10 0 10 8 8 6 0 6 10.5 9 13 1 14 6.5 10 4 0 4 5 11 10 3 13 2.5 12 3 5 8 13 13 12 4 16 12.5 14 9 l 10 3 15 17 1 18 22.5 IIIII’I‘II IIIIIIIIIII’ 77 5038 I 3 031 9 A III! N 1’ ”1 m3 M H