THE MMS CULTURE AND FIELD RELEASE OF ANAPHES FLAVEPES (FOERSTER) {HYMENOPTEM MYMARIDAE), AN EGG PARASITE OF THE CEREAL LEAF BEETLE. OULEMA MELANOPUS (L.) (COLEOPTERP: CHRYSO‘MELIDAE) Thesis for the Degree of M. S. MICHEGAN STATE UNIVERSITY LARRY CARSON; BARTON 1968 5". I. r v :4 r2 [Micki man Start: THE—S's University .EWW'L ' magma Iv “ HUM} & SDNS' 800K BINDERY INC. LIBRARY BIND£RS Quinn-1 Ina-nu- ABSTRACT THE MASS CULTURE AND FIELD RELEASE OF ANAPHES FLAVIPES (FOERSTER) (HYMENOPTERA: MYMARIDAE), AN EGG PARASITE OF THE CEREAL LEAF BEETLE, OULEMA MELANOPUS (L.) (COLEOPTERA: CHRYSOMELIDAE) by Larry Carson Barton Experiments have been conducted to improve the technique currently in use for mass culturing Anaphes flavipes (Foerster) (Hymenoptera: Mymaridae), an egg para— site of the cereal leaf beetle, Oulema melanopus (L.) (Coleoptera: Chrysomelidae). There are basically two tech- niques for rearing Anaphes: removing cereal leaf beetle eggs from plants upon which they are laid or leaving the eggs on the plants. Tests have shown that there is no dif- ference between the two techniques in terms of the number of parasitized cereal leaf beetle eggs to develop per female parasite used. The removal of cereal leaf beetle eggs appears to be better than the non-removal of the eggs because (1) less space is required during rearing and during storage of both parasitized and unparasitized eggs, (2) eggs are easily counted, (3) it is easy to ascertain the number of parasites needed to parasitize the eggs, and (4) field release of parasitized material is easily accomplished. Larry Carson Barton Attempts were made to improve the techniques used in the storage of both unparasitized and parasitized cereal leaf beetle eggs. Storage techniques tested included (1) the use of modified gas atmospheres at both 140 and 400 F, (2) a glycerol egg-yolk—citrate extender to allow storage in liquid nitrogen, (3) the use of fungicides on cereal leaf beetle eggs stored at 400 F and (4) storage of eggs at 400 F while inverted over water. During the spring and early summers of 1966 and 1967 field releases of Anaphes were made in various areas in southern Michigan. Although no overwintering adults were found in the spring of 1967, the releases in both 1966 and 1967 did indicate that Anaphes would successfully develop in the field and that possibly three generations could be obtained in one season. The 1967 releases indicated that Anaphes will dis- perse at least 430 feet from the release point in one season. The releases showed that Anaphes can disperse through alter- nating 80 foot—wide strips of wheat, oats, and corn with voluntary oats. ii THE MASS CULTURE AND FIELD RELEASE OF ANAPHES FLAVIPES (FOERSTER) (HYMENOPTERA: MYMARIDAE), AN EGG PARASITE OF THE CEREAL LEAF BEETLE, OULEMA MELANOPUS (L.) (COLEOPTERA: CHRYSOMELIDAE) BY Larry Carson Barton A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Entomology 1968 ACKNOWLEDGMENT I very gratefully thank my major professor, Dr. Frederick Stehr for his guidance and thoughtful assis- tance. Thanks also goes to Drs. James Bath and Everett Everson, both for serving on my committee and for their assistance and suggestions regarding the writing. I would also like to thank Dr. Gordon Guyer for serving on my com- mittee. I wish to thank Richard Connin not only for serving on my committee but also because he and his crew of cereal leaf beetle "growers" furnished the innumerable beetle eggs needed for this project. Harry Maltby and Gary Moorehead of the USDA Biological Control Station, Niles, Michigan must be thanked for their suggestions and for furnishing the para- sites for the 1967 releases. Dr. Harold Hafs of the Dairy Department deserves much thanks for his suggestions concerning controlled atmo- ~sphere and liquid nitrogen storage. Also, I am very much indebted to the personnel of the Michigan Animal Breeder's C00perative for the use of their facilities for several of my experiments. I extend thanks to the Insect Identification and Parasite Introduction Research Branch, Entomology Research Division, Agricultural Research Service, U.S. Department of Agriculture for financial assistance given to the research program through Cooperative Agreement No. 12-14-100-5628(33). In addition I am grateful for the financial aid given by the National Science Foundation via a traineeship. I would like to thank the many comrades-in-arms who listened to me and made numerous suggestions regarding the project. In particular I would like to single out Julian P. Donahue and John McPherson for the many suggestions and editorial assistance that they provided. Finally, I would like to give special thanks to my wife, Leslie, for having the patience to put up with a struggling graduate student. ii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . . l PARASITES AND PREDATORS OF THE CEREAL LEAF BEETLE . . 3 Egg Parasites . . . . . . . . . . . . . . . . 3 Larval Parasites . . . . . . . . . . . . . . . 4 Hyperparasites . . . . . . . . . . . . . . . . 5 Predators . . . . . . . . . . . . . . . . . . 5 MYMARIDS IN BIOLOGICAL CONTROL . . . . . . . . . . . . 7 LIFE HISTORY OF ANAPHES FLAVIPES . . . . . . . . . . . 10 DEVELOPMENT OF MASS CULTURING . . . . . . . . . . . . l4 Objectives and Review . . . . . . . . . . . . 14 Mass Culturing Used at Niles in 1967 . . . . . 16 Use of Hospital Talc on Fresh Eggs . . . . . . 18 Daily Feeding of Anaphes to Increase Progeny per Female . . . . . . . . . . . . . . . . . l9 Collecting Container . . . . . . . . . . . . . 23 Parasitization in the Dark . . . . . . . . . . 27 Containers Used for Mass Culturing Anaphes while CLB Eggs Are on the Plants . . . . . . 29 Discussion . . . . . . . . . . . . . . . . . . 37 STORAGE OF UNPARASITIZED EGGS . . . . . . . . . . . . 41 Storage over Water . . . . . . . . . . . . . 41 Use of Fungicides to Retard Growth of Fungus on Eggs during Storage . . . . . . . . . . . 48 Freezing Eggs for Long Term Storage . . . . . 58 The Use of Extender for Egg Storage . . . . . 60 Gas Atmosphere Storage of Eggs . . . . . . . . 67 STORAGE OF PARASITIZED EGGS . . . . . . . . . . . . . 78 Storage of Parasitized Eggs Earlier than the Black Pupal Stage . . . . . . . . . . . 80 iii Page COLD STORAGE OF ADULT PARASITES . . . . . . . . . . . 87 X—RAY TREATMENTS OF CLB ADULTS AND EGGS . . . . . . . 91 Adult Beetle Treatment . . . . . . . . . . . . 91 Egg Treatment . . . . . . . . . . . . . . . . 95 Discussion . . . . . . . . . . . . . . . . . . lOO FIELD RELEASES . . . . . . . . . . . . . . . . . . . . 103 Techniques . . . . . . . . . . . . . . . . . . . . 103 1966 Releases . . . . . . . . . . . . . . . . . 106 Hillsdale County . . . . . . . . . . . . . . . 106 Kalamazoo County . . . . . . . . . . . . . . . 110 Berrien County . . . . . . . . . . . . 111 1966 and 1967 Recovery Attempts . . . . . . . 114 Discussion . . . . . . . . . . . . . . . . . . 116 1967 Field Releases . . . . . . . . . . . . . . . 117 Clinton County . . . . . . . . . . . . . . . . 124 Jackson County . . . . . . . . . . . . . 127 Gull Lake (Kalamazoo County) . . . . . . . . . 137 Parasitization Rates . . . . . . . . . . . . . 142 Summary . . . . . . . . . . . . . . . . . . . 145 SUMMARY AND DISCUSSION . . . . . . . . . . . . . . . . 147 LITERATURE CITED . . . . . . . . . . . . . . . . . . . 152 iv Table LIST OF TABLES The effect of daily feeding and watering of Anaphes and daily transfer to fresh eggs on the number of offspring produced per female Anaphes . . . . . . . . . . . . . . . . . . The efficiency of the Anaphes flavipes self- collecting apparatus . . . . . . . . . . . The percent female offspring from Anaphes flavipes collected and mated in the collect- ing apparatus . . . . . . . . . . . . . . . . The parasitization of cereal leaf beetle eggs by Anaphes flavipes in total darkness (Test 2) . . . . . . . . . . . . . . . . . . . . A comparison of three different mass culture containers, using French (F) and Yugoslavian (Y) strains of Anaphes . . . . . . . . . . . . A comparison of rearing both Yugoslavian and French strains of Anaphes in petri dishes . . . The influence of the length of cereal leaf beetle egg storage while inverted over water on subsequent parasitization by the Yugoslavian strain of Anaphes (Test 1). . . . . . . . . . The influence of the length of cereal leaf beetle egg storage while inverted over water on subsequent parasitization by the French strain of Anaphes (Test 2) . . . . . . . . . The effect of Captan as a fungus inhibitor at the rate of l g (50% active ingredients) per 100 ml sterilized distilled water on cereal leaf beetle egg storage and subsequent para- sitization by Anaphes flavipes . . . . . . . Page 21 25 26 28 32 38 44 46 50 Table Page 10. The effect of Arasan as a fungus inhibitor at the rate of .1 g per 100 ml sterilized distilled water (750 ppm active ingredients) on cereal leaf beetle egg storage and sub- sequent parasitization by Anaphes flavipes . . 52 11. The effect of Phygon as a fungus inhibitor at the rate of .1 g per 100 ml sterilized dis— tilled water (500 ppm active ingredients) on cereal leaf beetle egg storage and subsequent parasitization by Anaphes flavipes . . . . . . 54 12. The effect of Semesan as a fungus inhibitor at the rate of .1 g per 100 m1 sterilized distilled water (286 ppm active ingredients) on cereal leaf beetle egg storage and sub- sequent parasitization by Anaphes flavipes . . 55 13. The effect of Captan as a fungus inhibitor at the rate of .1 g per 100 ml sterilized dis— tilled water (500 ppm active ingredients) on cereal leaf beetle egg storage and subsequent parasitization by Anaphes flavipes . . . . . . 56 14. The parasitization success of Anaphes flavipes on cereal leaf beetle eggs stored at -120 F for 2.5 and 14 hours, and 9, 20, 33, and 331 days . . . . . . . . . . . . . . . . . . . 59 15. Parasitization of extender-treated cereal leaf beetle eggs that were stored at 400 F for various periods of time. The eggs were either dipped in physiological saline for approx— imately 1 to 2 minutes before parasitization or they were parasitized without the physiolog- ical saline treatment . . . . . . . . . . . . . 61 16. Parasitization success of Anaphes flavipes on cereal leaf beetle eggs stored with non- glycerated and 7% and 14%1g1ycerated extender for 11 days, with a 5-3/4 hour rinse of physiological saline before parasitization . . 63 17. The effects on cereal leaf beetle eggs and parasitization success of Anaphes flavipes on the eggs after storage for 5 days in liquid nitrogen . . . . . . . . . . . . . . . . 65 Table 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. Parasitization success of Anaphes flavipes on cereal leaf beetle eggs after storage for 17 days in liquid nitrogen . . . . . . . . Parasitization success of Anaphes flavipes on unparasitized cereal leaf beetle eggs stored for 11 days at 400 F in gas atmospheres of air, 100% nitrogen (N ), 100% carbon dioxide (C02), and mixtures of 5% carbon dioxide, 95% oxygen (COZ/Oz), and 12% carbon dioxide, 79% nitrogen, and 9% oxygen (C02/N2/02)(25 eggs per flask) . . . . . . . . . . . . . . . . . Parasitization success of Anaphes flavipes on unparasitized cereal leaf beetle eggs stored for various time periods at 400 F in gas atmospheres of air, 100% nitrogen (N2), 100% carbon dioxide (C02), and mixtures of 5% carbon dioxide and 95% oxygen (C02/02), and 12% carbon dioxide, 79% nitrogen, and 9% oxygen (COZ/Nz/Oz) . . . . . . . . . . . . . Survival and parasitization success of para- sites emerging from Anaphes flavipes pupae stored at 400 F for 1-7 weeks . . . . . . . . . Survival and emergence of Anaphes flavipes after storage of parasitized cereal leaf beetle eggs with various aged developmental stages of the parasite (50 eggs per dish) Survival of 17 female and male Anaphes flavipes (French strain) stored at 400 F . . Survival of female Anaphes flavipes (French strain) stored at 400 F . . . . . . . . . . . . Survival of 10 male Anaphes flavipes (French strain) stored at 400 F . . . . . . . . . . . . Survival of adult male Anaphes flavipes (French strain) at room temperature and stored with and without honey at 400 F . . . . . . . . . . . Parasitization success of Anaphes flavipes in eggs from untreated female cereal leaf beetles mated with male cereal leaf beetles exposed to 2000 roentgens of X-ray . . . . . . . . . vii Page 67 7O 74 79 82 87 88 88 89 94 Table Page 28. The number of cereal leaf beetle larvae to hatch, number of parasitized eggs to develop black pupae, and the number of adult parasites to emerge from cereal leaf beetle eggs treated with 1000 and 2000 roentgens X—ray and untreated eggs (200 eggs for each test group) . . 96 29. The number of cereal leaf beetle larvae to develop and/or hatch from 50 eggs treated with 1000 roentgens and 50 eggs treated with 2000 roentgens X—ray that were not subjected to parasitization . . . . . . . . . . . 97 30. The number of cereal leaf beetle larvae to develop and/or hatch from untreated cereal leaf beetle eggs and from eggs treated with 3000, 4000, and 5000 roentgens X-ray that were not subjected to parasitization . . . . . . 98 31. Parasitization success of Anaphes flavipes in cereal leaf beetle eggs treated with 3000, 4000, and 5000 roentgens X-ray . . . . . . . . . 99 32. Parasitization success of F Anaphes flavipes females from cereal leaf beetle eggs treated with 3000, 4000, and 5000 roentgens of X—ray on untreated cereal leaf beetle eggs . . . . . . 100 33. The date, number of parasitized eggs, and location of Anaphes flavipes releases in 1966 . . . . . . . . . . . . . . . . . . . . . . 110 34. 1967 sampling results from Bohn's wheat field for overwintered Anaphes flavipes . . . . . . . . 115 35. The Anaphes flavipes release sites of 1967, dates of release, and the number of parasit— ized cereal leaf beetle eggs that were released . . . . . . . . . . . . . . . . . . . . 121 36. The number of cereal leaf beetle eggs taken at each Anaphes flavipes release site on the dates that samples were taken . . . . . . . . . . 123 37. Date, location, number of samples taken, and the number of samples containing Anaphes flavipes for the 1967 release sites . . . . . . . 125 viii Table 38. 39. 40. The minimum, maximum, and average daily temperatures for the city of Jackson, Michigan (9 June to 17 July, 1967) . . . . . The minimum, maximum, and average daily temperatures for Gull Lake in Kalamazoo County, Michigan (16 May to 14 July, 1967) The number of female and male Anaphes flavipes to emerge from parasitized field collected eggs and the percent parasitization of the eggs from the samples taken in Clinton County, Jackson County, and at Gull Lake in 1967 ix Page 135 143 . 144 Figure 10. 11. LIST OF FIGURES Page The egg of Anaphes flavipes, 250 x. (Photograph by Richard Snider, Department of Entomology, Michigan State University) . . 11 Container used for collecting newly emerged Anaphes . . . . . . . . . . . . . . . 24 The "large" mass culture box without its top . . . . . . . . . . . . . . . . . . . . . 31 The "small" mass culture box . . . . . . . . . 31 The mass culture containers: from left to right they are the "large," globe, and "small" . . . . . . . . . . . . . . . . . 34 Container used for storing cereal leaf beetle eggs inverted over water at 40° F . . . 43 Acceptability of CLB eggs to Anaphes when the eggs are stored over water up to 176 days . . . . . . . . . . . . . . . . . . . . . 47 The container used for storing CLB eggs in various gas atmospheres . . . . . . . . . . . 71 Storage of unparasitized CLB eggs at 400 F in various gas atmospheres . . . . . . . . . . 76 The results of tests to determine the optimum age of Anaphes flavipes for storage at 400 F. The eggs were stored 2, 4, 6, and 8 days after parasites were placed with the eggs . . . . . . . . . . . . . . . . . . . 85 Wooden stakes used for releasing Anaphes flavipes in the field. Note the sections of glass slides taped to the stakes. (Wheat field, Charles Bohn's farm, Galien, Michigan, 1966) . . . . . . . . . . . . . . . 105 Figure 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. Page Wooden stakes for the field release of Anaphes flavipes in position in Charles Bohn's wheat field (1966) . . . . . . . . . . 105 Milk carton release containers before being closed. In the second set of containers from the right in the front row, the wooden shelf in the top release container is visible. (Release site in Jackson County in 1967, looking south - oats) . . . . . . . . 107 The release cage used in Hillsdale County in 1966. Note the release stakes inside the cage and the woodlot in the background . . . . 108 The release cage in Hillsdale County after fine white cloth was placed over the cage . . 109 Release site in oats at Bohn's in 1966 (looking east). Note the stakes and the brush along the edge of the field . . . . . . 112 Charles Bohn's oat field in 1966 showing the woods along the south edge of the field (looking southwest). The circle marks the location of the release site . . . . 113 Clinton County release site (1 mm = 15 feet) . . . . . . . . . . . . . . . . . . . . 118 Jackson County release sites (1 mm = 15 feet) . . . . . . . . . . . . . . . . . . . . 119 Release sites at Gull Lake (1 mm = 15 feet). . 120 Sample points and recovery perimeter of Anaphes for 1 June, 1967 in Clinton County (1 cm = 40 feet) . . . . . . . . . . . . . . . 126 Sample points in wheat of Jackson County (1 cm = 40 feet) . . . . . . . . . . . . . . . 129 The Jackson release site in oats, looking northeast. Note the release containers and wooden stakes. The stakes mark the location of the sampling sites . . . . . . . . . . . . 130 xi Figure 24. 25. 26. Sample points and recovery perimeter of Anaphes in oats for Jackson County (1 cm=40 feet). . . . . . . . . . Sample points and recovery perimeter of Anaphes on the south side of B Avenue (Site 1) Gull Lake, Michigan (1 cm = 40 feet) . . . . . . . . . . . . . . . . Sample points and recovery perimeter of Anaphes on the north side of B Avenue (Site 2) Gull Lake, Michigan (1 cm = 40 feet) . . . . . . . . . . . . . . . . xii Page 131 138 141 INTRODUCTION The cereal leaf beetle, Oulema melanopus (L.), is native to the Old World and is found throughout most of Europe including England, Wales, and part of Siberia (Cooperative Economic Insect Report, 1958). In addition, it is found in Morocco, Tunisia, Iran, and Turkey (U.S.D.A. Program Aid 550, 1964). The cereal leaf bettle (hereafter referred to as "CLB") was first discovered in the United States in south— western Michigan. Although farmers were spraying for the CLB as early as 1959, it actually was not identified until 1962 from Berrien County, Michigan (Castro, 1964). Since then, the CLB has spread throughout the Lower Peninsula of Michigan and into Indiana, Ohio, and parts of Pennsylvania and Illinois (Cooperative Economic Insect Report, 1967). In addition the CLB has been reported in parts of Canada (YUn, 1967). The cereal leaf beetle, a serious pest because of the preference for cereals by both the larvae and adults, also feeds on many other grasses (U.S.D.A. Program Aid 550, 1964). Early in the spring, as the overwintering adult CLBs become active, they concentrate their feeding on winter cereals but move to the more succulent spring grains such as oats when they develop. Damage to winter grains is less severe because the plants are larger than the spring grain at the time of attack, and because of the movement of CLB adults to the spring grain. Damage to cereal crops in Russia has amounted to as much as 25 to 50% (Cooperative Economic Insect Report, 1958); the same source indicated that serious damage to cereal crops was recorded as early as 1891 in Hungary. Because of the CLB's potential to damage cereal crops, a great deal of work has been expended in its control including the use of chemicals, plant resistance, and para— sites. The work described herein has been conducted to develop and improve those techniques used for the mass cul- turing of Anaphes flavipes (Foerster) an egg parasite of the CLB. The work has included testing different containers, the storage of unparasitized eggs in modified gas atmospheres and under deep freeze conditions, storage of parasitized CLB eggs, and the use of fungicides on stored eggs. Tests were also conducted to discover whether Anaphes develops in eggs treated with up to 5000 roentgens X—ray. In addition, attempts to establish this exotic parasite have been made at several locations in Southern Michigan. PARASITES AND PREDATORS OF THE CEREAL LEAF BEETLE In 1963 Agricultural Research Service entomologists began search for parasites of the CLB in Europe, and the first shipments of parasites were made from Europe in 1964 (R. I. Sailer, unpublished report, 1967). Egg Parasites Anaphes flavipes (Foerster), and other forms of Anaphes with which this paper deals, are mymarid egg para- sites of the CLB. Anaphes flavipes also is known to attack the eggs of Oulema gallaeciana Heyd. and has been found in Italy, Spain, Yugoslavia, France, and Germany (R. I. Sailer, unpublished report, 1967). It appears that two species of Anaphes may be in- volved in the host-parasite relationship, as evidenced by unsuccessful crosses between the strains of Anaphes from France and YUgoslavia made by workers at the Agricultural Research Service Biological Control Station at Niles, Michigan, where Anaphes is mass—cultured for field release (Moorehead, personal communication). The workers at Niles were unable to obtain females from crosses of French males with YUgoslavian females and vice versa (Anaphes will pro- duce only male progeny if unmated). Pattasson valkenbergica Soyka was reported by Bakkendorf (1964) to have been recovered from the eggs of Oulema lichenis Voet (=gallaeciana) at Tours, France in 1963. Since Anaphes parasitizes Q. gallaeciana eggs, it is possi- ble that g, valkenbergica may attack CLB eggs, although this has not yet been proven. In addition, at least one and possibly two species of Trichogramma that parasitize CLB eggs occur naturally in Berrien County, Michigan. Larval Parasites The following is a summary of the larval parasites of Anaphes flavipes discussed by R. I. Sailer (unpublished report, 1967). Lemophagus curtus Townes, an ichneumonid originally called Hypersoter sp., has been found in Italy, France, YUgoslavia, Germany, Denmark, and Sweden. Its facultative diapause facilitates lab rearing, since a con- tinuous supply of parasites can be maintained. Diapause may furnish a good means of stock—piling Lemophaqus for release the following spring. Another ichneumonid larval parasite is Tersilochus carinifer Thompson, which has been collected in YUgoslavia, Italy, France, Germany, and Scandinavia. A different but closely related species has been collected in Denmark. ‘Tersilochus has an obligatory diapause. Tetrastichus julis (Walker) is another hymenopteran larval parasite of the CLB. This eulophid has been found at all localities in Europe where CLB larvae have been collected and reared. It appears to be more important in the northern part of its range than is Tersilochus. It has a facultative diapause. A tachinid, Meigenia mutabilis Fall has been reported by Petar Bjegovic (unpublished report, 1966) to emerge from CLB pupal cells in Yugoslavia. This fly para— sitized a number of chrysomelid larvae in addition to the CLB. Hyperparasites According to R. I. Sailer (unpublished report, 1967), Mesochorus is a hyperparasite known to attack Tersilochus and possibly Lemophagus. Predators Known predators of the CLB include Nabis feroides Romane, a hemipteran in the family Nabidae. In Europe prior to the build—up of Anaphes in the last part of May, 1966, Nabis feroides appeared to be very effective against CLB eggs (Petar Bjegovic, unpublished report, 1966). In 1967, however, very few N. feroides could be found (Dysart, per- sonal communication). Another nabid, Nabis ferus L., is known to be a predator of CLB eggs and larvae. “N. ferus is widespread in northern Europe, but does not appear to be abundant enough to reduce populations of the CLB (Petar Bjegovic, unpub- lished report, l966)~ According to Castro (1964), the lady beetle Coleomegilla maculata lengi Timberlake, feeds on the eggs of the CLB in Michigan in early spring when its preferred hosts (aphids) are not available. Other coccinellids have been seen in the field during the time that g, maculata lengi was active, but their roles as predators of the CLB are unknown. Castro also observed an unidentified pentatomid kill a CLB larva, a carabid beetle attack and kill adult CLBs on the ground, and a cantharid which attacked both adults and larvae. MYMARIDS IN BIOLOGICAL CONTROL Although not a mymarid, one of the earliest trials of an egg parasite in biological control was the use of Trichogramma pretiosa (Riley) against the currant sawfly, Nematus ventricosus [=ribesii (Scopoli)]. In 1882, a large number of leaves bearing parasitized eggs of the currant sawfly were collected and mailed to entomologists in various parts of the U.S.A. and Canada (Lintner, 1882). The leaves were to be pinned to currant bushes where the currant sawfly eggs are found. Nothing was said of the results of these mailings. One of the most successful examples of a mymarid used for biological control was the introduction of Anaphoidea nitens Girault into South Africa from Australia to control the eucalyptus snout-beetle, Gonipterus scutel- latus Gyllenhali (Tooke, 1953)° According to Tooke, the program to control 9, scutellatus "may be claimed to be successful, since out of 65 species of eucalyptus originally attacked, only two species, E, viminalis and E, insizwaensis, now suffer severe damage and these only in certain restricted areas." Tooke further states in regard to the use of A. nitens that "no other method of control would have proved so economical. . . ." According to DeBach (1964), E. nitens was also introduced into New Zealand in 1927 where it gave "substan- tial" control of g, scutellatus. He also reports that E. nitens gave "substantial" control when introduced into both Kenya and Madagascar. In Mauritius, where this parasite was introduced in 1946, control of g, scutellatus was complete (DeBach, 1964). Anagrus epos Girault is another example of a mymarid used in biological control, and provides a good example of the importance of a knowledge of an insect's ecology and habits. On grape in Napa and Sonoma Valleys in California, '5..gpg§ has provided commercial control of the leafhopper Erythroneura elegantula (Osborn). It was found that in those areas where this insect was not controlling the leaf- hopper satisfactorily (San Joaquin and Sacramento Valleys of California), there were no wild blackberry plants growing nearby (Doutt and Nakata, 1965). It was found that Anagrus must overwinter in the eggs of the leafhopper Dikrella cruentata Gillette, which is found on wild blackberries. This secondary host, which oviposits all year, is needed since the primary host, E, elegantula, does not oviposit in the winter months. Another species of Anaphes known to attack insect pests is Anaphes nipponicus Kuwayama, an important egg para- site of Lema oryzae Kuwayama, the rice leaf beetle (Kuwayama, 1932). Muhle and Fr6hlich (1953) reported that in Germany Anaphes brachygaster was found to parasitize the eggs of the weevil Legphloeus tessulatus found on Levisticum officinale, which is grown for its aromatic and medicinal properties. In addition, Anaphes ovijentatus (Crosby and Leonard) attacks the eggs of the mirid Lygus hesperus Knight, accord- ing to Romney and Cassidy (1945). One of the mymarids with the most interesting habits is Caraphractus cinctus Walker, a parasite of dytiscid eggs in Europe (Jackson, 1958). The female swims underwater by rapid vibrations of her wings and oviposits on the dytiscid eggs she finds. Although there are many known egg parasites, only a very small number of these have been studied in any great detail, and even fewer have been used in biological control. Several of those used in biological control have had rather spectacular results. From these successes, it appears that egg parasites do have great potential in the biological con- trol of pests. But before the potential is realized, much more work and study remains to be done with the known egg parasites as well as with those that will be discovered in the future. LIFE HISTORY OF ANAPHES A new generation of Anaphes begins when a female locates a suitable egg and oviposits in it ("stings“ it). The female Anaphes walks about tapping her antennae verti— cally in front until she finds a CLB egg; she then mounts the egg, searches for an appropriate site, and oviposits. While ovipositing, she stands motionless with her antennae held in front of her. Although she may stand on the end of the egg when ovipositing, she normally is situated on the "top" or side of the egg and is oriented parallel with the long axis of the egg. The egg laid by Anaphes flavipes is elongate and semitransparent, with a small "stem" on one end (Fig. l). Superparasitism of CLB eggs is normal, usually with from one to three parasites developing successfully from one CLB egg. At room temperature (70 — 800 F), the earliest a parasitized egg can be identified is from 2 to 3 days after the CLB eggs have been "stung." At this time, under the microscope, from one to several larvae, which appear as "spots" slightly darker than the surrounding yolk may be seen. Parasitized eggs can more definitely be identified at pupation, when compound eyes are formed from 6 to 8 days after parasitization, depending on temperature. By the 10 11 Figure l — The egg of Anaphes flavipes, 250x. (Photograph by Richard Snider, Department of Entomology, Michigan State University) 12 eighth to tenth day, the black pupae of the parasite are clearly visible; in fact, to the naked eye the entire egg appears black except for some small orange spots of meconium. Depending on the temperature, adults emerge on the ninth to thirteenth day after the parent parasites are placed on the eggs. Parasites can be reared the year-round in the lab- oratory without interruption because laboratory conditions have not induced diapause. It is not known whether Anaphes overwinters as an adult, as an immature within the egg of the CLB, or within the egg of some alternate host. In Europe, parasitized CLB eggs have been found as early as 20 May, and adult Anaphes have been found as early as 17 May (R. I. Sailer, unpublished report, 1967). Early in the season, eggs parasitized by Anaphes are difficult to find, but by the end of the CLB oviposition period the parasitization rate comes close to 100% in some fields (Petar Bjegovic, unpublished report, 1966). At present there are Anaphes cultures from various parts of Europe including Italy, France, and YUgoslavia (R. I. Sailer, unpublished report, 1967). The original Anaphes culture came to the United States from Paris, France in 1965 (R. I. Sailer, unpublished report, 1967). This cul— ture and the 1966 YUgoslavian culture are the two that have been used for all the work reported here. The original YUgoslavian culture was sent to the United States in 1966 13 through the cooperative efforts of Dr. Petar Bjegovic of the Laboratory for Biological Control, Banatska, Zemun, YUgoslavia, Dr. J. W. Butcher (Entomology Department, M.S.U.), Dr. R. I. Sailer (U.S.D.A., formerly head of the European Parasite Laboratory), and M. H. Brunson (Insect Identification and Parasite Introduction Research Branch, U.S.D.A., Moorestown, N.J.). DEVELOPMENT OF MASS CULTURING Objectives and Review According to DeBach (1964), "the goal of a mass- culture programme is to produce with minimum man hours and space the maximum number of fertile females of an entomoph- agous species in as short a time and as inexpensively as possible." In the mass culturing of insects, it is neces- sary to gain as much knowledge about both the host and para- site as quickly as possible. This information ranges from mating behavior under laboratory conditions to fecundity and longevity of the insect cultured (both the host and entomoph- agous insect). Factors such as superparasitism and cannibal- ism by either the host or entomophagous insect must be taken into consideration. The ideal host used in mass culturing should be readily accepted by the entomophagous insect to be reared (Debach, 1964). The host should: (1) be readily cultured, (2) have a rapid rate of increase, (3) present no serious mating problems, (4) not give off any undesirable by-prod- ucts, (5) be a general feeder, and (6) be highly resistant to disease. .According to Flanders (1949) the actual procedure in culturing the insects includes the segregation of operations, 14 15 operational factors, and handling of the populations. In the rearing of Anaphes the preparation of host-supporting medium and propagation of the host have been kept segregated from the culturing of Anaphes, either by rearing Anaphes in a different building or in a different room than that used for rearing the host supporting medium and the host. Opera— tional factors, such as temperature and humidity, have been arranged so that it is possible to complete a generation approximately every 10 days. Mass rearing Anaphes could possibly be accomplished using either of two basic techniques: (1) removing CLB eggs from plants and confining them with Anaphes or (2) parasit- izing CLB eggs on the plants upon which they were laid. The following section will describe the current method of rearing Anaphes as used at the Agricultural Re- search Service Biological Control Station at Niles, Michigan, which is responsible for mass rearing Anaphes for field release. The method used at Niles for rearing Anaphes in- volves the removal of CLB eggs from plants. Mass Culturing Used §E_Niles 12.1967 The eggs used for rearing Anaphes are generally a minimum of 24 hours old. While on the plants, the eggs are dipped in a suspension of Captan (.L%), a fungicide which effectively retards the growth of fungus on the eggs while they are being stored and during the time that the eggs contain developing Anaphes. Once the eggs have been treated 16 with fungicide, each individual egg is removed by placing a dissecting needle next to the egg, gently lifting it from the plant, and placing the egg on a glass coverslip (35 mm x 30 mm). Normally 150 eggs are placed on each coverslip in this manner. The coverslip, which is within a small (50 mm x 12 mm) petri dish, has petroleum jelly on the bottom of it so that the coverslip will not move about in the dish while the eggs are being transferred. The petri dish has moist filter paper on the bottom and a tight fit- ting top that helps keep moisture within the dish. Currently, Niles is developing a technique for egg removal from plants which eliminates the need for hand picking. This particular technique not only removes CLB eggs from plants but it also ”desticks" the eggs. When handling the parasites, a Thermolyne Laboratory Light, with a fluorescent tube mounted behind a plate of transluscent plastic, is used in a partially darkened room in which parasites are handled. The photopositive parasites are attracted to the light and can be easily handled with a small brush or vacuum-operated collecting pipette. The physical handling of Anaphes originally was done with a fine camel hair brush, but a soft sable hair brush is now used. The parasites become entangled in the bristles and in this manner can easily be transferred. A micro— aspirator is used also. The microaspirator consists of a piece of tygon tubing connected to a disposable pipette 17 (15 cm long). The end of the tubing that fits into the pipette is covered with a piece of nylon organdy cloth to keep parasites from being sucked into the vacuum generator, which is a milking machine. The parasites can mate within the pipette, and when females are wanted, the pipette's open end is oriented toward the light, and as the parasites leave the pipette they are picked up with the sable hair brush. Presently at Niles, the parasites receive no food or water before being placed on the eggs to be parasitized, and they remain there until they die. They receive water from the moist filter paper in the dish with the eggs to be parasitized. At Niles, unparasitized eggs are stored at 400 F until they are needed. At present, Niles does not use eggs that have been stored for more than 2 months, and they prefer to use eggs that have been stored for no more than 6 weeks. Older eggs are not used because their acceptability to Anaphes is reduced too greatly. The parasites may also be stored at 400 F in the black pupal stage which occurs from 1 to 2 days before the adult parasites emerge. Niles prefers to store Anaphes for no more than 2 weeks. This is because there appears to be a decrease in the activity of adult parasites the longer they are stored as black pupae. 18 Use 9£_Hospital Talc 23 Fresh Eggs In the laboratory rearing of Anaphes, when 1 or 2-day— old eggs that have been removed from plants are exposed to Anaphes for parasitization, the parasites may become perma- nently stuck to the eggs. Because of the stickiness of the eggs, tests were conducted using eggs treated with hospital talc in an attempt to eliminate the problem. In the first test, 200 eggs were used of which 100 were treated with talc. All eggs were placed in the same dish and then 28 female and 14 male Anaphes were confined with the eggs. When on the talc-treated eggs the parasites continued to get stuck, but they managed to free themselves. After leaving a cluster of talc—covered eggs, they seemed to lose balance for a short period of time and sometimes fell on their backs. The parasites were left in the dish until they died. Of the dead parasites, two males were stuck to talc-treated eggs and 23 parasites, ll of which were females, were stuck to the untreated eggs. The remaining 17 females were not in contact with the eggs. Before the parasites had completed development, the dish was accidentally destroyed. In the next test, 20 untreated eggs and 15 talc- treated eggs were placed in the same dish and one female parasite was added and observed. When she contacted the talc-covered eggs, she would get off the slide and clean 19 herself; after contacting the talc she sometimes staggered and even fell on her back, often having trouble righting herself. Probably the imbalance was caused by talc parti- cles attached to the insect's tarsi, but it is possible some form of toxicity might have been involved. Twenty parasites emerged from the untreated eggs whereas nothing developed from those treated with talc, indicating that parasitization of talc-treated eggs was not accomplished. Tests using talc were discontinued for several reasons. The activity of the parasites appeared to be adversely affected by the talc and it was found that allow- ing the eggs to air dry for several hours before parasitiza- tion would sufficiently decrease the stickiness of the eggs, especially when they had been stored at 400 F. Daily Feeding_g£_Anaphes 32 Increase Progeny per Female To test the possibility that the number of eggs parasitized by a given number of female Anaphes could be increased by feeding and watering the parasites every day, the following experiment was conducted. On day one, five French female parasites were placed on each of 50 test and control eggs. The following day, the live females on the test eggs were removed, placed in a dish with moist filter paper and honey for 1 hour and then placed on a fresh group of 50 eggs. This procedure was continued until all the parasites were dead. The females with the check eggs were 20 left there until they died. The eggs used in the tests were from 1 to 2-days old and never stored for more than 4 days at 400 F before use. Tests 6 to 9 were begun the same day and the females used emerged the same day; thus, only one group of 50 eggs was used as check for the four tests. The results of all the tests are presented in Table 1. There is an increase in the total number of eggs parasitized and progeny produced but it appears that the increase is not sufficient to warrant the extra work. Since there are fewer eggs parasitized each succeeding day, and larger numbers of eggs are unparasitized when compared with the use of fresh females on the eggs, this technique would probably be most valuable when there is a shortage of female Anaphes and an abundance of eggs. Tests 1, 2, 4, 7, 10, and 11 indicate that it is only necessary to mate Anaphes on the first day of testing to obtain an adequate F1 sex ratio. Tests 6, 8, and 9 appear to indicate the opposite. Since the controls for 6, 8, and 9 had slightly more males than females develop, the unsatisfactory female to male sex ratio of the tests is due to inadequately mated females at the beginning of the tests. 21 Table 1. The effect of daily feeding and watering of Anaphes flavipes and daily transfer to fresh eggs on the number of offspring produced per female Anaphes No. Progeny Percent No. No. Anaphes 9 d Parasit- Test Day Eggs BPa Used ization l l 50 4O 5 74 16 80 2 50 24 5 34 3 48 3 50 7 4 9 2 14 Total 150 71 117 21 47 Check 50 38 5 68 7 76 2 1 50 34 5 58 13 68 2 50 23 5 38 6 46 3 50 15 4 21 8 30 Total 150 72 17 27 48 Checkb 50 34 5 51 7 68 3 l 50 20 42 16 40 Check 50 7 9 2 l4 4 l 50 36 6 69 8 72 2 50 14 4 15 2 28 3 50 0 2 0 0 0 Total 150 50 84 10 33 Checkb 50 l 6 50 2 62 5 1 50 33 5 36 45 66 2 50 0 5 O 0 0 3 50 O 1 0 O 0 Total 150 33 36 45 22 Check 50 33 5 10 67 66 6 1 50 17 5 9 8 34 2 50 5 4 4 3 10 3 50 4 3 3 4 8 Total 50 26 16 15 17 22 Table 1--Continued No. Progeny Percent No. No. Anaphes 9 d Parasit- Test Day Eggs BPa Used ization 7 1 50 28 5 25 6 56 2 50 5 4 9 2 10 3 50 O 2 O O 0 Total 150 33 34 8 22 Check 50 28 5 22 24 56 8 l 50 38 5 25 35 76 2 50 14 5 7 15 28 3 50 2 4 O 3 4 Total 50 54 32 53 36 Check 50 28 5 22 24 56 9 l 50 11 5 O 6 22 2 50 5 4 7 4 10 3 50 4 2 0 7 8 Total 150 20 7 17 13 Checkb 50 28 5 22 24 56 10 1 50 34 5 58 ll 68 2 50 7 3 12 1 l4 3 50 O l O O 0 Total 150 41 70 12 27 Checkb 50 23 5 35 6 46 ll 1 50 15 5 l7 2 3O 2 50 1 4 2 0 2 Total 100 19 2 l6 Checkb 50 39 5 61 10 78 aBP = the number of parasitized eggs that developed black pupae. bCheck - the parasites were left with the original 50 eggs until the parasites died. 23 Collecting_Container An apparatus for automatically collecting Anaphes upon emergence from petri dishes was devised (Fig. 2). The apparatus consisted of a plastic funnel (inside diameter of 91 mm) with all but the neck painted black on the outside. The neck was removed and inserted near the open end of the funnel at a 450 angle. This was done because an earlier version of the device utilizing a vertical neck did not adequately collect the parasites. Possibly the movement of Anaphes within the vertical collecting device caused inter- ference to such an extent that they fell back into the petri dish. Into the neck, held in place by friction, was in— serted the widest end of a disposable glass pipette (15 cm long). The narrow end of the pipette was closed with a small piece of cork. A piece of black cloth was placed at an angle inside the funnel so that the highest point was above the exit opening where the neck inserted into the funnel. The glass slides with parasitized eggs in the black pupal stage were put into a plastic disposable petri dish (89 mm in diameter) that contained moist filter paper. The funnel was placed over the petri dish and taped down. The parasites that emerged were collected in the pipette. Table 2 presents the results of collecting-Anaphes with this device. Most of the parasitized eggs that had no parasites .mmnmmcm pmonEm ma3m: mafiuuwadbu How cons HmCHMucoo I N musmam rune “Hume ofiummam Hmmmm umuafln wmmB wmwm muuwmflm 25 Table 2. The efficiency of the Anaphes flavipes self- collecting apparatus No. No. of Dead No. Anaphes Anaphes Anaphes in Test No. in Pipgtte in Dish Dish b No. BPa 9 d 9 d 9 d’ Emergence l 101 122 83 18 29 O O 93 2 92 116 18 7 5 13 4 75 3 64 58 7 3 3 1 1 41 4 126 145 18 9 13 12 o 100C 5 32 39 4 0 2 2 O 20 6 34 47 6 2 O 3 4 29 7 61 81 6 4 13 3 O 50 8 60 71 19 5 3 1 O 50 Total 570 679 161 48 68 35 9 458 BP = the number of parasitized CLB eggs with black pupae. bThe number of parasitized eggs that had parasites emerge. CThe balance of parasitized eggs were removed before they could emerge. emerge were heavily covered with fungus. Ninety-three per- cent of the females and 70 percent of the males that emerged were recovered in the pipette. The male parasites are not as easily collected as are the females using this apparatus. Possibly the males are not as photopositive as the females or they may tend to stay close to the parasitized eggs so they can mate with the emerging females. Several tests were conducted to discover whether mating occurs within the collecting apparatus. The Anaphes 26 used in the test group were removed from the pipette and put directly on the eggs. combination of the following: pipette, placed in a mating capsule, The controls consisted of one or a Anaphes taken from the and then put on eggs; or Anaphes taken from a 50 x 12 mm petri dish, placed in a mating capsule, and then put on eggs. The Anaphes placed in mating capsules were left there for 1/2 to 1 hour. The results of these tests are presented in Table 3. Table 3. The percent female offspring from Anaphes flavipes collected and mated in the collecting apparatus Controla Dish to Capsule Test Control Directly from Pipette Pipette to Capsule Sex Ratio of Parents Sex Ratio of Parents in Pipette % Fem. in Pipette ‘% Fem. % Fem. (9:d) Offspring (9zd) Offspring Offspring 8:1 78.5 7:1 80.3 83.9 14:1 75.3 ... .... 0.0 6:1 28.3 4:1 0.0 ... 8:1 35.2 7:1 67.7 ... 2:1 78.8 1:1 82.3 ... 2:1 84.8 1:1 82.3 ... 11.5:1 52.8 10:1 90.0 ... aThe sex ratio used for these controls was not recorded. 27 The results of the tests indicate that mating does occur within the collecting device, although it is unknown whether it occurs within the dish, the pipette, or both. The percentage of females to develop from adults placed on eggs directly from the pipette is similar to the controls for tests 1, 5, and 6. The controls for tests 2 and 3 had no females develop even though females (75.3%.for test 2 and 28.3% for test 3) developed from eggs on which adults were placed directly from the collecting pipette. It appears that not only is a large number of the emerging females collected by this device, but that sufficient mating can occur within the pipette to preclude further measures to insure mating. Pagasitization_£g the Dark Several tests were conducted to discover whether Anaphes will parasitize CLB eggs in total darkness. A female and three male Anaphes were placed in a dish contain- ing 20 eggs which was then covered with a cloth and placed in a closed cabinet, to keep light from reaching the para- sites and eggs. The controls consisted of one female and three male French Anaphes on 20 eggs in daylight. No para- sites developed in the eggs that had been in darkness where- as 20 parasites were obtained from the eggs used as controls. The age of the eggs used in both the test and control groups was not known but both were from the same group of eggs so the age of the test and control eggs was the same. 28 Because of the inadequate number of parasites avail- able for the previous test, Test 2 was conducted, using pots with glass globes and eggs left on the plants. Although the age of the eggs was not known, it was the same for both the test and control groups. The control was left with access to daylight while the test group was covered with two layers of black cloth and placed in a cabinet. The space between the closed doors was sealed to insure total darkness within the cabinet. The cabinet was opened once, 2 days later, long enough to water the plants. Eighteen female and five male Anaphes (French) had been placed in each of the con— tainers at the beginning of the test. The results are presented in Table 4. Table 4. The parasitization of cereal leaf beetle eggs by Anaphes flavipes in total darkness (Test 2) No. No. No. No. Eggs Percent Females Males Test Eggs Parasitized Parasitized Emerged Emerged Darkness 104 15 14.4 8 6 Daylight 92 37 40.2 45 30 29 Although not conclusive, the tests indicate that Anaphes does not parasitize as effectively in total darkness as under normal photoperiods. Some of the parasitization . under "total darkness" for Test 2 could have taken place while the parasites were being added and before the con- tainers were placed in total darkness and/or when the plants were watered. Containers Used for Mass Culturing Anaphes while CLB Eggs Are .QE the Plants To save time in rearing Anaphes by leaving CLB eggs on the plants (and thereby avoiding hand picking), a number of rearing containers were tested. The procedures used with the various containers were similar. Wheat or barley was planted in soil placed in the container. Once the plants were tall enough (3 or 4 inches), beetles were confined on them and allowed to oviposit. Before the beetles were placed in the container, the soil was covered with white sand, facilitating removal of the beetles. After oviposi— tion, the beetles were removed, the eggs were counted, and parasites in a gelatin capsule were dropped into the con- tainer. After 7 or 8 days, the plants were removed one by one and those eggs that did not hatch were checked for parasitization. The eggs were allowed to develop, and those with black pupae were counted. The parasites were counted and sexed on emergence. 30 The first container used was a clear plastic box, 14 x 10.5 x 6.5 inches (Figs. 3 and 5). In the cover, also made of clear plastic, a hole approximately 10 x 8 inches had been cut and covered with a piece of fine white cloth to keep parasites from escaping (the top was taped down dur- ing usage). The bottom of the plastic container had six slits approximately 1/6 of an inch wide equally spaced along the length of the bottom to allow water into the soil within the plastic container. In Table 5 this container is referred to as the "large" container. Another plastic box used was similar to the above in that it was rectangular and the plants were allowed to grow in it (Figs.4 and 5). The bottom was a gray plastic flat (8.5 x 5.5 x 2.5 inches) while the top consisted of a plexi— glas box that fit inside the bottom. The top had three, 2-inch holes (one on the top and one on each end) covered with fine cloth to keep parasites from escaping. The top was placed over the plants and pushed into the soil of the plastic container, forming a tight seal. Beetles were placed in the plastic box by dropping them through the hole in the top. They were removed with an aspirator. Parasites were released in the small plastic box by dropping a gelatin capsule containing the parasites through the hole in the top which was thereafter covered with the fine cloth. In Table 5, this small plastic box is referred to as the "small" container. Although the dimensions of 31 Figure 3 — The "large" mass culture box without its top. Figure 4 - The "small" mass culture box. .N 006 um ammo owuwuwmmumm on» no ommuouu Shop mcoH on 056 663.00comH080 Happen GHNHAO ou ouudwmmm .60>Hmunb uo: oocomuoamw .6mx06 uo: 0H¢3 mcaummmuo 060836 .czbcxcs mmcv .ommnm xuman muHmmumm nuH3 mmmm mac «0 Hones: way an ammo mac mo Hones: Hmuou any mcwod>wo ha mafiamunb NH cowumufiudumumm ucmuuom used .Nacuouaou 0:» ca 606: vacuum 0Hmsvu Hem omuoso on uouwumudg Hm mo gonad: came 0:93 .ommam xUMHA camon>uu van» 6000 noNNuHumunm Mo Hones: can u mmm I32 .. o.HH .... ... ovN 6... NH N N . 6.. 66 wgoHo .. o.NN .... ... ooHH 6... 6H N 6 6.. 6N 666H6 to COOH 0000 0.. “Odd ”.00 OH h dd ”00 OH.” afiHw 66 H.6 66.6 6H N6 66 6H N 6H N 6N unoHo N6 ... 66.N 6N HNH NNH 6H N 6N N NNN ogoHo 6N ... 6N.e 6H 666 NvH «H N am N 6H6 onoHo HN H.N oH.m 6N N6 H6 6H N oH H 66H HHaam He ... No.6 NN mNHH mmH 6H N 6N H va HHNEN 6e ... HN.6 N 6N 66H oH N HN N 6HN HHNEN 6H o.N NN.H ow m 6N HH N 6H H N6H HHmsm 6N ... 6H.N H 66H Nm 6H N 6H H 66N HHNEN 6H 6.N NN.H HN 6H NN NH N NH H oNH HHNEN NN 6.N oN.N N NN NN NH N 6H N «NH , HHuam 66 N.NH 66.6 N6 66 66 6H N oH N HoH HHNEN 6m ... 6N.N ... N... N6 6 N 6N N NeH HHNEN NN 6.6 o6.N 6N N6H 66H N N on N 666 HHNEN oH ... HH.N 6 6H NH oN N N H 66H HHuam NN ... m6.6 N NHN oNH 6H N NN N NeN HHNEN 6N ... 6N.N N moN N6 NH N 6H H N6N HHNEN 6 m.N No.H NH HN 6N 6H N NN N N66 HHNEN 66 ... 66.6 ... 6... N6 oH N NH N NmH HHmam NN ... NN.6 ... N... mN NH N «H N NNN HHmam NH 6.H 66.H NH NH HN .6 N 6H N 6NH HHmsm NN 6.6 66.N ... 666H ooH 6H N 6N 6.. N66 omuma 6N N.N 6N.6 66 NoH 66H . 6 N mN 6.. 66H omumq NH N.N 6N.N N 6N 6N 6H N 6H N oNN «mung «e 6.6 oN.6 NN NvH 66 oH N 6N N HNH omumq 6N 6.N NN.6 NN ooH Nm NN N.NH N NoN omuoq HN 6.6H oH.N N6 NHN . NvH oH N 6N N ooN mmumH NN 6.NH 6N.6 NNN NN H6H 6N N 6H 6.. 666 omumH NN o.NN 6N.HH 66 NNN moN , 6N N 6H N 6H6 omumH 66 6.6 No.6 H6N NHN H6N NH N Nm 6 ocN omuaH N6 N.oH oN.6 .. 6666 Now oH N 66 6.. NNN «mama 6e o.oH oN.6 NNN N6N cmN NH N 66 N (oNN owuuH 6N 6.NH NN.N 6N ooH NoH 6N N NH N N6N omuaq usoHumuwunmumm Hm 66069 b a manna ouuoouum amuuum.vc6 Huhauv noon Hocwuucoo unmoumm .oznamu: «\Nm saws xOMHn sud: «\ume no: «:4 .oz vm‘ man man .02 omha «NNN .02 HN .oz ummu .0: ago .03 |llv mm£m6C4 mo mcwmuum Hwy cmwbmduompw 696 Aug nucouh Dada: .nuufiaaucoo ounuunu Inna acououuuv conga no Goadudmaoo 4 .m manna 33 this and the large plastic box are different, the area enclosed by both boxes is similar. This small plastic box has advantages over the first one in that a tight fit between the top and bottom is obtained without having to tape the top down and it is smaller and easier to handle. With the present cages used for rearing the CLB, the small plastic box is more conve- nient, since it can be placed within the beetle oviposition cages. In addition to the above plastic boxes, lantern globe-covered plastic pots were used (Fig- 5). Beetles were either confined on the plants using a glass globe or the pots were placed in oviposition cages. The soil was covered with white sand before oviposition. After oviposi- tion, the parasites (French) were placed in a gelatin capsule and dropped into the container through the globe's top which was then covered with fine cloth held in place by a rubber band. The glass globe was taped to the plastic pot to keep it from accidently being knocked off the pot. The effectiveness of the small plastic boxes versus the large plastic boxes without regard to the strain of Anaphes used was tested in terms of the number of parasit- ized eggs to develop per female Anaphes used. The Mann- Whitney U Test was used for analysis. Since the mass- culturing tests were conducted at different times, it is assumed that any generation of parasites is as good as the 34 Figure 5 — The mass culture containers: from left to right they are the "large," globe, and "small." 35 preceding and following generations. There is no reason to suspect that one generation of parasite is better than another unless separated by many generations. In such a situation there could be differences due to inbreeding and/or selection of a laboratory-favored gene pool. The same assumption has been made in regard to the CLB eggs that were used in the various trials. The analysis of the data revealed that there is a difference between the plastic boxes in favor of the large plastic boxes at the 5% level of significance but not at the F% level. The Mann-Whitney Test also was used to test for any significant difference between the YUgoslavian and French strains of Anaphes that were used in the large and small plastic boxes. At the 5% and 1% levels of significance the analysis revealed no differences in the strains in terms of the number of CLB eggs parasitized per female used. In addition to the above tests, the MannAWhitney Test was used to test the following parasite strain—plastic box combinations: (1) YUgoslavian strain of Anaphes in the large box vs. the Yugoslavian strain in the small box; (2) French strain of Anaphes in the large box vs. the French strain of Anaphes in the small box; (3) French strain in the large box vs. the YUgoslavian strain in the large box; (4) French strain in the small box vs. the Yugoslavian strain in the small box; (5) French strain in large box vs. the 36 YUgoslavian strain in small box; (6) French strain in small box vs. the YUgoslavian strain in the large box. The tests showed that at the 5% level of significance there was no difference between the pairs that were tested except the YUgoslavian strain in the two different plastic boxes (1) favoring the large box. The YUgoslavian strain in the large plastic boxes (6) was favored over the French strain in the small plastic boxes. The use of French Anaphes in lantern globes was tested against the use of the YUgoslavian and the French strain in both the large and small plastic boxes. The use of the lantern globe versus the use of the YUgoslavian strain of Anaphes in large plastic boxes was not analyzed because there were not enough samples to conduct the analysis. According to the MannNWhitney Test, at the 5%.level of significance there was no difference in the use of the French Anaphes in the lantern globes and the use of the French strain in either of the other plastic boxes. This is true also in the comparison with the YUgoslavian strain in the small plastic boxes. Visual examination of the data indicated that there was no difference for the comparison of the French Anaphes in the lantern globe and the YUgo- slavian strain in the large plastic boxes. 37 Table 6 includes the results of rearing both the Yugoslavian and French strains of Anaphes by the method that is currently in use at Niles; that is, in 50 x 12 mm petri dishes with tight-fitting lids. There are a few differences however. Niles uses 150 eggs and 10 female parasites per dish whereas the number of female parasites and the number of eggs used per dish in Table 6 varies from dish to dish. The data of Table 6 will be used to compare the effectiveness of the two basic methods of mass rearing Anaphes. The Mann-Whitney Test, indicated no difference between the Yugoslavian Anaphes and the French Anaphes reared in the 50 x 12 mm petri dishes in terms of the number of parasitized eggs per female parasite used. The same analysis also indicated that there was no difference between the use of the plastic boxes and petri dishes. Furthermore, the effectiveness of the Yugoslavian strain in the plastic boxes was compared with the Yugoslavian strain in the petri dishes as well as the effectivenss of the French Anaphes in the plastic boxes compared with the French Anaphes in the petri dishes. The MannAWhitney Test indicated no differ- ences in the comparisons. Discussion Although it is possible to increase the number of parasitized eggs per Anaphes female by putting them on fresh eggs each day, the effort does not seem to be warranted on the basis of the number of eggs used and wasted, especially 38 Table 6. A comparison of rearing both Yugoslavian and French strains of Anaphes in petri dishes The Number of Parasitized Eggs Per Tested Female of the Specified Strain Rank No. French Yugoslavia l .78 .82 2 1.50 .83 3 2.00 1.16 4 2.14 1.33 5 2.33 1.42 6 2.40 1.46 7 2.53 2.20 8 3.50 2.20 9 3.50 3.14 10 4.13 3.25 11 4.20 3.80 12 4.36 3.80 13 4.40 4.14 14 4.60 4.84 15 5.10 5.00 16 6.40 5.40 17 6.40 6.00 18 6.60 6.16 19 6.80 7.00 20 7.00 7.33 21 7.20 12.00 22 8.00 after the second day when very few eggs are parasitized. The only time this technique might be of value is when there are very few female Anaphes and an abundance of eggs. Hospital talc decreases stickiness of fresh eggs to parasites but it is not efficient since it causes erratic parasite behavior; i.e., they may stagger and fall on their backs. Furthermore, air drying eggs for several hours before usage decreases stickiness sufficiently. 39 The pros and cons associated with both techniques of rearing Anaphes (removing eggs vs. leaving them on plants) are numerous. The use of the plastic boxes or globes has the advantage that it is not necessary to handle the eggs. There appears to be no great problem in introducing para— sites to the eggs with the use of the plastic boxes or globes. It is also possible that with the use of the plastic boxes, feeding and watering of emerging parasites would be no problem since they may get what they need from within the boxes. Also, parasites probably do not become stuck to eggs as readily since they will not come into con- tact with the eggs as often as is the case within the petri dishes where the eggs are concentrated. In the case of the field release of parasitized eggs on plants, one would only need to place the leaves bearing the pupae into the field. On the other hand, there are drawbacks to the use of the plastic boxes and globes for mass culturing. Although the boxes and globes appear to be as efficient as the petri dishes in terms of the number of parasitized eggs to develop per female used, they take up much more space than do the petri dishes now in use. It is much more difficult to make accurate egg counts using the plastic boxes or globes. It would be necessary to have a table that would give an estimate of the number of eggs laid by a given number of beetles of a given age for a given length of time. With such a table, estimating the number of eggs within a plastic 40 box or globe would not be difficult. Related to this is knowing how many parasites to introduce into the box, but this could be overcome once a table as mentioned above is developed. One of the biggest drawbacks to the use of the petri dishes (the technique currently used for mass culturing Anaphes) is the great length of time necessary to remove the eggs from the plants by hand prior to parasitization. Thus, with the two techniques, the one best suited for use will be determined by the time and space requirements and by the manpower available. The development of a technique for non- manual removal of eggs from leaves would make the petri dish method (removal of eggs from plants) far superior to the use of the much larger mass rearing boxes or globes. Currently, using either the plastic boxes or globes or the petri dishes for rearing Anaphes, there are problems in storing both parasitized and unparasitized eggs. Fungus has been one of the big problems in the storage of CLB eggs. At present, it is not known how Anaphes overwinters: if this can be determined, the problems involved in storage of Anaphes may be greatly diminished. It must be learned whether the parasites overwinter as adults or immatures and whether or not an intermediate host is involved. STORAGE OF UNPARASITIZED EGGS CLB egg storage is important in the mass culture of Anaphes for several reasons. Anaphes develops in approx— imately 10 days; thus, to maintain an active culture, a continuous egg supply is needed. Since there may be fluctu- ations in the production of CLB eggs, there are times when sufficient fresh eggs are not available for the continuous rearing of Anaphes. Stored eggs must be used during these times until beetle oviposition increases. When mass production begins, a large number of eggs must be available to build up the parasite population. In most instances, this means stockpiling the unstung CLB eggs at least a month before the actual parasite population build-up. A rapid increase in the parasite population can be easily achieved when there is a sufficiently large number of eggs with which to work rather than having to wait for the day-to—day production of fresh eggs. Storage over Water During the storage of eggs on glass slides or glass coverslips desiccation may occur if enough moisture is not maintained within the storage dish. Consequently, the first storage tests involved inverting slides with eggs over water. This was done in the belief that water would condense on the 41 42 underside of the slide with the eggs, thus providing suffi- cient moisture for the eggs during storage and eliminating the need for the periodic addition of moisture to storage dishes. In Test 1, 576 unparasitized CLB eggs were put into storage at 400 F. The eggs were placed on slides that were inverted and mounted in the side of a vertical piece of balsa wood (Fig. 6). The mounted slides were then placed in a transparent plastic container (10.5 x 7.5 x 4 inches) with about 1 inch of sterilized distilled water in the bottom and covered with a tight fitting lid. The eggs, not treated with a fungicide, were removed at intervals and subjected to parasitization by Yugoslavian Anaphes. In Test 1g French female Anaphes were used due to the lack of Yugoslavian Anaphes. The results are presented in Table 7 and Fig. 7. For Test lg, there was a control consisting of 50, 2-day-old eggs that had been in storage 7 days at 400 F upon which were placed six female French Anaphes. From 34 eggs, 44 parasites emerged. This control indicates that the failure to obtain parasite development from eggs of Test lg was caused by some factor or factors other than the para- sites that were used. There were no other controls. The failure to obtain parasite development in Test ld were partly caused by the fungal growth since fungus may inter- fere with oviposition and/or emergence of parasites. 43 . . .6N66 oNH On a: umum3 umbo owuum>cH,mmmw 0Hummn mmmH Hmmnwu mCHHONm How 606: H0CH6ucou I m muanN 30H> uconm Hmum3 GOHAHNMHQ UONHHHHmum (illldmilrallllll ( . a E E noun _1 L 33> 66$ mmmm QQU null-ll." ""0 "m (.I €53 0313 60.9.6 16.1.1.- E E E NNCHMuCOO 0.3663 ucmummmcmun. km. E NNN. .m. 6003 6613 ace 44 Table 7. The influence of the length of cereal leaf beetle egg storage while inverted over water on subsequent parasitization by the Yugoslavian strain of Anaphes (Test 1) No. Eggs With Days Fungus When Percent Test No. Eggs Taken From No. 9 No. No. Parasit- No. Eggs Stored Storage Used BPa F1 ization la 74 15 .. 10 25 41 33.8 lb 75b 23 25 10 20 30 29.9 1c 98 35 7 10 29 37 29.6 1d 75C 42 48 10 0 0 00.0 1e 100d 69 20 8 16 25 16.3 1f 100e 82 21 10 2 2 2.5 1g 54 176 6 6f 0 0 00.0 aBP = the number of parasitized cereal leaf beetle eggs that developed black pupae. bEight eggs were not used because of too much fungus. CSeven eggs were removed because of fungus or because they had collapsed. dTwo of the eggs were not used because there was too much fungus on them. eTwenty—one eggs were not used (79 used) because of profuse fungus. fThe French strain of Anaphes was used. This set of tests, even without controls, indicates that eggs can be stored for at least 35 days at 400 F while inverted over water with only a slight decrease in the per— cent parasitization, but after 35 days of storage there is a rather sharp decrease in the number of parasitized eggs. 45 In Test 2, unparasitized CLB eggs were again inverted over water on glass slides with 50 eggs per slide. They were a maximum of 2—days—old at the time of storage. Six female French Anaphes were used on each set of 50 test and control eggs. The control eggs were either fresh or stored for a maximum of 6 days before use. The results are pre— sented in Table 8 and graphically in Figure 7. The results of Test 2 indicate a steady decline in acceptability of eggs to parasitization as the length of storage increases, although the results of storage for the first 60 days are within the range of results for the con- trols of Tests 2a to 2e. Both sets of tests indicate that CLB egg storage by inversion of eggs over water works well for the first 30 to 60 days. But fungicide treatment should be given before storage of the eggs if this technique is to be used since fungus may pose a problem as storage time increases. The results of the two tests indicate a difference between the Yugoslavian and French strain, and their accep- tance of stored CLB eggs. The eggs appear to be less accep- table to the Yugoslavian strain than to the French strain (Fig. 7). But there is a smaller decline in the Yugoslavian parasitization rate than there is for the French strain. Although it is not known whether the observed differences are due to actual differences between the strains or to experimental factors such as the female-to—egg ratio or the 46 .06050 MomHQ pmmoam>0p umnu mmmm maummn Mama H60H00 pmuHuHmmumm mo Hmflesm mzu H mm Nb 0 0m mm H m om N om om OOH mm mm mm 6H 00 0 ma mm ma om om mm UN mg mg 0H AM OH mm 0N 0N om om No UN mm mm mm 00 NH 00 mm mm om om m0 SN 00 on ma mm mm mm mv mm mm om mm mm U0¥MOHHGD .....mTH. .0 0 .0 @ COHMTHUCD Hmmfl. UQDMOHHCD #mmH. UTHOflm .OZ soHumquHmmumm ucmoumm pmummnucb umwa mQHHmmmmO .oz mam .OZ mmmm .OZ mmmn .oz umme AN ummev mmrmmc¢ mo sHmHum nosmHm man an COHHMNHuHmmumm usmsvmmnnm so Hmumz N0>o 60HH0>£H 0HH£3 mmmHOHm mam maummn mmma Hmmumo mo cpmsma may mo mosmsawsfi 038 .m magma 47 F’iZb 7 -' 5 ----- Control (Test 2) ’i 2e W23 60-; A26 - )2C a 45 "‘ 2’. -='.: r a . «‘5 g ’ la X r 304.. i‘ 20 _ - —Test 2 (French Strain) 15 1)- Test 1 (Yugoslavian Strain a-f) ------- P ‘ kl Lid. _ _ 1"" L . . . lg‘U‘rench) I ‘7 V I " 1 _‘ 10 50 100 150 180 Days of Storage Figure 7 - Acceptability of CLB eggs to Anaphes when the eggs are stbred over water up to 176 days. 48 slightly more fungus found on the eggs parasitized by the YUgoslavian strain, the mass culturing tests previously described indicate no differences between the French and Yugoslavian strains in terms of parasitized eggs per female used. E§§.9£_Fungicides.EghRetard Growth g§_Fungus 9g Eggs During,Storage When old eggs are stored for long periods of time, fungus begins growing on them. It was believed that the fungal growth may be so great as to prevent parasites from ovipositing in the eggs, or if they are able to oviposit, then the offspring are unable to emerge. Consequently, various fungicides have been used in an attempt to eliminate the fungus. Among the fungi identified on CLB eggs were Aspergillus sp., Alternaria sp., and two species of Penicillium (Dr. Thanassoulopoulos, personal communication). Captan, Semesan, Arasan, and Phygon have been used in the following storage tests. The use of these particular fungicides was suggested by Dr. Thanassoulopoulos. The eggs, mounted in groups of 50 per 1/3 section of cut glass slide, were dipped in a bath of the given fungicide for 10 to 15 seconds and then rinsed twice in sterilized distilled water. Then, they were placed in 50 x 12 mm plastic disposable petri dishes (50 eggs per dish) and allowed to dry for l to 2 hours. Before storage, sterilized distilled water was placed in the dish to a level near that 49 of the top of the glass slide (none of the water came in contact with the eggs), the top of the dish was replaced, and thecihfliwas stored at 400 F. The controls received no fungicide treatment but were dipped three times in steril- ized distilled water except in Test 1 in which the controls were simply brought from storage and subjected to parasitism without being put through sterilized distilled water rinses. Fungicide Test l_- Captan.-—The test solution con- tained 1 g of Captan per 100 ml of sterilized distilled water (5,000 ppm active ingredients). The eggs used were all from the same group of beetles, were a maximum of 2—days old, and were stored 1 day at 400 F without special treat- ment before being placed on slides. All slides had 50 eggs before they were placed in Captan, but during the dipping process a few eggs were washed from the slides; hence, dishes 1 to 8 did not have exactly 50 eggs. When test eggs were taken from storage a control group of 50 eggs was used. Six female parasites per 50 eggs were used. After dish 3, fresh eggs were used for controls, since it was necessary to know whether failure in parasite development was due to treatment and/or storage or to infe- rior parasites. Table 9 gives the results of this test. After 102 days, the number of eggs with black pupae in the test group is very much reduced compared to the con- trols, although three eggs were parasitized after 132 days .Umummuuss N .02006 .mosmmnwem usmsvmmadm on fiuHB mmmm pmuHuHmmHmm mo Hwnfiss 0380 .mmmsm MUMHQ pmmoH0>0© awn» mmmm mHummn mmmH H60H00 pmuHUHmmnmm mo Hmnfiss man u mmfl .COHumuHuHmmumm Hmumm mmmp OH 0668 mmB pasoo msmcsmm 50 0H o¢ o o o o mN o NH o m NNH mm mfi w 0 mm o m o O NH m mw 0 MN NMH 00 m0 N N ow o o N 0 Nm 0 Nm 0 H ONH om N¢ o 0 ON N- mH o N 0H NH 0% m N NOH om m0 m N fi H N o H 0 N mm 0 N mm H0 Nfi w H MH OH «N m o wH NH 0% 0 m0 mw 0m 00 m HH «m N mm m m mN ON ¢¢ H mH 0N om N¢ N mH ON mm mm H 0 N0 H0 mN o NH mH om mg H b a b m .ucoo umma .usou umma .ucou ummB .ucoo ummB 0.ucoo umme .oz .nsoo ummB 00000mu0Emrcoz 3mm .02 mmsmcsm suHB Umuoum Mmmm mo .02 anm muH5©¢ .oz mmmm .oz mmmm mama mmmH>mHm mmcmmc4 Na coHumNHuHmMHmm usmsvwmnsm can mmmuonm mom 0Hummn mmmH H60H00 so Hmu63 UmHHHume ©0NHHHH0um HE OOH H09 AusmHomumcH 0>Huom XOmV m H mo mumH 05» um HouHQHcsH msmcsm 6 m6 smummo mo uommmm 058 .m 0HQmB 51 of storage. This test indicates that fungus can be effec- tively controlled on stored unparasitized CLB eggs with 1% Captan. In the following tests, the eggs used were a maximum of l-day old when removed from oviposition cages. They were stored for 4 to 11 days at 400 F before being used (during this time, they received no fungicide treatment). The con- trols were stored the same length of time and brought from storage with the test eggs. In addition, some test dishes had a second control composed of eggs stored no more than 9 days at 400 F that checked the effectiveness of the para- sites. Five French female parasites were used in each dish of 50 eggs. Fungicide Test E_— Arasan.--For use in this test, .1 g Arasan 75 by Du Pont was mixed with 100 m1 of steril- ized distilled water (750 ppm active ingredients). Both the controls and test groups consisted of seven slides of 50 eggs each. The results of the test are in Table 10. Dishes 5, 6, and 7 had a second set of controls used. When the results (see Table 10) of the tests and first con- trols are compared with the results of the second controls it is evident that the failure of the test and the first control eggs to develop more black pupae is not the result of weak parasites but instead is due to some other factor. 2 5 .00msm 300H3 O0Q0H0>0© 3033 0OO0 0Hu003 mm0H H00H00 O0NHNH00H0Q mo H03E53 03p .0HOH3300 630000 033 How 0H0 03H000N 0390 .O0u00nucs .0660 U .0030OH0E0 3305000350 on 33H3 0OO0 O0NH3H00H0Q mo H03E03 0390 H me .COHumuHuHmmHmm N0um0 0>06 OH 0605 063 03500 msmcsmm OH OO H6 NN m Om 00NIN O O O O O O O O O6 O OO OO Om Om N OH mm O Om Om O OO 00OIN m OH O O O O O O Om OH OO OO Om OO O 6H O6 6N ON m mm 00mIN m OH O m O O m H O6 H MN mN O6 om m 6 NH N O O O O m mm 6 OO Om N6 om 6 OH N6 mH ON O H ON OH Om mH m6 m6 Om om m O HO N O O O ON O ON N ON ON O6 om N O OO N HO O N ON Hm ON O OH OH N6 O6 H b O b O .pcoo H009 .3300 3009 .3300 3009 .3300 3009 0.3300 3009 .02 .3300 3009 0030OH0EOIQOZ .oz mfiOdsm 33H3 O0Houm fimmOm mo .02 30HQ mustm .oz 3 0OOm .oz 0OOm 0N00 00MH>6HO 003QM3¢ N3 COHHMNHuHmmumm 3305000350 636 0O0Hou0 OO0 0Hu003 mm0H Hm0H00 so A0330HO0NO3H 0>H300 Ema OmNV H0303 O0HHH00HO O0NHHHH000 HE OOH H0O O H. mo 0umH 033 30 HouH3H3cH 05Onsm 0 00 3000H< mo 300mm0 039 .OH oHnmN 53 Although .1 g Arasan has controlled the fungus effectively, parasitized egg development has been consistently less than for the controls except for dish 1. Fungicide Test §_— Phygon.--In this test, .1 g of Phygon per 100 ml of sterilized distilled water was used (500 ppm active ingredients). Both the test and control groups consisted of seven slides of 50 eggs each. The results of the test are presented in Table 11. The results indicate that .l g of Phygon is not a very effective fungi- cide for this work in terms of fungus controlled. Fungicide Test 4_- Semesan.--In the fourth test, .1 g of Semesan per 100 ml of sterilized distilled water was used (286 ppm active ingredients). The results are pre— sented in Table 12. I The fungicide appears to have retarded fungal growth at least for the first 70 days of storage and at the same time allowed more eggs to develop black pupae and adults then the stored controls. But the test results are not good when dishes 4, 5, 6, and 7 are compared with the controls using fresh eggs. Fungicide Test E_- Captan.--In this test, .1 g of Captan per 100 ml of sterilized distilled water was used (500 ppm active ingredients). Ten slides were used with 50 eggs per slide for both the control and test groups. The results are presented in Table 13. 54 .0H033300 630000 033 303 030 03H0003 0390 .603003333 I . 3o 3 06 .0030O30E0 3303000330 03 3333 0OO0 60033300303 30 303333 0390 .00333 300H3 6030H0>06 3033 0OO0 0H3003 300H H00300 60033300303 30 303333 033 H mm 3 .30330033300303 30330 0N06 OH 0603 003 33300 05O3330 mH NO O m6 Hm H om 00N IN O O O O O O O O OO Om OHH OHH Nm Om N O Om O HN N O on 00O IN O O O O O O O O Om Om NO NO on OO O OH mm O Om Om O OO 00m IN O O O O O O O O Om m6 mO mO om OO m 63 O6 O 6N ON m mm 006 IN O O O O O O O H Hm 96 OO OO Hm Nm .6 N N O O O O m O HO HO m6 m6 Nm Hm m 6 NH N OH O O OH OH N6 Nm Om Om O6 mm N m Hm O m6 O O OH Hm 6m O6 OH OH Om Om H b m b O .3300 3009 .3300 3009 .3300 3009 .3300 3009 6.3300 3009 .02 .3300 3009 00030O30Emi3oz mm .02 003O3sm 3333 603030 .mmOm 30 .02 3033 03H063 .oz 3 0OOm .oz 0OOm 0N03 00m3>033 0033033 N3 30330033300303 3303000330 630 0O03030 OO0 0H3003 300H H00300 3o 303303603O33 0>3300 Ema OOmV 30303 60HH33036 6003333030 HE OOH 303 O H. 30 0303 033 30 303333333 03O333 0 00 3OON3O 30 300330 039 .HH 03309 55 .0H033300 630000 033 303 030 0333003 0390 .603003333 300 0 6 .003003030 3303000330 03 3333 0000 60033300303 30 303333 0390 .00333 300H3 6030H0>06 3033 0000 0H3003 300H H00300 60033300303 30 303333 033 H m m 3 .30330033300303 30330 0306 OH 0603 003 33300 0303330 mH NO O m6 Hm H om 00NIN O O O O O O O O om mm OHH OHH Nm Hm N O Om O HN N O Om 00OIN O O O O O O O O om O6 NO NO Om Om O OH mm O Om Om O Om 00mIN O O H OH O O O m Om mm mO mO Om mm m 63 O6 O 6N ON m mm 006IN m m N OH H O m HH O6 ON OO OO Om om 6 O m m m m O O 6 O6 Hm m6 m6 om Om m 6 6H NN Om H O HH mN om ON Om .Om om O6 N m O6 mH 6N N O ON mm mN 6H mH mH O6 Hm H b N b N .0000 0009 .0000 0009 .0000 0009 .0000 0009 6.0000 0009 .02 .3300 3009 00030OM0Eml3oZ 33m .02 0030333 3333 603030 .WOOO 30 .02 3033 03H363 .03 0003 .02 0003 0303 00mw>033 003M033 N3 30330033300303 3303000330 630 0003030 000 0H3003 300H H00300 30 303303603033 0>3300 E33 OONV 30303 60HH33036 6003333030 HE OOH 303 0 H. 30 0303 033 30 303333333 030333 0 00 3000300 30 300330 039 .NH 0H309 .0H033300 630000 033 303 030 03H3003 0383 .60030HH00 0303 300E 630 030333 3333 6030>00 3H3>003 0303 0000 HH0 0030003 333 303 003 3003 03390 .603003333 N .33006 .003003060 3303000330 03 3333 0000 60033300303 30 303833 0390 .00333 300H3 6030H0>06 3033 0000 0H3003 300H H00300 60033300303 30 303833 033 H 333 .30330033300303 30330 0306 OH 0608 003 33300 0303330 56 0Om 0Om OmH OmH Om Om OH mH NO O m6 Hm H om 30OIN O O O O O O O O om Om NHH NHH Om Om O OH OOH O m6 6 H Om 30OIN O O O O O O O O om Om mOH mOH Om Om O OH OO O H6 NN m Om 30NIN O O O O O O O O O6 O6 HO HO O6 O6 N OH mm O Om Om O Om 30OIN O O O O O O O O O6 Om NO NO O6 om O m mH O O O O O O O6 Om ON ON O6 Om m O O m OH O O O HH O6 O6 NO NO O6 Om 6 OH O6 mH Nm O O NN ON O6 OH O6 O6 O6 O6 m N mm m N6 O O mm mm N6 6H 6m 6m Nm O6 N m ON O O m H 6H m Hm m mH mH Hm Om H b O b O .3300 3008 .3300 3008 .3300 300a .3300 3008 6.3300 300B .02 .3300 3009 00033330E3|3oz 333 .02 0030333 3333. 603030 0003 30 .02 3033 03H363 .02 0003 .02 0003 0303. - . 003630H3 0033033 33 30330033300303 3303000330 630 0003030 000 0H3003 300H H00300 30 303303603033 0>3300 E33 OOmv 30303 60HH33036 6003H33030 HE OOH 303 0 H. 30 0303 033 30 303333333 030333 0 00 303300 30 300330 033 .mH 0H308 57 This concentration of Captan is fairly good in retarding fungal growth and allowing satisfactory parasitism for the first 48 days of storage. After this time the fungal growth increases greatly and the number of eggs to develop black pupae drops off sharply. To analyze the effectiveness of Phygon, Arasan, Semesan, and Captan (.l%) the test results were subjected twice to the two—way analysis of variance. In the first, the number of eggs to develop black pupae and the length of storage were analyzed; in the other, the number of eggs to develop fungus and the length of storage were analyzed. In both two-way analyses, the results of Arasan dish 5 were not used so that comparisons would be of equal size and of sim— ilar dates. In the other analysis, all dishes were assumed to have 50 eggs. The first Captan Test (1%) was not con- sidered in the analysis primarily because more female para- sites were used per 50 eggs and because the controls that were used were generally not in storage for the same period of time as the test eggs. In terms of the development of parasites in the stored eggs, analysis revealed at the 5% and T% levels of significance no difference between the tests and controls used. But in terms of fungal control, the analysis revealed that there is a highly significant difference (1%.level) between Arasan and the other treatments. Thus, from the test results, it appears that at the rates tested Arasan is 58 the best of the fungicides in terms of fungus control. Analysis also revealed no difference between Semesan and .l% Captan in terms of fungus control; Phygon proved to be the worst of the fungicides tested. Since the analysis indicated that none of the fungi- cide treatments decreased the development of parasites in unparasitized eggs held in storage at 400 F, and that fungus was effectively controlled at least by Arasan, .l% Captan, and Semesan, the failure to obtain larger numbers of para- sites from stored unparasitized eggs as their length of storage increased was not the result of fungus but was due to some other factor or factors. Furthermore, the results of these tests were not caused by "poor" female parasites being used since in those cases where a second control of fresh eggs was used, much larger numbers of parasites developed than in the test eggs. Freezing_Eggs for Long Term Storage Currently storage of unparasitized CLB eggs is conducted at 400 F. Possibly temperatures lower than 400 F might extend the CLB egg storage time. Unparasitized eggs, mounted on glass slides within petri dishes with moist filter paper, were put into storage at approximately -120 F. Eggs were stored 2—l/2 and 14 hours, and 9, 20, 33, and 331 days. There were control eggs for only those eggs stored 20 and 33 days at -120 F. Table 14 presents the results of the test. 59 Table 14. The parasitization success of Anaphes flavipes on cereal leaf beetle eggs stored at -120 F for 2.5 and 14 hours, and 9, 20, 33, and 331 days No. F1 Anaphes Time Eggs Stored No. Eggs Females Males 2.5 hrs 25 41 11 14.0 hrs 25 42 12 9 days 20 O O 20 days 20 O O 33 days 20 O O 331 daysa 80 0 O Unstoredb 25 22C aBecause 9 eggs collapsed during storage, only 71 eggs were actually subjected to parasitization. bThe unstored eggs were controls only for the eggs that were in storage 20 and 33 days. CThis is the total number of Anaphes flavipes that emerged. The females actively parasitized the eggs stored 2.5 and 14 hours; they reacted as if the eggs were fresh. Anaphes appeared to normally "sting" the eggs stored 9, 20, 33, and 331 days but parasites failed to develop in the eggs. It appears that CLB eggs may be suitable for parasite devel- opment after a very short time, a matter of hours, at -120 F but that for any length of time in storage, this temperature is of no value since no parasite development was obtained from eggs stored for 9 days or longer at -120 F. CLB eggs freeze at approximately 70 F (Dickler, personal communica— tion). 6O Thg_y§g_2£_Extender for Egg Storage Since direct freezing of eggs appears to show no promise for storing CLB eggs, the use of other materials to allow successful freezing of eggs has been tried. The basis of this storage has been the use of extender that is cur- rently used in the storage of bull semen (H. D. Hafs, K. T. Kirton, C. Desjardins, unpublished report, 1962). This material is an egg-yolk—citrate extender containing glycerol. The extender usually consists of a portion without glycerol and an equal volume of the same extender that contains twice the amount of glycerol that is desired in the final mixture. The final mixture used here contains 7%.glycerol. Tests were conducted to discover whether Anaphes would develop in eggs treated with the extender. The unpar— asitized CLB eggs were immersed in extender and stored at 400 F. When brought from storage the eggs were either allowed to dry and then subjected to parasitism or they were given 1 to 2 minute rinses in physiological saline before Anaphes were placed with them. The tests indicated that Anaphes could develop from extender—treated eggs but that development was not satisfactory. In all instances the con- trols gave satisfactory parasite development. The test results are summarized in Table 15. The tests indicated that a simple rinsing of eggs in physiological saline was not sufficient to allow satisfactory parasite development. Because of the results, treated eggs 61 .60630003 303 003 30H0>06 03 00>30H 30 303833 0386 .0H3003 300H H00300 H 3300 .00333 300H3 6030H0>06 3033 0000 0H3003 300H H00300 60033300303 30 303833 033 H 333 .306303x0 033 33 60030883 0H333 3 0O6 30 603030 0303 0000 033 0833 30 33030H 0800 033 03 03380 00030 3 0.. 0 0 0 0 mm 000 0030000330 063 0002 mm 0 3 m 0 00 003 0000000330 063 00030 3 0.. 0 0 o 03 00. 000 0000000330 03 0002 3m 0 m m 0 00 003 0000000330 00 000030 0 0m 0 0 0 m 06 30 0000000330 03 000030 0 m 0 0 o 6 mm mm 0030000330 0N 00030 3 0.. o 0 o 0 mm 000 0030000330n0oz 0002 m 0 0 o 0 00 003 00000003301002 330830038 0003083 30\630 b O 333 6003 0003 030630303 330830038 033H00 30H0>03 03 .IIIIII .oz mmmmmmfl. 30 33 03303 306303x3 00>003 030 .02 33 .03 .oz .02 330830033 033H00 H00300H030333 033 3303333 60033300303 0303 3033 30 30330033300303 030303 0033338 N 03 H 3H03083x03330 303 033H00 H00300H030333 33 603336 303330 0303 0000 038 .0833 30 0603303 030330> 303 3 0O6 30 603030 0303 3033 0000 0H3003 300H H00300 6030033I306303x0 30 30330033300303 .mH 0H308 62 were washed in the physiological saline for longer periods ofthm. In the next test, eggs on glass slides were put in 7% and 14% glycerated extender and stored for 11 days at 400 F. When brought from storage, they were immersed in physiological saline for 5-3/4 hours, then placed on filter paper to dry before transfer to new glass slides for parasit- ization. Each group of 25 eggs had five female French Anaphes put on it. The controls consisted of 25, l-day—old eggs that were in storage at 400 F for the same period as the test eggs. During storage, the controls had no moisture or extender added to them. All groups had some eggs develop parasites. The results are presented in Table 16. Fifty l—day—old eggs were covered with extender containing 7% glycerol and stored at 400 F for 16-1/2 hours; 50 untreated control eggs were also stored. When brought from storage, both the control and test eggs were immersed in physiological saline for 5 hours and then allowed to dry. Five female French Anaphes were used on each group of 25 eggs. From the test eggs five CLB larvae hatched and 31 developed black pupae producing 73 parasites. From the controls, 42 eggs with black pupae developed yielding 83 parasites. Test results indicate satisfactory parasite develop- ment in eggs treated with glycerated extender provided they are washed in physiological saline for 5 hours (although a 63 Table 16. Parasitization success of Anaphes flavipes on cereal leaf beetle eggs stored with non-glycerated and.7% and 14% glycerated extender for 11 days, with a 5—3/4 hour rinse of physiological saline before parasitization No. ’ No. Fl Test Eggs N0. Used BPa 9 6 Without glyceéol 25 5 7 9 1 With glycerol 25 5 22 31 28 With both portionsc 50 10 24 29 26 Untreated 25 5 20 26 32 aBP = the number of parasitized cereal leaf beetle eggs that developed black pupae. bOnly the glycerated portion of the extender (14% glycerol). CWith equal volumes of the glycerated and non—glycerated portion of the extender (7% glycerol). shorter time interval might be satisfactory) and then dried before parasitization. The extender tests were conducted to discover whether Anaphes would develop from the treated eggs so that if they did develop, storage of unparasitized eggs could be attempted using the method for storage of bull semen which with the use of extender (7% glycerated) is stored in liquid nitrogen (-l96O C). Since Anaphes can develop from extender- treated eggs, a number of eggs were treated with extender and stored in liquid nitrogen. Into each of ten, 1 ml vials used for storing bull semen were placed 25, l-day-old CLB eggs (stored at 400 F 64 1 day before use). In five vials, the eggs were left on pieces of the plant leaves and in the other five, three to four eggs were mounted on the inside wall of each vial with the remaining eggs mounted on those attached to the vial. The vials, when filled with 7%.glycerated extender, were heat sealed and allowed to equilibrate for approximately 20 hours. The vials were then stored in liquid nitrogen by Charles Cornell of the Michigan Animal Breeder's Cooperative utilizing their method for bull semen storage. Five days after storage, the five vials containing eggs mounted on leaves were removed from storage. The vials, left at room temperature for approximately 3 hours, were opened and the eggs immersed in physiological saline from 5-1/2 to 6—3/4 hours. Table 17 presents the results of the test. All the eggs were dried 2-1/2 to 3 hours after removal from saline and then the eggs were removed from the leaves and placed on glass slides. All eggs used looked fresh and ovipositor insertion was observed in two eggs of dish 4. The 25, l—day—old control eggs had been stored 2 days at 400 F before use. These eggs were immersed in physiological saline for approximately 3 hours and after drying, five Anaphes were placed on the eggs. Nothing developed from the test eggs although 14 control eggs were parasitized yielding 33 female and 5 male Anaphes. In 65 .pwcoumn omam wm>uma mao m “mmuflmmnmm mHmB mmmsep cam muson o How pmuflw ocu .mcflamm mo muQmEummnu m>Husommaoo oBu Cm>flm mHoB mmmm mmmne .maouucoo on» mum3 mmmm mmmneo .uson ¢\m “ow Honuo may a .Hsos «\H How “mayo msu paw muse: o How umuwm mnu .mcHHmm mo musmfiummuu m>au50mmcoo OBu sm>wm mHmB mmmm mmmzfim Umm m m mm .. .. mm 00 o Qv\muo m on m m mm m o mm\a-o m 0H m a mm a o o N OH OH m mm m o ¢\mnm m ea n v mm N 0 «\Hum m ma m a mm H mon>mQ mCflHmm bomb bomb mafiapcmm mmmuoum mmmm .OZ ou mm>umq CH musomfl m .02 mmmm .02 ca cmxoum ca mommaaoo .OZ nmflm maummm HO\Ucm mmmm .02 Cu mmmm .OZ mmnmmc< .oz cmmoupac pwsvfla CH mwmp m Mom mmmuoum Hmumm mmmm mnu so mmmfl>mam monmmcm mo mmmoosm coaumufluflmmumm pcm mmmm mabmmn mmma Hmmnmo so mpommmm one .NH magma 66 addition, five CLB larvae hatched from the controls and none hatched or developed from the test eggs. The remaining five groups of eggs were removed from liquid nitrogen 17 days later. All the eggs, left at room temperature for 1 day after storage, were then placed in physiological saline from 5-3/4 hours to 72 hours. The eggs were dried 15 minutes on filter paper and then placed on glass slides; after the eggs were completely dried, para- sites were placed with the eggs. The eggs not used in each dish were either destroyed in storage or in handling after storage. Table 18 lists the number of eggs used and the physiological saline treatment time for each set of eggs. Upon the 50 2-day—old unstored control eggs were placed five Anaphes. Dish 4 was dropped and the eggs destroyed. When checked 72 hours after immersion in physiological saline, all eggs of dish 5 were found to have collapsed. Nothing developed from the test eggs whereas 27 control eggs were parasitized and from which 22 female and 38 male Anaphes emerged. Although Anaphes develops from eggs treated with glycerated extender and held at 400 F, they do not appear to develop from eggs treated with 7% glycerated extender and then stored at -l96O C under the conditions used here. In addition, the eggs appear to be more fragile after nitrogen storage than do those that have not been in nitrogen storage. 67 Table 18. Parasitization success of Anaphes flavipes on cereal leaf beetle eggs after storage for 17 days in liquid nitrogen NO- 5m No. 9 and/or Dish No° No. Eggs Anaphes Hours in Beetle Larvae No. Eggs Used Used Saline to Develop l 25 7 2 5-3/4 0 2 25 12 2 5-3/4 0 3 25 10 2 5-3/4 0 4 25 9 ..b .. 5 25 O .. 72 .. 6C 50 50 5 o sod aThe balance of the eggs were either damaged during storage or in handling. bThe dish was dropped before the eggs could be dried. CThese are the control eggs and were not stored in liquid nitrogen. dThese were all Anaphes; no CLB larvae developed. Gas Atmosphere Storage 9f.§gg§' As early as 1821, M. Barard reported that storage in oxygen-free atmospheres could be used to preserve fresh fruits (Harvey, 1967). But according to Harvey (1967) it was not until almost 100 years later that work by Kidd and West in 1920 provided a base for the commercial development of controlled atmosphere storage. In 1940 the first commercial trials involving controlled atmosphere storage were begun in North America (Porritt, 1963). For the United States, controlled 68 atmosphere storage has lengthened storage life of certain apple varieties. In addition, a number of problems such as soft scald and brown core have been almost eliminated with the use of controlled atmosphere storage (Van Doren, 1961). Harvey (1967) states that as of 1965, 15 million bushels of apples were in controlled atmosphere storage in the United States. In addition to apples, controlled atmosphere tests have been conducted on strawberries, peaches, sweet cherries, lettuce, tomatoes, pears, and various other fruits and vegetables. Most controlled atmospheres are obtained by allowing the stored commodity to modify the atmosphere through respi- ration in a sealed room or container. When the desired level of oxygen is reached, enough outside air is admitted to maintain the oxygen at that level. When the carbon diox- ide reaches the desired level, the excess carbon dioxide is removed by passing the atmosphere through some material such as caustic soda that will absorb carbon dioxide. Both pro- cesses are carefully controlled to provide the proportion of oxygen and carbon dioxide that best maintain quality for the particular variety of fruit being stored (Harvey, 1967). Low-oxygen atmospheres, by slowing the respiration rates of fruits and vegetables, lengthen their storage or market life. High carbon dioxide levels, like low-oxygen levels, slow the respiration rate and are even more effec- tive than low oxygen in retarding decay. 69 Controlled atmosphere storage is a supplement to refrigeration but not a substitute since temperature is a controlling factor in determining the effects of controlled atmospheres used in storage. Fruits and vegetables such as apples and pears that are harvested green and ripen after harvest are most affected by controlled atmosphere storage whereas fruits such as grapes that do not ripen further after harvest do not respond significantly to controlled atmosphere. In addition each variety of fruit or vegetable responds differently to a particular atmosphere. Thus, in mixed loads no single combination of gases would provide optimum conditions for all commodities being stored. Because of the success in controlled atmosphere storage of apples and other fruit and vegetables, gas atmo- sphere were used in an attempt to develop a more satisfac- tory means of storing unparasitized CLB eggs. Storage was conducted at 400 F and lower temperatures using 100% carbon dioxide (C02), 100% nitrogen (N a mixture of 5% carbon 2), dioxide and 95% oxygen (C02/02)' and a mixture of 12% carbon dioxide, 79% nitrogen, and 9% oxygen (COZ/N2/02)' The first tests were pilot tests to see if Anaphes could develop from eggs stored in various gas atmospheres. In the test 25, 2-day-old eggs, were mounted on strips of plastic large enough to fit into either 25 or 50 ml Erlen— meyer flasks. The flask contained a piece of sterilized 70 cotton which was saturated with sterilized distilled water. After the plastic strip with the eggs was placed in the flask, it was flushed with the appropriate gas for 2 to 3 minutes before the top was sealed with a sleeve-type rubber stopper (see Fig. 8). The flasks were then placed in stor— age at 400 F. The results are presented in Table 19. Table 19. Parasitization success of Anaphes flavipes on unparasitized cereal leaf beetle eggs stored for 11 days at 400 F in gas atmospheres of air, 100% nitrogen (N2), 100% carbon dioxide (C02), and mixtures of 5% carbon dioxide, 95% oxygen (C02/02), and 12% carbon dioxide, 79% nitrogen, and %% oxygen (COZ/N2/02)(25 eggs per flask) No. No. Eggs with Fungus Flask No. offspring, After b 9 Days After No. Gas BPa 9 6 Storage Storage 1 Air 15 33 4 6 10 2 Air 22 43 6 1 7 1 N2 18 32 5 O 16 2 N2 15 3O 2 1 14 1 CO2 23 39 7 2 3 2 CO2 20 29 2 5 7 1 C02/02 6 13 1 O 5 2 C02/O2 19 4O 7 1 10 1 C02/N2/02 21 38 6 O 16 2 coz/N2 /02 14 19 3 2 22 aBP = the number of parasitized cereal leaf beetle eggs that developed black pupae. bThe fungus count was made at the time that the eggs were removed from storage. 71 P Sleeve Type | Stopper Plastic [6666 \ Strip Eggs Erlenmeyer Flask Cotton with @ Sterilized ~4’ Distilled water Figure 8 - The container used for storing CLB eggs in various gas atmospheres. 72 All the gases used except for COz/Nz/O2 came from large metal cylinders used for storing gases. The exception was in an inner tube fitted with a special valve. With COZ/NZ/OZ’ the flasks were flushed for 1 minute although the amount of gas actually in the flasks may be questionable due to the inner-tube apparatus used. The eggs exposed to N2 and C02/02 were in 50 ml flasks while the remaining eggs were in 25 m1 flasks. The results of the test indicated that for the first 11 days of storage at 400 F using the various gas atmospheres, parasites developed quite well from the stored eggs. .Since development did occur in the eggs, it was decided to conduct a series of tests that involved egg storage in the gas atmo- spheres for longer periods of time. In this series of tests a total of 2000 eggs were stored, 500 eggs to be brought out at monthly intervals. One-half of them were stored at 400 F and the other half at 140 F. The eggs, a maximum of l—day—old, were mounted in groups of 25 per strip of clear plastic, approximately 50 x 10 mm. Half of the eggs stored at each temperature were treated with Captan (.1 g/100 ml sterilized distilled water) before subjecting them to the gases. The Captan treatment consisted of dipping the eggs into Captan for about 15 seconds and then dipping them into two separate rinses of sterilized distilled water. After drying, the eggs were placed in 25 m1 Erlenmeyer flasks with a small piece of 73 sterilized cotton that had been saturated with sterilized distilled water. Within 24 hours from the time the eggs were mounted, the flasks were flushed with the appropriate gas, stoppered, and stored at 400 F. The flasks to be stored at 140 F were first stored for 6 days at 400 F. When stored at 140 F, they were placed within a styrofoam box which helped to maintain a constant temperature around the eggs. Beginning with Test 3 (stored approximately 100 days) a set of controls consisting of fresh eggs were used. The control eggs were used to test the Anaphes for their ability to produce offspring. These eggs were in 400 F air storage for no more than 12 days. The test results for eggs stored at 400 F are presented in Table 20. This set of tests indicate that for the first 30 days, eggs may be stored at 400 F in the various gas atmo— spheres tested and satisfactory parasite development can be obtained. The only exception to the gases used would be 100% CO which had about half the number of parasitized eggs 2 develop as did the other gases or air after 30 days of stor- age. Figure 9 presents graphically both the results of the pilot test and the succeeding gas atmosphere test. The slight differences in terms of parasitized eggs between the pilot test (11 days) and the test results after 30 days are probably due to the fact that these were different tests beginning at different times and using both eggs and para- sites from different groups. 741 Table 20. Parasitization success of Anaphes flavipes on unparasitized cereal leaf beetle eggs stored for various time periods at 40° F in gas atmospheres of air, 100% nitrogen (N2), 100% carbon dioxide (002), and mixtures of 5% carbon dioxide and 95% oxygen (COZ/Oz), and 12% carbon dioxide, 79% nitrogen, and 9% oxygen (C02/N2/02) Test No. Days Eggs No. No. Anaphes No. Eggs No. of CLB No. Gas Eggs Stored ‘Captan BPa 9 6 With Fungus Larvaec 1 Air 25 33 YEs 23 46 8 11 0 Air 25 33 No 14 20 4 4 8 N2 25 33 YEs 22 1 31 4 3 0 N2 25 33 No 15 23 5 l9 2 C02 25 31 YEs 8 11 2 0 0 C02 25 31 No 7 8 l 21 0 coz/o2 25 33 YES 23 31 6 1 o C02/02 25 33 No 10 19 3 6 2 COZ/NZ/Oz 25 32 YEs 20 25 10 0 0 COz/Nz/Oz 25 32 No 18 7 21 16 l 2 Air 25 67 YEs 9 14 l 16 0 Air 25 66 No 4 0 0 0 0 N2 25 67 YEs 6 7 1 0 0 N2 25 66 No 0 0 0 0 0 C02 25 65 YES 0 0 0 4 0 C02 25 64 No 1 2 o 25 o (302/02 25 67 Yes 0 0 0 O 0 C02/02 25 66 No 0 o o 25 o coz/Nz/oz 25 66 YEs 8 5 2 23 o COZ/NZ/o2 25 65 No 0 0 0 25 0 3 Air 25 105 YEs 0 0 0 12 0 Air 25 101 No ..d .. .. .. .. N2 25 105 YEs 0 0 0 O 0 N2 25 101 No 0 0 0 25 0 C02 25 103 YES 1 0 1 0 1. C02 25 99 No 0 0 0 25 0 C02/02 25 105 YEs 0e 0 O 6 O 002/02 25 101 . No l .. .. 25 0 COz/Nz/Oz 25 104 Yes 0 0 O 0 0 Control 1f 50 24 o 53 Control 29 25 20 38 5 4 Air 25 123 YEs ..d .. .. .. .. Air 25 - 122 No 0d 0 O 25 0 N2 25 123 Yes .0 o. o. o. 6. N2 25 122 No 0 o o 25 0 C02 25 . 121 YEs 0 0 0 O 0 002 _25 120 No 0 o o 25 o C02/02 25 123 YEs 0 0 0 0 0 COz/Oz 25 122 No ..d .. .. .. .. COZ/N2/02 25 122 YES 0 0 0 O O C02/N2/02 25 121 No 2 1 o 25 0 Control 1f 25 21 29 23 Control 29 25 9 9 3 a8? a the number of parasitized cereal leaf beetle eggs that developed black pupae. bThe fungus count was made 9 days after the eggs were brought from storage. cThe number of cereal leaf beetle larvae to develop (none hatched). dThe eggs were too heavily covered with fungus so Anaphes was not placed on the eggs. eNo Anaphes emerged from the one parasitized egg. fFresh egg (untreated) controls for the test eggs treated with Captan. gFresh egg (untreated) controls for the test eggs not treated with Captan. 75 The results indicated that after approximately 65 days of storage at 400 F some eggs from several of the gases tested still developed black pupae but the numbers were not as great as for the controls (air) and the controls did not do well (only 13 of 50 eggs developed black pupae). (See Fig. 9). Even after approximately 100 days storage at 400 F one parasitized egg developed from those eggs stored in each gas atmosphere containing C02 and 2 parasitized eggs were obtained from eggs stored 121 days in COz/NZ/Oz. At 140 F storage, the tests revealed that no gas mixture (including air) resulted in parasitized eggs (there was one exception: one egg stored in air for 33 days supported the development of parasites after storage). The testing further revealed that with this method of storage at either 400 F or 140 F fungicide treatment is probably necessary for fungal control with the various gas mixtures tested. Almost all the Captan-treated eggs stored at both 140 F and 400 F had much less fungus than did the eggs without Captan. Furthermore, treated eggs usually yielded more parasitized eggs than did the untreated eggs. The yield in parasitized eggs from those parasitized after storage at 400 F in gas atmospheres is no greater than from those eggs stored in air. The ability of eggs stored in gas atmospheres to support development of parasites as storage time increases is no greater than for eggs stored in air. Therefore the gas atmospheres tested offer no better 76 40" \ 30- Eggs Parasitized 201 Number of j I 30 60 90 120 Days of Storage ‘ -- - -Air ----Nitrogen ----- 10096 coz 5% coz, 95% 02 -—-12°/o coz. 79% N2, 9%02 Figure 9 — Storage of unparasitized CLB eggs at 40° F in various gas atmospheres. 77 means of storage than is presently used. But it is possible that other gases or proportions and/or temperatures might perform better than those used here. At 140 F, storage is definitely not desirable since almost no parasitized eggs developed after 30 days of storage. Assuming the eggs stored at 140 F had satisfactory development before 30 days of storage, this would be of no benefit since the current technique of storage at 400 F gives satisfactory development for 30 days and longer. Furthermore, the current storage technique is less complicated than the use of the gas atmOSpheres. STORAGE OF PARASITIZED EGGS Occasionally it is necessary to store parasitized eggs due to such factors as insufficient CLB eggs or para— sitization. Currently the only practical way to store Anaphes is as black pupae. This stage occurs within the CLB egg from 1 to 2 days before the adult parasite emerges. At this time the egg takes on the dark color of the adult parasite. CLB eggs containing black pupae were held in stor- age at 400 F for 7 weeks. The parasitized eggs, mounted on glass slides, were placed in petri dishes that contained filter paper to which sterilized distilled water was added every other day. Each week enough parasitized eggs were removed from storage to provide a minimum of 15 female Anaphes plus males for mating. The most vigorous individ— uals were used in the test (see Table 21). The females were placed with 100 CLB eggs. The results indicate that it is possible to hold pupae as long as 7 weeks at 400 F without completely elim— inating mating or viable sperm. But since there were no controls, it is not possible to say whether the unusually high proportion of male F1 is due to the storage of the parents as black pupae or due to insufficient mating time allowed the parasites after emergence. 78 79 Table 21. .Survival and parasitization success of parasites emerging from Anaphes flavipes pupae stored at 400 F for 1-7 weeks No. Adults to Emerge No. Adults No. Weeks After Storage, Used Eggs No. F] Total Stored 9 (f 9 d Used 9 6 Fl 1 17 9 15 9 100 36 70 106 2 23 10 15 7a 100 33 41 74 3 35 11 15 7a 100 53 40 93 4 27 12 15 6a 100 o 29 29 5 28 8 15 7a 100 28 49 77 6 31 7 15 5a 100 o 59 59 7 14 15 8a ea 100 35 23 58 a I Fewer were used than emerged because some were weak, dying, or dead. The results indicate that with an increase in the storage time of the parents as black pupae there is a gen- eral decline in the number of F to develop, although the 1 last test seems to negate this general trend since only eight females (instead of 15) were available. It is pos- sible that all eight females used in the last test were strong and the weak ones had died before any could be placed on the eggs. In the other tests, the most active and vigor— ous females were chosen, but it is possible that some of these were not as strong as they seemed and oviposited little or not at all. This would mean that, even though 15 females were present, only part of them were actually ovi- positing. 80 Adults emerging from pupae held up to 2 weeks in storage at 400 F appear to be as active as adults emerging from a continuous culture. Beyond 2 weeks there is some reduction in vigor and activity and beyond 4 weeks the reduc— tion is quite noticeable, resulting in specimens whose activ- ity is considerably less than adults emerging from unstored pupae. The loss of vigor and activity of adult parasites as the length of black pupal storage increases has been noted by workers at the Niles Lab (Moorehead, personal communica- tion). Storage of Parasitized Eggs Earlier than the Black Pupal Stage The general procedure used in storage of parasitized eggs consists of placing the eggs in petri dishes with moist filter paper and then storing at 400 F. Fungus is a problem in the storage and is partly aleviated by the use of fungi- cide on the unparasitized eggs since protection carries over to the eggs after parasitization. In an attempt to find a better means of storing parasitized eggs, it was thought that storing eggs in life stages earlier than the black pupal stage might be useful. Thus, eggs were stored at 400 F 2 days, 4 days, 6 days, and 8 days after adult parasites were put on the eggs. The parasitized eggs were stored in the following manner: the eggs, five rows of 10 eggs each per 1/3 section of cut glass slide were left in the petri dish (50 x 12 mm 81 disposable plastic petri dishes with tight fitting lids) in which they were stung. Filter paper was placed in the bottom of the petri dishes and then sterilized distilled water was placed in the dish to a level nearly equal to the height of the glass slide (the eggs were not submerged in the water). The dishes were then put into storage. In all but the first three tests, five females were used per 50 eggs. In the first three tests, six females were used. The females were confined with males in gelatin capsules for approximately 1 hour for mating, then watered and fed for 2 to 4 hours before being used. The results of the first three tests are not given because nothing emerged after the first set of parasitized eggs were brought from storage. The lack of emergence was apparently the result of fungus. After the first three tests were concluded, it became clear that a fungicide was necessary during storage to retard fungal growth. Thus, the eggs in the next test were treated with Captan 3 days after the parasites were placed on the eggs (1 g of Captan per 100 ml of sterilized distilled watervor 5000 ppm active ingredients). Treatment consisted of the eggs being dipped into the Captan solution and then into two rinses of sterilized distilled water. Eggs stored 2 days after being parasitized were treated with Captan an hour before they were stored. The results are presented in Table 22. Table 22. 82 Survival and emergence of Anaphes flavipes after storage of parasitized cereal leaf beetle eggs with various aged developmental stages of the parasite (50 eggs per dish) No. Days Eggs Exposed to No. No. of Parasites Days No. of Parasitized Before Eggs No. Offspring» Eggs with Dish Storage Stored BPa 9 o' No Emergence 1 2 15 15 14 4 3 2 2 29 20 15 10 4 3 2 43 28 26 5 9 4 2 57 32 10 4 22 5 4 17 11 12 2 O 6 4 33 2 2 O O 7 4 43 13 7 2 4 8 4 57 15 0 0 15 9 6 15 22 17 13 3 10 6 31 23 14 2 13 11 6 43 37 2 0 36 12 6 57 31 O O 31 13 8 15 10 .. .. .. l4 8 29 36 46 10 6 15 8 43 37 7 5 31 16 8 57 38 4 6 31 Control 1 0 35 40 23 0 Control 2 0 45 86 14 3 aBP = the number of parasitized CLB eggs to develop black pupae. bThe eggs dried out before the parasites could emerge. 83 The number of parasitized eggs with no parasite emergence in dishes 1, 2, 5, 6, 9, 10, 13, and 14 was primarily due to the collapse of the eggs by the time the remaining parasitized eggs had parasite emergence. .In dish 4, the eggs with black pupae that failed to complete development looked good, but for some reason adults did not emerge. In dish 7, the parasites that emerged appeared to be normal. In dish 8, none of the parasitized eggs had emer- gence although one egg had a female Anaphes that was alive—- she could be seen moving within the egg--but she failed to emerge. From dish 10, one of the females to emerge had mal— formed wings and antennae. Although no males emerged with her, she was placed with 14 eggs that were a maximum of l—day old and had been stored 5 days before use. The parasite did not appear to be interested in the eggs. Cereal leaf beetle larvae hatched from eight of the eggs and the other six collapsed with nothing developing. In dish 11, the eggs developed black pupae from 4 July to 6 July, 1967. As of 12 July only one of the eggs with black pupae had had adults emerge. There appeared to be no movement in the other eggs containing black pupae. Since it was unknown whether these black pupae were dead or in diapause, they were replaced in storage in an attempt to break diapause if this were the cause. At this time, 84 21 eggs with black pupae still looked good. The other eggs with black pupae were either covered with fungus or had collapsed. The eggs in dish 11 were periodically checked but after 3 months all the eggs had collapsed and had fungus covering them. In dish 12, the eggs with black pupae looked good but parasites failed to emerge. When dish 13 was brought from storage the top of the dish was accidentally left off and the eggs dried out before the adults could emerge. In dish 15 and 16 all the parasites that emerged had deformed wings. Figure 10 shows the results of the storage tests. As in the case with the storage of unparasitized eggs, there appears to be a definite period of storage after which para— site survival begins to decline rapidly; the time seems to be between 30 and 40 days. In addition, it is evident (Table 22) that as the storage time increases, there is also an increase in the number of parasites that do not emerge. The decreased emergence may be due to parasites going into diapause, but this has not been shown. The eggs with unemerged black pupae collapsed, both when left at room temperature and when replaced in storage and checked periodically. By the use of a two-way analysis of variance, at the 1% level of significance there is no difference between eggs stored 2 days after introduction of parasites and 6 days 85 100 - 1. 90"b ' °---- ZDays 801 *---4 Days 70'“ 0—-"8 Days L . E 60" E r O E 501'- L '5 ° 40*- 30-L 20-1- “\‘ 10¢. \\ \ . F \ \ \\ ‘ n n N: .L KAI Days of Storage Figure 10 - The results of tests to determine the optimum age of Anaphes flavipes for storage at 40° F. The eggs were stored 2, 4, 6, and 8 days after parasites were placed with the eggs. 86 after introduction and also between 6 days and 8 days after parasite introduction. Eggs stored 4 days after the intro- duction of the parasites had the fewest parasitized eggs develop and in terms of the number of parasitized eggs was significantly different from the eggs stored 2, 6, and 8 days after the introduction of parasites. Egg storage 2 and 4 days after parasite introduction is not as good as storage 6 or 8 days after parasite introduction. Since the eggs stored at 8 days after parasite introduction are close to or are black pupae, this group of stored eggs is similar to those that are regularly stored. COLD STORAGE OF ADULT PARASITES As an alternative to the storage of black pupae, an attempt was made at storing adult parasites. Into a petri dish containing moist filter paper were placed 12 female and 5 male French Anaphes. The top of the dish was taped to the bottom and then stored at 400 F. At the same time 10 female parasites that had previously parasitized eggs were stored in another petri dish under the same conditions. The adults were fed and watered each time the dishes were checked for live parasites. Tables 23 and 24 give the results of the tests. Table 23. Survival of 17 female and male Anaphes flavipes (French strain) stored at 400 F No. Live Parasites Days Stored 9 d 0 12 5 2 ll 4 4 11 4 6 9 2 8 ..a .. aThe dish was dropped. 87 88 Table 24. Survival of female Anaphes flavipes (French strain) stored at 400 F Days Stored No. Live Parasites 0 10 2 9 4 8a 6 8 8 4 ll 2 13 1 15 l 17 0 aOne parasite was lost while being transferred to a dish with honey and moisture. The same conditions were used in the next test. A petri dish with 10 males was stored at 400 F. The results are in Table 25. Table 25. Survival of 10 male Anaphes flavipes (French strain) stored at 400 F Days Stored No. Live Parasites 0 10 2 7 4 5 7 l 9 0 Three dishes with parasites were used in another test. One dish with 9 adult males and moist filter paper was left at room temperature and used as control. The second dish, stored at 400 F, contained moist filter paper 89 and 8 males. Nine males were stored at 400 F in the third dish which contained moist filter paper with honey. The results of this test are in Table 26. Table 26. Survival of adult male Anaphes flavipes (French strain) at room temperature and stored with and without honey at 400 F Number of Live Parasites Days Stored Room Temperature Without Honey With Honey 0 9 8 9 2 0 3 9 4 0 2 3 6 0 0 0 At 400 F in the laboratory about 50% female mortal- ity occurs after 1 week of storage and 50% male mortality about 3 to 4 days after commencement of storage. It appears as a result of these tests that adult Anaphes cannot be effectively stored for long periods of time under the condi- tions used. In those instances where parasites survived for a period of time in storage, they were much less active when brought out than are newly emerged adults. .Adult Anaphes storage is not an effective means of long term storage. 90 Laboratory cultures are not exposed to the environ- ment the way field populations are. Whether or not field conditioning would enable the parasites to survive the summer and winter as adults after CLB eggs are gone is unknown. X—RAY TREATMENTS OF CLB ADULTS AND EGGS X-ray treatments were given CLB eggs to determine whether CLB larval development could be terminated and at the same time obtain parasite development. If parasite development could be obtained, then in the field release of parasite pupae there would be no addition of CLB larvae to the CLB population already present in the field. Adult Beetle Treatment According to Brennan (1967) female CLBs are more sensitive to radiation than males. When females are treated with X-ray dosages of 2000 roentgens or greater, egg produc- tion is almost eliminated. In addition, Brennan states that when untreated females are mated to males that have been treated with 2000 rads (93 roentgens = 100 rads) of beta radiation about 87% of the eggs laid are infertile. Un— treated females mated to adult males treated at 1000 rads results in about 50% egg viability whereas at the 4000 rad level viability is almost reduced to zero (W. Myser, unpub- lished report, 1967). Egg production is not adversely affected by mating unirradiated females to males irradiated up to 8,000 rads (W. Myser, unpublished report, 1967). 91 92 Above 2000 roentgens, according to Brennen (1967) mortality of pre-diapause males begins to increase greatly. At the 2000 roentgens level mortality is much greater for the first 15 days after treatment of post-diapause beetles than for untreated males. Because irradiation of females appears to affect oviposition, it was decided to treat males with 2000 roent- gens of X_ray. In the test, all the beetles used were reared in the laboratory and in diapause storage for approx- imately 12 weeks before use. They were sexed, using the technique developed by Myser and Schultz (1967), as soon as they were removed from storage. Since females do not begin oocyte development until after the cessation of diapause (Hoopingarner, Kumararaj, and French, 1965) those used in the test were virgins. The males while confined in a plastic disposable petri dish were treated with a Maximar 250—III machine for 6.45 minutes at 310 r/min. The 50 treated males were divided into two groups of 25 each; each group was placed with 17 untreated females from the same storage container from which the males and controls came. All the eggs to be used in the testing were subjected to parasitism at room temperature. Fifty untreated and unsexed adults were used as controls. 93 T§§£_lf--In this test, there were 109 eggs from the treated beetles and 37 eggs from the controls. The eggs were a maximum of 3-days old and stored for 5 days at 400 F before they were used. . In the test group two petri dishes each had 50 eggs and a third dish had nine eggs. One petri dish contained control eggs. Filter paper was placed in the bottom of the dishes and kept moist with sterilized distilled water during the test. In the test group, five females were placed in each dish with 50 eggs and two were placed with the nine eggs. Five French Anaphes were used on the controls. The results are listed in Table 27. T§§E_2.—-In this test, there were 110 test eggs and 30 control eggs, a maximum of 2-days old. The eggs, without fungicide treatment, were stored for 3 days at 400 F before use. Two petri dishes contained 50 test eggs each and one contained 10 eggs. Five female French Anaphes were used in each dish of 50 eggs and two females were put in the dish with 10 eggs. Five French female Anaphes were used on the controls. Table 27 presents the results of this test. It is evident that parasites do develop from eggs laid by unirradiated female CLBs that have been mated with males treated with 2000 roentgens X—ray, but there may be some difference in the parasitization. The control eggs 94 .mmmsm MUMHQ pmmoam>mp pmnu mmmm maumon mmwa Hmmpoo UmNfluflmmpmm mo umnasc esp I mm m om.e m.m ea Ho ma hm mm mm mg gm N m om NH OHH m mn.m e.m 8 mm g mm me ha om mm m m hm NH moa a b m b m x. .02 8. .oz mmmm a mmmm m mmmm .oz pmme Honucoo umme Houpcoo ummfi Houusoo mo pmme Houucoo ummB Comb m\mm mmmmam mmm npflz mmmm mom Comb mmnmmc< mHmEom mo oflumm muasom 02 mo X wow .02 osm mmmm mo .02 manlx mo mcmm upcmou ooom ou oomOme mmauoon mama Hmmumo mama Cus3 pupae mmaummn mmma Hmmumo mHmEow Umummppcs Eonm mmmm CH mmmfl>mam mmnmmc¢ mo mmmooflm coHumuflpfimmnmm .hm Canoe 95 appear to be somewhat better for Anaphes development than are the eggs from the X-rayed adults, although the differ- ence between the two groups is small and possibly not significant. There are some undesirable aspects to this technique for obtaining sterile eggs. The sexing of the beetles, although not difficult, is time consuming. There is always the possibility that a female is not virgin, especially if the beetles are not sexed immediately after being taken from diapause storage. Egg Treatment To obtain "sterile" CLB eggs by radiation treatment, it would probably be more convenient to treat the eggs directly after they have been obtained from untreated parents than to treat the parents. Egg treatment would eliminate time-consuming sexing. According to Brennan (1967) exposure levels of 1000 rads of Beta radiation and above completely eliminate hatch- ing of CLB larvae. Consequently, 250 eggs that were a max— imum of l—day old were treated with 1000 roentgens X—ray and another 250 were treated with 2000 roentgens of X-ray. The eggs, 250 per plastic petri dish and stored at 400 F for 9 days before treatment, were treated with a Maximar 250~III machine at 310 roentgens per minute for 6.45 minutes while those treated with 1000 roentgens were treated for half that time. The eggs were stored at 400 F for 3 days 96 after treatment before being parasitized. The 250 control eggs were not subjected to X-ray. In this test, 13 female French Anaphe§_were placed on each group of 200 eggs (200 at 2000 r, 200 at 1000 r, and 200 without X-ray). One hundred eggs (50 from the two different treatments) were allowed to develop without parasites being present to see if any beetle larvae would develop and/or hatch. Table 28 gives the results of this experiment and Table 29 lists the results from the X-ray-treated eggs that had no parasites placed on them. Table 28. The number of cereal leaf beetle larvae to hatch, number of parasitized eggs to develop black pupae, and the number of adult parasites to emerge from cereal leaf beetle eggs treated with 1000 and 2000 roentgens X-ray and untreated eggs (200 eggs for each test group) Untreated 1000 r 2000 r No. cereal leaf beetle larvae to hatch 51 6O 38 No. cereal leaf beetle larvae to developa l9 17 43 No. eggs with nothing developing 69 56 65 No. eggs parasitized 61 67 54 No. female Anaphes 62 75 68 No. male Anaphes 21 17 32 aThe number of cereal leaf beetle eggs developing to larvae but not hatching. 97 Table 29. The number of cereal leaf beetle larvae to develop and/or hatch from 50 eggs treated with 1000 roentgens and 50 eggs treated with 2000 roentgens X-ray that were not subjected to parasitization 1000 roentgens 2000 roentgens No. of cereal leaf beetle larvae to hatch 34 26 No. of eggs with cereal leaf beetle larval developmenta 10 17 No. of eggs with no signs of larval development 6 7 aThe number of cereal leaf beetle larvae to develop but not hatch. Since the hatch of the CLB larvae was fairly great at the levels of 1000 and 2000 roentgens (contrary to Brennan's 1967 results), further tests were conducted using 3000, 4000, and 5000 roentgens. A total of 750 CLB eggs (l—day old and stored for 4 days before treatment) were treated with X—ray (250 eggs were treated at each level). The 250 eggs in each treatment (50 eggs per 1/3 section of cut glass slide) were in the same petri dish with the lid taped shut and moist filter paper on the bottom of the dish. Using the Maximar 250-III machine, the treatment levels were as follows: 3000 r — 9 minutes, 54 seconds at 330 r/minute; 4000 r - 12 minutes and 7 seconds at 330 r/minute; and 5000 r - 15 minutes and 9 seconds at 98 330 r/minute. From each of the treatments 50 eggs were not subjected to parasitization and 150 untreated eggs a max- imum of 2-days old were used as controls (100 with parasites and 50 without). Five female Anaphes were used per 50 eggs. Table 30 presents the results of the treated and untreated CLB eggs not subjected to parasitization and Table 31 pre- sents the results of those eggs subjected to parasitization. Table 30. The number of cereal leaf beetle larvae to develop and/or hatch from untreated cereal leaf beetle eggs and from eggs treated with 3000, 4000, and 5000 roentgens X—ray that were not subjected to parasitization Treated (50 eggs each) Untreated (50 eggs) 3000 r 4000 r 5000 r a b No. CLB hatched 39 O 0 O No. CLBa to develop but not hatch 3 ll 6 l6 aCLB = cereal leaf beetle larvae. bOne cereal leaf beetle larva had partially emerged but never completely escaped from the egg. 99 Table 31. Parasitization success of Anaphes flavipes in cereal leaf beetle eggs treated with 3000, 4000, and 5000 roentgens X-ray 3000 r 4000 r 5000 r Untreated (200 (200 (200 100 Eggs 2xa Eggs) Eggs) Eggs) No. BPb 68 136 144 108 89 Percent parasitism 68 68 72 54 44.5 No. female Anaphes 108 216 235 188 132 No. male Anaphes 22 44 43 35 19 Total Anaphes 130 260 278 223 151 No. BP not to emerge 2 4 ll 4 19 No. CLBC hatch 8 l6 0 0 0 No. CLBC to develop but not hatch 3 6 4 ll 14 aThese values are two times the values obtained for 100 untreated eggs so they can more easily be compared with the test groups. bBP = the number of parasitized cereal leaf beetle eggs that developed black pupae. cCLB = cereal leaf beetle larvae. Ten Fl females from each of the test groups to emerge were put on 50 unparasitized CLB eggs that were a maximum of 2—days old and had been stored at 400 F for 5 days before use. The females were mated for 1/2 hour and fed and watered for 1 hour before being used. The test was con- ducted to see if development from X-ray treated eggs affected the parasites that emerged. The results are presented in Table 32. 100 Table 32. Parasitization success of F Anaphes flavipes females from cereal leaf beetle eggs treated with 3000, 4000, and 5000 roentgens of X-ray on un- treated cereal leaf beetle eggs 3000 r 4000 r 5000 r No. BPa 33 44 40 No. females 28 99 75 No. males 40 17 16 aBP = the number of parasitized cereal leaf beetle eggs that developed black pupae. Discussion The X-ray tests of eggs indicate that 1000 or 2000 r is not great enough to effectively retard the development of CLB larvae as seen from the number of larvae that developed from both the treated eggs exposed to parasites and those that were not exposed to parasites. These results seem to be contrary to the findings of Brennen (1967). He found that at and above 1000 rads, CLB larval hatch was completely eliminated. The reason for this discrepancy is not known unless it is the result of experimental error or due to the difference in the type of radiation used (Brennen used Beta radiation). There is the possibility that the discrepancies are the result of improper dosimetry. With the equipment Brennen used, the dosage given would have been for about l/50th of a second; this short period of time may be diffi— cult to control with the result that dosages Brennen gave were actually greater than he stated. In addition, the 101 dosimeter used with the Maximar machine has not been calibrated for sometime, consequently the dosages used may not have been exactly those calculated. Although slightly fewer larvae hatched at the 2000 r level than at the 1000 r level there is very little differ— ence in terms of the total number of larvae to develop and/ or emerge. At levels of 3000 r and above larval hatch is completely eliminated and larval development is very much reduced. In addition, all levels of X—ray used on eggs do not prevent the development of Anaphes. The use of Chi Square shows no significant difference in terms of the black pupal development between controls, 1000 n and 2000 r of X-ray. Furthermore, the use of a two—way analysis of vari- ance indicates no difference between the 3000 r, 4000 r, and 5000 r levels of X—ray in terms of the development of black pupae. The raw data suggests no difference between the 3000 r level and untreated eggs, but there may be differ- ences with the higher levels of X—ray, especially at 5000 r. It appears that as the level of radiation used on the eggs increases from 3000 to 5000 r, there is a decrease in the number of eggs that develop black pupae. Females emerging from eggs treated with 3000 r parasitized fewer untreated eggs and produced fewer off- spring than those from 4000 r and 5000 r levels. Even so, 102 there probably is little difference in the effects of Anaphes development from eggs treated with 3000, 4000, and 5000 roentgens. Placing more CLB larvae into the field population by the mass release of immature stages of Anaphes is of no real danger since any CLB larvae that are going to hatch, hatch and die in the lab before the releases occur. The danger is eliminated since it takes 4 to 7 days for CLB larvae to hatch, but 8 to 10 days for the development of the Anaphes black pupal stage. To obtain sterile eggs for the development of Anaphes it is more economical to treat the eggs directly than to get them from unirradiated female CLBs mated with irradiated males. Although Anaphes develops from the eggs of irradi- ated parents, it is time—consuming to sex the parents before treatment and the females used must be virgin. It appears that in the direct application of X—ray to eggs, the 3000 r level of X—ray is best to obtain maximum parasite production and no CLB larval hatch. FIELD RELEASES The mass culturing of Anaphes has been conducted to provide sufficient numbers to attempt establishment in those areas where the CLB poses a threat to agriculture. Techniques There are essentially two means of field releasing Anaphes: either as adults or as black pupae. With one exception, all releases utilized the latter. There are several reasons for the selection of the black pupal stage for field release. Black pupae, since they can be stored effectively for at least 2 weeks, need not be placed in the field at the time they reach the pupal stage; adults must be released almost immediately since they only live from 1 to 4 or 5 days in the lab. When adults are released almost invariably there are some that do not emerge the day of the release, but instead emerge a day or even 2 days later. By releasing the parasites in the black pupal stage, there is a much better chance that these stragglers will get into the field, especially when releases are made at other than daily intervals. When releasing adults mating can be ensured before the releases. However, mating appears to be no problem in the release of black pupae since field recoveries indicate that when the pupal stage is released, 103 104 mating does occur after emergence and one obtains a satis— factory sex ratio in the field (approximately 2.3 females to one male). There have been two methods used in field releasing the black pupae: wooden stakes to which are taped the coverslips or glass slides with the parasitized eggs, and wooden stakes with milk cartons that shelter the parasitized eggs. The 1966 trials utilized the first method of release. The 1/3 sections of cut glass slides with the parasitized eggs were taped to one side of wooden stakes (23—3/4 x 1-1/2 inches) which in turn were placed in the ground at an angle of from 150 to 300 with the eggs on the lower side to pro- tect them from the elements (see Figs. 12 and 16). From four to six sections of slides with 50 to 100 eggs each were placed on each stake (see Fig. 11). This method is unsuit— able for release of large numbers of parasitized eggs since it is time consuming to tape each slide or cover slip to the stakes. The second container was used in the spring and summer of 1967. The release containers, patterned after those used by the Niles lab, consisted of wooden stakes to which were attached two, 1 quart milk cartons held in place with tacks. A piece of plywood was placed inside each carton to form a shelf. Thus, each carton could hold six 50 x 12 mm petri dishes (three on the shelf and three on the floor of the carton). The stakes were placed in the ground 105 Figure 11 - Wooden stakes used for releasing Anaphes flavipes in the field. Note the sections of glass slides taped to the stakes. (Wheat field, Charles Bohn's farm, Galien, Mich., 1966.) Figure 12 — Wooden stakes for the field release of Anaphes flavipes in position in Charles Bohn's wheat field (1966). 106 so the cartons were oriented parallel to the ground. The front of the cartons was kept closed (to keep rain out and the petri dishes in) with paper clips. The parasites escaped through a hole cut in the bottom of the front of the container (see Figs. 13 and 23). The 1966 Release The field releases were made in 1966 at four differ— ent sites in Michigan, one each in Hillsdale County and Kalamazoo County and two in Berrien County. Hillsdale County The release site in Hillsdale County (Section 31, T58, R3W) was about 2 miles west of Jonesville on land worked by Walter Kochendorfer. The release, less than 100 feet from the nearest woodlot, was in an area of very low CLB numbers. At this site, a pup-tent—shaped frame was placed in an oat field that was situated next to a wheat field. The frame, 4 feet long and 2 feet wide at the base and covered with window screen, had the legs and lower edges of the screen buried (Fig. 14). Cereal leaf beetles were swept up and confined in this cage (less than 100 beetles were swept up in several thousand sweeps from the field). The beetles were confined in the cage so oviposition would be concen- trated. Three days later, 195 parasitized CLB eggs were placed in the cage which was then covered with fine—mesh cloth so the emerging parasites could not escape (Fig. 15). 107 Figure 13 — Milk carton release containers before being closed. In the second set of containers from the right in the front row, the wooden shelf in the top release container is visible. (Release site in Jackson County in 1967, looking south - oats.) 108 Figure 14 - The release cage used in Hillsdale County in 1966. Note the release stakes inside the cage and the woodlot in the background. 109 Figure 15 - The release cage in Hillsdale County in 1966 after fine white cloth was placed over the cage. llO Ten days later, 183 parasitized eggs were released in the same field using the same techniques. Table 33 gives the dates and numbers of parasitized eggs released. Table 33. The date, number of parasitized eggs, and location of Anaphes flavipes releases in 1966 No. BPa Berrien County Bohn's Tobler's Jackson Kalamazoo Date County County Oat Wheat NE Corner SE Corner 19 May 195 ... 198 199 ... ... 23 May ... ... 200 216 ... ... 29 May 183 ... ... ... ... ... 4June 250b 15010 80 BP 10 June ... 456 ... ... ... ... 11 June ... ... 321 ... ... ... aBP = the number of parasitized cereal leaf beetle eggs released. bThese were adult Anaphes. Kalamazoo County The release site in Kalamazoo County was about 6 miles northeast of Vicksburg, on land owned by Earl Guyer (T3S, RlOW, Sec. 22). The release was made in an area of low to medium CLB populations. It was made close to a dead tree near the center of an oat field about 1/4 mile from the near- est woodlot. On June 10, 456 parasitized eggs were released. lll Berrien County In Berrien County one of the release sites was about 2 miles SSE of Galien, 1/2 mile from the Indiana line, on land owned by Charles Bohn (T88, R19W, Sec. 24). There were two different locations used here in the release of Anaphes. One location was in wheat just south of Olive Branch Road with a wooded area on the west side of the field (see Figs. 11 and 12). The other location was in oats about 1/4 mile south of the site just described (see Figs. 16 and 17). There was a woodlot about 25 yards away along the southern border of the field. At the first site, 415 parasitized eggs were released in two releases 5 days apart. In the oats, 719 parasitized eggs were released at three separate times (Table 33). Another site in Berrien County was also used. This site was about 2 miles SW of Galien, on land owned by Joe Tobler (T88, R19W, Sec. 17). The releases were made at the NE and SE corners of an oat field near a woodlot. Emerged adults were released here, and females were observed ovi— positing in CLB eggs shortly after release. The CLB popula— tion was high in this field and without our knowledge Joe Tobler sprayed with malathion 4 days after the parasite releases were made, leaving an unsprayed area about 50 feet in diameter around the release points. 112 Figure 16 - Release site in oats at Bohn's in 1966 (looking east). Note the stakes and the brush along the edge of the field. 113 Figure 17 - Charles Bohn's oat field in 1966 showing the woods along the south edge of the field (looking west). The circle marks the location of the release site. 114 1966 and 1967 Recovery Attempts Recovery was not attempted in Jackson County in 1966 or 1967 because of the very low CLB population and because of the very small number of Anaphes released. In Kalamazoo County on 1 July 1966 a single parasit- ized egg was recovered which contained three Anaphes. Very few eggs were present at this time. In 1967, one set of samples was taken from here. Twenty-five, 1 yard samples were taken 19 June from wheat at random within a radius of 25 feet from the 1966 release point. The 25 square yards of wheat plants yielded four eggs, three of which had already hatched when the samples were taken. The other egg had a CLB larva hatch in the lab. In Berrien County in 1966, 4 days after the last release was made in Bohn's wheat field, 18 eggs were col- lected within 2 yards of the release point. Six eggs were parasitized, from which 13 female and three male parasites emerged beginning 3 June. In addition, 20 days after the last release, eggs in the same area were checked and six were taken to the lab because they appeared to be parasit- ized. The next day parasites began emerging with a total of six female and four male Anaphes emerging. No additional attempts at recovery at either site in Bohn's fields were made so as to leave the maximum number of parasites in the field for overwintering. 115 In 1967, wheat was growing where oats had been the previous year on Charles Bohn's farm. To assure the pres— ence of beetles and eggs throughout the season at the 1966 release site, a strip of oats about 15 feet wide was planted running the length of the field on the east side. Samples were taken at this site on 28 May, 15 June, 27 June, and 7 July, 1967. The samples consisted of 25, one yard strips of grain cut at random from the oat strip. The samples were placed in plastic bags after which the open ends were stapled closed. The samples were then placed in storage at 400 F until they were checked for eggs. Table 34 lists the results of this sampling. Table 34. 1967 sampling results from Bohn's wheat field for overwintered Anaphes flavipes No. of Eggs to Have No. CLBa Emergence to Emerge No. of Total Before After Eggs No. of Sample No. of Sample Sample with No Anaphes Date Eggs Checked Checked Emergence to Emerge 28 May 277 139 91 47 0 15 Jun 46 6 8 32 0 27 Jun 217 125 40 52 O 7 Jul 53 47 4 2 0 aCLB = cereal leaf beetle larvae. 116 From the samples taken 27 June, 1967 one egg was found to have had three parasites emerge. These parasites were not Anaphes and were later identified as male Tricho- gramma minutum Riley, by Dr. B. D. Burks of the U.S.N.M. (The Niles laboratory has also recovered Trichogramma from Berrien County, but they have been identified as I, evanescens.) Because of the malathion treatment no recovery attempts were made in Tobler's field in 1966 or 1967. Discussion The field releases made in 1966 indicated that Anaphes will parasitize CLB eggs when they are released in the field. Also, it is possible that more than one genera- tion was obtained from the releases made in Charles Bohn's wheat in 1966. If the Anaphes that emerged from eggs col- lected 20 days after the last release in Bohn's wheat were not F they were F1 and would have parasitized CLB eggs to 2, start the F2 generation. Although the sampling of 1967 indicated that no Anaphes overwintered, the results of the sampling cannot be considered conclusive evidence that Anaphes did not over- winter. It is entirely possible that the sampling missed adults or progeny of overwintering adults. They could have been missed either because of the sampling method that was used, or because only a small number of parasites survived or a combination of these factors. 117 The sampling did indicate that there may be a native parasite that will parasitize the CLB eggs. Trichogramma minutum and/or evanescens are known as egg parasites of a variety of insects. It is possible that this parasite may find the CLB eggs so satisfactory for its development that it will build its population to the point where it may be of benefit in the control of the cereal leaf beetle. Even if this does not occur, it does indicate that there are native insects that may eventually find the CLB a suitable host. 1967 Field Releases Field release of Anaphes were made at three sites in Michigan in 1967. The first site was in Watertown Township (T5N, R3W, Sec. 4) of Clinton County (Fig. 18). The second site, in Jackson County, was on the farm of Harold Benn Jr. located northwest of Jackson in Sandstone Township (T28, R2W, Sec. 16). (See Fig. 19.) The third release site was in Ross Township (T18, R9W, Sec. 4 and 9) of Kalamazoo County at the Kellogg Gull Lake Farms of Michigan State University (Fig. 20). During the 1967 release period approximately 92,690 parasitized CLB eggs were released. Clinton County received 31,070 parasitized eggs, Jackson County 31,320 parasitized eggs, and 30,300 were released at Gull Lake (Table 35). The releases utilized milk cartons attached to wooden stakes for the field placement of parasitized CLB eggs (see Figs. 13 and 23). 118 Woods Wheat Oats \ I Releasé Point Figure 18 — Clinton County release site (1 mm 15 feet). Road 119 Benn Road / ESQ Alfalfa Corn Wheat Release ’,’P0int Area Sampled before Release" 7 in Oats Oats Clover NORTH Release Pom! ‘ s i ‘ ‘ f Figure 19 - Jackson County release site (1 mm = 15 feet). 120 NDRIH /J[Relzase PGlnt RT /_ Site 2 ll Area of Secondary \ / Growth /T\ \Release Pomt Site I , c w w c w c w o w o CZCM" C O: Oats W: Wheat Figure 20 - Release sites at Gull Lake (1 mm = 15 feet). 121 Table 35. The Anaphes flavipes release sites of 1967, dates of release, and the number of parasitized cereal leaf beetle eggs that were released Approximate No. Release of Parasitized Location Date Eggs Released Clinton County 17 May 2,570 Clinton County 21 May 2,570 Clinton County 25 May 3,240 Clinton County 30 May 4,000 Clinton County 1 June 3,340 Clinton County 10 June 3,290 Clinton County 13 June 12,060 Total 31,070 Jackson County, Wheat 19 May 2,570 Jackson County, Wheat 23 May 2,510 Jackson County, Wheat 26 May 3,190 Jackson County, Wheat 29 May 3,960 Jackson County,.Wheat 2 June 3,340 Jackson County, Oats 9 June 3,360 Jackson County, Oats 12 June 12,390 Total 31,320 Gull Lake, Site 1 19 May 2,570 Gull Lake, Site 1 23 May 3,290 Gull Lake, Site 1 26 May 3,190 Gull Lake, Site 1 29 May 4,000 Gull Lake, Site 1 4 June 3,440 Gull Lake, Site 1 9 June 3,290 Gull Lake, Site 2 16 June 10,520 Total 30,300 Grand Total 92,690 122 Samples were taken around the release points to determine the distance and directional dispersal of the parasites after release, and to obtain an indication of the egg density and parasitization rate. The sampling pattern used was similar at all the sites. It basically consisted of four lines intersecting at the release point. Lines radiated to the north-south, east- west, northeast—southwest, and northwest-southeast. Along the north-south and east-west lines samples were taken 10, 40, 80, 120, and 180 feet north, south, east, and west of the release point; along the other lines samples were taken 5, 20, 60, 100, and 150 feet from the release point. Any samples taken at distances greater than the above were taken at 50 foot intervals. Each sample taken during the survey period was approximately 1 square yard in size. After cutting, each sample was placed in a polyethylene bag, labelled, and stapled shut. This sometimes left the tops of tall plants exposed, but eggs are rarely found near the top or on the heads. Table 36 lists the range in total numbers of eggs taken per site and the average number of eggs per sample. After cutting, the samples were taken to the lab where they were stored at 400 F until examined. The samples were checked for eggs and if any were found, they were removed and placed in petri dishes with moist filter paper, one petri dish for each sample. The eggs were first checked 123 cm Hmcump pop .mmHQEmm ppm» mumsvm Doc mHmB mumo mpmucsao> CH cmxmu mmHmEmm mne .EOpcmH um Gmeu mucmam poo mo Hmnfis: UmuQDOUCU Q .Umuomaaoo mm3 mHmEmm mnu mafia map um pmmHmEm Um: mcflnuo: SUHCB Eonm mmmm moose l mmmm poowm o. n.6H Huo omlo o omN. mass on N monm .oxmq Haze m.m «.Ho onno omlm om moan sass N N mono .oxmq Haze m.oN s.ms Nona mmaam Ham mVON mono mN N mono .oxmq Haze N.NN o.mm mono NHHIO NNN New moss ma N oonm .oxoq Nose ... ... sno Norm s on mass on “mono snmoooao>v o.NN m.mo omno Naalm NNN Nos Nash N m moon .oxmq Haze o.NN o.mN smlo none 464 6N4 mono mN H doom .oxma Haze m.m N.mN onto ooalo osm mom mean no N open .oxmq Noam m.ON m.oN Nmauo Nmalo Nos Ham mean 6 N mono .oxmq Haze N. N.s Nuo oaro m non sass NH mono .moqsoo nemxomo m.N m.oN 6.0 were Nm oom mass m mono .Noeooo acnxomo m.o 6.6N more Nous omN mom mono 4N mono .socsoo 606x666 N.m m.6 oauo omuo om oNN moon ma mono .Nocooo 606x666 m. 6.4 mno mlo ma Ne moon NH noon: .mucooo consume 4. o.N ouo one NH mN moss N noon: .Noesoo acnxooo m. m. muo muo an 64 mass m momo .moasoo coonNNo H. s. Nlo muo m ma mass 6N noon: .monsoo acetone m. m. muo mlo oN NN mono N ommnz_.socooo concede mmmmm mmmm mmmmm mmmm mommm pmuomaaov Anomav muflm ooom Hmooe ooom Nance ooom mmmm moon mHmEmm\mUUm mo mHQEmm\mUUm mo Hmuoe .oz mmmum>¢ .02 Ca mmcmm cmxmp mnt mmHQEmm umzp mmumo mcu co mpflm mmmmamu mmdfl>maw mmcmmcm comm um cmxmu mmmm maummn mmma Hmmpmo mo Hogans mne .om mHQMB 124 for those that had had emergence before the samples were col— lected, and those found were counted and discarded. The re- maining eggs (= "good eggs" in Table 36) were observed in the lab for the development of Anaphes. As the parasites devel- oped, the number of parasitized eggs was counted, and after emergence the adults were sexed and counted. Samples were checked daily for emerged parasites, and every other day they were more closely checked for emerged CLB larvae, parasitized eggs, collapsed eggs or anything else that might be of inter— est. Table 37 lists the date, location, number of samples taken, and the number of samples with parasitized eggs. Clinton County In Clinton County, all releases were made in a wheat field with a small woodlot at the north end. There was an oat field adjacent to the east side of the wheat field but it was planted too late for any of the releases to be made there. Seven separate releases were made in Clinton County. Table 35 lists the releases and Figure 21 shows the sampling pattern used. This site was chosen primarily because of the low CLB population present (0-5 eggs/sq. yd.). Parasitized eggs were recovered at a maximum dis- tance of 40 feet north, south, and west of the release point. When the samples for 16 June were checked there was one egg in a sample taken 60 feet northwest of the release point that appeared to have had parasites emerge before the sample was 125 H NH Nash ea mono .N doom .mxmq Hasm s 6N Nana N mono .N ooflm .oxmq Haze Ha 6N moon MN mumo .N muflm .mxmq Haze m MN mCSb ma mumo .N mUHm .mxmq Haso "wucsoo OONmEmHmmr. H N mach ea CHOU CH mumo mnmocoao> .H moon .mxmq Haze ma 6N soon a mute .N ounm .oxmq Nose m 5H mGSb MN mumo .H muflm .mMmA HHSO 6 mm moon on mono w some: .N mono .mxmq Haze m mm mono v mumo 8 Damn: .H muflm .mxmq Hasw "wucsoo ooumfimamx N ma mash NH mumo .mucsou COmxomb ea mm mach m mumo .mucsoo COmxomb 8H am much gN mumo .mpcsoo comeMb 0 am mash ma mumo .mucsoo somxomb H hN mash NH ummfiB .mucdoo comxomh o 6N mash N ummnz .mpcsoo somxomb 0 mm mash m mumo .mucsoo coucHHU o mN mono 6H downs .Noaooo montage g mN mash H ummnz .mucsou coucHHU mmuflmmumm CDHB mmHmEmm omamEmm muflm mmmmamm mmHmEmm .oz .02 mqu mmuflm mmmmamu homa map How mmmfl>maw mmnmoc¢ mcflsflmucoo mmHmEmm mo Hmnfisc mnu paw .cmeu mmHmEmm mo HmnESG .coflumooa .mqu .h m magma Fence Row- - -.- VVheat 130 ’ v 150' ,, mo Regase 180' 120' 150' 0 180’ 126 NORIH 280' [A l -N- Oats ”230' 250' : 4——: 280' 230 .. 250' oSample sites 0 Sample sites with «230' parasite recovery absample site with possible L parasite recovery 280' x x0 Recovery perimeter Road Figure 21 - Sample points and recovery perimeter of Agaphes 'for 1 June 1967 in Clinton County (1 cm = feet). 4&3 127 checked. If this is correct then the maximum dispersal distance as shown by the samples was 60 feet. The circled sample locations in Figure 21 are those locations where parasites were recovered. In addition, one set of 35 samples was taken from the oats on 9 July to see if Anaphes had moved into them. None of the 16 eggs collected showed signs of Anaphes. The parasites that did emerge from the samples were first or second generation since the maximum development time from the original release to the time the adults emerged in the lab was about 20 days. In the lab, it might be pos- sible to get two generations in this time period (this would require an average daily temperature of approximately 800 F). It is difficult to determine whether these offspring are F1 or F2 due to the multiple releases made in this field. Jackson County The site at Jackson is actually two sites: a wheat field and an oat field. The first five releases were made in the wheat; once the oats were up and the beetles had moved into it the last two releases were made there. There is a woodlot about 290 feet south of the release point in the wheat. The west edge of the wheat field was bordered by clover and corn. 0n the west side of the clover was the oat field. The two release points were about 1225 feet apart. A list of the releases is found in Table 35. 128 The only sample from wheat in Jackson County to have parasites develop was taken 20 feet to the northeast of the release point. CLB oviposition was low in the wheat; conse- quently the number of eggs per sample ranged from 0 to 6 for 2 June and 0 to 8 for 12 June, 1967. In Figure 22 the circled sample site is where the parasitized egg was found. Before any releases were made in the oat field ten, l-yard strips of oats were taken from the northeast corner of the oat field opposite the release point in the wheat (Fig. 19). No Anaphes developed in any of the 64 eggs recovered, indicating that Anaphes may not have crossed from the wheat to the oats. The point of release in oats was 180 feet from the east side of the field and 40 feet from the south edge. There is a woods and stream located south of the point of release (Figs. 13 and 19). The maximum dispersal shown by the samples from the oat field of Jackson County is 430 feet directly north of the release point. In addition, a sample taken 280 feet to the west of the release point also had parasitized eggs. Figure 24 shows the sampling pattern used and the distribu- tion of parasitized eggs. The circled sample sites in Figure 24 are those that contained parasitized eggs. Little can be said about the dispersal of Anaphes in wheat on Benn's farm since only one sample contained parasit— ized eggs, but for oats several observations can be made. 129 Corn Wheat 0130' NORTH wo' a 3346 so - - - A V v v ' D 080' -‘\\Release 120 180 230 280 330 Point lwo uuo' uo' ~Samp1e site osample site with parasites (12 June dwo' 1957) Clover Woods Figure 22 - Sample points in wheat of Jackson County (1 cm = 40 feet). 130 Figure 23 — The Jackson release site in oats, looking north- east. Note the release containers and the wooden stakes. The stakes mark the location of the sampling sites. 131 Umo' ° Sample site Q Sample site with parasite u430' recovery Recovery perimeter , "IO 18 June 1967 €430 --—0 24 June 1967 a .... _ case 3 July 1967 ’63‘30. Clover ----- ._ .—-0 12 July 1967 .' '. O Parasite recovery at all ,I \ sample points {039' Oats -' it 230). NORTH ! 1 A1 300' l 55230.) ’N" ' , ' , 250' Release .4 - ,..-Point>( .’°. / \x " / , ,\ 'v'. A 9"" 3 //§) 4.2 \ ' . - £ : —: 330' 280' "Ed"“180‘—--120'-Z‘.T:.-.-=-fi I/Jlo' 80' 120' 180' Figure 24 - Sample points and recovery perimeter of Anaphes in oats for Jackson County (1 cm = 40 feet). 132 Anaghgs probably reached the woods to the south of the release site; if so, it should find an overwintering site if it overwinters as an adult. The first samples taken from the oats in Jackson on 18 June indicated a general movement to the northeast but when sampled on 24 June, the dispersal indicated movement to the north, northwest, and west. 0n 3 July, the sampling indicated that northern dispersal had become predominant with no greater movement in the other directions than had occurred on 18 June. The samples of 12 July indicated that the parasite movement to the north was maintained and that movement to the west had again occurred. Since the samples of 12 July were taken at 50 foot intervals at greater dis- tances than the previous samples, no samples were taken to the east and only one was taken to the northeast. Samples could not be taken any farther in these directions since the sampling was already near if not at the edge of the field. Consequently, nothing can be said for dispersal to the east or northeast for 12 July. For the distribution pattern of 12 July in Figure 24, the most distant sample sites that previously had Anaphes were used for the pattern to the east, northeast, northwest, and south. Although samples were taken as far as 250 feet to the northeast and 300 feet to the northwest, the maximum dis— tance that Anaphes was found to the northeast was 100 feet and only 60 feet to the northwest. If dispersal into the 133 woods to the south, southeast, or southwest occurred it was not detectable since the release was made only 40 feet from the south edge of the field. In the releases made in the oats, it is difficult to separate the generations present in the field for several reasons: (1) the two separate releases were made 3 days apart, (2) parasite emergence usually ranges over several days so that all the parasites released probably did not emerge the same day (this also holds true for the succeeding generations), and (3) females parasitize eggs over a period of several days. With the above factors in mind, an attempt has been made to determine the number of generations recov- ered in the field. Parasites emerging from the eggs recovered 18 June (Table 37) were F since no more than 8 days had elapsed 1 between recovery and the first release (parasites require 11 to 13 days to develop in the lab between 650 and 75° F). The adult parasites emerged in the lab 2 to 6 days after the samples were taken which means they were the equivalent of 9 to 5-day-old lab-reared parasites when collected in the field. Using the ratio, 9 da s 11 da s g—d—alYI—S- " —‘—)'{—Z—, X = 9.8 days 134 where 9 days = lab equivalent of the parasitized eggs when recovered from the field. 8 days = length of time the eggs were actually in the field, assuming the eggs were parasit- ized the day after the first release (10 June). 11 days = the normal development time for parasites in the lab at approximately 750 F. X = the estimated number of days for Anaphes to emerge in the field. Fl development in the field for the first release required about 10 days. Thus the F1 should have begun emergence in the field about 20 June. The F1 development is somewhat shorter than lab development at 750 F but the temperature in the field (Table 38—-it is assumed the temperature for Jackson is similar to that of the Benn farm which is 6 miles from Jackson), averaged 750 from 10 to 20 June and for the first 7 days the average was 790 F. The temperature in the field probably explains the rapid development. The parasites that emerged from eggs recovered 24 June required from 1 to 7 days to emerge in the labora- tory. Since it was calculated that the F1 of the first release emerged about 20 June, those eggs that required 7 days to develop were the offspring of the F1 that emerged 20 June. From the ratio %—%§§§ — ll—ggxg, X - 11 days 135 where 4 days = both the lab equivalent age of the parasit- ized eggs when recovered from the field and the total amount of time spent in the field (20 June to 24 June). 11 days = the normal development time for parasites in the lab at approximately 750 F. X = the estimated number of days for Anaphes to emerge in the field. one finds that the F2 would have emerged 11 days after 20 June or about 1 July. Table 38. The minimum, maximum, and average daily tempera- tures for the City of Jackson, Michigan (9 June to 17 July, 1967)a Temperature Temperature Date Min. Max. Aver. Date Min. Max. Aver. June 9 69 86 78 June 27 55 81 68 June 10 66 85 76 June 28 62 69 66 June 11 69 82 76 June 29 61 75 68 June 12 68 89 79 June 30 60 88 74 June 13 66 89 78 July 1 70 89 80 June 14 67 90 79 July 2 56 77 67 June 15 72 92 82 July 3 54 71 63 June 16 70 92 81 July 4 53 67 60 June 17 62 75 69 July 5 52 7O 61 June 18 58 76 67 July 6 50 79 65 June 19 50 76 63 July 7 57 82 70 June 20 58 83 71 July 8 63 86 75 June 21 55 73 64 July 9 72 87 80 June 22 60 76 68 July 10 72 80 76 June 23 58 80 69 July 11 66 88 77 June 24 60 86 73 July 12 6O 84 72 June 25 54 68 61 July 13 49 73 61 June 26 52 76 64 July 14 49 65 57 aData from Preliminary Climatological Data for Jackson, Michigan from the U.S. Weather Bureau. 136 The samples of 3 July required 1 to 7 days to emerge in the lab, thus in the field the parasites when recovered were the lab equivalent of 10 and 4 day old parasites. The eggs that were the equivalent of 10 day old lab-reared eggs were the F2 that were to emerge 1 July. Using the ratio, 10 da s 11 da 3 m = _—X—L_’ X = 14.3 days one finds that the F2 required about 14 days to develop and not 11 days, so that they emerged about 4 July. The F1 of the 12 June release emerged about 25 June. This date is obtained from the ratio: 10 da s 11 da 3 W = —')—{—L, X = 12.1 days Since the temperatures (see Table 38) present in the field during the development of the F2 for both the first and second releases are similar it is assumed F development of 2 the second release also required about 14 days, so they would have emerged about 9 July 1967. In addition, the samples of 12 July indicate the possibility of a third generation. The parasitized eggs required 3 days to emerge in the laboratory; hence, if para— sitized by the F that emerged 4 July, these would be F . 2 3 137 Gull Lake (Kalamazoo County) Sit§_l,--Site 1 was bordered on the east side by an area of secondary plant growth (Figs. 20 and 25). Next to this growth was a strip of oats about 80 feet wide, bordered on the west by a strip of wheat 80 feet wide. The release point in the center of the wheat strip was approximately 350 feet south of the road (B Avenue) which forms the north boundary of the field. On the west side of the wheat strip was a strip of oats after which came alternating 80 foot strips of wheat and corn. Six releases were made at this site. Table 35 presents a list of the releases. In the first two sets of samples taken from site 1 both the wheat and oats were sampled. After 19 June, only the oat strips were sampled because of the lack of eggs due to maturity of the wheat and movement of beetles from wheat to oats. In the samples of 4 June there was less than one egg per square yard of wheat and for 19 June there were only 13 eggs in 22, one square yard samples. Figure 25 shows the sampling pattern and dispersal of Anaphes at this site. At site 1 parasitized eggs were found in samples taken 150 feet northwest, northeast, and southwest from the release point. In addition, samples taken from voluntary oats in the corn strip on a line west of the release point showed that the parasites had crossed into the corn and were on both the east and west edge of the corn strip. This means that the parasites crossed an 80 foot strip of oats, III ills: ‘I JIIIII‘III‘ 5.11111 |.I \llill lil'. '13 I .ll Wheat Wheat Corn Wheat Oats (DISC Oats (with “lot?" mm 150' - "120.: 150' A l ' T o":' Ill ," 'N' ‘ 100' "80,, .. - q ' x ’ "EX'J’XX ' .ll ' : 6Q ‘ 40.0 X£ r" 60 1 ' x .’ . Release :l 14 July x '1 20 0 -4 ,vPolnt . $7—: 0 . ‘ " * * _'- 1' 40' [80" ‘120, 27 June 180 120 ‘3: 'I I all 1*‘ "/'l l *l . 'd‘. .12 / ‘ :1, 60 ._.} ** «.40 ,' ~50 // I ’9' ‘1‘). ’ ." ‘ "I 7‘, ** oz' ’ loo l;.¢/’*,,fl”’ '32 ’L” ' ' " , 150' 150 “120 ‘ V 180 Secondary Growth - Sample site 0 Sample site with parasite recovery - Recovery perimeter HO ll June 1967 no 19 June 1967 ---0 23 June 1967 ---01 July 1967 Figure 25 - Sample points and recovery perimeter of Anaphes on the south side of B Avenue (Site 1) Gull Lake, Michigan (1 cm = 40 feet). till-1151111111111 139 then crossed an 80 foot strip of wheat (only three unparasit- ized eggs were found in one square yard of this wheat on 4 June and two on 19 June), and also crossed 80 feet of corn (height of the corn was not recorded). The total distance covered was 275 feet. The circled sample sites in Figure 25 are those from which parasitized eggs were recovered. The lack of Anaphes recovery from wheat on 19 June was undoubt- edly due to the low number of eggs present in the wheat. The sampling showed that Anaphes moved out into those areas where eggs were present (in the oats). This is strikingly demonstrated in Figure 25 by the total lack of recoveries in wheat on the N—S sampling axis. Sampling was stopped at the end of the diagonals originally because the extent of Anaphes dispersal had not been anticipated at the time sampling began. In addition, since sampling_in wheat was discontinued after the second set of samples was taken, the diagonals were not extended even though Anaphes was found at the end of the southwest diagonal (150 feet from the release point). Furthermore, there were no other oat strips west of the diagonals into which to extend the sampling. The recoveries made in the corn were not placed in the dispersal pattern since these were separate samples taken by themselves, with no other samples taken at Site 1 at that time. The number of generations to develop at Site 1 cannot be calculated because the adults of the six releases 140 overlap one another and are spread over 22 days at 3 to 6 day intervals. Site 2.——After the beetles left the wheat and moved into the oats, the last release was made on 16 June on the north side of B Avenue which separates the fields. This is called Site 2. Here there were only corn and oat strips about 80 feet wide. The release was made in the center of an oat strip about 500 feet north of B Avenue which serves as the south boundary of the strips. Figure 26 shows the sampling pattern used. Two of the samples taken here on 19 June were in the oat strips on either side of the adjacent corn strips as were six of the samples taken on 23 June (three in each strip). The 11 samples taken 14 July were at greater dis- tances north and south of the release point than were the earlier samples. One sample taken 1 June from Site 2 showed that parasites had crossed the adjacent 80 feet of corn and moved into the next oat strip. This sample was taken 180 feet west of the release point. The distance included about 40 feet of the oat strip where the release was made, 80 feet of corn and 60 feet of the next oat strip. In addition, one sample taken on 14 July that was 430 feet south of the release point had parasites develop. With the exceptions of the recoveries of Anaphes 180 and 430 feet from the release point all recoveries were within 20 feet of the release 141 >180' ulzo' l80 lmd '30 20' NORTH mmm -’ 4 'mmmmmmMmmm ”N‘ Rune wO 180 \ Point 040' two m buo' 0180' Oats Corn Oats Corn Oats 3mm mm» 14 July 430' 480 - Sample site 0 Sample site with parasite recovery Recovery perimeter {-70 19 June 1967 no 23 June 1967 tome 1 July 1967 Figure 26 - Sample points and recovery perimeter of Anaphes on the north side of B Avenue (Site 2) Gull Lake, Michigan (1 cm = 40 feet). 142 point. The circled sample sites of Figure 26 are those from which parasites were recovered. The parasites emerging from samples taken 19 and 23 June were Fl since a maximum of 7 days had passed since the parent parasites were placed in the field as black pupae and the samples of 23 June were taken. Since one egg of the 1 July samples had Anaphes emerge the day they were first examined, it appears that development of the F required 1 about 14 days. Temperatures were similar from 16 June to 1 July and from 2 July to 14 July (Table 39), thus develop- ment times for the parasites recovered 14 July should be about 14 days, making the parasites recovered 14 July F2. Parasitization Rates Table 40 lists the parasitization rates for the various release sites on the dates the samples were taken. Two different rates are given. In one, the parasitization rate is based on the total number of eggs found in the samples. The other rate is based on the number of good eggs which are defined as those that had not hatched or had any- thing emerge by the time the samples were collected. When one looks at the parasitization of good eggs from oats at all the release sites, one finds there is a steady increase in the rate from the first set of samples to the last set. It is not at all obvious when one uses the total egg counts, but here one is unable to say definitely 143 Table 39. The minimum, maximum, and average daily tempera— tures for Gull Lake in Kalamazoo County, Michigan (16 May to 14 July, 1967)a Temperature Temperature Date Min. Max. Aver. Date Min. Max. Aver. May 16 34 62 48 June 15 71 88 80 May 17 43 69 56 June 16 72 88 80 May 18 48 75 62 June 17 66 83 75 May 19 54 75 64 June 18 57 77 67 May 20 38 64 51 June 19 50 76 63 May 21 38 68 53 June 20 61 80 71 May 22 39 62 50 June 21 55 79 67 May 23 36 60 48 June 22 57 77 67 May 24 47 77 62 June 23 56 80 68 May 25 51 83 67 June 24 67 82 75 May 26 46 82 64 June 25 50 70 60 May 27 57 81 69 June 26 51 78 65 May 28 54 76 65 June 27 54 80 67 May 29 ..b ..b ..b June 28 59 79 69 May 30 41 70 56 June 29 60 75 68 May 31 44 68 56 June 30 60 83 72 June 1 45 78 62 July 1 65 85 75 June 2 46 80 63 July 2 61 84 73 June 3 51 86 69 July 3 64 75 70 June 4 51 82 66 July 4 48 70 59 June 5 58 85 72 July 5 46 68 57. June 6 63 79 71 July 6 50 78 64 June 7 64 75 70 July 7 54 79 67 June 8 65 82 74 July 8 64 82 73 June 9 67 82 74 July 9 67 82 75 June 10 65 83 74 July 10 69 80 70 June 11 66 81 74 July 11 65 85 75 June 12 66 88 77 July 12 62 82 72 June 13 66 87 77 July 13 57 77 67 June 14 65 86 76 July 14 51 67 59 aRecord 9f_Climatoloqical Observations, W. K. Kellogg Biol. Station, Kalamazoo County, Michigan, Jan. to Dec., 1967, U.S. Weather Bureau. bTemperatures not given. ]J44 .pHmHm mHau CH mmmamamu o: mHmB mama» moaflm mmoHa> Hauou Ca ampsaocw uoa mua aoHuaooH mwau HON mmfiaa> maB o .omuunooo moammHmEm 020 .madsm xoaaa ommoam>mo uaau mmmm maumma mama Hamumo omuwuamanam mo umaEsc man u mmfl .pmxomao umHHm mumz mmHmEam mau cma3 moammHmEm on Caa uaau mmmm mHumma mama Hamumu mmoau mua mmmm ooooa ..... o o o o 6 HH eH honors omuoo .moesoo coucHHo Hum.N mom can w.mH m.o oam MHhN moom maauoe oaauo Hum.H OHH moH e.oa m.m oNH ooh mNH¢ Hauoa N muam .mxaq HHoo Huo.H o H o.mN moo. H v oma hoIeHrb muao .N muam .mxaq Haso Hue.H 6H NN H.HN S.H ma om mafia morals muao .N muwm .mxaq Haze Hum.H Ho Hm n.6H ¢.m mo Ham m¢ON hoIMNlo muao .N muam .mxaq HHDU Hum.H mm Ho N.mH m.¢ hm HmN Nun normalo muao .N muam .mxaa daze Hun.N moH ome v.6H w.oa th vmma emmN Hauoe H muam .maaq Haso Hum.N Hm HoN w.hh m.mH oeH mma Non nonalh nuao .H muHm .maaa Haze H.N.N mN No o.mN ¢.mH km omH 6N4 soumNuo . memo .H muHm .mxaq HHso Hum.N we mHH m.o~ H.HH on com mmm soumHlo Home; a memo .H ouHm .mxaq HHsm Hum.e N m woo. 600. 0 Non HHm bolero uamaz a muao .H muam .maaq HHso Hum.N no moH h.wN o.m mm mom oeNH Hauoa muao .huasoo coaxoab Huo.N H N 6.00 m.H N m 50H holNth muao .hucsoo coaxoab H"6.N mH Hm n.6m m.m om Nm com mounts muao .hucsoo conxmab Huv.N do mOH m.mN m.0H om eoH hem hoIvNIw muao .huasoo COmxoab HHN.N m m ¢.HH m.¢ HH om oNN normalo muao .hucsoo COmeah ..... ... ... o.¢ m.H H mN no Hauoa mamas .huasoo cemxoah ..... ... o... N.N o.N H MH .Na NouNHuo yams: .Noesoo consume ..... o o o o 6 NH mN NouNuo some: .muesoo eomxooo Hum.m N n h.HN o.MH m mN mm Hauoa .mucsoo GOucHHo ..... o o o o o m mH NouoHuo Hoodoo :oueHHo Hum.m N n o.mN m.mN m 0N HN hoIHlm hucdou COuGHHU oHuam b a ammmm mmmm mm auNB ammmm mmmm muao muwm “w .02 .oz @000 Hauoe mmmm .oz @000 .oz coHuaNHanauam .oz Hauoa ucmoumm hood aw mama dado ua oca .muasou COmeab .auasou coucHHO CH cmxau mmHmEam mau Eouw mmmm may no Goduauauwmauam ucmuumm man tca mmmm pmuumaaoo mama“ omuauamauam Eouw mmumEm ou mmaw>aam mmadaad mane Cam maaamm mo Hmaasc mas .ov maaae il'lltlll'ill ll. 145 how many eggs had previously had CLB larvae hatch and how many had parasites emerge. In actuality, the real parasitization rate probably lies somewhere between the 6.3% "total egg rate" and the 18.8% "good egg rate." It is probably higher than the total egg rate since some of the spent eggs may have had Anaphes develop. If Anaphes developed from any of the spent eggs than the rate would be higher. Likewise, the true rate is probably lower than the good egg rate since it does not con- sider any of the eggs that had emergence before the samples were checked. Summary The sampling of 1967 showed that Anaphes can spread into adjacent strips of oats and wheat when the strips are as wide as 80 feet. It also showed that barriers of 80 foot wide corn strips can be crossed by Anaphes at least when. voluntary oats are present in the corn. The sampling also showed that Anaphes can move at least 430 feet from the release point in one season. In addition, it was found that at least two and possibly three generations of Anaphes can be recovered from the field. The 1967 release containers proved to be far superior to the stakes used in 1966 for releasing Anaphes as black pupae for several reasons: (1) they can be used more than once, (2) the placement of the parasitized eggs in the field is relatively easy and quick, (3) the containers protect the 146 eggs from the elements better than the stake method and (4) the carton's confined space probably allows more opportunity for mating by temporarily confining the emerging parasites together in closer quarters. Black pupal release has several advantages over adult releases. The black pupae do not need to be placed in the field at the time of development as is true with adult releases, so when adverse weather conditions prevail on the release date, the pupae can be stored at 400 F until more favorable conditions occur. In addition, by releasing pupae, any stragglers that emerge a day or two later than the major- ity of the parasites will also get into the field; this is especially true if the releases are made at other than daily intervals. SUMMARY AND DISCUSS ION In the mass culturing of Anaphes flavipes there are two basic techniques: removing CLB eggs from plants upon which they are laid or leaving the eggs on the plants. There is no difference between the two methods as evidenced by the number of parasitized eggs to develop per female used. Although no difference occurred between the basic techniques of rearing Anaphes, at this time the disadvan- tages outweigh the advantages of the non-removal of CLB eggs from plants. The number of eggs within the plastic boxes (approximately 600 cubic inches in size) equals the number of eggs contained in three or four, 50 mm x 12 mm petri dishes used when eggs are removed from plants for parasitiza— tion. In other words, the containers require about 94 times as much space as do the petri dishes. In addition, the lack of an accurate method of estimating the number of eggs present in the plastic boxes and globes poses much of a problem to their use. Consequently, the use of petri dishes (removal of eggs from plants) is probably the better tech— nique for mass rearing Anaphes even with the time consuming disadvantages of egg removal. When a technique for non- manual removal of eggs is developed the use of petri dishes will be far superior to the use of the larger mass culture containers. 147 148 With the use of petri dishes for rearing Anaphes an apparatus for collecting emerging adults has been developed. With the device about 93%.of the emerging females are col- lected. Only 70% of the males to emerge are collected indicating that they may not be as attracted to light as are females. It is also possible that the males at emergence remain in the vicinity to mate with emerging females. In the storage of unstung CLB eggs there appears to be a period of time after which they are no longer suitable for Anaphes development. At 400 F this appears to be approx- imately 6 to 8 weeks. It was originally believed that the presence of fungus on stored eggs was responsible, but tests with fungicide-treated eggs proved fungus did not cause the lack of egg suitability after 6 to 8 weeks of storage. With the use of gas atmospheres for CLB egg storage at 400 F the 6 to 8 week critical period is not increased, but neither is it decreased. At 140 F storage in gas atmospheres, develop- ment of either CLB larvae or Anaphes does not occur after 30 days of storage. Although storage of unstung CLB eggs in various gas atmospheres has proved to be no better than storage in air at 400 F it does not mean that this technique will not pro- long storage life of eggs. Since storage in gas at 400 F and storage in air at 400 F were similar perhaps further testing with other proportions and/or gases may prove suc- cessful. In the case of fruit and vegetable storage in tillllll 149 controlled atmospheres, conditions favorable for one fruit or vegetable may not be favoralbe for another or even for varieties of the same fruit or vegetable. Thus, this tech- nique is potentially useful for long term storage of insects, and further tests should be conducted, not only with differ— ent gases and proportions, but also at temperatures other than 140 F and 400 F. Parasitized eggs also are storable. Eggs in the black pupal stage can be stored for at least 7 weeks without eliminating viable sperm or mating of the resulting adults. However, after 2 to 4 weeks of storage, the resulting adults appear to be less active than those emerging from unstored eggs. In parasitized eggs stored 2, 4, 6, and 8 days after parasite introduction, it was noted that as storage time increased, more and more parasitized eggs failed to have emergence. One of two reasons may explain the failure to emerge: (l) the parasites were dead or (2) they had gone into diapause. Since the parasitized eggs collapsed without anything emerging, it is not known whether the parasites would have emerged. Anaphes can develop from CLB eggs laid by untreated female CLBs previously mated with males treated with 2000 roentgens X-ray. In addition, Anaphes develops from CLB eggs treated with as much as 5000 roentgens X-ray. Of treated eggs, those treated with 3000 roentgens appear best 150 for maximum parasite production and no CLB larval hatch. Furthermore, it is much easier working with eggs treated directly with X-ray than those eggs resulting from treated adults. With the direct treatment of eggs, the sexing of beetles is eliminated as well as the need for virgin females to be mated with the treated males (virgin females are needed to eliminate the possibility of previous matings with untreated males). The 1966 field release of Anaphes indicated that it would develop in the field when released. Also, recoveries showed that when parasites are released as black pupae, mating does occur in the field. Sampling in 1967 of 1966 release sites did not reveal the presence of overwintered Anaphes, but this cannot be construed to mean they did not overwinter. It is possible that too few survived the winter to be recovered in the sampling. The 1967 release results indicated that Anaphes will disperse at least 430 feet from the release point in one season. Furthermore, the release showed that alternating 80 foot-wide rows of wheat and oats did not prevent the movement of Anaphes. It was also found that 80 foot—wide rows of corn did not act as a barrier to Anaphes, partly due to the presence of CLB eggs on voluntary oats in the corn. 151 Currently, there is in use at the Niles laboratory techniques that permit the production of sufficient para- sites for field release in an attempt to establish Anaphes flavipes as a biological control agent of the cereal leaf beetle. It now remains to be seen whether the conditions present in those localities where Anaphes releases have been made are sufficient for its establishment. The location of its overwintering site(s) is the most important single piece of information needed to better estimate whether establish- ment of Anaphes will be possible (especially if an alternate host is necessary). LITERATURE CITED Bakkendorf, O. 1964. Notes on Pattason Walker, Anaphes Hal., and Cleruchus detritus n. sp. (Hym., Mymaridae). Entomophaga 9(1):3-17. Brennan, P. A. 1967. 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The controlled atmosphere storage - local and national. Wash. State Hort. Assoc. Proc. 57th Ann. Meeting 57:133-136. Yun, Y. M. 1967. Effects of some physical and biological factors on the reproduction, development, survival, and behavior of the cereal leaf beetle, Oulema melanopus (Linnaeus) under laboratory conditions. Ph.D. Thesis, Mich. State Univ. 153 p. M ' ililllllllfilllll llllll lilllilillllllllllll lllllalllllllpll ES 3 1293 03082 8200