' MSU LIBRARIES m RETURNING MATERIAL§z Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. INTERSPECIFIC RESPONSES OF DIPRIONID SAWFLIES TO NATURAL AND OPTICALLY ACTIVE AYNTHETIC PHEROMONBS BY Julius Ipadeola Olaifa A DISSERTATION Submitted to Michigan State Univeristy in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Entomology 1984 :i/VL/e’v) © COPYRIGHT Bv JULIUS IPADEOLA OLAIFA I984 ABSTRACT INTERSPECIFIC RESPONSES OF DIPRIONID SAWFLIES TO NATURAL AND OPTICALLY ACTIVE SYNTHETIC PHEROMONES BY Julius Ipadeola Olaifa In this study» the responses of diprionid sawflies to mixtures of optical isomers of 3,7-dimethylpentadecan-2-yl acetate or proprionate were studied both in the field and by electroantennogram (BAG). Various aspects of behavioral response of male sawflies to sex pheromones were studied in the field and greenhouse. The nature of chirality of the natural pheromones from the females of different species was determined by gas liquid chromatography (GLC). The field studies show that (1) all the fall-flying species respond mainly to the acetate while the spring/early summer-flying species respond either to acetate (designated as 'A') or proprionate (designated as 'P'); (2) the (28,38,78)-3,7-dimethylpentadecan-Z-yl acetate (28,38,7S-A) or proprionate (28,38,78-P) isomer was active against all Nggdiprign_species, but 28,3R{7R-P was the active isomer for 1212111211 8111111148 and W Wm (Fabricius): (3) the effectiveness of the 28.38.78 isomer was significantly im- proved by addition of varying concentrations of 28,3R;TR or 28.3R.7S isomer. Only 1L annulus annulus and H. lemtei Julius Ipadeola Olaifa appeared not to respond synergistically to mixtures of isomers. Through BAG analysis. synergistic interaction was found among the optical isomers only when submicrogram amounts of the compounds were used to stimulate the male antennae. 'The synergists appeared to increase the maximum response value infirm: hummus. andmninstum. By using capillary GLC analysis on samples purified on TLC and preparative GLC it was found that the erythro (i.e.. 28.38 or 2R.3R) isomers can be separated from the threo (i.eu' 28.3R or 2R.3S) isomers, with the threo isomers having longer retention time. Analysis of the natural pheromone showed that only 28.38.78 isomers were detectable in N. leggntei. N. nanulns annulus. and H. W. However. in D. similia and N- Rlnfitum.two isomers 28.38.78 and 28.3R.7R were detectable. The same set of isomers is believed to be present in n. .uzatti bankfiianae. Two peaks corresponding to 28.38.78 and 28.3R.78 are indicated in N,;11;g1niann§, Natural phero- mones of two other species. H. mm: and N. W appeared to have these isomers. There were variations in the amount of pheromone extracted from different species. Behavioral response to pheromone source is similar in Q. aimilia. N. gigginianna and N..sertifer, The optimum height at which males respond to pheromone on the host plant is about 3.5 m. To my children: Bolarinwa Tiwalola. Olubunmi Ikeolape and Olufunmilola Eniola ii ACKNOWLEDGEMENTS Conducting this study has been a challenging and en- lightening experience. Dr. Fumio Matsumura. my major pro- fessor provided valuable guidance and example of academic excellence. The support given by Mrs. Teruko Matsumura to my family is also appreciated. My thanks to both of them. Appreciation is extended to other members of my guid- ance committee. Dr. Gary Simmons. Dr. Donald Farnum. Dr. George Ayers and Dr. Jim Miller for their guidance and instruction throughout the course of study. I thank Dr. Sonny Ramaswamy of Mississippi State University for reading the draft of this thesis and the help of Dr. R. C. Wilkinson for the field test at the University of Florida. Gainesville is well appreciated. This study would not have been com- pleted without some important samples provided by Dr. H. C. Coppel of the University of Wisconsin. Madison. I also enjoyed the c00peration of Dr. Mark Kraemer of Virginia State University. Petersburg. I also thank all the members of Dr. Matsumura's group past and present. especially Dr. B. V. Madhukar. Dr. Keiji Tanaka. Dr. Yoshihisa Ozoe. Dr. John Quensen. Yoshihisa Watanabe. Tadashi Kikukawa (T.K.). Dr. K. Yoneyama. Dr. John Marshall Clark. Jeff Scott. Dr. Ateef Kadous. Dr. Chris iii Mullin. Kevin Blair. David Grant. Ignatius Hadioetomo. Dr. 8. M. Ghiasuddin. Dr. Subhash Rashtawar. Petros Charalambous. Gen Tsushimoto and Jay Coach for the good times together while working. playing golf or fishing some- where in Michigan. Special thanks also go to all the mem- bers of Dr. Jim Miller's group. especially Marion Harris. Paul Weston. John Behm. Jim Keller and Joan Harlin for being very friendly all the time. Mr. Jose Varlade provided a lot of assistance in getting my work done around the greenhouse. my special thanks to him. .All the administrative staff in Dr. Matsumura's office are thanked for their assistance all the time. I also thank all the members of Dr. Mathew Zabik and Dr. Dick Leavit groups for their assistance in the gas liquid chromatograph works. Appreciation is particularly extended to my parents Olaifa and Aina and my brothers Iyiola and Olawuwo and sisters Faboyin. Fadunke and Irawoola for making the in- numerable sacrifices during my early years in school. I thank the University of Ife. Nigeria and the Federal Govern- ment of Nigeria for providing me the support for the train- ing. My cousin Chief 8. O. Kolade has always been my mentor. he deserves my special thanks. I have also enjoyed the friendship of Tunde Kolade. Larry Powell. Barry Cooley. Ayo Walker. Nath Ejelonu. Segun Yerokun. Bode Osofisan. Segun Matanmi. Richard Nsiah-Yeboah and Dave Baker here in the 0.8.A. The assistance and communication maintained with Nigeria through my friends Ayo Akinsanya. Revd Femi Oladele. iv Adewale Olawale. Kunle Taiwo. Titilola Sule. Abidemi Opalola. Role Adeboyeku. and Ifelodun Youth Club. Ilora was a significant inspiration to the successful completion of this study; Finally» I thank my wife Janet for her love. understanding and dedication to the course of completion of this study. TABLE OF CONTENTS LIST OF TABLES O O O O O I O O O O I O O O O O O O O 0 LIST OF FIGURES O O O O O O O O O O O O O O O O O O 0 CHAPTER ONE: Field Evaluation of Chiral Isomers of the Sex Pheromone of the European Pine SaWflYrWW o o o o o 0 INTRODUCTION . . . . C . C . C . . . . . . . . . MATERIALS AND METHODS O O O O O O O O O O I O O 0 Field TeStS I O O O I O O O I O O O O O O 0 Synthetic Pheromone . . . . . . . . . . Partial Purifications of Natural Pheromone from ROE-Treated Body. . . . Purification of 28. 3S. 7S-A by Gas Liquid Chromatography (GLC) . . . . . . . . . . RESULTS 0 O O O O O O O O O O O O O O O O O O O O D I SCU SS ION O O O O O O O O O O O O O O O O O O 0 REFERENCES 0 I O O O O O O O O O O I O O O O O 0 CHAPTER TWO: Response fo Male Red Pine Sawfly. nanulua,nanulus.(Hymenoptera: Diprionidae) to Natural and Mixtures of Optically’Active Synthetic Sex Pheromone 3.7-dimethylpentadecan-2-ylacetate . . . INTRODUCTI ON 0 O O O O O O O O O O O O O O O O 0 MATERIALS AND METHODS I O O O I O O O O O O O O 0 Field Tests 0 I O O I O O O O O O O 0 Laboratory Rearing of Larvae and Pupae . . Synthetic Pheromones . . . . . . . Extraction of Natural Pheromone for Field Tests 0 O O O O O I I O O O O I O O 0 O 0 RE SULTS O O O O O O O O O O O I I O O O O O O O 0 vi xii bbUNNNl-‘H l-‘H ant-a 15 15 16 16 18 18 21 DISCUSSION 0 O O O O O O O I O O O O O O O O O 0 REFERENCES CHAPTER THREE: INTRODUCTI MATERIALS RESULTS . Response of Male Jack Pine Sawfly (Eymen- optera: Diprionidae) to Mixtures of Optical Isomers of the Sex Pheromone 3,7-dimethylpentadecan-Z-ylacetate . . ON C O I O O O O I O O O O O O O O O C AND METHODS O O O O O O O O O O O O O 0 DISCUSSION 0 O I O C O O O O O O O O O O O O O 0 REFERENCES CHAPTER FOUR: INTRODUCTI MATERIALS Field Field Response of Male Red-headed Saw- fly Mediation 1W (Hymenoptera: Diprionidae) to Mixtures of Optical Isomers of the Sex Pheromone 3,7-di- methylpentadecan-Z-ylacetate . . .... ON 0 O I O O O O O O O O O O O O O O 0 AND METHODS O O O O O O O O O O O O O O Tests 0 O O O O O O O O O O O O O O 0 Synthetic Pheromone .. . .. . .. . .. . RESULTS . DISCUSSION 0 O O O O O O O O O O O I O O O O O 0 REFERENCES CHAPTER FIVE: INTRODUCTI Field Response of Male Neodiprign (Eymenopteraz Diprionidae) to Mixtures of Optical Isomers of the Sex Pheromone 3,7-dimethylpentadecan-2- ylpropionate . . . . . . . . . . . . . ON 0 O O O O O O O O O O O O O O O I 0 vii 33 36 38 38 39 40 45 52 54 54 56 56 57 58 58 64 65 65 MATERIALS AND METHODS . . . Field Tests . . . . . Natural Pheromone . . Synthetic Pheromone . Purifications of Synthetic Charcoal-Celite Column RESULTS 0 O O O O O I O O 0 DISCUSSION . . . . . . . . REFERENCES 0 O O O O O O 0 CHAPTER SIX: Nggdipzign Isomers Synergistic Response to Mixtures of Optical Isomers of the Sex Pheromone 3,7-dimethylpentadecan-2— yl. acetate INTRODUCTION . . . . . . . MATERIALS AND METHODS . . . Field Tests . . . . . Synthetic Pheromone . Purification of Synthetic Isomers by Charcoal-Celite Column RESULTS 0 O O I O O O I O I DISCUSSION . . . . . . . . REFERENCES 0 O O O O O O 0 CHAPTER SEVEN: Attractancy of Optically Active Iso- mers of Pheromone 3,7-dimethylpentade- can-Z-yl propionate for Male 12132191 Diprionidae) on similia the Field INTRODUCTION . . . . . . . MATERIALS AND METHODS . . . Field Trapping . . . . Natural Pheromone . . Synthetic Pheromone . (Hymenoptera: Purification of Racemic Isomer by Charcoal-Celite Column . . . Preparative GLC Fraction Collection viii a”... ,7 69 69 70 70 71 83 87 90 90 91 91 93 94 98 103 105 105 106 106 106 107 108 108 RESULTS I O O O O O O O O O O O O O O O O O O O O DI SCUSS ION O O I I O I O O O O O O O O O O O O 0 REFERENCES 0 O O O O O O O O O O O O O O O O I 0 CHAPTER EIGHT: Gas Liquid Chromatograph (GLC) Separa- tion of the Optically Active Synthetic and Natural Sex Pheromone of Diprionid Pine Sawfly Species . . . . . . . . . I NTROD UCTI ON 0 O I O O O O O O 0 O O O O O O O 0 MATERIALS AND METHODS O I O C O O O O O O O O O O Preparative GLC Fraction Collection Capillary GLC Analysis . . . . . . . . . . . RESULTS 0 O O O O O O O O O O O O O O O O O O O 0 DISCUSSION 0 O O O O I O O O O O O O O O O O O 0 REFERENCES 0 O O O O O O O O O O O O O O O O O 0 CHAPTER NINE: Electroantennogram Responses of Dipri- onid Pine Sawflies to the Natural and Optically Active Synthetic Pheromones and to TLC Fractions of Body Extracts IMRODUCTION O O O O O O O O O O O O O O O O O 0 MATERIALS AND METHODS Q Q Q 0 O O O O O O O O O 0 Preparation of Antenna for EAG . . . . Electrodes and Recording Instruments . Pheromone Delivery System . . . . . . Response Evaluation Method . . . . . . Synthetic Pheromones . . . . . . . . . RESULTS 0 I I O O O O O O O O O O O O O O O O O O DI SCUSS ION O O O O O O O O O ‘ O O O O I O O O O 0 REFERENCES 0 O O O O O O O O O O O O O O I O O 0 ix 109 125 131 133 133 135 136 137 137 152 156 158 158 159 159 160 161 162 162 163 176 181 CHAPTER TEN: Behavior Response to Optical Isomers of Sex Pheromone and Development of Non- Saturating Trap for Diprionid Sawflies . INTRODUCTION 0 O O O O O O O O O O O O O O O O 0 MATERIALS Am METHODS O I O O O I I O 0 O I O O O Courtship Behavioral Studies . . . . . . . . Effect of Trap Height . . . . . . . . . . . Trap Designs . . . . . . . . . . . . . . . . RESULTS 0 I O O O O O O I O O O I O O O O O O O O Pheromone Source Parameters . . . . . . . . Courtship Behavior of Male n, virginianna in the Field . . . . . Courtship Behavior of Male 5, similis in the Field . . . . . . . . . . Courtship Behavior of Male n, sertifer Trap Height . . . . . . . . . . Trap Designs: H. zirsinianua . . Trap Designs, 2, similia . . . . Trap Designs, u, virginianna and nanulus - . DISCUSSION 0 O O O O O O O O I O O O O O O O O 0 Location and Recruitment of a Mate . . . . . Courtship Behavior of Male Diprionid Saw- flies in the Field 0 O O O O O O O O I Trap Design . . . . . . . . . . . . . . . REFERENCES 0 O O O O O O I O O I O O I O O O O 0 APPENDIX 1: Field Response of Male Abbott Sawfly, M (Hymenoptera: Dipri- onidae) to Mixtures of Optical Isomers of the Sex Pheromone 3,7-dimethylpentadecan- 2-y1acetate or propionate . . . . . . . . INTRODUCTION 0 o o o I o o o o a o o o o '0 o o 0 MATERIALS AND METHODS o o o o o o o o o e o o o 0 Field Tests 0 o o o o e o e o o o o o e o 0 Synthetic Pheromones . . . . . . . . . . . Purification of Synthetic Isomers by Charcoal-Celite CODE!!! 0 o o o o o o o o RESULTSOOOOOOOOOIOOOOOOOOOOO 183 183 186 186 188 189 192 192 198 204 207 210 213 216 217 217 217 222 223 226 229 229 231 231 232 233 234 DISCUSSION . . . . . . . . . . . . . . . . . . . 240 REFERENCES . . . . . . . . . . . . . . . . . . . 245 APPENDIX II: Cuticular Hydrocarbon of Nine Species of Pine Sawfly .. . .. . .. . .. . .. 247 INTRODUCTION . . . . . . . . . . . . . . . . . . 247 MATERIALS AND METHODS . . . . . . . . . . . . . . 249 RESULTS . . . . . . . . . . . . . . . . . . . . . 250 DISCUSSION . . . . . . . . . . . . . . . . . . . 265 REFERENCES . . . . . . . . . . . . . . . . . . . 270 BIBLIOGRAPHY O O O O O O O O O O O O O O O O C O O O O 272 xi 1-3 1-5 LIST OF TABLES Comparison of N. aertifgr trap catch between original 28,38,7S-A prepartions and that puri- fied by GLC, September 19 to October 26, 1981 at Rose Lake, Lansing, Michigan .. . .. . .. Stimulatory effect of trace amount of 28,38,7R— A on effectiveness of trap catch by 28,38,7S-A. The test was conducted at Rose Lake, Michigan. September 19 to November 9, 1981 . . . . . . . Stimulatory effect of different threo isomers on 28,38,7S-A in attracting male N; a:xti£:z.on the field. Test conducted at Rose Lake, Michi- gan, September 25 to October 19, 1982 .. .. . Importance of configurations at C-7 of threo isomers on effectiveness of trap catch by 28,38,78-5. The test was conducted at Rose Comparison of field effectiveness of natural and synthetic pheromone of N. sentiifizi Rose Lake, Michigan, October 9 - November 9, 1981 . Field response of male N. annulus, annulus, to the increasing concentrations of two isomers. 28,3R,7R/S-A and 28,3R,7R-A on 23,38,7S-A isomer. Test was conducted in Roscommon County between September 4 and October 16, 1981. . . . Field response of N. nanulus ninnlna to the increasing concentration of two isomers 28,3R,7R/S-A and 28,3R,7R-A on 28,38,7S-A iso- mer. The test was conducted at two locations in Michigan from September 4 - October 16, 1981 Field response of N. annulus annulus to 28,38,7S-A and mixtures of 28,38,7S-A with serial dilutions of 28,3R,7R—A isomer. Test conducted at Vogel Center, Michigan, September 10 to catcher 7' 1983 0 O O O O O O O O O O O O xii 10 12 22 23 24 2-8 3-1 0 3-3 Comparison of field effectiveness of synthetic and natural pheromone of N. annulus annulus. Test conducted at three locations in Michigan between September 5 and October 16, 1981 . . . Comparison of field effectiveness of the syn- thetic and national pheromone of nnnulus_nnnu; lus. Test conducted at Vogel Center, Michigan, September 16 to October 16, 1981. Three repli- cates randomized three times . .. . .. .. . Threshold of the synthetic pheromone 28,38,7S-A isomer from two sources and comparison with natural pheromone on the field against N. annulus annulus at Vogel Center, Michigan, September 18 to October 17, 1982 . . . . . . . Field effectiveness of the TLC fractions of alcohol and ester from the female N. annulus annulus. Test conducted at Vogel Center from September 16 through October 16, 1981 . . . . . Effect of 28,38,7S-diprionol on the field ef- fectiveness of 28,38,7S-A against N5 annulus, annulus. Test conducted at Vogel Center, Mich- igan, September 18 to October 17, 1982. . . . . Response of males of N. annulus annulus and N. sending to different combinations of optical isomers of 3,7-dimethy1pentadecan-2-y1 acetate. Test conducted in a red pine stand at Vogel Center, Michigan, October 16-81, 1982 . . . . . Field response of males N. arnttl bnnlssinnns to traps baited with synthetic pheromones. The test was conducted from September 13-October 9, 1980 at Hartwick Pines, Michigan . . . . . . . Field response of N. m1 banksianae males to traps baited with synthetic pheromones or admixing of synthetic pheromones. The test was conducted from September 13-October 9, 1980 at Hartwick Pines, Michigan .. .... .. . .. . Synergistic effect of the 38, BR, 7R/S-A on 23, 38, 78-13 isomer on attraction of male N. bnnksinnns in the field. Test was Pratti conducted September 26-October 16, 1981 at Hartwick Pines, Michigan . . . . . . . . . . . xiii 26 2'7 29 30 31 32 42 43 44 3-4 4-2 4-3 5-2 5-4 EffeCt Of the 28'3R17R-AG’ 28'3R'7R-A} 28,3R,7S-A and 28,3R,7R/S-A isomers on the 28,38,7S-A isomer in stimulation of male N. W bnnkslnnns attraction to baited traps in the field. Testing was done August 30-November 14, l982.at Hartwick Pines, Michigan . . . . . Threshold of the synthetic pheromone of N. . Test was conducted from mtti bankaianae September 27-October 16, 1982 at Hartwick Pines, Michigan . . . . . . . . . . . . . . . . Catches of first generation male N. lsannnsl by traps baited with 28,38,7S-A/2S,3R,7R-A mixture at 3 locations in Florida. Test conducted on slash pine stand mid-May through June 1, 1983 . Catches of first generation male N. lnngntsl by traps baited with 28,38,7S-A/ZS,3R,7S-A mixture at 3 locations in Florida. Test conducted on slash pine stand mid-May through June 1, 1983 . Catches of third generation male N. lnsnnasj. by traps baited with 28,38,7S-A/28,3R,7R-A mixture at 3 locations in Florida. Test conducted on sand pine stand mid-November through mid- December1983................. Field response of N. ulnalnlnnus to optical isomers of 3,7-dimethy1pentadecan-z-yl propionate or acetate. Test conducted at 5 locations in Roscommon, Crawford and kalkaska counties, Michigan, May lS-July 10, 1981. Two replicates at 4 locations and 3 replicates at one location, all randomized twice . . . . . . Field response of N. 11minlnnus to mixtures of optical isomers of 3,7-dimethylpentadecan-2-y1 propionate. Test conducted at Mio, Michigan, July 21-August 4, 1981. Three replicates randomized three times . . . . . . . . . . . . Field response of N. amininnus to mixtures of candidate optical synergist-isomers. Test conducted at Sharon, Michigan, May 14-June 20, 1982. Three replicates randomized three times. Field response of N. W to mixtures of three best candidate synergist-isomers. Test conducted at Sharon, Michigan, May lS-June 20, 1982. Three replicates randomized three times. xiv 46 48 59 60 61 73 75 76 77 5-5 5-6 5-8 5-10 6-2 Field response of N. ulnnlninnus to varying concentrations of 28,3R,7S-P and 28,3R,7R-P mixed with major pheromone isomer. Test conducted at Hartwick Pines, Michigan, June 10- July 14, 1983. Three replicates randomized three times . . . . . . . . . . . . . . . . . . Synergistic effect of admixing 28,3R,7S-P’and 28,38,7S-P isomers in attracting male N. xinglnlnnus on the field. Test conducted at Sharon, Michigan, June 10-15, 1983. Three replicates randomized three times . . . . . . . Field response of N. ulnalnlnnus to the front, center, and back charcoal-celite column fractions of 28,3R,7R/S-P* mixed with 28,38,78- P. Test conducted at Sharon, Michigan, June 30-July 23, 1982. Three replicates randomized three times 0 O I O O O I O I O I O O O O O O I Field response of N. 1.1.;31nlnnus to the crude and the front, center, and back fractions of 2S,3R,7R/S-P isomer mixed with the major pheromone 28,33,7S-P isomer.* Test conducted at Sharon, Michigan, August 2-31, 1982. Three replicates randomized three times . . . . . . . Species specificity and ester moiety preference of N. ulnlnlnnus. Test conducted at Sharon, Michigan, May 14-August 13, 1982. Three replicates randomized three times . . . . . . . Threshold limit of the cynthetic pheromone of N. W- Test conducted at Sharon, Michigan, June 29-August 13, 1982. Three replicates randomized three times . . . . . . . synergistic effect of the 28,3R,7R/S-A isomer on the effectiveness of 28,38,7S-A isomer at attracting wild male N. ainntum at Higgins Lake State Forest Michigan, May 15-July 20, 1981 on White Pine 0 O O O O O O O O O O O I O O O I 0 Field response of male N. ainetum to traps baited with isomers of acetate or prepionate diprionol. Test conducted at Siren, Wisconsin, HaYIS-June3o'1981ooooooooooooo XV 79 80 81 82 84 85 95 96 6-5 7-4 Comparison of three threo isomers 28,3R,7R-A, 2S,3R,7S-A and 28,3R,7R/S-A in synergizing the effectiveness of 28,38,7S-A isomer against N. ninstnm males. Test conducted at Higgins Lake, Michigan, May 7-June 10, 1982 . . . . . . . . . Comparison of three threo isomers 28,3R,7R-A: 2S,3R,7S-A and 2R/S,3R,7S-A in synergizing the effectiveness of 28,38,7S-A isomer against N. males. Test conducted in Kalkaska, M1Chigan' May 5-June 10' 1982 o o o o o o o o 0 Determination of the Optimum blend of the mixture of 28,38,7S-A and 28,3R,7R-A isomers in attracting N. ninfiium males at Kalkaska, Michi- gan, June 29"Ju1y 7' 1982 o o o o o o o o o o 0 Determination of the optimum blend of the mixture of 28,38,7S-A and 28,3R,7R/S-A isomers in attracting N. ainstum males at Siren, 771800118111, May lS-June 30' 1982 o o o o o o o 0 Field response of n. slmllls to three enantiomers of 3,7-dimethy1pentadecan-Z-yl propionate at five locations in Michigan and Siren, Wisconsin. Test conducted June 6-July 20, 1981. Three replicates at each location . Field response of D slnllls to crude and puri- fied 28,3R,7R/S-P isomer. Test conducted at Higgins Lake and Kalkaska, Michigan, May 15- July 20' 1981 O O O O O O O I O O O O O O I O 0 Response of 12. 111111115 to a mixture of 28,3R,7R-P with four other isomers of diprionid pine sawfly sex pheromone. Conducted May 29- July 23, 1982 in Kalkaska, Michigan . . . . . . Response of D.- almills to a mixture of 2R/S,3R,7R-P and 28,3R,7R-P isomer. Test conducted June 30-August 31, 1982 at Kalkaska, niobigan O O I O O O O O O O O O O I O O O O 0 Response of D. similia.to synthetic 2R/S,3R,7R- P and a mixture of 28,3R,7R-P and 2R,3R,7R-P. Test conducted at Ralkaska, July 23-August 13, 1982 O O O O O O O O O O O O O O O O O O O O 0 Field response of n. simills to varying concentrations of 2R/S,3R,7R-P isomer and natural pheromone. Test conducted in Kalkaska, Michigan, June 9-July 23, 1983 . . . . . . . . xvi 97 99 100 101 111 113 114 116 117 118 7-10 7-11 7-12 7-13 10-1 10-2 10-3 Response of n. slmllis to synthetic 2R/S,3R,7R- P and a mixture of 28,3R,7R-P and 2R,3R,7R-P. Test conducted at Kalkaska, July 23-August 13, 1982 O O C O O O O O O O O O O O O O O O O O 0 Field response of D. slmllls to mixtures of isomers 28,7R,7R-P front/28,35,7R-P and 28,3R,7R-P front/28,38,7S-P’at varying ratios. 33st czflfucted in Kalkaska, Michigan, June 15- ' 19 C O O I O O I I O O O O O O I I O O 0 Threshold limit and comparison of the synthetic with the natural pheromone of D.- Himma. Test conducted in Kalkaska, Michigan, June 21-July 30' 1983 O O O O O O O O O O O O O O O O O O 0 Response of D. almilis.to mixtures of 28,3R,7R- P with six isomers at varying concentrations. Test conducted in Kalkaska, Michigan, June 15- 28' 1983 O O O O O O O O O O I O O O I O O O O Synergistic interactions of three isomers on 28, BR, 7R- P isomer in attracting male D.- simllis, Test conducted at Kalkaska, Michigan, {Hillel-16,1984................ Synergistic interaction of 28,38,7R-P isomer with 28,3R,7R-P isomer in attracting male D. slnills on the field. Test conducted at Ralkaska, Michigan, June 7-16, 1984. Three replicates randomized twice . . . . . . . . . . Synergistic interaction of 28,38,7S-P isomer with 28,3R,7R-P isomer in attracting male n. slallls on the field. Test conducted at Kalkaska, Michigan, June 7-16, 1984. Three replicates randomized twice . . . . . . . . . . Descriptive data for trap designs . . . . . . . Effect of different types of pheromone sources on response of wild N. annulus annulus in the field within 40 min. observation period with 23H3s7s-A(1oug)oooooooooooooo Effect of grass leaves an host pine needles* as background for the pheromone source** in males of N. 1:11:1st and N. annulus annulus on the field. Test conducted for 60 minutes at Rose Lake and 30 minutes at Vogel Center respectively for N, ssntlfsn and N. annulus annulus xvii Bass 120 122 123 124 126 127 128 191 193 195 10-4 10-5 10-6 10-7 10-8 10-9 10-10 10-11 Effect of the nature of background object on wild N. annulufi nuaulun males when the pheromone source was glass petri dish. Test conducted for 50 minutes with 10 ug 28,38,7S-A. Effect of mesh size of the mating cage on the response of wild N. annulus annulus males to 10 ug 28,38,7S-A. Observation was made for 60 minUtes O O O I O O O I I O O O O I O O O I I 0 Effect of background color of pheromone source on the response of wild N. ssntlfsa males on the field. Each treatment contained 10 ug 28,38,7S-A/23,3R,7R-A (5:0.003) and observation lasted 80 minutes 0 O O O I O O I I O O O O O I Field response of male N. ssanlfsa and N. annulus annulus to different colors of sticky trap Pherocon II baited with sex pheromone. Test for N. ssntlfsa conducted at Rose Lake (October 2-5, 1982) and for N. annulus annulus at Vogel Center, Michigan (September 25-October 16, 1982). Three replicates randomized 3 times Effect of distance of synthetic pheromone source used for courtship»behavior of male N. sssnifn;,on the field from the nearest scotch pine stand. Test conducted at Rose Lake, Michigan, 10/8-10/15, 1982 and at Pinckney, Michigan 10/16/82. Pheromone source was 40 ug 530.003 28,38,78‘A/28'3R'7R"A m1Xture o o o o 0 Effect of trap height on the response of male N. snrtifsa to synthetic pheromone. Traps placed on scotch pine stands and artificial poles erected at 12 mbor 3 m from.the nearest host stands. Test conducted at Rose Lake, Lansing, Michigan, September 25-October 6, 1983 Effect of trap height on the response of male N. annani bnaksinans to pheromone. Traps were placed on jack pine, replicated 3 times at Hartwick Pines, Michigan in 1981 and 1983 . . . Field effectiveness of five trap designs against male N. girgininaus. Test conducted at Sharon, Michigan, June 10-July 15, 1983. Three replicates randomized three times . . . . . . . xviii 196 197 199 200 201 212 214 215 10-12 AI-l AI-2 AI-3 AI-4 AI-S AI-6 AII-l Field effectiveness of Inverted Cup Trap (ICT) and Pherocon II traps against two species of pine sawfly. Test conducted for N. annulus annulus at Vogel Center, and for N. zirgininnus at Sharon, Michigan. Three replicates random- ized three times .... . . .. . .. . .. . . Field response of males N. nabaaal to four optical isomers of acetate or prOpionate diprionol. Test conducted.at Hartwick Pines, MIChigan, May ls-JUIY 10' 1981 o o o o o o o a Field response of males N. 312119.131 to different optical isomers of acetate or prOpionate diprionol. Test conducted at.Hartwick Pines, Michigan, June 6-July 20, 1981 . . . . . . . . Field response of males N. nbhnttl to mixtures of optical isomers of acetate diprionol. Test conducted at Hartwick Pines, Michigan, June 5- July 11' 1981 I I I I I I I I I I I I I I I I I Field response of males N. AthLLi to mixtures Of 28'3S'7S'A With 28'3R'7R-A; 28'3R'7S’A and 2R/S,3R,7S-A isomers. Test conducted at Fletcher Road near Sharon, Michigan, May 7-June 10' 1982 I I I I I I I I I I I I I I I I I I I Synergistic effectof the 28,3R,7R/S-A isomer on the field effectiveness of 28,38,7S-A against N. am. Test conducted at Hartwick Pines, 14161119811, May ls-AuguSt 4' 1981 o o o o o o o 0 Field response of male N. nbaattl to a varying mixture of 25,38,7S-A and 28,3R,7S-A isomers. Test conducted at Fletcher Road, Garfield Township, Michigan, May 25-August 13, 1982 . . Grouping of a species of sawfly based on degree of occurrence of identical GLC peaks.of their cuticular hydrocarbon . . . . . . . . . . . . . xix 218 235 237 238 239 241 242 266 8-1 8-2 LIST OF FIGURES GLC fractionation of 28,38,7S-A peak into I front, II center, and III back fractions on carbowax 20M column . . . . . . . . . . . . . . A scheme for the preparation.of diprionol for field tests I I I I I I I I I I I I I I I I I I Stimulation and inhibition of the field response of N. 21:31:17.1 muslin“ males to the major sex pheromone isomer 28,38,7S-A by the 28'3R'7R-Aisomeroooooooooooooso Scheme for the purification of isomers of diprionol by charcoal-celite column . . . . . . GLC pattern of the 28,3R,7R-P on 5% carbowax 20M column. The fractionation pattern into front and back is shown by arrows . . . . . . . Capillary GLC separation of 28,38,7S-A / 28,3R,7R-A mixture. (a) Ratio 3:2, 80 ng, using DES column, (b) Ratio 2:3, 80 ng, using carbowax 20M column .. . .. . .. . .. . . Capillary GLC separation on DES column at iso- thermal 180°C. (a) 28,38,7S-A / 28,3R,7S-A mixture, 100 as: (b) 28,38,7S-A / 28,3R,7R-A miXture' 100 mg I I I I I I I I I I I I I I I I Capillary GLC separation on D85 column tempera- ture programmed 130-190°C at 4°/min. (a) 25,38,7S-A / 28,3R,7S-A mixture 100 ng. (b) 23,35,7S-A / 25'3R'7R’A mixture 50 n9 0 o o o o (i) Capillary GLC recordings of acetylated natural pheromone: A. NEW lngantsl 20 Female equivalent (FE), B. N. annulus annulus 60 PE. 0. N. ssrtifer 6 FE, D. N. anti-.1 banksiaaas 10 FE. 13. N. ruuifrnna 16 FE. F. N. niuruanutunll FE, G.D.inriunsimilis10PE. H. N.xiruinianusBFE. Ion-magnum FE (ii) Capillary GLC recordings of acetylated natural pheromone of species A-I spiked with 28'38'7s-A I I I I I I I I I I I I I I I I I I XX 20 47 72 110 138 140 141 143 9-2 9-3 9-6 10-1 10-2 10-3 Wave form of sex pheromone induced EAG in D. slmllis. (a) 5 ug 28,38,7R-P. (b) 5/5 ug ZR/SBR'7R-Po ooooooooooooooooo Response of male 12. similis to optical isomers. (a) additive effect of 28,3R,7R-P / 2R/S,3R,7R- P mixture. (b) inhibitiion of 28,3R,7R-P by 28,3R,7R diprionol. (c) inhibition of 28,3R,7R-P by various isomers. (d) effect of fractionation by GLC peak of 28,3R,7R-P. . . . Relative EAG response of male of 3 species of diprionid sawfly to various isomers of sex pheromone . . . . . . . . . . . . . . . . . . . Response of female diprionid sawflies to sex pheromone. (a) N. ssrtifsr. (b) N. W- (C)N.1irainianus. (d)N.exnitaas.. . .. . Mean EAG response of 3 species of sawfly to a range of concentration of sex pheromone . . . . Synergistic interactions of iptical isomers in EAG of 3 species. (a) 28,3R,7S-P as synergist of 28, as, 7S-P in N. xiruinianus. (b) 23,311,712- A as synergist of 28, 3S, 78-A in N. seating. (c) 28, BR, 7R-A and 28, BR, 78-A as synergists of 23H387S‘A1nnonm00000000000 EAG response of male N. ssntifnx. to the TLC fractions of the body extract from female N. nannlusnaaulusandu.ssr.tifer........ Trap designs for sawflies (all measurements in cm.). (a) Pherocon on cut trap (POC). (b) Double funnel trap (DFT). (c) Inverted pyramid trap (IPT). (d) Inverted cup trap (ICT) . . . Average time (secs.) male spent within circle 2 cm. radius (L) or time mael spent with dead N. ssnalfsa male treated with chemicals by climbing up the pin holding the specimen. . . . Copulation in 12. 81mm. The male is copulat- ing a restrained live female at the top pic- ture: copulation below is with a dead solvent washed female that is pheromone treated. Note that in both pictures, male did not copulate with the pheromone treated septum at the back- ground. However the male climbed the septum first I I I I I I I I I I I I I I I I I I I I I xxi 164 166 168 170 172 173 175 190 202 206 10-4 AII-l AII-2 AII-3 Model of location of female and courtship behavior of wild male diprionid sawfly . . . . GLC chromatogram of cuticular hydrocarbon of ten diprionid sawfly species . . . . . . . . . Qualitative differences among GLC peaks of the cuticular hydrocarbon of ten species of sawfly. Qualitative differences among GLC peaks of the cuticular hydrocarbon of ten species of sawfly. xxii 211 251 262 264 CHAPTER ONE Field Evaluation of Chiral Isomers of the Sex Pheromone of the European Pine Sawfly, Wm INTRODUCTION A female-produced sex pheromone component of the red- headed pine sawfly Nsadlaalaa lsgaansl_(Fitch), has been isolated and identified as the acetate of 3,7-dimethy1penta- decan-2-01 (Jewett et a1. 1976). They observed, by using nuclear resonance spectroscopy, that the pheromone has an erythro configuration of the adjacent optically active car- bons (i.e., carbons 2 and 3). Electroantennographic studies have shown that males of many species of diprionid sawflies are responsive to either the acetate or proprionate of diprionol isolated from Nnadlpalnn snxtlfnr (Geoffroy) or N. lanannnl females. However, the responses of Dinalaa,smallis (Hartig) and fillalaln faunnuaaum (Fabricius) males appeared different from those belonging to the Nnadlaalan genus (Jewett et a1. 1976). Matsumura et a1. (1979) found that the chiral arrangement of the pheromone for N141suuntei.were 28,38. The chirality of all the carbons of the sex attrac- tant of Nsadiaaiaa,alnsnum,(Norton) was also established as the acetate of 28,38,78-diprionol. This was the only active erythro isomer for that species (Kraemer et a1. 1979). In 2 the present study, we have extended our work to N. ssatifna, a common species in the Great Lakes Region. MATERIALS AND METHODS Main All chemicals were dissolved in 1 m1. of n-hexane and stored in sealed glass ampules. In the field, the ampule was broken and its contents were poured into a 2.5 cm. cotton wick held with a pair of forceps. The wick was then attached to the inside roof of a Pherocon II trap (Zoecon Corp., Palo Alto, California). To avoid contamination, the forceps were washed with alcohol after each use. All the field trapping of N. again; took place in Rose Lake Wild- life Research Station, Sec. 33, Bath and at Arboretum, Lansing, Ingham County, Michigan. Larval colonies were collected at these locations on scotch pine, Pinus syluss; “is L. Rose Lake, where the majority of the studies were carried out, covered an area of about 8 square kilometers. Stands of scotch pine were mostly wind breaks separated by grasses, hardwoods and patches of other pines, notably, red Pine Finns resinnsa A11: and white pine Pinus strnhus L. W The acetate of (28,38,78)-3,7-dimethy1pentadecan-2-01 (28,38,78-A) was obtained from Dr. Mori (Mori et a1. 1978). From the same chemist was obtained 2R,3R,7R-A from which we synthesized 2R/8,3R,7R-A by the method described by Kikukawa et al. (1982a). The 28,3R,7R/8-A was obtained from Dr. Tai 3 (Tai et a1. 1978). NMR analyses of these compounds showed that each erythro isomer was free from contaminations by the other. On the other hand, 28,3R,7R/S—A contained approxi- mately'5% of 38 contamination. 'The 28,3R,7R-A: 28,3R,7S-A and one isomer synthesized through a Grignard reaction des- ignated 28,3R,7R-AG were obtained from Dr. Tai's laboratory (Kikukawa et al. 1982b). W WW Two hundred N. sen-Jinx, virgin females were immersed in ether overnight. The solvent was decanted and the insects were washed two more times with the same solvent. The solvent was evaporated by a rotary evaporator under reduced pressure. The residue was hydrolyzed by the addition of l g ROH in 50 ml. methanol. This mixture was refluxed for 5 hrs. The methanol was removed by rotary evaporation. The organic material was separated in a separatory funnel (hexane-water). The hexane fraction was dried over Na2804 to give the crude alcohol fraction. The hexane was removed by rotary evaporation and the residue was esterified by adding 2 m1. pyridine and 1 g. of acetic anhydride. The reaction was left overnight, in the dark and at room temper- ature. The crude ester fraction thus obtained was purified by TLC (activated silica gel HF254.356 with hexane-ether (4:1) mobile phase). The most field-active fractions (Rf - 0.65 - 0.39) was further purified by a second TLC system using n-hexane first and benzene second as the mobile phase. 4 211W W The original 28,38,78-A from Mori et a1. (1978) was subjected to GLC with a carbowax 20 M (4m) column on chromo- sob AW. In this system, erythro isomers come slightly ahead of threo isomers. To achieve a partial purification, the front, center, and tail parts of the peak due to 28,38,78-A were separately collected (Figure l-l). These fractions were separately biossayed on the field. RESULTS Kikukawa et a1. (1983) tested many isomers of diprionol and established that (1) males of N. sagging respond best to 28,38,78-A (2) TLC-charcoal purification of 28,38,78-A improves field effectiveness (3) such purifications alone could not bring the level of field effectiveness of the synthetic pheromone to that of natural pheromone and (4) female N. ssanlfsa has about 10 ng of pheromone shortly after adult emergence. The original 28,38,78-A preparation (Mori et a1. 1978) when separated into fractions through GLC (Figure 1-1) and field tested, the separated fractions were relatively more effective than the original 28,38,7S-A (Table 1-1). The central portion was most active, as 0.5 ug of this prepara- tion attracted more males than 10 ug of the original 28,38,7S-A. The front and tail portions of the same concen- tration as the central were less active, but those prepara- tions attracted similar numbers of males as did 10 ng of the Figure 1-1. GLC fractionation of 28,38,78-A peak into I front, II center, and III back fractions on carbowax 20M column. (a RECORDER RESPONSE k...) k. 10 20 30 TIIE (min) Table 1-1. Comparison of N. angling trap catch between original 28,38,78-A preparations and that puri- fied by GLC, September 19 to October 26, 1981 at Rose Lake, Lansing, Michigan. Amount Mean/Trapc Preparations (ug/trap) i 8.8. 28,38,7S-A original“ 10.0 5.0 i 0.3 GLC peak front 0.5 3.7 1 0.8 GLC peak center 0.5 7.7 i_1.4 GLC peak back 0.5 3.7 1 1.0 a 28,38,78-A contaminated by impurities. b The front, center and back portions of the 28,38,78-A peak were collected separately as shown in Figure l. c Means of 3 replicates. cant. Analysis of variance insignifi- 7 original 28,38,7S-A. Despite improved performance of this GLC purified 28,38,78-A, it is clear from this table that purification alone cannot drastically increase the effec- tiveness of 28,38,7S-A over the original 28,38,78-A. In a series of experiments summarized in Table 1-2, I have explored a possibility that the presence of a trace amount of a threo isomer in the purified 28,38,78—A prepara- tions could increase the effectiveness of the trap catch. The experiment was conducted in Fall 1981, when a new prepa- ration 28,3R,7R-A became available. It was shown by three independent tests that the presence of 0.01-0.003 ug of 28,3R,7R-A in 5 ug of 28,38,78-A was advantageous in terms of effective catching of N. anal-Jinx, males. In the next test the synergistic effectiveness of several threo prepara- tions containing 28,3R,7R-A component was compared at a fixed ratio of 5:0.003 ug. Among them 28,3R,7R-A and 2R/8,3R,7R-A were most synergistic followed by 28,3R,7R/8—A and 2R/8,3R,7S-A. In 1982, four threo isomers were mixed with 28,38,7S-A and field tested. The results (Table 1-3) showed that 28,38,7S-A/2S,3R,7R/S-A, 28,38,7S-A/ZS,3R,7S-A and 28,38,7S-A/28,3R,7R-AG mixtures at blend ratio 5:0.003 were more effective than 28,38,7S-A alone. At this ratio, the importance of chirality at 7-carbon of the synergists were tested (Table 1-4). The result indicates that the chirality of the 7th carbon is not important. In view of the high effectiveness of these mixtures, an attempt was made to compare the potency of naturaIly Table 1-2 0 Stimulatory effect of trace amount of 28,38,7R-A on effectiveness of trap catch by 28,38,7S-A. The test was conducted at Rose Lake, Michigan, September 19 to November 9, 1981. Series C8 28,38,78—A & Series Aa Series Ba 28,3R,7R-A 9/19 - 11/9 9/27 - 11/9 10/9 - 11/9 tug/trap) (x :1: SE) (x :t 53) (x 3: 88) 5/0 c 5.0 i 1.2 2.7 i 2.2 5/o.005 c 4.7 1 0.9 2.3 1 0.9 5/0.001 c 4.0 3; 1.0 5.0 i 0.6 5/0.003 87.7 i 39.1b 13.7 i 1.519 7.0 i 3.2b 5/0.01 43.7 3: 16.8b 24.3 i 13.9b 12.7 i 4.8b 5/o.03 7.7 1 4.7 4.7 i 2.7 3.0 i 1.0 5/0.1 22.3 1 4.3 14.3 .1: 5.0b 2.7 i 0.9 5/0.3 1.7 i 0.3 1.3 3: 0.3 0.0 i 0.0 5/1 0.7 i 0.7 0.7 :9; 0.7 0.7 3: 0.7 a The result of catches by three sets of traps for each concentrat ion. b All means significantly different from others by Duncan's multiple range test at 5% level. c These traps were inadvertently left out of Series A. During the same period and at the same locations, a trap baited with 20 ug of 28,38,7S-A alone caught 37 males. Table 1-3. Stimulatory effect of different threo isomers on 28,38,78-A in attracting male N. ssztlfs; on the field. Test conducted at Rose Lake, Michigan, September 25 to October 19, 1982. Amount 23,33,75-1/a 23.33.73.“a 2s,3s,7s-ua 2s,3s,75/a (us/trap) 23,33,73/3-1 23,33,75-1 23.33.73.40 ZR/S,3R,7R-A 5/0 3 3 10 6 5/0.003 14 7 13 2 5/0.03 5 2 2 2 5/0.3 0 0 0 1 5/1 0 0 0 0 a The total by three sets of traps for each concentration. 10 Table 1-4. Importance of configurations at C-7 of threo isomers on effectiveness of trap catch by 28,38,7S-A. The test was conducted at Rose Lake '10/5 - 11/5/81 and 9/22 - 10/19/82. Series A3 Series Ba 10/5 - ll/g/Bl 9/22-- 10/18/82 Pheromone (x i SE) (x): 8E) 23,38,7S-A / 28,3R,7S-A 2.6 i 0.9 2.3 1 0.8 28'33'7S-A / 23'3R'7R-A 4.3 i 101 4.3 i 103 a The result of catches by three-sets of traps for each treatement. b Analysis of variance insignificant and no significant difference among the means by Duncan's multiple range test. ll occurring diprionol which was purified as earlier described. As a result of a comparison test (Table 1-5), it was estab- lished that the synergized preparation containing 28,38,7S-A and a trace amount of 28,3R,7R-A at a ratio of 5:0.003 was roughly as active as the naturally occurring pheromone preparation. DISCUSSION This work demonstrates that a trace addition of threo acetate containing 28,3R,7R-A configurations certainly in- creased the field effectiveness of 28,38,78-A. A question must be raised as to the optical purity of these synthetic epimers themselves. At this stage there is no convenient method to measure the optical purity of each active carbon in the final synthetic product per se. NMR spectroscopic analysis, however, gives information as to the diasteroemet- ric arrangement on 2-carbon and 3-carbon (Jewett et a1. 1976). As judged by this approach, none of the erythro isomers provided by Dr. Mori's group is contaminated with threo isomers. The limit of detection is approximately 1%. On the other hand, the threo compounds 2S,3R,7R/S-A, synthe- sized via Wittig reaction (Tai et a1. 1978) contained ap- proximately'5% of epimerization product at carbon 3. The 28,3R,7R-A, 28,3R,7R-AG and 28,3R,78-A were synthesized through Grignard reaction and are known to contain no opti- cal impurity (Kikukawa et al. 1982b). Meanwhile, it appears from our data that the addition of 3R to 38 of 28,38,78-A is 12 Table 1-5. Comparison of field effectiveness of natural and synthetic pheromone of N. W: Rose Lake, Michigan, October 9 - November 9, 1981. Amount Replicates Pheromone (ng/trap) A B C Total Mean/Trap Synthetic Mixture 28,33'7S-A/ 10.0 0 7 5 12 4.0a 3.0 2 3 10 15 5.0a 1.0 0 3 5 8 2.7 Natural Pheromone (acetate) , 3.0 4 6 15 25 8.3a (. 0.3 FE) 1.0 1 0 8 9 3.0a (- 0.1 FE) 0.3 0 0 0 0 0.0 (. 0.03 FE) a Means significantly different by Duncan's multiple range test at 5% level from other values. 13 the crucial point in the case of field attraction of N. snrllfnn males, as mixing of a small amount (0.06%) of 28,3R,7R/8-A, 2R/S,3R,7R-A, 28,3R,7R-A and 28,3R,7S-A gave an equally stimulatory effect. In conclusion, the current work established that 28,38,7S-A is the chiral configuration most preferred by males of N... W. The same configuration is reported for males of mm (Kraemer et al. 1979) and N. lssaa; tnl (Rraemer et al. 1981). The most intriguing aspect of our finding is that small amounts of 28,3R,7R-A stimulates the field effectiveness of the 28,38,7S-A preparation. The significance of this is rather early to predict, but this signals that sex pheromones of diprionid sawflies might involve mixtures of Optical isomers. REFERENCES Jewett, D. M., F. Matsumura and H. C. Coppel. 1976. Sex pheromone specificity in the pine sawflies: inter- change of acid moieties in an ester. Science, 192: 51- 53. Kikukawa, T., F. Matsumura, M. Kraemer, H.C. Coppel and A. Tai. 1982a. Field attractiveness of chirally defined synthetic attractants to males of Dlaglaa slmllls and 5111211113 Wm. J. Chem. Ecol., 8: 301-314. Rikukawa, T., M. Imaida and A. Tai. 1982b. Synthesis of the sex attractant of pine sawflies (Diprion species): (28,3R,7R) and (28,3R,78)-3,7-dimethylpentadecan-Z-Ol. Chem. Lett., 1982: 1799-1802. Kikukawa, T., F. Matsumura, J. Olaifa, M. Kraemer, H. C. Coppel and A. Tai. 1983. The nature of the major sex pheromone of the European pine sawfly, 11149919111911 SELL: ling. J. Chem. Ecol., 9: 673-693. Kraemer, M., H. C. Coppel, F. Matsumura, T. Kikukawa and K. Mori. 1979. Field responses of the white pine sawfly Nnadlaglna alnnnun to optical isomers of sawfly sex pheromones. Environ. Entomo1., 8: 519-520. Kraemer, M. E., H. C. COppel, F. Matsumura, R. C. Wilkinson and T. Kikukawa. 1981. Field and electroantennogram responses of the red-headed pine sawfly Nnadlaglan lnnaannl (Fitch) to optical isomers of sawfly sex pher- omones. J. Chem. Ecol., 7: 1063-1071. Matsumura, F., A. Tai, H. C. Coppel and M. Imaida. 1979. Chiral specificity of the sex pheromone of the red- heeded Pine sawfly: Neediuinn lsunntni. J. Chem. Ecol., 5: 237-249. Mori, E., 8. Tamada and M. Matsui. 1978. Stereocontrol led synthesis of all of the four possible stereoisomers of erythro-3,7-dimethy1pentadecan-2-ylacetate and propio- nate, the sex pheromone of pine sawflies. Tetrahedron Lett., 10: 901-904. Tai, A., M. Imaida, T. Oda and H. Watanabe. 1978. Synthe- sis of optically active common precursor of sex phero- mone of pine sawflies. Chem. Lett., 1978: 61. 14 CHAPTER TWO Response of Male Red Pine Sawfly, Nnadlpglna annulus annulus (Hymenoptera: Diprionidae) to Natural and Mixtures of Optically Active Synthetic Sex Pheromone 3,7-dimethylpentadecan-2-y1 acetate INTRODUCTION The red pine sawfly, Nnadlaglaa_anaulus annulus Schedl is an important native defoliator of the red pine (Plans gnslnasn Ait), jack pine (P. bnnkslnan Lamb.) and lodgepole pine (2.‘naanngnn,Dougln). The range of the latter two host species overlap, making N. annulus annulus a cosmopolitan species throughout North America. Red pine is the predomi- nant conifer being planted in the Great Lakes region. Ac- cording to one reported case, one complete defoliation of red pine caused a 21% reduction in height growth. Two successive defoliations reduced as much as 64% (Kapler and Benjamin, 1960). Yet such defoliations are uncommon in Michigan where this study was done. Monitoring tools for this pest at the present consist of scouting for the larval stage and estimating the injury level. Jewett et a1. (1976) isolated and identified dipri- onid sawfly sex pheromone as 3,7-dimethylpentadecan-z-ol (diprionol), the acetate or proprionate ester of which was active in attracting males on the field. Kraemer et a1. (1983) reported that the optical isomer (25,3s,7sr—3;r- 15 16 dimethylpentadecan-Z-yl acetate (28,38,7S-A) and proprionate (28,38,7S-P) as well as (28,38,7R)-3,7-dimethylpentadecan-2- yl acetate (28,38,7R-A) were highly attractive, but 28,38,7S-A was significantly superior to others at lower concentrations. The 28,38,7S-A thus appeared to be the major pheromone in N. annulus annulus as in N. plannum (Kraemer et a1. 1979), N. lnnaannl (Matsumura et a1. 1979: Kraemer et a1. 1981). However, in another sympatric species N. sngnlfng where 28,38,7S-A is the major pheromone, combi- nation with small amount of (28,3R,7R)-3,7-dimethylpentade- can-2-yl acetate (28,3R,7R-A) stimulates the field effec- tiveness of the 28,38,7S-A preparation (Kikukawa, et a1. 1983). Here we report results of field studies conducted to examine if such interaction of optical isomers extends to N. nanulus.nanu1us. MATERIALS AND METHODS £1£1d_TESLS The red pine sawfly trapping was carried out in Vogel Center, Sec. 32, Aetna Loop, Misaukee Co., Michigan. A few trappings were also done at two other locations-Sec. 7, Manistee River, Garfield Township, Kalkaska County, and at Sec. 36, Higgins Lake, Old I-27, Beaver Township in Roscom- mon Co., Michigan. The main experimental area, Vogel Cen- ter, is about 1.2 Kilometer square. The red pine stand is surrounded by farm lands such that within 13 Kilometers radius only scattered plots of red pine could be found. 17 Ground cover is mostly grasses. Outbreak conditions in 1981 left many pine trees completely defoliated. In all the test areas, the characteristic injury of larvae«consuming just three-fourths of the needles leaving stubs 1.9-0.6 cm. long (Wilson, 1970) were clearly evident. On several occasions in late spring and early summer 1981, 1982, and 1983, larval colonies of N. a. annulus were collected from these sites. Pherocon IIR traps were used in all tests. Traps were hung at a height of 2.0 to 2.5 m. and spaced at no closer than 15 m. within and between trap rows. Observations were made weekly or at least twice per month during which the traps are rotated randomly within and between rows. Phero- mones were prepared in.l:m1. hexane solvent and sealed in glass ampules before taken to the field. At the test site, the contents of the ampules are dispensed onto a 4.0-5.0 cm. dental cotton roll which is positioned at the center on one side inside the Pherocon trap. The randomized block experi- mental design was used in all tests with at least three replicates of each treatment. Data were analyzed by analy- sis of variance, and the means compared by Duncan's multiple range test at 5% level. W W The female insect from which pheromone was extracted were reared from larvae and pupae collected from the test sites. Larvae were reared outdoors under shade near the greenhouse. The larvae were fed fresh host twigs dipped in 18 water to maintain turgidity. At pupation, the last larval instar made cocoons under the heap of fecal droppings inside the cage. Such cocoons, as well as field collected cocoons, were separated by sex according to size, the larger cocoons being females. Individual cocoons were then placed in plas- tic capsules and left outside in cages under shade till adult emergence. Individual virgin females were placed in 3x5 cm. vials with strips of teflon simulating pine needles for a few days before being frozen till use. SynthetiLEhernmnnes Two groups of chemists from Japan synthesized and sup- plied us with the optical isomers used in this study. Mori's group synthesized the 28,38,7S-A and the correspond- ing alcohol (Mori et a1. 1978). These materials contained some unknown percent of optical impurities, but is believed to be less than 1%, the approximate limit of detection by PMR assay. Tai's group synthesized the 28,3R,7R/S-A with a known 5% erythro contamination (Tai et a1. 1978). The same group later synthesized 28,38,7S-A (unpublished) and 2S,3R,7R-A and 28,3R,7R-AG (Kikukawa et a1. 1982). In this instance the 28,38,7S-A and 28,3R,7R-A contained less than 1% optical impurities but 28,3R,7R-AG synthesized through Grignard was greater than 99.9% optically pure. W We Virgin females were immersed in ethyl ether overnight. After decanting the solvent, they were rinsed with ether two 19 times and combined. The combined solvent was evaporated in a rotary evaporator and the residue was hydrolyzed by the addition of 5-10 m1. of 2% KOH in methanol. This mixture was refluxed.for 5 hrs. The methanol was then removed by rotary evaporation. The residue was picked up in hexane and washed with water. The hexane fraction was dried over Na2804 to give the crude alcohol fraction. The hexane was removed by rotary evaporation and the residue was esterified into acetate by adding 2 ml. pyridine and 1 gm. of acetic anhydride and refluxing for 5 hrs. The reaction mixture was then dissolved in n-hexane and extracted with water. 'The hexane phase was dried over MgSO4 and evaporated. The residue was spotted on preparative TLC [silica gel 606 HF254+366 (MC/B Manufacturing Chemists Co., Cincinnati, Ohio): 2 mm. thick] which was first developed to 1 cm. of the top edge in an acetone tank to remove impurities. The plates were developed until 15 cm. from the origin with a mixture of hexane and ether (4:1). The ester zone corre- sponding to Rf 0.45-0.56 was collected and eluted with ether. After the ether was removed, the residue was spotted on another TLC plate and developed first with n-hexane and then with benzene. Thereafter, the zone corresponding to Rf 0.75-0.85 was removed and eluted with ether. The residue remaining after evaporation of ether was then adjusted to female equivalents as necessary for field testing. The scheme for this extraction procedure is depicted in Figure 2-1 0 Figure 2-1. A scheme for the preparation of diprionol for field tests. 20 Virgin females ether overnight Body extract 1 ROM Crude alcohol fraction 1 Esterification Crude ester fraction TLC n—hexane:ether 4:1 \ Inactive fraction: (1 Active fraction TLC (1) n—hexane (2) benzene \ Inactive fractions Active fraction Rf 0.33-0.75 Field test 21 RESULTS The data summarized in Table 2-1 indicate that the 28,38,7S-A isomer alone was more active than any other combinations of 28,3R,7R/8-A or 28,3R,7R—A. At lower con- centrations, 28,3R,7R/S-A and 28,3R,7R-A appeared to in- crease the effectiveness of 28,38,7S-A isomer, however, the increase was not statistically significant (Table 2-2). One possibility is that the effectiveness of the synergist may not express at high pOpulations as this particular tests caught as many males. Lower concentrations of 28,38,78-A was used in 1983 so that fewer males might be attracted in order to bring on clear differences if any, among the treat- ments. The result (Table 2-3) clearly shows that the traps baited with 28,38,7S-A alone attracted more males than mix- tures. This again confirmed the results of Tables 2-1 and 2-2 that the 28,38,7S-A alone is the major pheromone of N. annulus annulus. Also, these data agree with the work of Kraemer et a1. (1983). However, since these workers found 28,38,7R/S-A as active as 28,38,7S-A, these two isomers were compared in a side by side set up on the field. With a total catch of 640 males by 28,38,78-A baited traps to 253 males in 28,38,7R/S-A baited traps, we proved the superi- ority of 28,38,7S-A isomer over 28,38,7R/S-A and the impor- tance of S on 7-carbon on the major pheromone. Based on apparent injury level at three locations in Roscommon County, Michigan, and the level of larvae popula- tion sampled on twenty 78 cm. branches, Manistee (with low 22 Table 2-1. Field response of male N. annulus annulus to the increasing concentrations of two isomers, 28,3R,7R/S-A and 28'3R'7R-A on 28,38,7S-A 18°- mer. Test was conducted in Roscommon County between September 4 and October 16, 1981. 28,35,78'A 28,38,78'A + + Amount 25'3R'7R/S-A 25' 3R'7R'A (ug/trap) (x 1,8.E.)* (x 1,8.E.)** 10/0 582.5 3; 9.1 98.0 g; 5.63 48/2 92.5 1 3.7 35.3 3: 3.0al 6/4 98.5 1 4.5 35.7 :1; 3.3a 4/6 107.5 1 3.8 9.3 :1 1.6b 2/8 60.5 :I: 3.5 18.7 3; 2.6b 0/10 69.5 :1: 2.3 18.6 1 2.5ID * Mean of 2 replicates. ** Mean of 3 replicates. Means followed by same letter not significantly different at 5% level by Duncan's multiple range test. 23 Table 2-2. Field response of N. annulus annulus to the increasing concentration of two isomers ZS'BRp-IR/S'A and 25'3R'7R-A on 28,38,7S-A 18°- mer. The test was conducted at two locations in Michigan from September 4 - October 16, 1981. 28,38,7S-A 28,38,7S-A + + Amount 28'3R'7R/S.A 25'3R'7R-A (ug/trap) (3* i S.E.)** (3* i S.E.)*** 10/0 413.7 1 10.28” 47.0 1 4.53 10/0.1 383.3 1 8.831” 81.7 1 5.3aI 10/0.3 440.3 1 9.5” 87.0 1 5.9‘1 10/1 524.3 1 11.9a 50.7 1 3.8a 10/3 64.5 1 4.5° 18.3 1 2.23 10/10 261.3 1 10.1albc 20.0 1 2.23 10/30 19.7 1.2.4a ** *** Mean of 3 replicates: means followed by same letter not significantly different at 5% by Duncanls multiple range test. Tested at VOgel Center, Misaukee County, Michigan. Tested at Higgins Lake, Roscommon County, Michigan. 24 Table 2-3. Field response of N. annulus annulus to 28,38,7S-A and mixtures of 28,38,7S-A with ser- ial dilutions of 28,3R,7R-A isomer. ‘Test con- ducted at Vogel Center, Michigan, September 10 to October 7, 1983. 28,38,78-A / 28,3R,7R—A Mean Catch/Trap (ug/trap) (1 8.E.)a 5/0 41.0 1 2.9 5/0.001 16.3 1 0.9 5/0.003 27.3 1 1.9 5/0.01 16.3 1.1.3 5/0.03 ~ 30.0 1 2.1 5/1 18.3 1 1.4 5/5 26.0 1 2.0 5/30 17.7 :t 1.7 i 3.0 5/100 33.0 a Means of 3 replicates. Analysis of variance not significant at 5% level. 25 population), 0.3 1 0.7 SD/branch, Roscommon (with medium populations), 14.5 1 10.9 SD/branch, and Vogel Center (with high populations), 28.05 1 8.7 SD/branch, were chosen to compare field effectiveness of the synthetic mixture and natural pheromone. At these three locations, trap catches increased with increasing concentration of the synthetic compound (Table 2-4). That agrees with the suggestion that an attractant with a correct optical configuration shows a dose-related response (Carde et a1. 1977). Traps baited with the natural pheromone, however, could not catch any adults in Manistee and Roscommon, apparently'due'to lower population. The effectiveness of the synthetic and natural pheromone may be computed. If one assumes that one female N. annulus annulus contains about 10 ng. pheromone as female of N. sngnlfng,(xikukawa et a1. 1983), any valid comparison between the synthetic and natural pheromone can not be made with the data in Table 2-4 because the synthetic pheromone concentration tested was not low enough. At lower concen- tration, the effectiveness of the synthetic was comparable to the natural pheromone (Table 2-5). One noticeable fea- ture of the natural pheromone is that it was not dose re- lated. Catches were generally lower at higher concentra- tions. There is a possibility that the extracts may contain certain impurities which at higher concentration was inhibi- tory. In 1982, all the natural preparations were purified one step further after acetylation.by passing through one extra TLC system-preparative TLC HF254+356 and developed 26 Table 2-4. Comparison of field effectiveness of the syn- thetic and natural pheromone of N. annulus anau_-_ lus. Test conducted at three locations in Mich- igan between September 5 and October 16, 1981. Amount ___lo.catians____ Mean! Pheromone (us/Trap) Roscommon Manistee vogel Total Trap Synthetic mixture 28,33,7S-A + 28,3R,78/S-A 3.00 162 22 663 848 282.7 (5:1) 1.00 123 16 429 568 198.3 0.30 29 5 124 158 52.7 0.01 12 1 55 68 22.7 Natural Pheromone (acetate) 1.00 FE 0 0 6 6 2.0 0.30 PE 0 0 3 3 0.7 0.10 PE 0 0 u 4 1.3 0.03 FE 0 0 18 18 6.0 27 Table 2—5. Comparison of field effectiveness of the syn- thetic and national pheromone of N. annulus annulus. Test conducted at Vogel Center, Michi- gan, September 16 to October 16, 1981. Three replicates randomized three times. Amount Mean Catch/trap* Pheromone (ug/Trap) (1 S.E.) Synthetic 28'3S'7S.A '7’ 283315, 7R/S’A (5:1) 3.000 70.7 1,2.9a 1.000 33.0 1 0.8b 0.300 19.3 1 1.71” 0.100. 5.7 1 0.8cd 0.030 3.0 10.8d 0.010 7.0 1 1.4Cd 0.003 3.0 1 1.0(1 0.001 5.0 1 1.2‘1 Control 0 0.0 1 0.06 Natural pheromone (acetate) 3.000 88 3.0 1 1.0‘1 1.000 as 1.7 1 0.6d 0.300 as 15.7 1 2.285 0.100 rs 19.3 1 2.2be 0.030 PE: 11.3 1 2.0“ 0.003 FE 1.7 1 0.96 * Means followed by same letter not significantly different at 5% level. 28 with 20% ether in hexane as the mobile phase (Kikukawa et al. 1983). The synthetic 28,38,78-A (Mori et a1. 1978) was also purified through a charcoal-celite column. Pure form of 28,38,7S-A prepared by different routes of synthesis by another group of chemists (Kikukawa et a1. 1982) was also purified. The 28,38,7S-A from the two sources established threshold at 0.003 ug. (Table 2-6). .Also, both the synthet- ic and natural pheromones were favorably comparable. To determine indirectly the form in which natural pher- omone is stored in the females in nature, the ester and alcohol fractions of N. annulus annulus females were first purified by TLC, the latter fraction was acetylated and then tested for relative activity on the field. The data indi- cate that the alcohol fractions was more active by a factor of 4m5 (Table 2-7). To test whether unesterified diprionol itself might have any effect on effectiveness of synthetic pheromone, varying amounts of 28,38;7S-a1cohol was added to a fixed amount of 28,38,7S-A isomer. It is clear from these data that alcohol is inhibitory and the inhibition was dose related (Table 2-8). The responses of males of N. annulus annulus and N. sngnlfng to different combinations of 28,38,7S-A and 28,3R,7R-AG were different from each other (Table 2-9). .As observed earlier (Table 2-2), the 28,3R,7R- A isomer was not synergistic or inhibitory to the field effectiveness of 28,38,7S-A isomer against N. annulus anau: lus. Males of N. sngtlgng, however, showed a preference for 29 Table 2-6. Threshold of the synthetic pheromone 28,38,7S-A isomer from two sources and comparison with natural pheromone on the field against N. anau: lus annulus at Vogel Center, Michigan, September 18 to October 17, 1982. Mean* Trap Catch in Pheromone Amount/Trap Three Replicates 1 8.5:. (ng) 25,38,7S-A** 28,38,7S-A*** Synthetic 10.000 131.0 1 2.411 298.7 1 2.7a 5.000 93.0 1 2.1b 195.0 1 4.1b 1.000 39.0 1 1.9c 119.7 1 3.59 0.300 37.7 1 3.2c 26.0 1 2.26 0.100 5.3 1 1.2‘fl 6.0 1 0.8‘3 0.030 0.7 1 0.46 2.3 1 0.8‘3l 0.010 1.0 1 0.66 0.3 1 0.4‘3 0.003 0.7 1 0.6‘1 1.0 1 0.6‘3 0.001 0.0 1 0.0‘3 0.0 1 0.0d Control 0 0.0 1 0.06 0.0 1 0.06 Natural pheromone . (acetate) 10.000 re 1.7 1 1.06 0.3 1 0.4‘il 3.000 rs 1.0 1 0.6(1 0.0 1 0.0d 1.000 r2 1.7 1 0.66 2.3 1 0.7‘3 0.300 rs 1.0 1 0.86 0.3 1 0.4‘3 0.100 rs 0.0 1 0.0‘1 0.0 1 0.0‘1 * Means followed by same letter not significantly dif- ferent at 5% level by Duncan's Multiple Range Test. ** From Mori et a1. 1978. *** From Rikukawa et a1. 1982 (unpublished). 30 Table 2-7. Field effectiveness of the TLC fractions of alcohol and ester from the female N.‘anaulus annulus. Test conducted at Vogel Center from September 16 through October 16, 1981. TLC Total fractions Female Replicates trap Mean Catch/ testeda equivalents A B C catch trap 1 8.8. Ester 1 2 1 1 4 1.3 1 0.6 Alcoholb 1 4 7 7 18 6.0 1 1.0 a values for each fraction in this TLC system was alcohol fraction 0.07-0.30 [corresponding to band between (cinnamyl alcohol and cinnamyl acetate), ester fraction 0.39-0.65 (corresponding to 10 (E)-dodecen-1-yl acetate)]. b Acetylated before tested on the field. 31 Table 2—8. Effect of 28,38,7S-dipriono1 on the field effec- tiveness of 28,38,78-A against N. annulus anau: lus. Test conducted at Vogel Center, Michigan, September 18 to October 17, 1982. 28,38,78—A / 28,38,7S-OH Mean catch/trap (mg) 1 S.E.* 5/ 0.000 107.7 1 3.73 5/ 0.001 73.0 1 3.51l 5/ 0.003 46.0 1 3.6b 5/ 0.010 55.3 1 2.4"1 5/ 0.030 83.3 1 2.13 5/ 0.100 66.3 1 1.63 5/ 0.300 40.7 1 2.3b 5/ 1.000 46.0 1 3.9b 5/ 5.000 12.3 1 1.8b 5/10.000 7.7 1 1.3b * Mean catch of 3 replicates. Means followed by same letter may not be significantly different at 5% level by Duncan's Multiple Range Test. 32 Table 2-9. Response of males of N. annulus annulus and N. sngnlfng to different combinations of Optical isomers of 3,7-dimethy1pentadecan-Z-yl acetate. Test conducted in a red pine stand at Vogel Center, Michigan, October 16-31, 1982. 28’3S'7S’A/ 28,3R,7R’AG (us/trap) N. annulus annulusa N. seriaifnx;a 5/o.ooo 2 2 5/0.001 0 22 5/0.003 4 9 5/0.010 3 10 5/0.030 4 3 5/0.100 1 0 5/0.300 6 0 5/1.000 4 0 5/5.000 2 0 a Total catch of 3 replicates. 33 the combination of 28,38,7S-A and 28,3R,7R-A between ratio 5:0.0'01 and 5:0.01. DISCUSSION One intriguing aspect of males of N. annulus annulus response to synthetic pheromone is that while 28,38,7S-A undoubtedly is the pheromone of this species, addition of 28,3R,7R/S-A, 28,3R,7R-A, and 28,3R,7S-A was not inhibitory or synergistic to the effectiveness of 28,38,7S-A. This fact was evident in Table 2-2 for 28,3R,7R-A and 2S,3R,7R/S- A and when varying concentrations of 28,3R,7S-A were com- bined with 28,38,7S-A in 1982, no treatment was superior. The importance of the pheromone as a monitoring tool for this insect is indicated by the fact that the synthetic pheromone, either as 28,38,78-A alone or as mixtures, was as effective as the natural pheromone extracted from females (Table 2-5). The red pine, no doubt, is not a preferred host for N. sngslfng. However, when the traps baited with synthetic pheromone were placed in a red pine stand in mid October 1982, both N. annulus annulus and N. sngnlfng were caught. The males of N. ssgtlfng were recognized by the black abdom- inal tip ventrally. They are also larger and longer than males Of N. annulus annulus. There was a distinct range for optimum catch of N. snnslfng around isomer ratio 5:0.001, 5:0.003 and 5:0.01 of 28,38,7S-A to 28,3R,7R-A, while N. annulus annulus at the same time showed no preference for 34 any isomer combination. Catch for N. annulus annulus was low in this test (Table 2-9) due to nearness to end of adult flight season which leaves us with the data of Table 2-3 for comparison in this instance. The 28,38,7S-A alone was supe- rior to combinations Of isomers in attracting N..anaulus_ annulus (Table 2-3) while addition of submicrogram concen- tration of 28,3R,7R—A significantly improved the effective- ness of 28,38,78-A against N.‘sngslfng,(Tab1e 2-9). This result agrees with our earlier findings on N. sngnlfing (Kikukawa et al. 1983) and supports the theory that the 28,3R isomer may be used as a chemical discriminator against alien species while the 28,38,78 remain the major pheromone of Nnadlpglaa species (Olaifa et a1. 1984). Also in 1979 at two N. sngtlfng infested locations near Lansing, Michigan, it was found that traps baited with natural pheromone of N. annulus annulus caught no male sawflies while those baited with natural pheromone of N. sngnlgng caught 38 male N. sngtlfng. In the same experiment, addition of natural pher- omone of N. annulus annulus to that of N. snulfng decreased the catch of male N. sngnlfng,(kikukawa et a1. 1983). It is thus clear when natural pheromones were used as above and when synthetic pheromones were used as in this study that critical species specific blend of optical isomers is the major mechanism for reproductive isolation in diprionid sawflies. So far, it is in N. annulus annulus that the threo isomers do not appear to play any major role in the pheromone system. From the catch of two species in the same 35 trap, even though response of N. sngslfng appeared more specific to a particular isomer combination, it can be speculated that where chemical discrimination alone fails to separate species reproductively, other isolation factors-- such as copulatory behavior--may come to play. Two incipient races have been reported in N. annulus annulus, one race feeding on jack pine and the other on red pine, but no morphological differences have been detected in the adult populations (Knerer and Atwood, 1973). To invest- igate whether these two races respond to synthetic phero- mones, two active isomers 28,38,7S-A and 28,38,7R/S-A were used in traps set out at the same time at two locations, Vogel Center where N. annulus annulus eggs, larvae, and pupae were collected from red pine and along highway M-SS near Houghton Lake, Michigan, were N. annulus annulus larvae were collected from jack pine. At Houghton Lake, the 28,38,7S-A traps with a total catch of 154 males were more active than the 28,38,7R/8-A traps. At Vogel Center, simi- lar results were obtained as described earlier. The results indicate that both incipient races of N. annulus annulus respond to the same sex-pheromone. REFERENCES Carde, R. T., C. C. Doane, T. C. Baker, 8. Iwaki and S. Marumo. 1977. Attractancy of Optically active phero- mone for male gypsy moths. Environ. Entomol., 6: 768- 772. Jewett, D. M., F. Matsumura and H. C. Coppel. 1976. Sex pheromone specificity in the pine sawflies: inter- change of acid moieties in an ester. Science, 192: 51- 53. Kapler, J. E. and D. M. Benjamin. 1960. The biology and ecology of the red pine sawfly in Wisconsin. For. SCiI' 6: 253-2680 Kikukawa, T., M. Imaida and A. Tai. 1982. Synthesis of the sex-attractant of pine sawflies (Diprion species): (28,3R,7R)- and (28,3R,7S)-3,7-dimethy1pentadecan-2-Ol. Chem. Lett., 1799-18020 Kikukawa, T., F. Matsumura, J. Olaifa, M. Kraemer, H. C. Coppel and A. Tai. 1983. The nature of the major sex pheromone of the European pine sawfly, Nnadlaglga sent; 13‘. JI Chemo ECOlq 9: 673-6930 Knerer, G. and C. E. Atwood. 1973. Diprionid sawflies: polymorphism and speciation. Science, 179: 1090-1099. Kraemer, M., H. C. Coppel, F. Matsumura, T. Kikukawa and K. Mori. 1979. Field responses of the white pine sawfly, Nnadlaglna alannum to Optical isomers of sawfly sex pheromones. Environ. Entomol., 8: 519-520. Kraemer, M. E., H. C. COppel, F. Matsumura, R. C. Wilkinson and T. Kikukawa. 1981. Field and electroantennogram responses of the red-headed pine sawfly, Nnaalaglan lnnnnnnl (Fitch), to optical isomers of sawfly sex pheromones. J. Chem.'Ecol., 7: 1063-1071. Kraemer, M. E., H. C. Coppel, T. Kikukawa, F. Matsumura, H. A. Thomas, L. C. Thompson, and K. Mori. 1983. Field and electroantennogram responses to sex pheromone opti- cal isomers by four fall-flying sawfly species (Hymen- optera: Diprionidae, Nnndlarlna). Environ. Entomol., 12 (5): 1592-1596. 36 37 Matsumura, F., A. Tai, H. C. COppel and M. Imaida. 1979. Chiral specificity of the pheromone of the red-headed Pine sawfly. Marian lssnntei. J. Chem. Ecol., 5: 237—249. Mori, E., 8. Tamada, and M. Matsui. 1978. Stereocontrolled synthesis of all of the four possible stereoisomers of erythro-3,7-dimethylpentadecan-Z-yl acetate and pro- pionate, the sex pheromone of pine sawflies. Tetra- hedron Lett. No. 10, pp. 901-904. Olaifa, J. I., T. Kikukawa, F. Matsumura and H. C. Coppel. 1984. Response of male jack pine sawfly, Nnndlarlaa annual anakslnann (Hymenoptera: Diprionidae) to mix- tures of optical isomers of the sex pheromone 3,7- dimethylpentadecan-Z-yl acetate. Environ. entomol., (in press). Tai, A., M. Imaida, T. Oda and H. Watanabe. 1978. Synthe- sis Of Optically active common precursor of sex phero- mone of pine sawflies. Chem. Lett., 1978: 61. CHAPTER THREE Response of Male Jack Pine Sawfly Nnadlaglan,agnunl anakslnann (Hymenoptera: Diprionidae) to Mixtures of Optical Isomers of the Sex Pheromone, 3,7- dimethylpentadecan-Z-yl acetate INTRODUCTION The major sex pheromone Of various diprionid pine saw- fly are either acetate or propionate esters of 3,7-dimethyl- pentadecan-Z-ol (diprionol) (Jewett et a1. 1976). In field testing the synthetic pheromone of the redheaded pine sawfly Nnadlaglaa lnnnannl (Fitch), it was recognized that the racemic synthetic counterpart was not as effective as the purified natural pheromone. The difference in the optical configurations between these two preparations was suspected (Matsumura et a1. 1979). With three asymmetric carbon cen- ters in the structure ofldiprionol, eight Optical isomers are possible. Various optical isomers were synthesized (Tai et a1. 1978; Mori et a1. 1978: and Kikukawa et a1. 1983) and sup- plied to us for use in various field studies (Matsumura et a1. 1979; Kraemer et a1. 1979, 1981, 1983:.Kikukawa et a1. 1983). These studies established that (a) most Neodiprion species respond to 28,38,78-3,7-dimethylpendadecan-2-yl ace- tate or propionate (abbrev. 28,38,7S-A or P) depending on the species, and (b) two Old World species Dlaglna slmllls 38 39 and Gllplaln igunnnagum most favorably responded to propio- nate and acetate respectively Of 28,3R,7R-diprionol (Kikukawa et al. 1982a). Another major improvement in our knowledge of pine sawfly pheromone was the study by Kikukawa et al. (1983), which established that the presence of a small level of 28,3R,7R-A in 28,38,7S-A drastically in- creased the latter'sleffectiveness in the field. In this study we have examined such interplay of optical isomers in the field against 1119511211911 nrntti banksinnnn Rohwer an- other important defoliator of jack pine in North America. MATERIALS AND METHODS All the field tests were conducted in a jack pine stand at Hartwick Pines, Michigan. This area is located in the middle Of 40 hectares of State Forest. Jack pine comprises approximately 90% of the forest composition. The test area, 1.6 kilometer square, consists of pure jack pines, including a virgin forest. Nnndlpglan,pgnnnl is a complex with several subspecies. In the test area only N. agnnsl anakslnnnn and N. agnsnl pngnaaglnus Ross, have been recorded. However, despite several attempts, we have found only N. pgnnnl,annkslnann in this location as judged by the larvae collected and reared to the adult stage in our laboratory. The females from such laboratory reared larvae were extracted for their pheromone (Kikukawa et a1. 1982). Trap preparation and field distri- bution have been described (Matsumura et al. 1979: Kikukawa 40 et a1. 1983). The number of males caught in each trap was recorded. They were removed from the traps, washed with n- hexane to remove the sticky material, and stored in 70% alcohol. Each trapped male was examined microscopically and compared to laboratory reared specimens. The synthetic pheromones used were from Japan. They came either as pure, racemic isomers, or mixtures. We refer to carbon 2,3 and 7 of diprionol as either R or S, or R/S when racemic. All the isomers were esters. Four chiral isomers 2R.3R,7R; 2R.3R,7S: 28,38,7R: and 28,38,78 were synthesized by Mori et a1. (1978). Tai et a1. (1978) syn- thesized 28,38,7R/8 and 28,3R,7R/S used for this study. The same group (Kikukawa et al. 1983) later synthesized and supplied 28,38,783 28,3R,7R and 28,3R,7S isomers. These isomers probably contained varying levels of optical impuri- ties. In one instance 28,3S,7R/S there was a known 5% contamination with 28,3R,7R/8 isomer on the basis of PMR (Matsumura et a1. 1979). Other isomers contained less than 1% of erythro in threo or threo in erythro contamination as this was the approximate limit of PRM assay. RESULTS In a series of field tests involving twelve propionate and acetate isomers, the males of N. pgnnsl anakslnnnn were attracted to 28,38,7S-A as with N. alanLum and N. lnnaannl (Kraemer et a1. 1979, 1981 and Matsumura 1979) and N. sngtl: fing,(xikukawa et al. 1983). That 28,38,78-A was more active 41 than 28,38,7R-A established the importance of S at the 7- carbon for the major pheromone for this species. No optical isomer containing 2R or 3R was active. In the second series Of experiments, traps baited with 28,38,7R/S-A showed an outstanding catch of males (Table 3-1). Several replicates comparing 28,38,7S-A with 28,38,7R/S-A were later tried. As 28,38,7R/S is made of two isomers-28,38,7S and 28,38,7R in the ratio 1:1 (determined by PMR), we first considered that the mixture of these isomers might be important. Several combinations of 28,38,7S-A and 28,38,7R-A were tried. This was done by holding 28,38/7S-A constant and by varying 28,38,7R-A. However, none of the combinations was active. Even at the ratio 1:1 the mixture was not effective. Another possibility is that the 28,38,7R/S-A preparation contained either 2R or 3R impurities. Several possible combinations of synthetic pheromones were tried by increas- ing the proportion of R at 2 or 3 carbon positions (Table 3- 2). The results indicated that none of the mixtures of 28,38 and 2R,3R were significantly attractive. However, several combinations where 28,38 was mixed with isomers containing 3R were highly attractive. The most males were caught when 28,3R,7R/8-A was added to 28,38,7S-A. An at- tempt was made to determine which of the 28,3R isomers and at what concentrations would provide maximum activity. Al- though flight season was almost over when the traps were set out, the data (Table 3-3) indicated that 28,3R,7R/S-A acted as a synergist to 28,38,7S-A. The 28,3R,7R/8-A isomer used 42 Table 3-1. Field response of males N. agnssl annkslnnnn to traps baited with synthetic pheromones. The test was conducted from September l3-October 9, 1980 at Hartwick Pines, Michigan. Replicates Optical Amount iQntchLTrnnl Isomer (ug) A B Total 28,38,7S-A 5 25 19 44 28,3R,7R/S-A 10 0 l 1 28,38,7R/8-A 10 226 252 478 28,38,7R/S-P 10 0 9 9 28,3R,7R/8-P + 28,38,7S-A 10/5 41 24 65 28,3R,7R/S-A + 28,38,78-P 10/5 1 2 3 43 Table 3-2. Field response of N. pgnnnl anakslnann males to traps baited with synthetic pheromones or admixing of synthetic pheromones. The test was conducted from September 13-October 9, 1980 at Hartwick Pines, Michigan. Optical Amount Mean Catch/Trap Isomer (ug) .1 S.E.* 28,38,7S-A 5 2.0 1 0.6b 2R/S,3R,7R-A 10 0.0 1 0.0b 2R/S,3R,7S-A 10 0.3 1 0.4b 212.311.7341 + 28,38,7S-A 5/5 1.3 1 0.4b 2R,3R,7S-A + 28,38,7S-A 5/5 1.0 1 0.8b 2R/S,3R,7R-A + 28,38,7S-A 10/5 38.3 1 3.15 2R/S,3R,7S-A + 28,38,7S-A 10/5 8.7 1 1.2’D 28,3R,7R/S-A + 28,38,7S-A 10/5 31.7 1 1.71’ Control (no pheromone) 0 0.0 1.0.0b * Means of 3 replicates. Means followed by same letter not significantly different at 5% level by Duncan's multiple range test. 44 Table 3-3. Synergistic effect of the 28,3R,7R/8-A on 28,38,7S-A isomer on attraction of male N. anakslnnnn in the field. Test was con- ducted September 26-October 16, 1981 at Hartwick Pines, Michigan. Optical Amount Mean Catch/Trap Isomer (ug) 1 S.E.* 28,38,7S-A 5 0.0 1 0.0ID 28,3R,7R/S-A 5 0.0 1 0.0b 28,3R,7R-A 5 0.0 1 0.0b 28,38,7S-A + 28,3R,7R/S-A 5/1 4.0 1 0.0a 28,38,7S-A + 28,3R,7R/S-A 1/5 0.0 1 0.0b 2S,38,7S-A + 28,3R,7R-A 5/1 0.0 1 0.0b 28,38,7S-A + 28,3R,7R-A 1/5 0.3 1 0.4b * Means of 3 replicates. Means followed by same letter not significantly different at 5% level by Duncan's multiple range test. 45 in this particular experiment, however, still contained about 5% of 38. In 1982 a new 28,3R,7R-A preparation was synthesized through a Grignand preparation reaction and was designated as 28,3R,7R-AG (Kikukawa et al. 1982b). 'This preparation contained +99.9% optical purity at carbon 3. By varying concentrations of 28,3R,7R-AG: 2S,3R,7R-A; 28,3R,7S-A and 28,3R,7R/S-A against 28,38,7S-A (Table 3-4 and Figure 3-1), we showed that inhibition and stimulation were caused by the same combinations. Using the synthetic 28,38,7R/S-A and the 5:1 optimum combination of 28,38,7S-A and 28,3R,7R-AG, the response threshold concentration was determined. The threshold con- centrations of 28,38,7R/S-A was high (100 ng.) whereas the 5:1 combinations of 28,38,7S-A and 28,3R,7R-AG gave a threshold of 1.0 ng. (Table 3-5). Expressed as female equivalent, this threshold of 1.0 ng. corresponds to 0.1 female equivalent assuming N. arntti bnakslnnnn contains a 10 ng. pheromone per female as does N. sng:l£ng,(xikukawa et a1. 1983). DISCUSSION Several Nnadlaglaa,species.not only feed on the same host plant, but their males fly at the same time and respond to the same ester of 28,38,78 isomer as their major phero- mone component. These sawfly species apparently'maintain reproductive isolation and there is evidence that the males 46 Table 3-4. Effect of the 28 ,3R,7R-AG; 28 ,3R,7R-A: 28 .38.78-- A and 28, 3R ,7R/S-A isomers on the 28, 38, 7S-A isomer in stimulation of male N. nann attraction to baited traps in the field. Testing was done August 30-November 14, 1982 at Hartwick Pines, Michigan. Mam1txap=o< o>:u< o>:u< 2... as :- 25 2:3: no-3 00.? «Ton _ _ _ A:_E\_Euévocuxos r; 52:0 89— A.u._ Eu N x03 5.5.3 :3: 2_.oo-_08._o.__u vcaanoU ut¢£t>m C>=UDC_ u A... 23 an - F 23:02“. 73 Table 5-1. Field response of N. W to optical isomers of 3,7-dimethylpentadecan-Z-yl prOpio- nate or acetate. Test conducted at 5 locations in Roscommon, Crawford and Kalkaska counties, Michigan, May lS-July 10, 1981. Two replicates at 4 locations and 3 replicates at one location, all randomized twice. Amount Total catch Isomer (ug/trap) per treatment 2R,3R,7R-P 20 0 2R,3R,7S-P 20 0 28,38,7R-P 20 0 28,3R,7R-P 20 0 28,3R,7R/S-P 20 0 28,38,78‘A 20 0 25,35,7R‘A 20 0 28,38,7S-A / 2R/S,3R,7S-A 20/20 0 74 the lower peninsula, Michigan. There was an indication also, that the 28,3R,7R-P isomer at the concentration tested might be inhibitory to the 28,38,7S-P isomer. Actions of different propionate isomers on the major isomer were then tested (Table 5-2).' Four pure or racemic isomers (mixtures not included) were found to be active in combination with the 28,35,7S-P isomer. The data also showed traps baited with 2R/S,3S,7S-P catching significantly more males than the major isomer. However, when 2R,3S,7S-P was added to the major isomer at ratio 1:1, there was a slight-but-insignificant reduction in catch. It must be noted that apart from 2R/S,3$,7S-P isomer, apparently be- cause of similarities to major isomer, three other isomers which improved effectiveness of the major isomer were not active by themselves. Three other isomers 28,3R,7R-P; 28,3R,7S-P and 2R,3R,7S-P and those that were earlier found to improve effectiveness of the major isomer were compared for their synergistic activity (Table 5-3). Of these 7 candidate synergists, 3 of them, 28,3R,7S-P; 28,3R,7R/S-P and 2R/S,2R,7S-P isomers, were superior in increasing field effectiveness of the major isomer. A side by side compari- son of these 3 best synergists at four concentrations showed that 2S,3R,7S-P isomer was the superior synergist at a blend ratio 1:2 of 28,38,7S-P and 28,3R,7S-P (Table 5-4). These data also showed a dose (synergists) related response (trap catch) especially with ZS,3R,7S-P and 2R/S,34,7S-P isomers. Table 5-2 0 75 Field response of N. giggininnns to mixtures of optical isomers of 3,7-d methylpentadecan-Z-yl propionate. Test conducted at Mio, Michigan, uly 21-August 4, 1981. Three replicates r andomized three times. Optical Isomers Amount _ (ug/trap) x male/trap 25,35,7s-p 2R/S,3$,7S-P za/s,3s,7n-p zx/s,3n,7s-p 25,3R,7R/S-P 23,33,7n/s-p 25,33,73/5 / 23,33,73/3 / 2R,3S,7R/S / 25,35,7s-p / 25,35,7s-p / zs£§s§7s-p / ' 5,7R/S-P 25 3s 75-? / fix in '7R/S-P 25 as 75-? / £3.55 5- ,7R/ 28,38,7S-P / 23,35,7s-p / 2s,3s,7s-p / 23,33,75-9 / 25,38,7S-P / 25,33,7s-p / Control 20 8.7 20 30.7a 20 0.0 20 0.0 20 0.0 20 0.0 2R,38,7R/S-P 20 0.0 2R,3R,7R/S-P 20 0.0 25,35,7R/S-P 20 0.0 28,3R,7R/S-P 20 45.0a 2R,38,7R/S-P 20/20 1.3 25,33,73/5 / - 20/20 14.0a 25,33,73/5 / 20/20 8.0 23,35,73/5 / 9 20/20 3.0 zn/s,3a,7s-p 20/20 30.7a 2R/S,3S,7R-P 20/20 0.0 2R/S,BS,7S-P 20/20 20.3a 2R,3R,7R-P zo/zo ° 13.3a 23,73,7s-p 20/20 2.7 25,35,7n-p 20/20 0.0 o 0.0 a Mean significantly different from others at 5% level. 76 Table 5-3. Field response of N. xixginisnus to mixtures of candidate optical synergist-isomers. Test con- ducted at Sharon, Michigan, May l4-June 20, 1982. Three replicates randomized three times. Amount i catch/trap Optical Isomers (ug/trap) 1.8.8. 2R/S,3S,7S-P 10 1.3 1 0.7 28'3SI7S-P / 28,3R,7S’P 1.0/10 5807 i 3.13 25,3s,7s-p / 28,3R,7R/S-P 10/10 65.3 1 4.3al 23,38,7S-P / 2R/S,3R,7S-P 10/10 33.3 1 2.93 28,38,7S-P / 2R/S,38,7S-P 10/10 4.7 i 1.0 28'38'7S-P / 2R,3R,7R‘P 10/10 5.3 i 1.2 a Significantly different from other means at 5% level. 77 Table 5-4. Field response of N. gigginianus to mixtures of three best candidate synergist-isomers. Test conducted at Sharon, Michigan, May lS-June 20, 1982. Three replicates randomized three times. Amount 2s,3s,7s-p / 23,3s,7s-p / 25,35,7s-p / (ug/trap) 28,3R,7R/S-P 2R/S,3R,7S-P 28,3R,7S-P 5/ 0.0 0.3 5.7 0.0 5/ 0.1 2.0 5.3 0.7 5/ 1.0 10.3 1.3 3.3 5/ 5.0 30.7 5.0 33.3 5/10.o 18.3 24.0 71.03 a Significantly different from other means at 5% level. 78 The Optimum blend for 28,38,7S-P and 23,3R,7R/S-P appeared to be at 1:1 ratio, even though not significant. In 1983, the 28,3R,7S-P and 28,3R,7R-P isomers were further investigated regarding their synergistic influence on the major isomer. The data derived from two locations show that traps baited with a 1:2 blend of 2S,38,7S-P and 28,3R,7S-P significantly caught more males (Tables 5-5 and 5-6). Also, 28,3R,7R-P isomer exhibited a subtle broad synergism to 25,38,7S-P with the highest catch at ratio 10:0.6. This type of synergism was not dose specific as 28,38,7S-P/28,3R,7S-P mixture. The next approach was analytical: purifying the syner- gist isomer ZS,3R,7R/S-P through a charcoal-celite column and collecting fractions as described in Figure 5-1. Frac- tion nos. 34-42 designated as 25,3R,7R/S-P front was generally more active than the 28,3R,7R/S-P'center (fraction nos. 43-60) and 28,3R,7R/S-P back (fraction nos. 61-63) (Table 5-7). Also the 5:5 blend ratio for 28,38,78- P/2S,3R,7R/S-P front significantly attracted more males than any other fractions or blends. This result not only con- firmed the results of Table 5-2 and 5-3 concerning 28,3R,7R/S-P, it also indicated the presence of synthetic contaminant(s) which eluted with the 28,3R,7R/S-P center and 28,3R,7R/S-P'back. .Further evidence for this inhibitor is presented in Table 5-8 where the blend with 28,3R,7R/S-P front attracted significantly more males than other blends. The ratio for an optimum catch was again found to be 1:1. 79 Table 5-5. Field response of N... W to varying concentrations of 28,3R,7S-P and 28,3R,7R-P mixed with major pheromone isomer. Test conducted at Hartwick Pines, Michigan, June 10- July 14, 1983. Three replicates randomized three times. Amount (“9) ZS'BS'7S-P/ZS'3R'7S‘P* ZS'BS'7S-P/2S'3R'7R-P* 10/0 5.7b 1.0b 0/10 1.3c 4.3b 10/0.002 1.7c 7.0a 10/0.006 5.7b 17.3a 10/0.02 15.0b 8.7a .10/0.06 28.3b 6.0b 10/0.2 2.7c 9.0a 10/0.6 4.7c 34.7a 10/2 7.3b 30.03 10/10 56.3a 33.3a 10/20 66.3a 3.0b 10/100 4.0b 1.3b * Means followed by same letter not significantly different at 5% level by Duncan's new multiple range test. 80 Table 5-6. Synergistic effect of admixing 28,3R,7S-P and 28,38,7S-P isomers in attracting male N. giggin: iunus_on the field. Test conducted at Sharon, Michigan, June 10-15, 1983. Three replicates randomized three times. Amount 5 catch/trap* Optical Isomers (ug/trap) ‘1 8.8. 25,35,7s-p 5 5.0 1 1.4b 25,311,754» 5 2.3 1 0.7c 30 9.7 1 1.8b 25,3s,7s-p / 28,3R,7S-P 5/5 47.7 1 2.8” 5/10 69.7 1 4.1!ll 5/30 23.7 1 2.5b 5/100 12.3 1 1.7c * Means followed by same letter not significant. 81 Table 5-7. Field response Of N. Wm to the front, center, and.back charcoal-celite column frac- tions of 28,3R,7R/S-P* mixed with 28,38,7S-P. Test conducted at Sharon, Michigan, June 30-July 23, 1982. Three replicates randomized three times. Amount/trap mixed with 5 ug 25,3R,7R/S-P 28,3R,7R/S-P 28,3R,7R/S-P 28,38,7S-P (front) (center) (back) 0.0 1.0 0.3 0.3 0.1 2.0 0.3 2.0 1.0 11.0 2.3 2.3 5.0 147.3a 5.3 2.3 10.0 56.0 5.0 9.7 * Fractions as described in Figure 1. a Mean significantly different from others at 5% level. 82 Table 5-8. Field response of N. uiminisuus to the crude and the front, center, and back fractions of 23,3R,7R/S-P isomer mixed with the major phero- mone 28,38,7S-P isomer.* Test conducted at Sharon, Michigan, August 2-31, 1982. Three replicates randomized three times. Mean Amount catch/trap Preparations (ng/trap) 1,8.8. Control (no pheromone) 0 0.0 1 0.0 ZS'BS'7S-P / ZS'BR'7R/S-PCIUde 1.0/10 903 i 1.9 25,3s,7s-p / 28,3R,7R/S-Pfront 10/10 43.3 1 2.5a 28,38,7S-P / 23'3R'7R/S'Pback 10/10 5.7 1 0.7 * Fractions as described in Figure l. a Significantly different from other means by Duncan's mul- tiple range test at 5%. 83 The data in Table 5-9 indicate that N. uimiuiunus males were attracted by natural pheromone of N. duuiusus another member of the N. 1W complex. The data also show that propionate ester moiety is preferred. Using the opti- mum blend of the synthetic pheromone 28,38,7S-P / 2S,3R,7R/S-P mixture, the male catch showed a dose response relationship with threshold limit being 10 ng. (Table 5-10). DISCUSSION The sex pheromone of N. singinisnus is a mixture of 28,38,7S-P and 28,3R,7S-P isomers. This represents the most complex pheromone system in the Ngudiprign species because of divergence from the acetate moiety. This two-component pheromone also illustrates the natural significance of synergistic response to pheromone optical isomers of provid- ing means of reproductive isolations between sympatric spe- cies. Three isomers 28,38,783 28,3R,7S and 28,3R,7R appear to be common components in the pheromone system of Neudi; 21:19.11 species with one species utilizing a combination of 28,38,78 and any one of the other two isomers. In N. 8:111:12 is; and N. W W 28,3R,7R appeared to be the preferred second component (Kikukawa et al. 1983 and Olaifa et a1. 1984). Analysis by capillary GLC indicates that the natural pheromone is comprised of 28,38,78 and 28,3R,78 isomers in N. Wm. The details of GLC analysis is published elsewhere. The abbott sawfly N. W (unpub— lished) and N. W appear to utilize 28,3R,78 84 Table 5-9. Species specificity and ester moiety preference of N. gigginisnus. Test conducted at Sharon, Michigan, May l4-August 13, 1982. Three repli- cates randomized three times. Female Total Natural pheromone equivalents catch Series 1 (May l4-June 20, 1982) N. ruuifrons (acetate) 1 0 N. uinetum (acetate) 1 0 N. dubigsus (proprionate) l 4 N. dubiusus (acetate) 1 0 Series 2 (June 29-August 13, 1982) N. dubiosua 2 6 l 2 0.3 0 Blank 0 0 85 Table 5-10. Threshold limit of the synthetic pheromone of N. 11.1mm. Test conducted at Sharon, Michigan, June 29-August 13, 1982. Three rep- licates randomized three times. Amount ng/trap Mean catch/trap (28,38,7S-P / 28,3R,7R/S-P 1:1) 1 5.8.* 20,000 111.3 1 3.8“ 10,000 92.7 1 1.9“l 2,000 47.7 1 3.4b 1,000 22.3 1 2.2ID 200 14.0 1 1.6° 100 9.7 1 l.6° 20 8.0 1 1.6c 10 0.3 1 0.4c 2 0.0 1 0.0c 1 0.0 10.00 0.2 0.0 1 0.0° 0.1 0.0 1 0.00 0 0.0 1 0.00 * Means followed by the same letter not significantly different at 5% level by Duncan's new multiple range test. 86 isomer. However, attractant blends that are highly species specific appear to play a major role of species isolations despite common usage of common components. Such method Of species isolation has been described among the sympatric tortricine moths (Carde et al. 1977) which utilize geometric isomers. Sympatric fina:huts1shus_species also utilize crit- ical ratios of R and 8 sulcatol for species isolation (Borden et a1. 1980) but that involved mixtures of one enantiomer and its antipode. In the Nsudipziun species, the major pheromone component is the 28,38,78 isomer, its anti- pode 2R,3R,7R has never been active or synergistic. The utilization of critical ratios of 28,38,78 with a threo isomer 28,3R,7S or 28,3R,7R or 28,3R,7R/A for species isolar tion as evidenced in mm (Kikukawa et a1. 1983), N. 2:41:11 banksianae (Olaifa, et al. 1984) and n. uiruinianus in this study is novel to science. Further studies will be needed to completely elucidate the mechanism of reproductive isolation among species complexes. REFERENCES Atwood, C. E. 1961. Present status of the sawfly family Diprionidae (HymenOptera) in Ontario. Proc. Ent. Soc. 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Sex pheromone specificity as a reproductive isolating mechanism among the sibling species W and A. W and other sympatric tor- tricine moths (Lepidoptera: Tortricidae). J. Chem. Ecol., 3 (1): 71-84. Kikukawa, T., M. Imaida and A. Tai. 1982a. Synthesis of the sex attractant of pine sawflies (Diprion species): (28,3R,7R) and (28,3R,7S)-3,7-dimethyl-pentadecan-2-Ol. Chem. Lett., 1982: 1799-1802. Kikukawa, T., F. Matsumura, M. Kraemer, H. C. Coppel and A. Tai. 1982b. Field attractiveness of chirally defined synthetic attractants to males of 2121:1911 8111111313 and W frutstorum. J. Chem. Ecol., 8: 301-314. 87 88 Kikukawa, T., F. Matsumura, J. Olaifa, M. Kraemer, H. C. COppel and A. Tai. 1983. The nature of the major sex pheromone of the European pine sawfly, Neudimgn sen; Lin. J. Chem. Ecol., 9: 673-693. King, 1.. L. and D. M. Benjamin. 1965. The effect of photo period and temperature on the development of multi- voline populations of Nsudimgn sum Middleton. Proc. North Central Branch E.S.A., 20: 139-140. Kraemer, M., H. C. Coppel, F. Matsumura, T. Kikukawa and K. Mori. 1979. Field responses of the white pine sawfly. pinsnum to optical isomers of sawfly sex pheromones. Environ. Entomol., 8: 519-520. Kraemer, M. E., H. C. Coppel, F. Matsumura, R. C. Wilkinson and T. Kikukawa. 1981. Field and electroantennogram responses of the red-headed pine sawfly, Neudipriun ism (Fitch), to Optical isomers of sawfly sex pheromones. J. Chem. Ecol., 7: 1063-1071. Kraemer, M. E., H. C. Coppel, T. Kikukawa, F. Matsumura, H. A. Thomas, L. C. Thompson and K. Mori. 1983. Field and electroantennogram responses sex pheromone Optical isomers by four fall-flying species (Hymenoptera: Dip- rionidae, Marius). Environ. Entomol., 12: 1592- 1596. Matsumura, F., A. Tai, H. C. Coppel and M. Imaida. 1979. Chiral specificity of the sex pheromone of the red headed pine sawfly: Nam W. J. Chem. Maxwell, D. E. 1958. Sawfly cytology with emphasis upon the Diprionidae (HymenOptera: Symphyta). Proc. 10th Intern. Congress Entomol., 2: 961-978. Middleton, W. 1933. Five new sawflies of the genus Nadia; mu Rohwer. Can. Ent., LXV (1): 77-84. Mori, K., S. Tamada and M. Matsui. 1978. Stereocontrolled synthesis of all Of the form possible stereoisomers of erythro-3,7-dimethylpentadecan-Z-yl acetate and pro- prionate, the sex pheromone of pine sawflies. Tetra- hedron Lett. #10: 901-904. Olaifa, J. 1., T. Kikukawa, F. Matsumura and H. C. Coppel. 1984. Response of male jack pine sawfly, hummus: (Hymenoptera: Diprionidae) to mix- tures of Optical isomers of the sex pheromone 3,7- dimethylpentadecan-Z-yl acetate. Environ. Entomol., (In press). 89 Ross, H. H. 1955. The taxonomy and evaluation of the sawfly genus Wan. Forest Science, 1: 196-209. Tai, A., M. Imaida, T. Oda and H. Watanabe. 1978. Synthe- sis Of Optically active common precursor of sex phero- mone of pine sawflies. Chem. Lett., 1978: 61. Wilson, 1.. F. 1970. A guide to insect injury of conifers in the lake state. USDA Forest Service, Agriculture Handbook #501. CHAPTER SIX : Synergistic Response to Mixtures of Optical Isomers of the Sex Pheromone 3,7-dimethylpentadecan-Z-ylacetate INTRODUCTION The sex pheromone of diprionid pine sawflies was de- scribed as the acetate or propionate 3,7-dimethylpentadecan- 2-01 (diprionol) (Jewett et al. 1976). However, when tested on the field against three diprionid sawfly species, the synthetic compound was not as active as the natural phero- mone from the field. When Marian W (Fitch) showed significant response to (28,38,7R/S)-3,7-dimethyl- pentadecan-Z-ylacetate (28,38,7R/S-A) on the field (Matsumura et al. 1979), it then became clear that only certain Optical isomers of diprionol was active. This Ob- servation was also confirmed in N. W (Kraemer et a1. 1979) which showed response to 28,38,7S-A, but not to 2R,7R,7R-A, and 2R,3R,7S-A. The 28,38,7R-A isomer also showed some activity. This study was the first to show that a mm species responded to 28,38,7S-isomer, although this has been speculated when 28,38,7R/S-A baited traps attracted male»N..lesuntgi.on the field (Matsumura et a1. 1979). Further investigations by the same group revealed that N. W. N. nanulus nanulus Schedl, N. ssrtifsr 90 91 (Geoffroy) and N. unsung lingnnis Ross respond mainly to 28,38,78 isomer (Kraemer et a1. 1981 and 1983), while two Old World species Elysian sinilis (Hartig) and Gilpinin Wm (Fabricius) prefer the 28,3R,7R isomer (Kikukawa et al. 1982a). Another field studies in 1980 at Higgins Lake Michigan by Kikukawa and Matsumura (unpublished) estab- lished that N. pinstum_respond mainly to 28,38,7S-A and that males respond better to the acetate than the propionate isomers. However, no definite conclusion was made by their data as well as those of Kraemer et a1. (1979) as to the effect of mixing isomers. This study reports interaction Of Optical isomers in field response of N. gingham, an impor- tant defoliator of Pinus strnnus Linnaeus in North America. MATERIALS AND METHODS £1£1d_IESLS Field trapping of N. ninetum was carried out in the States of Michigan and Wisconsin. In Michigan, two tests areas, Sec. 25 and 30, Higgins Lake State Forest, Roscommon County and Sec. 33, McGee on Highway M66, Kalkaska County, were used. Both consisted Of pure white pine stands, each about 1.6 kilometer square. The ground cover consisted of grasses and fern, Mum mm (1’...) Kuhn. In Wis- consin, pure white pine stands 2 miles west Of Siren were used. The larval colonies of N. pinstum were collected by Dr. Mark Kraemer of University of Wisconsin and reared to adulthood in the laboratory. These laboratory reared adults 92 from Wisconsin were morphologically identical to the field trapped N. pinstum from Michigan. Pherocon II traps were used throughout this study. Trap preparations have been described (Kraemer et a1. 1979). Randomized complete block design was the experimental design used with the block choice based on rows Of clearings at the two locations. At least one re-randomization of treatments within and between blocks was done, and the record of trap catch taken weekly. The data so collected are evaluated with analysis of vari- ance and the differences among means graded at P s 0.05 according to Duncan's new multiple range test. Trapped males were removed from the traps, washed with n-hexane to remove the sticky material and stored in 70% alcohol. Each trapped male was examined microscopically and compared to laboratory reared specimens. SynthetiLFhsromsns Four isomers, 28,38,78-A and 28,38,7S-P: 28,3R,7R/S-A; 28,3R,7R-A and 2S,3R,7S-A were used in this study. The R or 8 refers to chirality on 2-carbon, 3-carbon and 7-carbon of the structure of diprionol. The first two isomers were from the laboratory of Dr. Kenji Mori (Mori et al. 1978). These isomers were later synthesized by Kikukawa et al. (unpub- lished). The synthesis of 28,3R,7R/S-A has been described (Tai et al. 1978). The first synthesis of threo isomers in optically pure forms was made by Kikukawa et al. (1982b) who supplied us with 28,3R,7R-A and 28,3R,7S-A. These compounds 93 are prepared in desired concentrations in 1 ml n-hexane, sealed in glass ampules until use on the field. RurifluatimLSmthstiu WW Culumn Thirty-six gm of charcoal (Norit A., J. T. Baker Chemi- cal Co., New Jersey) pre-heated for 2 hrs. at 180°, was mixed thoroughly with 9 gm celite (Fisher Scientific Co.) and washed with 20 ml acetone in a funnel with Whatman NO.:2 filter paper. The charcoal-celite obtained from above was stirred into a 200 ml beaker with 140 ml 10% ether in hexane using a glass rod. The slurry obtained was packed in a 50 cm x 2 cm i.d. glass column. A 20 ml solvent was first used to wash the slurry down the walls of the column followed by 80 ml in 2 equal volumes to stabilize the column. A maximum of 7 mg sample was introduced to the column and was eluted with 10% of ether in n-hexane at 0.2:ml/min of flow rate. Recovery was about 60%, with samples less than 500 ug it was less. Sixty-three 10 ml fractions were collected using a micro fractionator (Model FC-80K, Gilson Medical Electron- ics, Inc., Middleton, Wisconsin). A small aliquot from each fraction was injected into a gas liquid chromatograph (GLC) equipped with a flame ioniza- tion detector (FID). Fractions 33-63 contained the sample, but fractions 33-42 and 61-63 contained the sample and impurities while fractions 43-60 contained the pure sample. It should be noted that columns less than 35 cm in height failed to purify the samples in this manner. 94 RESULTS Combination of 28,38,7S-A and 28,3R,7R/S-A at ratio 1:2, a significant synergism of male catch was recorded (Table 6-1). Synergism was directly related to the concen- tration of the 28,3R,7R/S-A isomer. The result of a similar experiment in Siren, Wisconsin (Table 6-2) proved the same point. The synergist 28,3R,7R/S-A, in each case, was not active by itself. It is not clear from Table 6-2 the role of 28,38,78-P which was not active alone but which appeared to improve effectiveness of 28,38,7S-A. In this study, the traps were re-randomized once but vandalism Of some of the traps made a definite conclusion of the role of 28,38,7S-P impossible. As indicated in previous studies (Kraemer et a1. 1979) and the unpublished data in 1980 by Kikukawa and Matsumura it is not likely that both prOpionate and acetate occur in one species as natural pheromones, and hence, 28,38,7S-P was not used in subsequent studies. Traps baited with mixtures of 28,38,7S-A and 28,3R,7R-A recorded a significant trap catch (Table 6—3). This experi- ment compared two pure isomers 28,3R,7S-A and 28,3R,7R-A with the racemic 28,3R,7R/S-A which was synergistic in the earlier results. The synergistic effect of 28,3R,7R-A was manifested when mixed with 28,38,7S-A from both Mori et al. (1978) and Kikukawa et al. (unpublished). Because of the superior performance of 28,38,7S-A from Kikukawa (Table 6- 3), we utilized this preparation in subsequent experiments. In Kalkaska, the males of N. ninetum were also attracted to 95 Table 6-1. Synergistic effect of the 28,3R,7R/S-A isomer on the effectiveness of 28,38,7S-A isomer in at- tracting wild male N5 pinstnm at Higgins Lake State Forest Michigan, May lS-July 20, 1981 on white pine. Mean male Amount catch/trap* Preparation (ug/trap) i S.E. 28,38,7S-A 20 7.0 1 14°59 28,3R,7R/S-A 20 0.0 1 0.0e 28,38,7S-A / 28,3R,7R/S-A 20/5 7.7 1 1.2Cd 28,38,7S-A / 28,3R,7R/S-A 20/10 8.7 1 1.76 28,38,7S-A / 28,3R,7R/S-A 20/20 18.7 1 1.1b 28,38,7S-A / 28,3R,7R/S-A 20/40 30.3 1 1.4a1 Control 0 0.0 1 0.0e * Means followed by same letter not significantly different at 5% level. Means of 3 replicates. 96 Table 6-2. Field response of male N. ninetum to traps baited with isomers of acetate or propionate diprionol. Test conducted at Siren, Wisconsin, May lS-June 30, 1981. Total catch Amount in 3 repli- Preparations (ug/trap) cates 28,38,7S-A 12-5 4 28,38,7S-A 25 6 28,38,7S-P 12-5 0 28,3R,7R/S-A 25 0 28,38,7S-A / 28,3R,7R/S-A 12-5/25 44* 28,38,78-A / 28,38,7S-P 12-5/12-5 40** * ** Total in one trap; 2 other traps vandalized. Total of two traps, 1 other trap vandalized. 97 Table 6-3. Comparison of three threo isomers 28,3R,7R-A, 28,3R,7S—A and 28,3R,7R/S-A in synergizing the effectiveness of 28,38,7S-A isomer against N. Rinfitum males. Test conducted at Higgins Lake, Michigan, May 7-June 10, 1982. Preparation Amount Mean catch/ of isomers (ug/trap) trap 1,8.E. 25,35,7s-Aa 5 0.0 1 0.0 28,38,7S-Ab 5 1.3 1 0.6 28,3R,7R-A 5 0.0 1 0.0 28,3R,7S-A 5 0.0 1 0.0 28,3R,7R/S-A 5 0.0 1 0.0 28,35,7s-Aa / 28,3R,7R-A 5/10 5.0 11.00 2s,3s,7s-Ab / 28,3R,7R-A 5/10 6.3 1 1.1c 25,33,754.b / 28,3R,78-A 5/10 1.0 1 0.6 25,38,7s-Ab / 28,3R,7R/s-A 5/10 0.3 1 0.4 Control 0 0.0 1 0.0 a 28,38,7S-A form Mori et al. 1978. b 28,38,7S-A from Kikukawa et al. (unpublished). c Means significantly different from others at 5% level by Duncan's multiple range test. 98 1a mixture of 28,38,7S-A and 28,3R,7R-A at 1:1 and 1:2 ratios but the latter ratio was superior (Table 6-4). The 28,3R,7S-A and 2R/S,3R,7S-A were not active as synergist in this experiment. By holding the concentration of 2S,3S,7S-A constant at 5 ug and varying the concentration of the syner- gist isomer 2S,3R,7R-A from 0.001 to 20 ug, it was estab- lished that maximum effectiveness of the mixture was achieved at a concentration of 10 ug of the synergist. This implies that a 1:2 ratio gave the most active blend. When the ratio increased to 1:4, a significant reduction in catch was recorded (Table 6-5). Using the 28,3R,7R/S-A as the synergist of the 28,38,7S-A isomer in Siren, Wisconsin, we again demonstrated significant effectiveness of the mixture at a blend ratio of 1:3 of 28,38,7S-A to 28,3R,7R/S-A (Table 6-6). DISCUSSION The 28,38,7S-A isomer again proved to be the major pheromone of N. ninstum. This time not only because it is more effective than other isomers tested singly, but because other isomers 28,3R,7R/S-A and 28,3R,7R-A acted as syner- gists to improve its effectiveness in a dose-related manner. This study also showed that the mixture of the major and synergist isomers at the right combination was better than the major isomer alone. The chiral differences between the pheromone of Neudi: 911.011 species and the Old World species must play a major 99 Table 6-4. Comparison Of three threo isomers 28,3R,7R-A 28, 3R, 7S-A and 2R/8, 3R, 7S-A in synergizing the effectiveness of 28, 3S, 7S-A isomer against N. males. Test conducted in Kalkaska, Michigan, May 5-June 10, 1982. Amount Mean Catch/Trap Preparation (ug/trap) .1 8.E.* 25.35.7541 5 0.0 1 0.0'3 25,35,75-11 / 28,3R,78-A 5/5 0.0 1 0.0b 28,38,7S-A / 38,3R,7S-A 5/10 0.0 1 0.0'3 28,38,7S-A / 28,3R,7R-A 5/5 2.7 1 1.18 25,35,75-11 / 28,3R,7R-A 5/10 4.7 1 1.49 28,38,7S-A / 212/5,311,754 5/5 0.0 1 0.0” 28,38,7S-A / 211/5,311,754 5/10 0.0 1 0.09 Control 0 0.0 1 0.0b * Mean of 3 significan Test. replicates. Means followed by same letter not tly different at 5% by Duncan's Multiple Range 100 Table 6-5. Determination of the optimum blend of the mix- ture of 28,38,7S-A and 28,3R,7R—A isomers in attracting N. pinstnm males at Kalkaska, Michi- gan, June 29-July 7, 1982. Mean male Isomer Amount catch/trap* preparations (ug/trap) 1 8.E. 28,38,7S-A 5 0.0 1 0.0c 28,38,7S-A / 28,3R,7R-A 5/0.001 0.0 1 0.0° 5/o.01 0.0 1 0.00 5/0.1 0.0 10.0c 5/1 0.0 1 0.0c 5/5 10.3 1 0.99 5/10 21.0 1 1.6'3| 5/20 18.3 1 1.6ab * Means followed by the same letter not significantly dif- ferent at 5% level. Means of 3 replicates. 101 Table 6-6. Determination of the optimum blend of the mix- ture of 28,38,7S-A and 28,3R,7R/S-A isomers in attracting N. pinshum.males at Siren, Wisconsin, May 15-June 30, 1982. Mean male Isomer Amount catch/trap preparations (ug/trap) 1 S.E. 28,3S,7S-A* 10 0.0 1 0.0 28,38,7S-A** 10 0.3 1 0.4 28,38,7S-A* / 28,3R,7R/S-A 10/0.001 1.0 1 0.6 10/0.01 1.0 1 0.6 10/0.1 2.3 1 0.9 10/1 1.7.1 0.7 10/3 0.7 i 0.4 10/10 3.0 1 0.8 10/30 15.0 1 1.76 * 28,38,7S-A from Mori et a1. (1978). ** 2S,38,7S-A from Kikukawa et al. (unpublished). c Mean significantly different from others by Duncanfls multiple range test at 5% level. 102 role in species recognition. Thus, the N. ninetum and n. sin111s_which.are the two species particularly feeding on white pine, must be separated by this mechansim since N. Rinfltum do not respond to 28,3R,7R isomer alone as demon- strated in Table 6-1 and 6-3. One other important mechanism of species recognition between these two species is the preference by D.- similis for the propionate (Jewett et a1. 1976) and acetate by N. ningtum_(xraemer et a1. 1979). Such chiral differences have not been observed among Nggdipninn species and as such cannot explain their mechanism of spe- cies recognition. Using purified mixture of optical isomers on the field, Kikukawa et a1. (1983) demonstrated that a small addition of the 28,3R,7R-A isomer significantly im- proved the effectiveness of 28,38,7S-A isomer against N. sgrtifen. That study was the first to demonstrate interac- tion of optical isomers in field response of diprionid sawflies to their sex pheromone. A similar interaction of optical isomers was also observed in field response of male N. unatti banksinnns Rohwer to synthetic pheromones (Olaifa et a1. 1984). One important difference in these two species is that while the former species utilizes a 5a0.003 combina- tion, the latter utilizes a 5:1 combination of 28,38,78- A:28,3R,7R-A. A demonstration of 5:10 combination of the same set of optical isomers in N. m in this study, is a clear indication that interaction of optical isomers is a mechanism of species recognition among Ngndinrinn species. REFERENCES Jewett, D. M., F. Matsumura and H. C. Coppel. 1976. Sex pheromone specificity in the pine sawflies: inter- change of acid moieties in an ester. Science, 192: 51- 53. Kikukawa, T., F. Matsumura, M. Kraemer, H. C. Coppel and Akira Tai. 1982a. Field attractiveness of chirally defined synthetic attractants to males of Man 31ml: 11s and 911% Mm. J. Chem. Ecol., 8: 301- 314. Kikukawa, T., M. Imaida and A. Tai. 1982b. Synthesis of the sex-attractant of pine sawflies (Diprion species): (28,3R,7R)- and (28,3R,78)-3,7-dimethylpentadecan-2-ol. Chem. Lett., 1799-1802. Kikukawa, T., F. Matsumura, J. Olaifa, M. Kraemer, H. C. COppel and A. Tai. 1983. The nature of the major sex pheromone of the European pine sawfly, Neudinninn SELL: ifsr. J. Chem. Ecol., 9: 673-693. Kraemer, M., H. C. Coppel, F. matsumura, T. Kikukawa and K. Mori. 1979. Field responses of the white pine sawfly, Rinfitum to optical isomers of sawfly sex pheromones. Environ. Entomol., 8: 519-520. Kraemer, M. E., H. C. Coppel, F. Matsumura, R. C. Wilkinson and T. Kikukawa. 1981. Field and electroantennogram responses of the red-headed pine sawfly, Nsndinninn 1mm (Fitch), to optical isomers of sawfly sex pheromones. J. Chem. Ecol., 7: 1063-1071. Kraemer, M. E., H. C. Coppel, T. Kikukawa, F. Matsumura, H. A, Thomas, L. C. Thompson, and K. Mori. 1983. Field and electroantennogram responses to sex pheromone Opti- cal isomers by four fall-flying sawfly species (Hymen- Optera: Diprionidae, Wun). Environ. Entomol., 12 (5): 1592-1596. Matsumura, F., A. Tai, H. C. Coppel and M. Imaida. 1979. Chiral specificity of the pheromone of the red-headed pine sawfly: W lssnntsi. J. Chem. Ecol., 5: 237-249. 103 104 Mori, E., S. Tamada, and M. Matsui. 1978. Stereocontrolled synthesis of all of the four possible stereoisomers of erythro-3,7-dimethy1pentadecan-2-yl acetate and pro- pionate, the sex pheromone of pine sawflies. Tetra- hedron Lett. NO. 10., pp. 901-904. Olaifa, J. I., T. Kikukawa, F. Matsumura and H. C. Coppel. T31; 1984. Response of male jack pine sawfly banksinnng_(Hymenoptera: Diprionidae) to mix- tures of optical isomers of the sex pheromone 3,7- dimethylpentadecan-z-yl acetate. Environ. Entomol. (in press . A., M. Imaida, T. Oda and H. Watanabe. 1978. Synthe- sis of optically active common precursor of sex phero- mone of pine sawflies. Chem. Lett., 1978: 61. CHAPTER SEVEN Attractancy of Optically Active Isomers of Pheromone 3,7-dimethylpentadecan-2-yl propionate for Male 01mm similis (HymenOptera: Diprionidae) on the Field INTRODUCTION The major sex pheromone of Dinnign s1n1115_(Hartig) was stereochemically established by field tests as the propio- nate of (28,3R,7R)-3,7-dimethylpentadecan-2-ol (28,3R,7R-P) (Kikukawa et al. 1982a). That conclusion, however, was based on field response of males to racemic compounds 28,3R,7R/S-P; 28,3R,7R/S / 2R,3S,7R/S-P and 2R/S,3R,7R-P isomers. Of these racemates, the 2R/S,3R;TR-P was the most active but not significantly above the others. In the same study, it was found that the natural pheromone was far superior, the difference being in the neighborhood of 2500 fold. The discrepancy between the natural and synthetic was attributed, among others, to the racemic nature of the synthetics. . ' There is evidence for enantiomeric specificity in the pheromones of many scolytid Coleptera (Hedden et a1. 1976; Wood et a1. 1976: Borden et a1. 1976ra 1980). It is.also hypothesized that racemates elicit response-inhibitory ef- fects on the receptor system of insects which naturally respond to one enantiomeric form only (Vite et a1. 1976). 105 106 Experience with other diprionid sawflies showed that species respond mainly to one enantiomeric form (28,38,7S)-3,7- dimethylpentadecan-Z-yl acetate (28,38,7S-A) but such re- sponse may be synergized by another one enantiomeric form 28,3R,7R-A or 28,3R,78-A depending on species (Kikukawa et al. 1983: Olaifa et a1. 1984). This study reports on the field response of D. similis to the racemates and one enan- tiomeric forms as well as mixtures of one enantiomeric forms of sex pheromone of diprionid sawflies. MATERIALS AND METHODS Waning Most of the field trappings took place on*white pine stands at Sec. 33, McGee on M-66 highway, Kalkaska County, Michigan. All the white pine stands at this location are shrubby and fully exposed to sun. Undergrowths are mainly grasses and sweet fern, Cunntunn W (L.) Coult. Also used on a few occasions were Sec. 25 and 30, Higgins Lake State Forest in Roscommon County and at Sec. 33, Rose Lake Wildlife Experimental Stations at Bath Township in Ingham County, Michigan. At all these locations, eggs, larvae, cocoons as well as adult D. slums were collected. Methods of bioassay have been described (Kikukawa et a1. 1982). On a few occasions, Siren, Wisconsin was used as a trapping site. 107 NatuLaLFheromuns The adult females reared from field collected larvae and pupae were extracted of their pheromone by the method described by Kikukawa et a1. (1982). Purification was by TLC HF254 + 366 developed 15 cm first in hexane:ether 4:1. After the pheromone fractions (Rf 0.21-0.33) has been re- moved and esterified to propionate, it is purified further by a second TLC HF254 1 366 developed first in n-hexane and then benzene. The zone corresponding to Rf 0.75-0.85 con- tained the purified propionate of natural pheromone Of D.- similis. The diprionol band comes right between the band of cinnamyl alcohol and cinnamyl acetate, while the propionate diprionol comes about 5.0 mm'above cinnamyl acetate. These two standards were chosen to take advantage of UV light detection. SynthetiLRhsrnmonss The propionate esters of 28,38,783 2R,3R,7R; 2R,3R,7S and 28,38,7R-diprionol were synthesized by Mori et a1. (1978). Synthesis of the racemic compound 28,3R,7R/S-P and 28,3R,7R/S / 2R,38,7R/8-P was by Tai et a1. (1978). By the method described by Kikukawa et al. (1982a), the 2R/S,3R,7R- P and 2R/S,38,7S-P were modified from one enantiomeric forms from Mori et al. (1978). Kikukawa et al. (1982b) synthe- sized for the first time one enantiomeric forms of dias- tereomers 28,3R,7S-P and 28,3R,7R-P. 108 W Chaznnalznelitfi_flnlumn Thirty-six gm of charcoal (Norit A., J. T. Baker Chemi- cal Co., New Jersey) preheated for 2 hrs at 180°C, was mixed thoroughly with 9 gm celite (Fisher Scientific Co.) and washed with 20 ml acetone in a funnel with Whatman No. 2 filter paper. The charcoal-celite Obtained from above was stirred into a 200 ml beaker with 140 ml 10% ether in hexane using a glass rod. The slurry Obtained was packed in a 50 cm x 2 cm i.d. glass column. A 20 ml solvent was first used to wash the slurry down the walls of the column followed by 80 ml in 2 equal volumes to stabilize the column. A maximum of 7 mg sample was introduced to the column and was eluted with 10% ether in n-hexane at 0.2 ml/min flow rate. Recov- ery was about 60%, with samples less that 500 ug it was less. Sixty-three 10 m1 fractions were collected using a microfractionator (Model FC-80K, Gilson Medical Electronics, Inc., Middleton, Wisconsin). .A small aliquot form each fraction was injected into a FID GLC (stainless steel columns 7.2 m x 3 mm packed with 3% S.E. 30 and 6 m x 3 mm 10% carbowax 20 M) for purity. The active fractions 43-60 were free of any impurity by GLCIdetection and was used as pure compound for the field test. W A Varian 1700 preparative gas chromatograph fitted with stainless steel column 6 m x 3 mm packed with 10% carbowax 20 M on 80-100 mesh chrom 0 were used for fractions 109 collection of seven isomers. The fractions consisted of the front and back of GLC peaks of 28,3R,7R-P: 28,3R,78-P; 28,38,7S-P; 28,35,7R-P; 2R,3R,7R-P; 2R,3R,7S-P and 2R/S,3S,7S-P as depicted in Figure 7-1. A 9:1 post column splitter was used which directed 90% of the injected mate- rial for collection and 10% to the detector. A 15 cm pyrex tubing bent into a WP at the middle was used for collec- tion. Hexane moistened glass wool was used to plug the distal end of the tubing and the bent portion of the tubing contained a small amount of hexane solvent to monitor the flow of carrier gas. Using the solvent also increased trapping efficiency of the collecting tube. The unplugged proximal end of the tubing was inserted into the metal outlet from the detector oven at the appropriate retention time being monitored by a Linear chart recorder. The ur tubing was placed in ice during collection and the collected material in the tube was washed with hexane. Trapping efficiency was about 80%. The front and back fractions were bioassayed separately on the field. RESULTS When compared with two racemic isomers that were found active earlier (Kikukawa et al. 1982a), the 28,3R,7R-P at- tracted more males. However, there was no significant dif- ference among the treatments (Table 7-1). The discrepancy between the activity of the synthetic and natural pheromone of n. sunilis was partly attributed to either some impurity Figure 7-1. GLC pattern of the 28,3R,7R-P on 5% carbowax 20M column. The fractionation pattern into front and back is shown by arrows. 110 14 TIME. mmZOnmmm cmooowc MIN. Table 7-1. 111 Field response of D. similis to three enantiomers of 3,7-dimethylpentadecan-Z-yl propionate at five locations in Michigan and Siren, Wisconsin. Test conducted June 6-July 20, 1981. Three replicates at each location. Guile Isaners Lake Kalkaska Mio Lansing Siren Total (20 ug/ trap) Michigm Michigan Michigan Michigan Wisconsin catch 28,3R, 7R-P 2 13 11 0 0 26 28 ,3R, 7R/S-P 0 14 0 l 0 15 ZSJHLTRGB/ 2R,38,7R/S-P 0 3 5 l 4 13 112 in the synthetic compound or to a synergist in the natural preparations which they could not find or to the racemic nature of the synthetics used (kikukawa et al. 1982a). First, an attempt was made to purify 28,3R,7R/S-P. In the results shown in Table 7-2, one can see a surprising finding that the crude 2S,3R,7R/S-P performed better than the pure 28,3R,7R/S-P (purified through a charcoal-celite column; fraction nos. 43-60). In a 2-day field study using another racemic isomer 28,3R,7R/S / 2R,38,7R/S-P, purified in the same way as 28,3R,7R/S-P, it was found that crude prepara- tions caught 2 males, traps with pure preparations did not catch males. The impurity of 28,3R,7R/S / 2R,38,7R/S-P which represented the impurities contained in fraction nos. 33-42 was further concentrated and collected on GLC as described earlier, and added to 28,3R,7R/S-P and 2S,3R,7R-P isomers. In this trial, the 28,3R,7R/S-P with a catch of 14 males performed better than 28,3R,7R-P plus impurity with 2 males and 1 male in the 28,3R,7R/S-P plus impurity. How- ever, the activity of impurity appeared to increase with dose. It appears to be that there is a synthetic synergist of which retention volume in the celite-charcoal system used in this study comes around fractions 33-42. Four isomers were mixed with 28,3R,7R-P but no apparent synergistic in- teractions were present (Table 7-3). Because of low catch in this trial, it can only be speculated that increased concentrations of 28,38,7S-P and 2R,3R,7R-P inhibit the activity of 28,3R,7R-P. A similar conclusion may be made 113 Table 7-2. Field response of D. similis to crude and puri- fied 28,3R,7R/S-P isomer. Test conducted at Higgins Lake and Kalkaska, Michigan, May 15-July 20, 1981. 28 1 3R, 7R/S-P preparation (40 ug/trap) Series Aa Series Bb Total Catch Crude 7 24 31 Pure0 l 4 5 a Total of 3 replicates at Higgins Lake, May lS-July 20, 1981. b Total of 4 replicates at Kalkaska, July 2-August 4, 1981. c Purified by charcoal-celite column, fraction NO. 43-60. 114 Table 7-3. Response of D. similis to a mixture of 28,3R,7R- P with four other isomers of diprionid pine sawfly sex pheromone. Conducted May 29-July 23, 1982 in Kalkaska, Michigan. Amount 28,3R,7R-P/ 28,3R,7R-P/ 28,3R,7R-P 28,3R,7R-P/ (“ya”) ZS I 38 ' 7S-Pa 25 3 3R, 7S-Pa ZS ,3S,7R-Pa 2R,3R, 7R"Pa 2m“) 6 3 5 5 ‘WQO 0 0 0 0 2&0. 0 2 0 1 Zafli 1 8 3 2 ZQUD 3 2 0 0 ”V20 1 4 0 0 ZWMO 7 2 0 0 atknal é’ a?.._ (.9 3 fl? hm" 23.33.1341 nan-v Islam-I 2am] 8.81841! 3.1117" mm W ma. can «a! mug) "16’ um" mi‘ “15‘ asni‘ was: .- ..... 9 a—cus .-' g » Goals-(1:2) I—I mum :o' 174 one-half and even one-quarter of this concentration. This result suggested that the mechanism by which 28,3R,7S-P elicited synergism is probably to increase the maximum re- sponse by additive effect. In two other species X. m (Figure 9-6b) and H. mm (Figure 9-6c) addition of optimum amount of the synergist 28,3R,7R-A in the former species and 28,3R,7R-A and 28,3R,7S-A in the latter species to 28,33,7S-A increased the maximum response. The response of the mixture at ratio 1:0.002 and 1:0.006 in n. m was greater than that of 28,38,7S-A alone at all the doses tested. Similar observations were made for the mixtures over 28,38,7S-A alone in n. uinetum at all the doses tested. The Optimum combination of 28,38,7S-A / 28,3R,7R-A and 28,38,7S-A / 28,3R,7S-A was 1:2 as determined by field test, the result of which is published elsewhere. It should be noted that the synergists by themselves elicited low EAG .response. Using the TLC fractions of the natural body extract of female h1- sguifer and n. nunulua nunuluu: the antennae of male n. M; were stimulated with these preparations singly or in combination with major pheromone isomer of n. W. The EAG response to the fractions from n. annulus ' annulus and n. W was almost identical (Figure 9-7). The acid, alcohol, aldehyde, and ester fractions from n. annulus annulus, did not elicit any response on the antennae of n. afinim. Only the hydrocarbon fraction showed about 10% response relative to the response produced by 25,38,7S-A Figure 9-7. EAG response of male M. 5:11:12; to the TLC fractions of the body extract from female n. nanulus nanulus and n. ssrtifer. 175 00000000 E E + 25. 3S , 751mm,.) 0. + 00000000000000000000000000 00000 0 O C O O O O C O O O O C O O C .0 O O O Q 0 O O C O O O O O C O O O O O O O O O . . I O O Q C C Q C O O O O O O O O O .0 ‘ .‘ oddoloo 660. 0.00: + + I ..... ... . .......................».......0.0.. ...o. 0...0.0.0.0.0 0. 0 .. 0. o. o . . o 0 . o 0 00 0 0 0 .0 e. 0. 0 .0..0 I (0 151-0) 00000 I = -l $.E. — !.udifer m acid alco. ddo- esterHC 8.15.750 - 0 b P b m w m m m 3:2—3“ .5. a: 176 isomer. The ester and hydrocarbon fractions from female u. sertifier produced some responses. All these fractions when added to the 28.38.7S-A isomer proved inhibitory. The acid, aldehyde and ester fractions from u. sentifgr were more inhibitory than those from n. nanuluu nanulua. One observa- tion made three out of six times the stimulations were made was the fact that the hydrocarbon fraction increased re- covery time of the depolarization from 0.4 m sec to 1.0 m sec, however, this fraction did not increase the amplitude. DISCUSSION Two interpretations have commonly been given for the diphasic (positive and negative) impulses produced by chemi- cal stimulation of antannae as in Figure 9-1 a a b. One is that the positive (hyperpolarization) and negativer(depolar- ization) phases of the impulses have independent origins. The positive phase is generated on or near the cell body and spreads in both directions along the axon and back up along the dendrite towards the constriction at the base of the hair. When the impulse passes through the constricting space and enters the lumen of the hair. the opposite situa- tion prevails and the impulse appears negative. The transi- tion from the positive to the negative phase is very rapid, reflecting the short time that the impulse takes to pass the small distance of the constriction (Wolbarsht and Hanson, 1965). This interpretation precludes the presence of two or more specific receptor types existing on each or most of the 177 neurons which is the second interpretation proposed by Pfaffman (1959) and Boeckh (1967). The latter investigator also proposed that receptor cell is capable of responding either by excitation or by inhibition depending upon the qualitative nature of the stimulus. However, for this in- terpretation to be valid, single sensillum recordings will be necessary. The interpretation of Pfaffman (1959) and Boeckh (1967) appeared more attractive in the present inves- tigation. It is suspected that hyperpolarization.was in- duced by one or some of enantiomers of the diastereomers used in this study. With regards to optical purity. the critical carbon center prone to contamination is the 3- carbon, in*which case with 23,3R,7R isomer, there could be contamination of 3waith.3sx and in 28.38.75 isomer, there could be contamination of 38 with 3R due to epimerization in the Wittig reaction during synthesis (Matsumura et al. 1979). Fortunately, the erythro isomers (i.e., 28,38 and 2R,3R) can be separated from the threo»(ime., 28,3R, and 212.33) isomers by the GLC using D35 and carbowax 20 11 columns. The details of this separation is described else- where. When the GLC peak of the optical isomers were frac- tionated into the front and back, the ideal behind this was that the back fraction should elicit greater EAG response if indeed 28,3R,7R-P is the pheromone of 12. 31min (Kikukawa et al. 1982a). The results show that 28,3R,7R-P GLC back was indeed better than 28,3R,7R-P GLC front but not signifi- cantly. This was a circumstantial evidence to suggest that 178 the receptors on the antennae of n. similis and probably the other diprionid sawflies are keyed to the same compound. Here, the enantiomers were acting as competitive blockers at the receptor cells. Also, as these results show enantio- meric interaction at the receptor cells, this is another circumstantial evidence that in these species a multicom- ponent pheromone is precluded. Differential response of male sawflies to different Optical isomers of sex pheromone is not new (Kraemer et a1. 1979, 1981 and 1983). However, it is novel that males respond to natural sex pheromone of other species of sawfly. It is also reported here for the first time that diprionid sawfly females respond to synthetic sex pheromone and that such response in most cases is mainly to the enantiomers of major isomers of the male counterpart. The interpretation of this observation is not clear at present. However, one implication of this female enantiomeric specific response could be that perhaps males also do produce long or close range pheromone or aphrodisiac which may be optically active and chemically related to the female produced sex pheromone. This needs to be investigated in view of presence of abdomi- nal intertergal gland in the male n. “mfg; (Hallberg and Lofqvist, 1981). It is also not known whether female BAG responses could translate to behavioral responses. On a few occasions. few female 11. m were caught in traps baited with 28.38.7541 / 28,3R,7R-OH; 28.38.7841 / 28,3R,7S- A and 28.38.7841 / 28,3R,7R-A at various combinations. One 179 other observation in this study is the superior response obtained from stimulation of the female antennae by natural pheromone of different species. Also, when two or more female are confined, they always engage in fights which often result in breaking of legs. Such fights are observed in 20 ml to 4 liter rearing jars and fights occur irrespec- tive of species. The strong response to other species pheromone by the females could therefore be interpreted to mean that perhaps a means of avoiding breeding sites of sympatric species or signal for dispersal is by chemical means. This may be worthy Of future pursuit. Using submicrogram concentration of Optical isomers, synergistic response to mixtures Of Optical isomers is meas- urable. This raises the question of how did such synergism occur with mixing of optical isomers. There could be four possibilities. One, the synergist isomer may lower the response threshold but this does not appear to be the case as response curves of 28,38,7S-A alone and the mixtures tended to rise at about the same concentration (Figure 9-6 b a c). Two, there could be no quantitative differences on EAG but qualitative recognition of Optical isomers may be the key. This possibility does not appear attractive at present. Three, the synergist isomer may act to increase the maximum response of the major isomer. This possibility seems to be part of the answer as evidenced in Figure 9-6. Since most tests were done with such high concentrations (Figure 9-2, 3 a 4), they also measure the maximum response. 180 Therefore, these Observations agree with those in Figure 9- 6, that the maximum response is important. The fourth possibility is that the synergist isomer may provide additive effect to the major isomer. Our data also support this possibility because synergists by themselves show little activity. It can therefore be concluded that the ' synergists increased the maximum response by additive effect with the main isomer. REFERENCES Boeckh, J. 1968. Odor specificity and reaction range of a single Olfactory receptor cell. In Theories of Odor and Odor Measurement, N. N. Tanyolac (Ed.), Circa Publication Inc., Pelham, New York. Hallberg, E. and J. Lofqvist. 1981. Morphology and ultrastructure of an intertergal pheromone gland in the abdomen of the pine sawfly Hendiprion agrtifer (Insecta, Hymenoptera): a potential source of sex pheromones. Can. J. 2001., 59: 47-53. Jewett, D. M., F. Matsumura and H. C. Coppel. 1976. Sex pheromone specificity in the pine sawflies: interchange of acid moieties in an ester. Science, 192: 51-53. Jewett, D. M., F. Matsumura and H. C. Coppel. 1977. A single system for recording electroantennograms for male pine sawflies. J. Electrophysiol. Tech. 5: 24-28. Kikukawa, T., F. Matsumura, M. Kraemer, a. C. Coppel and A. Tai. 1982a. Field attractiveness of chirally defined synthetic attractants to males of Dipriun,similia and Gilninia frutstnrum. J. Chem. Ecol., 8: 301-314. Kikukawa, T., M. Imaida and A. Tai. 1982b. Synthesis of the sex attractant of pine sawflies (Diprion species): (28,3R,7R) and (28,311.78)-3,7-dimethylpentadecan-Z-ol. Chemistry Lett., 1982: 1799-1802. Kikukawa, T., F. Matsumura, J. Olaifa, M. Kraemer, H. C. Coppel and A. Tai. 1983. The nature of the major sex pheromone of the European pine sawfly W m- J. Chem. ECOlop 93 673-6930 Kraemer, M., H. (C. Coppel, F. Matsumura, T. Kikukawa, and K. Mori. 1979. Field responses of the white pine sawfly. Neodipgign pinetum to optical isomers of sawfly sex pheromones. Environ. Entomol., 8: 519-520. Kraemer, H. E., B. C. Coppel, F. Matsumura, R. C. Wilkinson and T. Kikukawa. 1981. Field and electroantennogram responses of the red-headed pine sawfly, Heudipuign leguntgi (Fitch) to optical isomers of sawfly sex pheromones. J. Chem. Ecol., 7: 1063-1071. 181 182 Kraemer, H. E., H. C. Coppel, T. Kikukawa, F. Matsumura, H. A. Thomas, L. C. Thompson and K. Mori. 1983. Field and electroantennogram responses to sex pheromone optical isomers by four fall-flying sawfly species (Hymenoptera: Diprionidae, Neodiprion). Environ. Entomol., 12 (5): 1592-1596. Matsumura, F., A. Tai, H. C. Coppel and M. Imaida. 1979. Chiral specificity of the sex pheromone of the red- headed pine sawfly. Neodiprion lfisuntai. J. Chem. Ecol., 5: 237-249. Mori, K., S. Tamada and M. Matsui. 1978. Stereocontrol led synthesis of all of the four possible stereoisomers of erythro 3,7-dimethy1pentadecan-Z-yl acetate and propionate, the sex pheromone of pine sawflies. Tetrahedron lett. No. 10: 901-904. Oakley, B. and R. Schaffer. 1982. Experimental neurobiology: A laboratory manual. The University of Michigan Press, Ann Arbor, p. 367. Olaifa, J. I., T. Kikukawa, F. Matsumura and H. C. Coppel. 1984. Response of male jack pine sawfly, Nggdipnign_ pratti hanksianae (Hymenoptera: Diprionidae) to mixtures of optical isomers of the sex pheromone 3,7- dimethylpentadecan-Z-yl acetate. Environ. Entomol. (In press). Pfaffman, C. 1959. The sense of taste. In Handbook of Physiology: Neurophysiology. J. Field, 8. W. Hagoun and V. E. Hall (eds.), American Physiological Society, Washington, Sec. 1, p. 507. Roelofs, W. 1976. The scope and limitations of the electroantennogram technique in identifying pheromone components in crop protection agents. N. R. McFarlane (ed.), Academic Press Inc. (London) Ltd., pp. 147-165. Tai, A., M. Imaida, T. Oda, and H. Watanabe. 1978. Synthesis of optically active common precursor of sex pheromone of pine sawflies. Chem Lett., 1978: 61. Visser, J.EL 1979. Electroantennogram responses of the Colorado beetle. W decemlineata to plant volatiles. Ent. Expt. et. Appl., 25: 86-97. Wolbarsht, M. L. and F. E. Hanson. 1965. Electrical activity in the chemoreceptors of the blowfly III Dendritic action potentials. J. Gen. Physiol., 48: 673-683. CHAPTER TEN Behavioral Response to Optical Isomers Of Sex Pheromone and Development of Non-Saturating Trap for Diprionid Sawflies INTRODUCTION Courtship behavior is believed to have been conditioned by selection pressure to maintain species integrity in in- sects (Grant et a1. 1975). In the lepidopterous species where a lot of work has been done in this regard, courtship behavior is found to be species specific and chemical stimu- li plus other stimuli (visual and tactile) in some cases were necessary to initiate these stereotype behavior (Shorey and Gasteon, 1970; Carde et a1. 1975: Carde 1981: Grant 1981: and Teal et a1. 1981). Studies on courtship behavior of pine sawflies have been virtually'neglected. Researchers on sawfly have directed much attention to the biotaxonomic studies and recently to the chemical elucidation of the female sex pheromone. Chemical behavior can contribute to the yet unsolved taxonomy of sawflies and can shed more light to the role of interacting isomers of the sawfly pheromone. Coppel and Benjamin (1965) reported that males approach the females in zigzag pattern with forward pro- gress, gradually approaching to within about 1 fts, and then it appears that a visual stimulus becomes predominant. They 183 184 also reported end-to-end mating position and a mating period of 30 min. or more. Since their report represented the state of art in courtship behavior of pine sawflies, it became necessary as part of this study to prepare a full descriptive courtship behavior of three species of pine sawfly N. W. N. W: and D.- similis based on direct Observation on the field. This study also tested visual stimuli and various fractions of the female natural extract as they affect courtship behavior. Thomas 1983, evaluated three types of traps and two types of pheromone dispensers for the introduced pine saw- fly, D. similis. The author found that larger sticky traps and cigarette filter baited traps caught more males than the Pherocon II traps and the dental roll baited traps. Another important component Of traps is the trap placement on the field. For example, experience with many species of sawfly in this study showed that traps placed on the host plant in the Open always caught more males than traps placed in a closed canopy. Another aspect of trap placement is the height above the ground. In a field trapping experiment against the webspinning larch sawfly W 13112191111: Wacht utilizing vertical board traps affixed at heights of 0-0.5, l, 2 and 4m to four trees 20m apart, Borden et al. (1978) found that traps placed at 0-0.5m performed better than other heights by a factor of about seven. The use of sex attractant baited traps has become a conventional method of monitoring population trends and 185 estimating population density of many lepidopterous pests and bark beetles (Ryan and Molyneux, 1981; Vite 1970: Cards, 1979 and Sanders, 1981). Such use of traps for pine sawfly is still in its infancy due to recent development of sex attractants for this group of insects. ‘The experience we have gained over the years on the use of sticky traps Phero- con II for testing pine sawfly sex pheromone has led to the realization of inherent disadvantages Of using sticky traps for sawfly species. 1First, sticky traps are saturated by target and non-target species and this introduced errors into estimating population density or effectiveness Of at- tractants used. The flower of pine trees are wind polli- nated therefore sticky traps set out on the field for summer flying sawflies are often heavily saturated by debris of pollens and abborted flowers, but this problem is hardly noticeable in the fall season or in white pine stands which do not flower heavily like the jack pine. Second, trapped males are Often mired by the sticky material to the extent that to remove them for further investigations without los- ing the leg, wings or antennae becomes almost impossible. Our experience in cleaning the insects removed from sticky traps also showed that only benzene could thoroughly remove the sticky substance but that means dealing with a carcino- gen hazard. The third, and most pressing problem during the present investigations was the need to conduct a chemotaxo- nomic study in terms of pattern of cuticular hydrocarbon in pine sawfly species. Vegetable wax constituted one of the 186 major ingredients of the sticky substance in Pherocon II traps (Zoecon CON, personal communication) therefore, males removed from sticky traps could not be used for such studies because of similarities of the waxes from plants and insects (Kolattukudy, 1976). There was therefore, a need for the develOpment of nonadhesive traps which would preserve trapped males for chemotaxonomic investigations in view Of chaos in sawfly taxonomy which has not yet been resolved. Such traps eventually may have to replace conventional sticky traps for field population studies on summer species of sawfly in view of aforementioned problems. The present study also describes efforts at developing a cost effective non-sticky traps with evaluation concentrating on number of male sawfly trapped, the durability of the traps and their ability to exclude non-target insects and debris. MATERIALS AND METHODS WWW Tests were conducted in the middle of a pine stand for the species concerned. Test dates were chosen such that the weather was bright with low winds. A 40 ug pheromone in 1 m1 hexane was used in all the species tested to attract the male sawflies to the vicinity of the test site. The phero- mone was dispensed in an open 7.5 cm glass petri dish: the petri dish was placed on top of a 1.5 m aluminum pipe pole erectedxl meter from.a pine stand. Sometimes 2 or 3 such petri dishes were used in a single experiment. As soon as 187 up to 15 males are sighted, the petri dishes are covered and removed into an air tight polythene bag. The open source is replaced with a point source chemical stimuli either from a rubber septum decoy cut approximately to the size of a female sawfly or a pinned live female or a pheromone treated solvent-washed dry female sawfly pinned onto a mating cage. The mating cage (15 x 15 x 15 cm) with one side Open was made from aluminum mesh and painted green. The paint at some areas of the top of the cage was thick to allow anchor- age for the pinned decoys. The cage was put on a 18 x 18 cm ground glass plate on the top of the aluminum pole. The account of the courtship behavior on the field was recorded from the time the male was first sighted approaching the source for N. m. N. umninnua, and D.- aimilia. For completeness, the pre-copulatory and post-copulatory be- haviors of N. sentifiuz were recorded in a set-up in the greenhouse where males were kept in a giant screen cage (2.4 m x 2.4 m x 2.4 m) made with a plastic mesh. A young potted scotch pine 2 m tall placed at the center inside the cage served as the vertical silhouette where the males rested. Also recorded in this set-up was another experiment testing the effect of the acid, aldehyde, and parafiin TLC fractions of the natural extracts form female N. seztliez. The point source pheromone was presented as in the field. To test the effect of color on the response of N. W and N. anal: lug annulus, the ground glass plates on which the cages stood were painted white, gray, black, yellow, orange, red, 188 green, and blue colors. These colors served as a background to the cage. Different screen sizes and colors of cages were also tested against N. annulus annulus. The effect of color on N. aunulua nunulua and N. agntifg; was further tested on the field test using sticky traps Pherocon II painted black, blue, green, gray, orange, red, white and yellow. W In order to test the importance of trap height in response of male sawflies to pheromone, five heights 1.5, 2.5, 3.5, 5.5, and 8.0 m were chosen. The choice of heights was based on the fact that the ground cover, mainly grasses, were of height, 1.5 m in N. 33.21113; trapping site in Rose Lake, Michigan: ground cover at banksianag trapping site at Hartwick Pines, Michigan was generally, 1 m. The height 2.5 m represented the height by which most trapping had been done in this study. The 8 m represented the height of > 90% of the scotch pine and jack pine stands on which this study was carried out. The heights 3.5 m, and 5.5 m were randomly’ picked. The sawfly species tested were N. am and N. annual banksinnag because of formerfls abundance and its important ancestral relationship to many species (Ross 1955) and latter's notoriety for attacking the trees Of all sizes (Wilson, 1970). For N. sertifer. the trap heights were simultaneously tested on both the host plant and angle steel pole erected first at 12 m away from the nearest host plant 189 and then 3 m away after one week at the first location. Trapping lasted two weeks with one week each for each dis- tance of angle steel pole from the host plant. The Pherocon II traps baited with 20 ug Of 28,38,7S-A / 28,3R,7R-A di- prionol mixture (5:0.003) was used at each height. At the end of the first week, the number of males trapped at dif- ferent heights on both the angle steel pole and scotch pines was counted. There were three replicates for each treat- ment. After then the poles were moved to 3 m from the host plants. Trap heights were also simultaneously tested on both the host plant, the jack pine, Pinua_anakaianag_Lamb and the angle steel pole erected 3 m from the host plant. Duration of study was 11 days and Pherocon II traps baited with 20 ug of 28,35,7S-A / 28,3R,7R-A diprionol mixture (5:1) were used. manna There were 4 traps of our design which were compared with three other traps--Pherocon IC and Pherocon II, Zoecon Corp. Palo Alto, California, and the covered funnel trap CFT developed by Ramsawamy and Carde (1982). The Pherocon IC trap was also modified in this study by replacing the base with non-sticky top. Thus, only one trap, the Pherocon II trap was sticky. The 4 new designs are described in Figure 10-1 and Table 10-1 gives a detailed description of all the traps tested. Three of the designs (DFT, IPT, ICT) were expOsed in two ways and are such that will generate an elongated plume Figure 10-1. Trap designs for sawflies (all measurements in cm.). (a) Pherocon on cup trap (POC) (b) Double funnel trap (DFT) (c) Inverted pyramid trap (IPT) (d) Inverted cup trap (ICT) , .1 "D 1 l 190 1 (b) OFT 191 Descriptive data for trap designs Entry space Weight Model Material (sq. cm) Color (9) POC Cover - coated card- 250 cm2 near white 118 board top and Funnel - plastic sky blue Cup - plastic bottom Polyethylene funnel Plastic body DFT Polyethylene funnel 15 cm2 clear 226 Plastic body IPT Cardboard body stapled 80 cm2 white 45 together with duct tape side base of entry hole ICT 3202 plastic drinking 18 cm2 sky blue 52 cup Pher- Coated cardboard 250 cm2 near white 95 ocon IC Pher- Coated cardboard 96 cm2 near white 31 ocon II CFT Polyethylene funnel 70 cm2 near white 52 with plastic dish cover cover, clear bottom 192 with a visual tracer (Lewis and Macaulay, 1976). Such design might be more relevant for pine sawfly whose males are great fliers (Coppel et a1. 1960). The POC opens round and produced erratic plume of a visual tracer (Lewis and Macaulay, 1976). Perhaps sawfly males would take advantage7 of these characteristics because a more erratic odor plume is more likely to be easily detected by males presumed to be flying at random. Each Of the traps was baited with 20 ug of pheromone applied as a 1 ml aliquot in hexane onto a 4 cm cotton dental wick. The wick was held with a paper clip inside a cylindrical aluminum screen (4 cm x 2.5 cm diam» eter), attached to the center room inside the IPT and ICT. No screens were used for POC and DFT but, in all cases, the wick was held with a clip and was aligned with the openings. Dichlorvos strip, Raid , 36 sq. cm was used as the killing agent. Two Nnndinrlna species N. W and N. annulus annulus were trapped with the different designs at Sharon and Vogel Center, Michigan respectively. RESULTS Wars Open sources of pheromone attracted more males N. nnnu: lus annulus to land than a point source (Table 10-2). Bow-_ ever, all the sources appeared to attract the males to their vicinity. The open source (petri dish) when placed on the mating table covered with grasses attracted more males to land. In another experiment involving two species and 193 Table 10-2. Effect of different types of pheromone sources on response of wild N. annulun nnnulun in the field within 40 min. observation period with 28,38,7S-A (10 ug). Pheromone sources # malesb 4 Malesa landing on visiting source Point source (head of pin) Watch glass (5 cm diam.) Glass petri dish (7.5 cm diam.) Glass petri dish (7.5 diam.) 1;!“ painted green Glass petri dish (7.5 cm d on mating table (0.75 m covered with grasses 3 O 4 1 2 2 4 2 4 5 a Males visiting were those that made some circular flight around the source. b Males landing were those that actually landed on source. 194 grasses and host pine needles compared as background, the redpine needles attracted more males of N. annulus annulus to land than grass or plain glass background alone (Table 10-3). The males of N. 5311:1121. however, did not exhibit any preference and were equally attracted to the 3 back- grounds. Also, in another experiment with the former spe- cies, the effects of other types of background for pheromone source (Table 10-4) were tested. The mating table covered with grass and green ground grass plates attracted more males to land. Even though transparent glass had more males visiting but could not attract equally high landing of males. The legs of the male sawfly are adapted for walking on pine needles. Their overly long and folding tarsi makes this possible but at the same time makes them a poor walker on flat surfaces. It therefore became necessary to develop a surface on which the males could land and walk easily to the pheromone source in order to gain useful knowledge on how males approach the pheromone source. Wire nettings were considered apprOpriate for this purpose and different types were tested against N. annulus annulus (Table 10-5). Cross- ing aluminum 0.5 cm mesh green color attracted more males to land and was as good as transparent glass in the number of visiting males. This aluminum mesh was therefore made into the mating cage which was used in all the courtship behavior studies Of N. annulus annulus. N. m and D.- mun. Background color did not appear to be important to N. nestling when 8 different background colors were compared 195 Table 10-3. Effect of grass leaves and host pine needles* as background for the pheromone source** on males of N. sestifnr and N. annulus annulus on the field. Test conducted for 60 minutes at Rose Lake and 30 minutes at Vogel Center respectively for N. annular. and N. annulus annulus # Visiting _nad_lnndias__ NO N. n. N. N. n. Nature of background sertifer nanulus sertifer nanulus Ground glass only A 0 2 6 0 B 5 3 6 0 Ground glass covered with grass A 6 3 4 8 B 11 3 4 0 Ground glass covered with host pine needles A 8 7 2 13 B 4 8 7 10 * Scotch pine needles for N. snatlfnn red pine needles for N. a... annulus. ** Each source in petri dish, 10 ug 28,38,7S—A/25,3R,7R-A 5:0.003 for N. snutlfinLJ 10 ug 28,38,7S-A for N. a. annulus. 196 Table 10-4. lEffect of the nature of background object on wild N. annulus annulus males when the pheromone source was glass petri dish. Test conducted for 50 minutes with 10 ug 28,38,7S-A. 4 visiting Source background # visiting and landing Transparent glass 12 4 Mating table covered with grass 2 15 Metal plate 6 5 Green ground glass plate 3 7 Translucent ground glass plate 3 2 197 Table 10—5. .Effect Of mesh size of the mating cage on the response of wild N. annulus annulus males to 10 Observation was made for 60 ug 28,35,7S-A. minutes. # visiting Mesh type # visiting and landing Non-crossing steel 2 cm mesh A 8 8 B - - Crossing aluminum 1 cm mesh A l 8 B - - Crossing aluminum 0.5 cm mesh gray color A 2 4 B l 4 Crossing aluminum 0.5 cm mesh green color A 4 12 B 12 10 Crossing aluminum 0.5 cm mesh brown color A 4 10 B 2 4 NO cage (transparent glass) A 5 4 B 11 6 198 (Table 10-6). There was no clear cut differences among the number of male visiting or landing with all the background colors tested. Each background color was made of a ground glass plate (18 x 18 cm). However, when sticky traps with different colors were compared for N. snrtifn;_(Table 10-7), traps with the blue, green, orange, white and yellow colors significantly attracted more males. Yellow traps with no pheromone did not catch any males. The red color caught the least number of males. However, for N. annulus annulus, there was no significant difference among catches with dif- ferent colors, but the colors with the highest catches were yellow, green, and white. The results in Table 10-8 show that pheromone source placed on mating cage at the height of 15 m attracted more males of N. ssntlfn; when source was 1 m away from the nearest distal end of scotch pine branch. Fifteen to twenty year old stands were used. In the court- ship behavior studies, the mating cage was therefore placed 1 m from the host plant. It was also found that males of N. snnnlfnn on the average spent the longest time with the source when parafin fraction of body extract of virgin females was added to the pheromone source (Figure 10-2). The aldehyde fraction by itself showed a little activity but the number of males tested was small therefore no definite conclusion can be made in the regard. NW The first male was sighted about 5 minutes after the petri dish containing pheromone was Opened at the test site 199 Table 10-6. Effect of background color of pheromone source on the response of wild N..snntl£nu males on the field. Each treatment contained 10 ug 28,38,7S-A/28,3R,7R-A (5:0.003) and observation lasted 80 minutes. t visiting Background color 4 visiting and landing White 4 2 Gray 5 3 Black 4 3 Yellow 4 2 Orange 5 4 Red 5 4 Green 1 2 Blue 2 3 Table 10-7 . 200 Field response of male N. sentifnr. and N. annu: lus annulus to different colors of sticky trap Pherocon II baited with sex pheromone. Test for N. snnnifns conducted at Rose Lake (October 2-5, 1982) and for N. annulus annulus at VOgel Center, Michigan (September 25-October 16, 1982). Three replicates randomized 3 times. W1 3431* Trap color N. anztlinz** N. annulus.anaulus*** Black 24.3 i 1.0ID 09.0 1 4.53 Blue 32.7 i 2.9a 75.3 i 3.09 Green 37.7 a: 2.63 122.3 1 3.9° Red 23.3 1 2.39 104.7 1; 2.743‘ White 53.0 :1: 2.13 115.0 1 3.9a Yellow 37.0 a: 2.5‘31 152.3 1 0.0a Gray 24.3 a: 1.0b *1" Orange 38.0 1: 1.9b 111* Yellow (blank) 0 1 0c 0 1 ob * Means followed by same letter not significantly differ- ent. ** Traps baited with 10 ug 28,38,7S-A/2S,3R,7R-A 5:0.003. *** Traps baited with 5 ug 28,38,7S-A. **** Inadvertently left out. Table 10-8. 201 Effect of distance of synthetic pheromone source used for courtship behavior of male N. snrnlfnr. on the field from the nearest scotch pine stand. Test conducted at Rose Lake, Mich- igan, 10/8-10/15, 1982 and at Pinckney, Michi- gan 10/16/82. Pheromone source was 40 ug 5:0.003 28,38,7S-A/28,3R,7R-A mixture. Pheromone place- x males Observed x males that landed meat from nearest landing on nearest on mating cage pine tree (m) scotch pine within 30 min.* 0.1 6.3 1 0.0 0.5 1 0.4b 0.5 6.8 1 0.6 1.5 1 0.6b 1.0 7.3 1 0.6 5.0 1 0.6“El 5.0 6.5 1 0.5 0.5 1 0.4b * Means followed by same letter not significantly different at 5% level by Duncan's new multiple range test. Figure 10-2. Average time (secs.) male spent within circle 2 cm. radius (L) or time male spent with dead N. ssntifnn female treated with chemicals by climbing up the pin holding the specimen. 202 Time (sec-) 4 8 12 16 20 ' I t I I L ************¢*(Is’) Phermne c -------(.=‘) . L Paraffin C . L Ac I d C L ial: It (I=2) Aldehyde C Pheromone L * * * “'36) Pata'fln 0 -----------------(I85) 2.03 in Sharon, Michigan. The male at first made a fast circular flight around the upper halfof the tree nearest to the cage holding the decoy. After about one or two such turns, the male made a vertical flight down and landed on the pine branch about 1 m away from the decoy. Wing fanning started immediately after landing while the insect walked from the base to the tip of each needle. Up to 10 needles were searched. One male out of 10 even walked around the apex Of the jackpine flower. Also, abdominal tip was raised inter- mittently for about 1 to 5 secs. The males were observed engaged in short flights to land on the cage. There were two patterns of short flights: the first of the two was zigzag and only about 60-66 cm in length, the second type was 30-36 cm and was direct, precise on the target (septum or pheromone treated solvent washed N. annulus annulus fe- male) and with an apparent increase in speed. After land- ing, ambulation in search of the pheromone source followed. Copulation attempts with the source started within 5-10 secs (x - 5.9 1; 2.5 secs S.D.: n - 10) upon landing on either the septum or the dry N. annulus annulus female. The male could find the pheromone source within 30 sec if landed on the cage. Copulation attempts with septum on the average lasted only 2.62 1, 0.83 mins. S.D. Mating with the female took a different form from the septum's in that the male attempted mating with every part of the female. The frons, cervix of neck, underside abdomen, abdominal terga, sides of abdomen and the abdominal tip. Mating with female lasted 32.3 min. 204 ;|-_ 9.7 min. S.D. During copulation attempts, there was intermittent wing fanning, abdominal stroking, leg stroking, but no rear leg holding. Two males were observed to leak pheromone vials with their mouth parts. After copulation, the male walked down the insect pin holding the decoy. The male walked about 5 cm away from the decoy before making about 1 m vertical flight onto the canopy of pine tree. There post copulatory activities continued with cleaning of lst the legs, then the antennae and lastly the wings. INO more wing fanning this time and after the cleaning the male remained motionless for about 2 minutes before flying away. Where two males making ambulation met, they fought momentar- ily before each continued its searching. One Of them often took Off immediately after that. W “Weld The first male arriving is usually sighted about 510 away from the source flying a zigzag pattern likely in a positive anemotactic flight towards the source. The male landed on pine branch about 1 m away from source. The wings were spread, and fanning and abdominal tip raised. Then the male made some 2 or 3 short flights from on twig to the other. It raced up and down the pine needles, usually walked from base to about 2/3 up each needle. Sometimes the male jumped to another needle from there or traced its path down in a sort of reversing ambulation. .All along, wing fanning continued until the source is discovered. .Source 205 was discovered after about 2 minutes when the source was a pheromone treated rubber septum. The male then climbed the pin holding the septum to the top and remained on it for about 20 secs, motionless, no mating. All the 10 males Observed showed this arrestment. Two males also walked all around the septum i.e., top, side, underside Of the septum. There was no copulation Of the septum but a momentary copu- lation was observed when the pheromone was a mixture of 2S,3R,7R-P nd 2R,3R,7S-P isomers in ratio 40:0.1. The pher- omone used for this study was 28,3R,7R-P. However, when a live female was used (female pinned with #3 minuten insect pins to the cage), or a dead, solvent washed female that is treated with pheromone, the male climbed the pin onto the female and quickly positioned himself rear to rear with the female (Figure 10-3). The positioning was completed with 3 secs. Following this was a series of tactile movements. First, the male made 3 to 4 strokes on the lateral side of the abdomen with its rear legs. This was followed by leg strokes, the two meta- thoracic legs of both male and female were involved in this leg stroking. The male using its metathoracic legs to stroke from femoro-tibial joint to as far down as tarsus of the female rear legs. This was supposed to acquiesce the female as the female remained motionless upon stroking. The stroking continued intermittently throughout the copulation period which lasted on the average 9 minutes 29 secs 1 4.4 secs SJL). Occasionally during copulation when the female Figure 10-3. Copulation in D. slmllls. The male is COpu- lating a restrained live female at the top picture; copulation below is with a dead sol- vent washed female that is pheromone treated. Note that in both pictures, male did not copu- late with the pheromone treated septum at the background. However the male climbed the septum first. 206 207 showed some movement, the male fanned wings, followed by abdominal stroking, followed by leg stroking. Where the female was motionless, no stroking occurred but the male and female rear legs were touching. Immediately after copula- tion, the male disengaged himself and flew away rapidly. It was observed that there was no antennal touch during copula- tion and the wings remained wide Open and motionless during this time. There was wing fanning when copulation proved difficult. The pro and mesothoracic legs of the couple were essentially used for support. COURTSHIP BEHAVIOR 0? MALE N. m EresaaulntQL¥_Bshnxinr In the greenhouse where the precopulatory behavior was studied the latency period was about 10 secs. before a quiescent male made a quick turn of 180° facing the direc- tion of the source. Antennae movement followed and then a brief rubbing of the back with metathoracic legs. About 1 min. after the pheromone was present, the male started to move from its resting site on the scotch pine twig towards the source. Having covered the whole length Of a pine needle, the male took a somewhat zigzag flight towards the source. On the field, the male was first sighted when making a circular flight at the upper half of the vertical silhouette closest to source which was always a scotch pine about 3-4 meters in height. The circular flight was followed by a 208 vertical flight downwards and landing about 1 m on the pine twig away form the source. Like N. ulnulalnaus, the N. ssnnlfsn searched the pine needles around it by making a series of reversing ambulation covering the entire length of pine needles on the twig. A short flight, slow but zigzag always took the male to the cage, the center top of which was holding the pheromone source--the septum (SP), washed dry N. ssntlfsn female treated with synthetic pheromone (W3 P) or a N. snntlfnn female, unwashed but dry (0W2. ). What- ever the source and whether field or greenhouse, approach to the source upon landing on the cage was the same. This involved a zigzag ambulation with intermittent wing fanning every 2-3 secs and abdominal tip raising and an occasional attempt to copulate the cage. It took about 1 min to dis- cover the source when the source was synthetic pheromone i.e., SP and WEP, but only 30 secs when the source was natural pheromone awn . mummies Even though male N. snutlfinr. would climb SP and W92, only momentary copulatory attempts were made on these de- coys. However, when the decoy was an UWQ, , there was copu- lation which lasted an average 18.2 min. with about 4 breaks. The longest copulation time at a stretch observed was 10 minutes. The males Often mated several times. How- ever, it is not known whether multiple mating is a common behavior of live females. Certain stereotyped male be- haviors were clear from this study. Upon climbing the pin “—— 209 holding 0W3. , the male first made a wing stroking of the female with his forelegs. Then the male quickly dropped into a rear-to-rear position making genital contact. The male stroked the abdomen with its rear legs: this was fol- lowed by leg stroking of the female rear legs by the male's and the male held the female firmly with his mesolegs. Probably another series of stereotyped behavior was gener- ated in the mating male after about 5 min. copulation. The male then copulated the dorsum of abdomen of the female momentarily and stroking the wings of the female with his rear legs at the same time. It quickly changed to rear-to- rear position making genital contact again and mated for varying lengths of time from 1 min. to 10 min. This inter- change of genital mating and the dorsal abdominal mating went on for 3 or 4 times. Another Observation with other males was that instead of dorsal abdominal mating some males used the lateral side of female abdomen to interchange with normal rear-to-rear genital mating. 'There was no mixture of dorsal abdominal with lateral abdominal mating in a single male as Observed in N. Mainnus. The abdomen of the male is bent inward for male to assume a WP shape in carrying out this dorsal abdominal or lateral abdominal mating. Probing wings of the female with its prolegs however, was a common feature of the two styles. When another intruding male approached during copulation, the copulating male stopped mating to ward off the intruder, after which he came back to resume copulation. 210 2nst.£oaulntnl¥_nstluities After copulation, the male Often traced its way back the pin holding the female, then walked about 10 cm down to the side of the mating cage or sometimes to the surface of the ground glass on which the cage was placed. There was no more wing fanning, no more raised abdominal tipvthis time, the male halted on the ground glass, first cleaned its antennae with the aid of its forelegs for about 2 minutes. It spent another 2 minutes to clean its wings with its hind legs and lastly spent another 2 minutes to clean its anten- nae the second time before it walked a small distance and flew away. The courtship behavior of the male N. 11min; lnaus, D. slmllls and N. units; is summarized in Figure 10-40 Wt Traps placed at height 3.5 m on the host, the scotch pine Linus sylunstnls L. caught more males than those placed at 1.5, 2.5, 5.5 or 8.0 m (Table 10-9). Next to that was traps at 2.5 m, the commonly employed height of trap place- ment in diprionid sex pheromone research (Kikukawa et al. 1983). The number of males caught decreased with trap height on the host plant. These data also demonstrate that the optimum height of trapping on the host plant and artifi- cial poles placed the open grass field near the pine stand was not the same. Traps placed at.l.5 m above the ground on the angle steel caught significantly more males than traps at higher heights. 'The results also show that trap catch on Figure 10-4. Model of location of female and courtship behavior of wild male diprionid sawfly. 211 Quiescence or appetitive flight Orientate towards vertical silhouette Vertical flight upwards Circular flight around upper half of silhouette Vertical flight downwards Landing on silhouettes I Wing fanning: abdominal tip raised, reversing ambulation Positive consumatory flight to land on mating cage Zigzag ambulation: wing fanning: cage cOpulation attempts 4 Climb pheromone treated septum or virgin female Copulatory activities Wing 5 leg stroking Wing spread Rear-to-rear positioning Dorsal or lateral abdomen mating ! Post copulatory activities Fly away 212 Table 10-9. Effect of trap height on the response of male N. ssztlfnr to synthetic pheromone. Traps placed on scotch pine stands and artificial poles erected at 12 m or 3 m from the nearest host stands. Test conducted at Rose Lake, Lansing, Michigan, September 25-October 6, 1983. height ___Trnas_nu.tesl_nale*___ TraPB 011* (m) 12 m from host 3 m from host scotch pines 8.0 0.3 1 0.4b 0.3 1 0.4ID 24.0 1 3.2a 5.5 0.7 1 0.4b 0.7 1 0.4b 21.0 1 2.43 3.5 1.7 1 0.6b 2.3 1 0.6b 57.0 1 2.73 2.5 1.7 1 0.6b 3.3 1 0.7b 38.7 1 1.4a 1.5 8.3 1 1.1“ 9.0 1 1.2a 32.3 1 1.43 * Means of 3 replicates. Means followed by same letter not significantly different at 5% level by Duncan's new multiple range test. 213 the artificial pole decreased with height above the ground. There was more catch at heights 1.5, 2.5, and 3.5 10 when the artificial pole was nearer (3 m) the host plant. Table 10- 10 showed that trap catch Of male N. mill anakslnnns at heights 1.5, 2.5, and 3.5 m was significantly superior to catches at 5.5 m or 8.0 m in 1983. There was no significant difference among the mean trap catches at 1.5, 2.5 and 3.5 m and no catch at all on traps placed on artificial pole. Also in 1981, catches at these 3 heights were superior to traps at 5.5 and 8.0 10. W The result of this testing (Tableth-ll), showed that Pherocon II traps caught significantly more sawfly males than any of the other designs. The CFT and POC caught only 2 males while DFT caught only one. However, the funnels in two of the three replicates of DFT were lost as a result of high winds. Pherocon IC suffered the same fate from winds as in field trapping I. Pherocon II traps caught a total of 153 non-target species mainly dipterous and few lepidop- terous: the POC 15, CFT 20 and Pherocon IC zero. With regards to debris, Pherocon II topped other designs with tangled twigs, pine needles and flowers. Other designs just had traces of debris. In these two series, the two traps designed to collect males without contamination with the sticky substance, POC and DFT, did not appear to perform well. To continue research in this direction. we came up with two other designs, the IPT and ICT (Figure 10-1). Table 10-10. 214 Effect of trap height on the response Of male Nb Dznttl Dnnkslnnnn to pheromone. Traps were placed on jack pine, replicated 3 times at Hartwick Pines, Michigan in 1981 and 1983. Meaninntshltrny 3‘ SLE- Trap Traps on height .stsnl_anls__ (m) 9/26-10/16/81 10/7-10/18/83** 10/7-10/18/83 8.0 0.7 1 0.6 0.7 1 0.5ID 0 1 0 5.5 0.3 1 0.4 0.7 1 0.5b 0 1 0 3.5 1.0 1 0.8 3.3 1 0.43 o 1 0 2.5 1.3 1 0.7 2.3 1 0.4an o 1 0 1.5 1.3 1 0.7 2.3 1 0.6a 0 1 0 * ** Means of 3 replicates. Means followed by same letter not significantly differ- ent at 5% by Duncan's new multiple range test. 215 Table lO-ll. Field effectiveness of five trap designs against male N. unulnlnnus. Test conducted at Sharon, Michigan, June lO-July 15, 1983. Three replicates randomized three times. Mean catch/trap Design 1 S.E.* poc 0.7 1 0.6b DFT 0.3 1 0.4b CFT 0.7 1 0.6b Pherocon II 16.0 1 2.1a Pherocon IC ' 0 10b * Means followed by same letter not significantly different at 5% level by Duncan's new multiple range test. 216 MW Four trap designs, IPT, ICT, CFT, and Pherocon II were compared. The POC, DFT, and Pherocon IC which were ineffec- tive in earlier studies were drOpped. Each trap was baited with 20 ug purified 28,3R,7R-P. The purified preparations of this isomer was effective on the field. This was a one replicate experiment designed not only to test the efficacy of sawfly trapping of the designs but also to study the efficiency of each design and to study the behavior of the approach of sawfly into the traps. For all designs, the males landed less than 1 m from the pheromone source. With 2—3 short flights, the male landed on the pine needles closest to the entry space Of the traps. From there, the male walked towards the trap, then onto the trap and finally' entered the trap. In this trial, the ICT caught 2 males, Pherocon II 1, while the IPT, Pherocon IC and CFT did not catch any males. The 2 males trapped by ICT represented only about 16% of all the males that landed within the vicinity of the trap. The 1 male caught by the Pherocon II represented about 10%. The IPT and CFT trapped no males, even though 10-12 males hovered around and landed less than 1 m from these traps. The DDVP strip has a strong penetrat- ing odor which might have negative effect to the approach of male into the trap. It was also Observed that some males entered into the traps baited with DDVP strip and then escaped alive. This killing agent either needs to be more concentrated or be changed for a more effective one. 217 Increasing active ingredient of DDVP implied using a wider strip. Another approach was to find a means of either increasing the length of time the males have to stay inside the trap once it entered or to design the trap such that the vapor pressure concentration inside the trap is high enough to kill the male once it entered. This approach was pursued in succeeding series of field trapping that follows. TIna_aesnuuhnlh_xirslninaus nad.N.umunUmm_nnnulus The ICT with 9 sq cm entry space was the most effective among the 3 types of ICT tested against male N. ulxglainaus and N. annulus annulus. However, the insertion of pine twigs clearly improved the catch for ICT l8 and ICT 36 but not for ICT 9. The ICT with the best performance was about 19% and 15.4% as effective as the sticky Pherocon II trap reSPectively for N- uiruiainaus and N. annulus annulus. (Table 10—12). DISCUSSION Lnsntiaa_nad_Bssruitmsat_uf_n_Mnts The adult life of diprionid sawflies is specialized to propagate progeny. Adults hardly feed and do not diapause. In order to perform their reproductive function efficiently, the female has develOped a potent chemical stimuli and the male in turn has developed an equally elaborate olfactory system to perceive the chemical stimulus from the female. 218 Table 10-12. Field effectiveness Of Inverted Cup Trap (ICT) and Pherocon II traps against two species of pine sawfly. Test conducted for N. annulus annulus at Vogel Center, and for N. 1118112. lnnus at Sharon, Michigan. Three replicates randomized three times. :l'. S-E-* Model ghW 1}th ICT 9 4.7 1 10.09 5.3 1 0.7b 1cm 18 0.3 1 0.49 o 1 0'3 ICT 36 o 1 0ID o 1 0b ICT 9 + pine twig 4.3 1 0.7b 5.0 1 1.2b ICT 18 + pine twig 4.3 1 0.4b 5.7 1 1.7b ICT 36 + pine twig 1.0 1 0.6b 6.6 1 1.5" Pherocon II 25.0 1 3.0’1 41.0 1 2.9al * Means followed by same letter not significantly different at 5% level by Duncan's new multiple range test. 219 This chemical stimuli is the sex pheromone, acetate or propionate 3,7-dimethylpentadecan-2-ol (diprionol) (Jewett et a1. 1976). During calling, the pheromone is released from the exoglands at the intertergal of the abdomen. While doing this the female flaps the wings to drive the pheromone into the prevailing winds. 'This study shows that the males, having perceived the pheromone, obey certain rules or are stereotyped to certain behavior. First, response appeared tolbe dose related as open source attracted more males to visit and land. The implication of this is that a female which produces more pheromone is more likely to be favored with more males. Second, as Observed in the results of pheromone source parameters, the pine needles play some role in landing of males. Perhaps, volatiles from pine needles are involved in directing males to land on a suitable host plant. Color appeared not to influence response of males to pheromone probably because of the well-developed chemical communication system in these species. In a study of sexual activity of the gypsy moth on the field, Richerson et al. (1976) reported that all males oriented to vertical silhouettes such as trees, stumps, shrubs and upright boulders whether females are present or not. Using synthetic pheromone source, we also found this to be true to a great extent for male sawflies. The first response of males when pheromone is perceived is to move out from the thicket to the open. Evidence for this comes from the fact that pheromone traps set in the thicket or bole or 220 branch with a lot of overstories never catch.males; only traps set in the open do. In moving out of the thicket, the males fly just a few centimeters above the ground cover and orient towards a vertical silhouette. Evidence for this is seen in the significant catch of male N. snntlinu at height 1.5 m on artificial poles (Table 10-9) where ground covers were approximately 1 m in height. For sawflies, only the host plant appeared to be the major vertical silhouette to which the males orient for the location of the virgin fe- male. Evidence for this is also seen in the superior catch of male N. ssntlfnn and N. aunttl hnnkslnnnn at all the heights tested on the scotch and jack pines respectively compared to catch on the angle steel poles (Tables 10-9 and 10-10). This fact is also supported by the fact that trap catch on the artificial poles did not increase drastically whether the poles were near the host plants or far from them (Table 10-9). The third step in locating the female having approached the vertical silhouettes is for the male to make a vertical flight to cover the whole height of the sil- houette. This is similar to the situation in the gypsy moth where males are known to spend more time of their appetitive flight in tree oriented vertical flight (Elkinton and Carde, 1983). Circular flights, which are essentially cross wind flights to pinpoint the approximate location Of female on the host canopy, presumably starting from top and proceeding downwards, are the logical searching strategy. Indeed, circular flights were Observed in D. slmlllsr N. snrtlfnl 221 and N. glulalnaus males. Vertical flights downwards are also observed in these species. It can be speculated that males are probably able to measure the height of a particu- lar host tree silhouette using a series of vertical and circular flights and aided by optomotor cues to know the approximate locus of female location on the host tree. This proposition may be logical in view of turbulence in forest habitat to explain why more males are trapped at a particu- lar height (3.5 m) on the host tree. It is suspected that female sawflies too are evolutionarily guided to sit at this height to release the pheromone and the males too evolu- tionarily guided to locate the calling females. According to Cardé and Baker (1984) , to an organism responding to sex pheromonequroximate cues and orientation mechanisms not- withstanding, such outcomes are the result of evolutionary selection. .Kuenen and Baker (1982) interpreted the pref- erential capture of males at a particular Optimum height to reflect pre-pheromone stratification of flying males in the population, or may be indicative Of Optimal positioning of plumes over visual patterns for the average wind velocities of an area. It is not known whether the latter explanation is applicable to diprionid sawflies but the former explana- tion does not seem attractive for male sawflies. The males are hardly seen flying except when pheromone is present. Appetitive flight may be physiologically expensive for these non-feeding adult males. 222 MW DiannaidmuflisLiaJhLEisld Studying courtship behavior in the wild removes the artificial nature that is perrenial to such study in the laboratory. However, it must be mentioned that it is rather circumstantial for us to do this because both the laboratory reared and field collected male sawfly did not respond to either synthetic pheromone or virgin female in the wind tunnel. Once released, they fly straight to the side or top of the wind tunnel and stay there even when translucent screens were used to diffuse the lightings. There are striking similarities among the three species tested in their courtship behavior (Figure 10-4). However, certain close range copulatory behaviors were evident. First, ambu- lation locus Of the searching maleeon.the twig nearest to pheromone source appeared species specific. Both male N. W and N. ulnainlnnus cover the whole length of one needle to and fro before taking on another, male D. simills only covers about 67% of any particular needle. Second, D. slaills, despite repeated attempts, failed to mate septum despite the fact that septums are always the first that the males climb when presented with two choice pheromone sources--live female and septum or solvent washed pheromone treated' female and septum. The Nnndlnrlan species tested were not as discriminating as 12. slmllis This difference can not be due to pheromone because males of all species were able to execute all acts involved in courtship as well as behavior which facilitated copulation using the most 223 active synthetic pheromone by each species. Also, the same pheromone that was applied to the septum was also applied to the solvent washed female (which are sometimes of different species from the male being studied, and sometimes without legs or antennae) which are mated by male D.- simllis (Figure 10-3b). Rather, tactile cues are likely to be involved, it is probably more develOped in D. slmllls, intermediate in N. snaffle; and least develOped in N. ulaglninaus. Thirdly, within 1 m from pheromone source, male N. Wnaus ap- peared to jump precisely onto the source: this precision was lacking in the other two species studied. It is suspected that visual cues play a rolerin close range courtshiptbe- havior in diprionid sawflies, and perhaps this cue is more developed in N. simulnaus in this instance. Fourth, the lateral or tergal abdominal mating of N. ssulm and the mating of every part of the female body of N. Lininlnnus were lacking in D. simllls. The dry nature of the dead females or lack of response from females on which these acts were performed cannot be the cause, because male D. slallls also mated dead females. Rather, it is suspected that the act is species specific courtship behavior designed to stim- ulate the female. mansion Trapping experiments with ICT and Pherocon II traps show that the latter capture significantly more males than the former. The reason for this is that DDVP does not kill quickly. This was obvious from the fact that the ICT design 224 with the least entry space performed best. The smaller entry space not only limits escape but also keeps the vapor concentration of DDVP inside the trap higher because of limited access of winds. Also supporting this fact is that catches increased drastically when pine twigs were inserted into designs with wider entry spaces, ICT l8 and ICT 36. With twigs, males are retained inside longer for DDVP to effect a knockdown. It is observed on the field that more males at rela- tively less time arrive at the ICT 9 than the Pherocon II traps. The key to better efficiency of ICT 9 is to use a killing agent that act faster than DDVP. As it is now, ICT 9 can be very useful as survey traps for new species and as monitoring tool for delimitation of known populations. ICT 9 can also be modified to collect live males when DDVP or any killing agent is not used. Such modification is done by fitting a necked cone shaped wire mesh about same size as ICT into a 3 cm diameter hole at the center of ICT lid. Males N. snrtlfsn were trapped live with this modified ICT 9 in fall 1983 at Rose Lake, Lansing. This modified trap had an efficiency of about 6-8% of the sticky Pherocon II traps. Added to these are the advantages that ICT 9 had no debris and non-target species caught in them are minimal, 0- 3 in most cases as against 9-10 in the sticky traps. The trapped males inside the ICT 9 preserved well before collec- tion. The plastic cup used for ICT 9 is available in any food stores and it requires less than 8 minutes to make one 225 ICT 9 in the laboratory. Strong winds and many thunder- storms at the time of this study left the ICT intact, and the traps used for 3 trappings in spring, summer, and fall are reusable for another year. It is a design with a lot of potential in sawfly research. REFERENCES Borden, J. H., D. J. Billany, J. W. S. Bradshaw, M. Edwards, R. Baker, and D. A. Evans. 1978. Pheromone response and sexual behavior of W lnnlslnnlln Wachtl (Hymenoptera: Pamphilidae). Ecol. Entomol., 3: 13-23. Cardé, R. T., T. C. Baker, and W. L. Roelofs. 1975. Ethological function of components of a sex attractant system for oriental fruit moth mnlnstn (Lepideptera: Tortricidae). J. Chem. Ecol., l: 475- 491. Cardé, R. T. 1979. Behavioral responses of moths to female-produced pheromones and the utilization of attractant-baited traps for population monitoring. In movement of highly mobile insects: concepts and methodology in research, Rabb, R. L. and G. G. Kennedy (Eds.), North Carolina State University Press, Raleigh, No C., pp. 286-3150 Cardé, R. T. 1981. Precopulatory sexual behavior of the adult gypsey moth. In the gypsy moth: Research towards integrated pest management, Chp. 6-4, Doane, C. C. (ed.), USDA Tech. Bull. #1584. Cards, R. T. and T. C. Baker. 1984. Sexual communications with pheromones. In Chemical Ecology of Insects, W. J. Bell and R. T. Carde (eds.), Chapman and Hall London and Sinauer Associates, Inc., Sunderland, Massachusetts, Po 355-3830 Coppel, H. C., J. E. Casida and W. C. Dauterman. 1960. Evidence Of a potential sex attractant in the introduced pine sawfly. Elysian slmllls (Hymenoptera: Diprionidae). Annals. Ent. Soc. Amer., 53: 510-512. COppel, H. C. and D. M. Benjamin. 1965. Bionomics of the neartic pine-feeding diprionids. Ann. Rev. Ent., 10: 69-97. Elkinton, J. S. and R. T. Carde. 1983. Appetitive flight behavior of male gypsy moths (Lepidoptera: Lymantridae). Environ. Entomol., 12: 1702-1707. 226 227 Grant, G. G., E. B. Smithwick and J. B. Brady. 1975. Courtship behavior 0 phycitid moths. II. Behavioral and pheromonal isolation of Flourin interauastslln.and Cndrn ananslln in the laboratory. Can. J. Zool. 53: 527-832. Grant, G. G. 1981. Mating behavior of the white marked tussock moth and role of female scales in releasing male copulatory attempts. Anals. Ent. Amer., 74 (1): 100-105. Jewett, D. M., F. Matsumura and H. C. Coppel. 1976. Sex pheromone specificity in the pine sawflies: interchange of acid moieties in an ester. Science, 192: 51-43. Kikukawa, T., F. Matsumura, J. Olaifa, M. Kraemer, H. C. Coppel and A. Tai. 1983. The nature of the major sex pheromone of the European pine sawfly, Nsnnlnnlna ssstlfnn. J. Chem. Ecol., 9: 673-693. Kolattukudy, P. E. 1976. Introduction to natural waxes. In Chemistry and Biochemistry of Natural Waxes, P. E. Kolattukduy (edJ, lst Edition, Elsevier, Amsterdam, Pp. 1-15. Kuenen, L. P. S. and T. C. Baker. 1982. Optomotor regulation of ground velocity in moths during flight to sex pheromone at different heights. Physio. Entomol., 7: 193-202. Lewis, T. and E. D. M. Macaulay. 1976. Design and evaluation of sex-attractant traps for pea moth, Cydln alulgnan (Steph.) and the effect of plume shape on catches. Ecol. Ent., 1: 175-187. Ramsawamy, S. B., and R. T. Carde. 1982. Nonsaturating traps and long life attractant lures for monitoring spruce budworm males. J. Econ. Ent., 75: 126-129. Richerson, J. V., E. Cameron and E. A. Brown. 1976. Sexual activity of the gypsy moth. Am. Midl. Nat., 95: 299-312. Ross, H. H. 1955. The taxonomy and evolution of the sawfly genus Nnndlprina. Forest Science, 1: 196-209. Ryan, L. and D. H. Molyneux. 1981. Nonsetting adhesives for insect traps. Insect Sci. Applic., 1: 349-355. 228 Sanders, C. J. 1981. Sex attractant traps: Their role in the management of spruce budworm. In Management of Insect Pests with Semiochemicals: Concept and Practice, E. R. Mitchell (ed.), Plenum, New York and London, pp. 75-91. Shorey, H. H. and L. K. Gastron. 1970. Sex pheromone of noctuid moths: xx. Short range visual orientation by pheromone-stimulated males of Txlnnnpnusln al. Anals. Teal, P. E. A., J. R. McLaughlin and J. H. Tumlinson. 1981. Analysis of the reproductive behavior of Hsllnthls ulnnsssns,(Fu) under laboratory conditions. Anals. Ento SOC. Amerop 74: 324-3300 Thomas, H. A. 1983. Field evaluation of trap components for the introduced pine sawfly, Dinnlua slmllls (Hymenoptera: Diprionidae). Great Lake Entomologist, 16: 13-15. Vite, J. P. 1970. Pest management systems using synthetic pheromones. Contrib. Boyce Thompson Inst., 24: 343- 350. Wilson, L. F. 1970. A guide to insect injury Of conifers in the Lake State. USDA Forest Service Agriculture Handbook #501. APPENDI CES APPENDIX I Field Response Of Male Abbott Sawfly, (Hymenoptera: Diprionidae) to Mixtures of Optical Isomers Of the Sex Pheromone 3,7-dimethylpenta- decan-Z-ylacetate or propionate INTRODUCTION One of the potentially destructive diprionid sawflies, because of multi-generation (up to three in Virginia) occur- rence in one season, is the Abbott's sawfly Nnndlanlnn sum (Leach). It has been recorded in Ontario, Canada, Wisconsin, Virginia, North Carolina, South Carolina, Georgia, and Florida (Atwood and Peck, 1943) and is present- ly recorded in Michigan. The larvae have a pale spot on the front of the dark head (Atwood and Peck, 1943: Ross, 1955). The N. nbbnttl complex consists of N. alnrnssutum Middleton, EL gnmpnn,(Leach) and N. nhbnttl. These are regarded as the most primitive offshoot Of the lecontei group (Ross, 1955). The larvae of N. nannttl feed on various hard pines includ- ing Finns earrings. 2. eshinatn. P. batsmahxlln. 2. salad: ms, 2. 11:51am. and 2. min (Atwood and Peck, 1943) and 2. banksinan (Atwood, 1961). The new record of N. nannnnl in Michigan is made at Sec. 15, Hartwick Pines State Forest, Crawford County, from where larvae and rows of eggs were collected. Each row has 15 closely spaced eggs. The eggs, however, failed to hatch 229 230 in the laboratory. The larvae collected in that vicinity .are yellow green with two broad dark green stripes dorsally and a narrower pair latero-ventrally. The black head has a faint light spot on the frons. All the six larvae spun cocoons from which adults emerged. The larvae of N. algae: snunum and N. namnnn, the members of the nannnnl complex differ respectively by having reddish brown to black head without a frontal spot and dark brown head with additional white marking on the lower part Of the face (Wilson, 1970). No other diprionid pine sawfly larval headcapsule, except fillplnln magnum (Fabricius) with a frontal black tri- angular marking on a brown headcapsule, appears close to N. W. With this apparent expansion of distribution of this currently inconspicuous species whose injury is often confused with those Of other abundant species, sex pheromone baited trap could be a useful monitoring tool. Casida et al. (1963) first reported the presence of a female produced pheromone in diprionid sawflies which is capable of attracting males from a few hundred meters away. Jewett et a1. (1976) purified and identified the precursor of the pheromone as 3,7-dimethylpentadecan-Z-Ol (diprionol), the acetate or propionate of which is active against the males.of:many sawfly species. Synthesis of this compound around the time that this report was made produced a racemic ~compound (Jewett et a1. 1976: Kocienski and Ansell, 1977). When tested on the field, these synthetic pheromones caught sparingly few males and was far less active as well as being 231 inhibitory to natural pheromone (Jewett et a1. 1978). How- ever, when optical isomer Of this compound were tested, various field studies established that N. lnnnannl (Fitch) are attracted by (28,38,78) 3,7-dimethylpentadecan-2-yl acetate (28,38,7S-A) (Matsumura et al. 1979: Kraemer et al. 1981) while males of ulnnlna,slmllls,(Hartig) are mainly attracted by (28,3R,7R) 3,7-dimethylpentadecan-2-yl propio- nate (28,3R,7R-P) (Kikukawa et al. 1982a). About five other Neodiprion species N. plannum (Norton) Kraemer et al. 1979), N. snnnlfnn (Geoffroy) (Kikukawa et a1. 1983), N. arntti complex, N. unsung llnnnsls Ross and N. annulus annulus Schedl are known to be mainly attracted by 28,38,7S-A iso- mer. Through an extensive field investigation (Kikukawa et al. 1983) found that addition of a small amount of 28,3R isomer was synergistic to the field effectiveness of 28,38,7S-A against N. sen-Jinn. The research reported here was undertaken to define the stereospecific response of N. nannnnl and to elucidate the effect of mixtures of optical isomers in attracting males on the field. MATERIALS AND METHODS EinlsLTssts The field tests were conducted mainly in a mixed pine stand at Hartwick Pine, Michigan. The red pine, £5 rnslansn is the predominant species, with the white pine P. stmhus and jack pine P. bnakslnan found scattered around the edges. A few trappings were also done in a jack pine stand on 232 Fletcher Road in Roscommon County, Michigan. The egg and larval collections were made on jack pine twigs on the edges of clearings in the middle of Hartwick Pine State Forest. Pherocon II traps were used throughout this study. Trap preparations have been described (Kikukawa et al. 1983). Traps were set out On the field in a randomized complete block design, with the block choice based on past trapping experience. Throughout the trapping season, fre- quent re-randomization of treatments within and between blocks were done usually at the time the number of males caught in each trap was being recorded. The data so col- lected are evaluated with an analysis of variance and with differences among means graded at P I 0.05 according to Duncan's new multiple range test. Trapped males were re- moved from the traps, washed with n-hexane to remove the sticky material and stored in 70% alcohol. Each trapped male was examined microscopically'and compared to laboratory reared specimens. W The synthetic pheromones used came either as pure, racemic isomer or mixtures. We refer to carbon 2, 3, and 7 of diprionol as either R or S or R/S when racemic. All the isomers were esters. Four chiral isomers 2R,3R,7R: 2R,3R/7S: 28,38,7R: and 23,38,78 were synthesized by Mori et a1. (1978). Tai et al. (1978) synthesized 28,38,7R/S and 28,3R,7R/S and a mixture of the two isomers used in this study. Kikukawa et al. (1982b) synthesized an supplied 233 28,3R,7R and 28,3R,7S isomers. The 2R/S,3R.7R and 2R/S,3R,7S were modified from 2R,3R,7R and 2R,3R,7S by the method described by Kikukawa et al. (1982b). These isomers contained varying levels Of synthetic impurities which were removed by the method described below. The isomers from Tai et a1. (1978) contained a known 5% contamination of optical impurities and those from the other two chemists less than 1% of erythro-in-threo or threo-in-erythro contamination. PurifinntinaJLsmnstis WWW Thirty-six gm of charcoal (Norit A, J. T. Baker Chemi- cal CO., New Jersey) pre-heated for 2 hrs. at 180°C, was mixed thoroughly with 9 gm celite (Fisher Scientific Co.) and washed with 20 ml acetone in a funnel with Whatman No. 2 -filter paper. The charcoal-celite Obtained from above was stirred into a 200 ml beaker with 140 ml 10% ether in hexane using a glass rod. The slurry Obtained was packed in a 50 cm x 2 cm i.d. glass column. A 20 ml solvent was first used to wash the slurry down the walls of the column followed by 80 ml in 2 equal volumes to stabilize the column. A maximum of 7 mg sample was introduced to the column and was eluted with 10% of ether in n-hexane at 0.2 ml/min of flow rate. Recovery was about 60%, with samples less than 500 ug it was less. Sixty-three 10 ml fractions were collected using a micro fractionator (model FC-80R, Gilson Medical Electron- ics, Inc., Middleton, Wisconsin). 234 A small aliquot from each fraction was injected into a gas liquid chromatograph (GLC) equipped with a flame ioniza- tion detector (FID). Fractions 33-63 contained the sample, but fractions 33-42 and 61-63 contained the sample and impurities while fractions 43-60 contained the pure sample. It should be noted that columns less than 35 cm in height failed to purify the samples in this manner. RESULTS Of the four isomers tested in Table AI-l, traps baited with 28,38,7S-A caught the most males. The 28,3R,7R/S-P and 2S,3S,7R/S-A also showed some activity, about one third and one quarter respectively that of 28,38,7S-A. However, the corresponding propionate 28,38,7S-P was not active though 28,3R,7R/S-P showed some activity. This was our first en- counter with a species responding to 28,38,7S-A and 28,3R,7R/S-P but not to 28,38,7S-P. In the case of N. snnnlfn; (Rikukawa et al. 1983) and N. annnnl anakslnann (Olaifa et al. 1984), while responding to 28,38,7S-A as the 4 major isomer, these species were also attracted to a little extent by 28,38,7S-P and we interpreted that to mean an overriding stereospecific response to the 28,33,73 isomer. Also, in Wisconsin, N. nrnnnl annual which responded to 2S,3R,7R/S-P also showed activity towards 2S,3S,7S-P (Kraemer et al. 1983). The unexpected activity of 28,3R,7R/S-P prompted a further investigation of other available acetate and propionate diprionol isomers against 235 Field response of males N. nuhuttl to four Optical isomers of acetate or propionate diprionol. Test conducted at Hartwick Pines, Michigan, May lS-July 10, 1981. Optical isomers (25 ug/trap) Mean catch/trapa j: S.E. 28,38,7S-A 28,38,7R/S-A 28,38,7S-P 28,3R,7R/S-P 45.3 i 4.5 11.0.: 2.2 0.0 i 0.0 16.3 i 2.3 a Means of 3 replicates. cant. Analysis of variance not signifi- 236 this species (Table AI-2). Here, 25,38,7S-A again showed an outstanding activity towards N. nasal-.13.. The 28,38,7R/S-A isomer also showed some activity, but 2S,3R,7R/S-P this time caught only one male. The result also demonstrates the importance Of acetate isomers for this species and the fact that propionate isomers were not active against N. nasal-ll. Also demonstrated from this table is the importance Of S configuration on 7-carbon as 28,38,7S-A was active, 28,3S,7R/S-A with little activity and 28,38,7R-A inactive. It is thus established that 2S,38,7S-A is the major phero- mone of N. W, a characteristic shared with many Nnndl; man species as described earlier. To test whether some combinations of threo isomers, i.e., 28,3R or 2R,3s isomers, give high biological activity, five threo acetate isomers occurring as either pure or racemic forms were added to 28,38,7S-A (Table AI-3). Three isomers in increasing order of effectiveness in increasing the activity of the major isomer towards this species were 2R/S,3R,7R-A, 28,3R,7R/S-A and 28,3R,7R-A. Because of rela- tively large variance among the replicates for the 28,38,78- A plus 2R/S,3R,7R-A treatment, the data of the two other active synergists were regarded more reliable. In another test during the following year, three. threo isomers were compared and the importance of chirality on 2-carbon and 7- carbon was tested. The results show that 2S,3R,7S-A isomer was a better synergist of 28,35,7S-A than either 2S,3R,7R-A or 2R/S,3R,7S-A (Table AI-4). At a combination ratio of 1:2 237 Field response of males N. naustnl to different Optical isomers of acetate or propionate diprionol. Test conducted at Hartwick Pines, Michigan, June 6-July 20, 1981. Amount Replicates Optical isomers (ug/trap) A B Total 2R,3R,7R-P 20 0 0 0 2R,3R,7S-P 20 O 0 0 25,38,7R-P 20 0 0 0 28,38,7S-P 20 O 0 0 28,3R,7R/S-P 4O 1 0 1 28,3R,7R-P 20 O 0 0 2S,38,7R-A 2O 0 0 O 23,38,7S-A 20 27 26 53 28,35,7R/S-A 40 4 3 7 Control 0 0 0 0 238 Table AI-3. Field response of males N. nbbnttl to mixtures of optical isomers Of acetate diprionol. Test conducted at Hartwick Pines, Michigan, June 5- July 11, 1981. Amount Mean catch/trap* Preparations (ug/trap) .1 S.E. 28,38,7S-A 20 3.3 1 0.7 28,38,7S-A / 28,3R,7R/S-A 10/15 6.3 1,0.8 23,35,7S-A / 28,3R,7R-A 10/15 11.7 1 1.7 28,38,7S-A / 2R/S,3R,7R-A 10/15 5.3 1 1.7 28,38,7S-A / 2R/S,3R,7S-A 10/15 1.6 1 0.8 28,38,7S—A / (28,3R,7R/s / 2R,38,7R/S-A) 10/15 1.3 1 0.6 Control 0 0.0 1 0.0 * Mean catch of 3 replicates. Analysis of variance not significant. Table AI-4. 239 Field response of males N. nhhuttl to mixtures Of 23,38,78’A With 28,3R,7R-A, 28'3R37S-A and 2R/S,3R,7S-A isomers. Test conducted at Fletcher Road near Sharon, Michigan, May 7-June 10, 1982. Amount Mean catch/trap Preparations (ug/trap) 1 S.E. 28,38,7S-A 5 5.3 1 1.3 28,33,7S-A / 28,3R,7R-A 5/5 1.0.: 0.8 5/10 1.0 i 0.6 23,33,7S-A / 2S,3R,7S-A 5/5 6.7 1 1.7 5/10 6.3.1 1.4 28,38,7S-A / 2R/S,3R,7S-A 5/5 1.0 1,0.0 5/10 3.0 1.0.9 a Means of 3 replicates. Analysis of variance not cant. signifi- 240 28,38,7S-A:2R/S,3R,7R-A, more males were attracted than 28,38,7S-A:28,3R,7R-A.at the same ratio. It then appeared that S configuration at both 2-carbon and 7-carbon was preferred by this species. In two separate tests, one at Hartwick Pines in 1981 where 28,3R,7R/S-A was the synergist, the other at a low population area on Fletcher Road, Crawford County, in 1982 where 28,3R,7S-A was used, the effectiveness of these iso- mers as synergists Of 2S,38,7SeA was confirmed (Tables AI-S and 6). A significant synergism at a blend ratio of 1:1 was achieved when 28,3R,7R/S-A.was the synergist VTable AI-5). Also, at the same ratio, 2S,3R,7S-A isomer increased the effectiveness of the major isomer. Beyond this optimum ratio, inhibition appeared to set in. DISCUSSION Our data indicate that males Of N. nhbntnl respond mainly to 28,38,7S-A, but increased response is obtained by employing 1:1 mixture Of 2S,3S,7S-A and 2S,3R,7S-A, the latter isomer serving as a synergist. It has become clear that most Nnndlpnlna species utilize combinations of Optical isomers as their pheromone. This is the first report of a 1:1 combination. Other combinations are: a 5:0.003 for N. any-“tin; (Kikukawa et a1. 1983) and 5:1 for N. m finals: slnnnn (Olaifa et al.1984). Further studies are required for other species to identify the isomer combinations of their sex pheromone. It can only be speculated at the 241 Table AI-5. Synergistic effect of the 28,3R,7R/S-A isomer on the field effectiveness of 28,38,7S-A against N. nbhuttl. Test conducted at Hartwick Pines, Michigan, May lS-August 4, 1981. Amount Mean catch/trap* Preparations (ug/trap) .1 S.E.** 25,35,75-1 20 9.7 1 l.1° 2S,3R,7R/S-A 20 0.0 1 0.09 25,35,754; / 25,311,711/5-11 20/5 5.3 1 l.l° 20/10 29.7 1 3.6b'c 20/20 77.0 1 2.7'1 20/40 59.0 1 2.2a1b Control 0 0.0 10.00 * Means of 3 replicates. ** Means followed by same letter not significantly different by Duncan's multiple range test at 5% level. 242 Table AI-6. Field response of male N. nbbntti to a varying mixtures of 28,38,7S-A and 28,3R,7S-A isomers. Test conducted at Fletcher Road, Garfield Township, Michigan, May 25-August 13, 1982. Amount A5 85 Total Preparations (ug/trap) I II I II III catch 28,38,7S-A 5 0 0 0 l 0 l 23,38,78’A / 28,3R,7S-A 5/0.001 l l l 2 0 5 5/0.003 0 0 0 0 0 0 5/0.01 l 0 l 0 0 2 5/0.03 0 l 0 0 2 3 5/0.l l 0 0 0 l 2 5/0.3 l 0 0 0 0 1 5/1 0 0 0 l 2 3 5/3 1 l 0 0 0 2 5/5 3 3 2 0 2 10 5/10 2 l 1 0 2 6 5/30 3 0 -° -° -° 3 Control 0 0 0 0 0 0 0 a Test conducted May 25-June 19, 1982. b Test conducted June 30-August 13, 1982. c Inadvertently left out. 243 moment that second component would be serving an important isolation mechanism for the sympatric species and for the members of complexes. It will also be worthwhile to inves- tigate the occurrence of these isomers in the female pro- duced sex pheromone. Unlike other conspicuous species like N. salute; and N. annulus annulus, it was impossible to obtain enough females of N. nannnnl from the field for extraction.of pheromone. Thus, comparison of the natural and synthetic pheromone was not done in this study. The current study, however, established that the 1:1 mixture of 2S,38,7S-A and 28,3R,7S-A is a potent attractant of N. nhnustl. Apart from its pheromone being unique with the 1:1 blend of 2S,3S,7S-A and 28,3R,7S-A isomers, this species appears to be the only spring flying Nnndlanlna species caught in non-white pine area with acetate. The Swaine sawfliesum limalnaus. N. suninsl. N. mums. and N. duhlnsus--respond to the propionate. D. slallls and N. plannum are strictly white pine species and the former responds to the propionate while the latter is attracted with acetate. The only possible mixing up is N. alnntua. However, the male N. alnntum.is generally smaller and thin- ner than male N. nnbuttl. Male nhhnttl is more robust than any other spring flying Nnndlpnlnn,species so far encoun- tered in Michigan. Male N. abbnttl is reported to be more plump than males of known Nnndlnnlna species in eastern Virginia (Hetrick, 1956). Freshly caught maletNL alnshum.is 244 light orange at the lower abdomen while male N. shunts}. is dark orange. Thus, by size, shape and abdominal coloration, male N. alnntum and N. nbhntnl can be separated. Separation can also be achieved by species stereospecific blend 1:2 28,38,7S-A to 28,3R,7R-A for N. nlnntum. However, until the nature of the sex pheromone of other members of nannnnl complex and other species that have been reported in Michi- gan--such as N. mnunus Rohwer--is known and the males of these species caught and compared, we cannot be fully sure of the identification of the present species. REFERENCES Atwood, C. E. 1961. Present status of the sawfly family Diprionidae (HymenOptera) in Ontario. Proc. Ent. Soc. Ont., 91: 205-214. Atwood, C. E. and O. Peck. 1943. Some native sawflies of the genus Nnnnlnrlna attacking pins in eastern canada. Canadian Journal Of Research, 21 (5): 109-144. Casida, J. E., H. C. Coppel and T. Watanabe. 1963. Purification and potency of the sex attractant from the introduced Pine sawfly, ulnnlnn slmllls. J. Econ. Ent., 56: 18-24. Hetrick, L. A. 1956. Life history studies Of five species of Neodiprion sawflies. Forest Sci., 2: 181-185. Jewett, D. M., F. Matsumura and H. C. Coppel. 1976. Sex pheromone specificity in the pine sawflies: Interchange of acid moieties in an ester. Science, 192: 51-53. Jewett, D. M., F. Matsumura and H. C. COppel. 1978. Preparation and use of sex attractants for four species of pine sawflies. J. Chem. Ecol., 4: 277-287. Kikukawa, T., F. Matsumura, M. Kraemer, H. C. Coppel, and A. Tai. 1982a. Field attractiveness of chirally defined synthetic attractants to males of Dianna slallls and Gllnlnln.£zutntnsum. J. Chem. Ecol., 8: 301-314. Kikukawa, T., M. Imaida and A. Tai. 1982b. Synthesis of the sex attractant of pine sawflies (Diprion species): (2S,3R,7R) and (28,3R,7S)-3,7-dimethy1pentadecan-2-ol. Chem. Lett., 1982: 1799-1802. Kikukawa, T., F. matsumura, J. Olaifa, M. Kraemer, H. C. Coppel and A. Tai. 1983. The‘ nature of the major sex pheromone Of the European pine sawfly, Nnnulnrlnn snrnlfnn. J. Chem. Ecol., 9: 673-693. Rocienski, P. and J. Ansell. 1977. A synthesis of 3,7- dimethylpentadecan-Z-yl acetate, the sex pheromones of the pine sawfly Nnndlnnlna lnnnnnnl. J. Org. Chem., 42: 1102-1103. 245 246 Kraemer, M., H. C. Coppel, F. Matsumura, T. Kikukawa and K. Mori. 1979. Field responses of the white pine sawfly ° Rinntum to Optical isomers of sawfly sex pheromones. Environ. Entomol., 8: 519-520. Kraemer, M. E., H. C. Coppel, F. Matsumura, R. C. Wilkinson and T. Kikukawa. 1981. Field and electroantennogram responses of the red-headed pine sawfly, Nnndlnnlnn lnnnnnnl (Fitch), to optical isomers of sawfly sex pheromones. J. Chem. Ecol., 7: 1063-1071. Rraemer, M. E., H. C. Coppel, T. Kikukawa, F. Matsumura, H. A. Thomas, L. C. Thompson, and K. Mori. 1983. Field and electroantennogram responses to sex pheromone optical isomers by four fall-flying sawfly species (Hymenoptera: Diprionidae, Nnudlnnlua). Environ. Entomol., l2 (5): 1592-1596. Mori, R., S. Tamada and M. Matsui. 1978. Stereocontrol led synthesis of all of the four possible stereoisomers of erythro-3,7-dimethylpentadecan-Z-yl acetate and propionate, the sex pheromone Of pine sawflies. Tetrahedron Lett. #10: 901-904. Olaifa, J. I., T. Kikukawa, F. Matsumura and H. C. Coppel. 1984. Response of male jack pine sawfly, nunnnl annkslnnnn (Hymenoptera: Diprionidae) to mixtures of optical isomers of the sex pheromone 3,7- dimethylpentadecan-Z-yl acetate. Environ. Entomol. (In press). Ross, H. H. 1955. The taxonomy and evolution of the sawfly genus Nnnnlpninn. Forest Science, 1: 196-209. Tai, A., M. Imaida, T. Oda, and H. Watanabe. 1978. Synthesis of optically active common precursor of sex pheromone of pine sawflies. Chem Lett., 1978: 61. Wilson, L. F. 1970. A guide to insect injury of conifers in the Lake State. USDA Forest Service, Agriculture Handbook #501. APPENDIX II Cuticular Hydrocarbon of Ten Species Of Pine Sawfly INTRODUCTION Hydrocarbons are often an abundant component of cuticu- lar lipid of insects (Jackson and Arnold, 1977). There is a great variation in hydrocarbon composition between species and along with other components of cuticular wax, these compounds are known to be highly conserved during evolution therefore, they must serve a function (Jurgen Jacob, 1978). In insects apart from the primary physiological role of forming a water impermeable barrier on the surface of a cuticle and performing a communicative role as semiochemi- cals (Howard. 1982), the'cuticular hydrocarbon along with other wax components also protect insect from abrasive damage, serve as a barrier to the penetrating microorganisms and affect the absorption of insecticides and other chemi- cals from the environment (Blomquist and Jackson, 1979). Another function currently being assigned insects cuticular hydrocarbon is chemotaxonomy (Jackson and Blomquist, 1976: Lockey 1976). The basis for that is still being debated. In considering hydrocarbon composition in chemotaxonomy work in insect, several insects have been examined and interesting qualitative differences have been found between 247 248 families of Orthoptera (Nelson and Sukkestad, 1975) and between different species within the same genus of Ennl: nlnnnnn (Jackson, 1972). The complex nature Of the hydro- carbon composition and the interesting patterns of methyl branching in many insects suggest that such possibility might exist as previously suggested for fungi and higher plants (Jackson and Blomquist, 1976). It is also clear in many insects examined that only the branched.hydrocarbons might be relevant for chemotaxonomy in insects because by using labelled precursors, Blomquist and Jackson (1979), confirmed the view that insects synthesize branched alkane fraction of the hydrocarbon. The same authors also found that in insects, the n-alkane constituents of cuticular lipids are not synthesized but are mainly derived directly from the diet. In the light of that, the n-alkane pattern of a cuticular lipid will be related more to the insect's diet than to its genotype so that a correlation between a- alkane pattern and systematic grouping is not to be expected (Lockey, 1976 and 1980). Other workers (Kolattukudy, 1976) confirmed this Observation by finding large amount of n- alkanes in acridids to be common constituents Of plant waxes. It is believed that hydrocarbon biosynthesis is under hormonal control (Armold and Regnier, 1975), occurs in oenocytes (Wiggleworth, 1976) and the enzyme concerned in the synthesis are determined by the genotype. It could therefore be reasoned that the genotypic differences between 249 the species of sawfly should be reflected in differences between their branched alkane patterns. MATERIALS AND METHODS The adult males reared in the laboratory from the field collected larvae were used for this study. The males of N. suniasl. N. lemtsi. N. uni-Li unrndnxinus (with black lower abdomen) and N. pannnl_anrndnxlnus_(with red lower abdomen), N. ruuifrnns and N. alnntun were secured from the laboratory of Dr. H. C. Coppel, University of Wisconsin. The other Species N. annulus annulus: N. arntti baaksinnnn. N. snutlins, and D. slmllls were reared in our laboratory. A number of extraction procedures have_been described in the literature (Jackson and Armold, 1977), most of them involved some type of a short solvent wash utilizing commonly hexane, methylene chloride, diethyl ether or chloroform. The point of disagreement among various authors is the length of the washing time, some believed that short extraction periods may not remove the cuticular lipid quantitatively and others argued that long extraction periods may extract internal lipids. .All the species used for this study were extracted for 10 hours in 2 ml hexane per species. For each species, 2-4 males were used. After the 10 hours the hexane was removed and was partitioned with 2 ml acetonitrile. The hexane phase was then purified of the polar components by running through florisil column chromatography, packed dry 7 cm tall inside glass pipette 0.8 cm i.d. and 1.1 cm O.d. 250 Florisil was activated by heating in oven at 230° for 1 hr. before use. lThe column was eluted with hexane and the first 10 m1 fraction was collected. The solvent was removed with dry nitrogen gas to a final volume of 0.1 ml. Of this final volume, 0.05 ml was injected into a 20 ft. 1/8' i.d. stain- less steel 3% S.E. 30 column, at column, injector and detec- tor oven temperatures of 260°, 300°, and 310°, using a FID varian GC model 2400 with nitrogen carrier gas flow'of 30 ml/min. H2 30 ml/min. and air 300 ml/min. Linear recorder was used at 30 cm/hr. Attempt to clean the extracts of males collected through sticky traps from the field first by activated florisil and second by a 1:1 mixture of activated florisil and charcoal columns failed. Therefore, no males from sticky traps was used for this study. No attempt was made in this study to identify any of the cuticular hydro- carbon for any of the species. This study only compared the gas liquid chromatographic spectra of nine species of pine sawfly. This was done by first developing a spectrum for each species and then compile a sort of master spectrum comprising all the maximum peaks obtainable from all the spectra. All the peaks are renumbered according to the retention time. Identical peaks have identical numbers in all species. RESULTS Figure AII-l were the chromatogram of the cuticular hydrocarbon of the species examined. One obvious Figure AII-l. GLC chromatogram of cuticular hydrocarbon of ten diprionid sawfly species. 251 .3283.» nozoa no: and ”flanged a .a no nonsense»: nun—53:0 mo ecumoucEOuso 040 I; hfll’dd m9 U/\J’Wt\yx\lis/OJ J .- . fl. 0. ht... 370‘:“ I. — . -..-—--—.- - .. .ennmnc enemas 252 Acme—owns mesoH x003. and and an .a mo conuuooOcms uoHsOausO no soumouoeoso one a. = e .pauana enemas \ . V7. _ 253 .3 .2 mo conuooodcmn ”316350 no anemones—OE". 0.5 r/ . n./\ d— .oHnHH¢ unseen 1).... 254 .33 {a NO GODHUOOHOsnS HQHDOuuflo MO EOHDOHGEOHSO UAU .oaunac one... 255 .4343 .a mo conuuooOuhe Lea—503:0 mo scumousEOuso 0.5 .omlnnd capo: \ 3 r; «. . J... 256 .33 .2 no conucOOucmn usasojso uo ecumouceozo 0.5 .anHH¢ magmas 257 u so on mousaOu O son a: unaccuusu u no conucoo .2 mo .udduuduqu 3 ma Hue unseen 258 .333: and: .z no conusOOL—XE 23:03.6 «0 Eamon-cease 0.5 .aauaue unease 259 .duudduud.qz no conuoooucas u¢u50quso mo '. scumouosouso one .«HIHH‘ Gunman 260 .3 .d MO GODUGOOh—Qfifl uGHn-Odnwflu HO EQHOOHQEOHSO UGO R r n. .finnnue ensues 261 observation is the striking similarities of most of the peaks and quantitative differences among species was hardly noticeable. One quantitative difference however, separated the genus nipnlna_from the genus Nnndlnnlna: in all Nnndln: ulna species examined, peak 8 was lower than peak 7 while in Minn slmllls, the reverse was true. For the D. slallls three other replicates were prepared from another group of males and similar situation was Observed. Another notice- able quantitative difference which grouped the nine species examined into two was the ratio of peaks 12 and 13: the ratio was in favor Of peak 12 for N. n. annndnxlsus (red), N. duhinsus. N. suniael. N. ninetum and a. slmllis but in favor of peak 13 for N. n. anrndnxlnus (black), N. 11min: inaus. N. lscnatsl. N. ruuifraas and N. annulus annulus. These two quantitative differences clearly presented a cir- cumstantial evidence for separation of N. glxulainnus from N. dublqsus on one hand and N. ruglfimns from N. dublqsus on the other. Also separated by the same reasoning was N. a. .nnnnduxlsus (red abdomen) from its black abdomen counter- part. To bring out any qualitative difference among species, the chart (Figure All-2) was prepared to compare the hydro- carbon peaks. From this chart it is clear that peaks num- bered l, 2, 3, 5, 6,‘7, 8, ll, l2, 13, 15, 19, and 20 were common to all the species. 'These peaks therefore, could not be considered for chemotaxonomy for the pine sawfly species studied. The other peaks which showed qualitative 262 CI =E_u. Elm: o... _. 39:94: . ZIZ unseat .Eszsmi macs—Emmerm .83392 elegaiau. .2 32. 9mg...“ :83. .30. Sputum—.2 O OO 1 Peak 00 00 OO 00 OO 00 23 O O O O O O O O 10 O O O O O O 12 13 15 17 18 19 20 22 23 24 26 27 OPeak land 0 Peak not com: the cuticular hydrocarbon of ten species Of Qualitative differences among GLC peaks of sawfly. Figure AII-2. 263 differences among species are tabulated in Figure AII-3. In order to show similarities among species, a method of scor- ing was developed where one species is taken at a time, the number of peaks that species has is scored and that number is used as a denominator for the number of peaks in another species common to the denominator's. The bigger that frac- tion was towards integer l the closer was this second spe- cies towards the first. An example of this scoring system is seen in N. n. madnxlnus (black abdomen) with 11 peaks, compare N. a. anndnxluus (red abdomen) to this with 6 common peaks: score - 6/11 . 0.545. Other species, N. dunlnsus 3/11 - 0.273: N. sunlnnl 3/11 - 0.727: N. unnla; lnnus 4/ll - 0.364: N. ninnLum 3/11 - 0.273: N. a. annulus l/ll :- 0.091: N. lsnuntnl 3/11 - 0.273 and D. slmllls 6/11 . 0.545. Using N. a. pnrndnslcus (red) as denominator: other species compared to it as follows: N. a. mnduxlnus (black), 0.857: N. duulnsus, 0.429: N. suninnl, l: N. 11;; aiainaus. 0.571; n. aiastun, 0.429: n. a. annulus, 0.143: n. Luulmns, 0: N. lsnnntsl, 0.286: and N. slmllls, 0.571. In the same way, comparing others to N. duhlusus there appeared 3 groups: 11. a. anrnunxisus (black). n. a- anrndnxinus (red), and N. sunlnnl with 1 each: N. alnntum formed another group with 0.667 and N. a. annulus. N. lemtni, N. 11min; lnaus, and Q. slmllls with 0.333 each. Using N. sunlanl classified the nine species into 4 groups, N. a. anrndnxlnus (black and red) with 0.888 and 0.777 respectively as one steep: N. duuinsus, N. zlrniainaus and N. ninetum with 264 E 00 $5 3:9,: c 5 .~7°?9'¥T 35' .'-. 21 an 2123 21:21 am :znzzl 6: Peak 1 2 3 4 o o 5 6 7 8 9 D D U Do 0 'm 0 0 I: O o o o 11 12 13 1‘ D D D O o 15 “a D O O O o O 11 0 18 O o 19 20 21 o 22 D O a 2‘ O O o o 25 o 29 O o D U 27 o o 23 o Figure All-3. D Peeks showing qualitative diflerances Qualitative differences among GLC peaks of the cuticular hydrocarbons of ten species of sawfly. 265 0.333, 0.444 and 0.333 respectively as another group: N. a. annulus with 0.111 as one group and D. simuls with 0.555 as the last group. Other species were so compared to produce Table AII-l. The final grouping is based on the number of occurrence together in the same group of one species with other spe- cies. If the occurrence together of two species was greater or equal to 3 times, they are put in the same group. On the basis of such classification, 6 groups are formed from the nine species examined. Group 1: comprised of N. n. nnrnunxlnus (black): N. a. anrndnxisus (red) and N. sunlanl. Group 2: comprised of N. dumsus and N. W. Group 3: comprised of N. annulus annulus and N. ml; frnns. Group 4: comprised of Dianna slmllls. Group 5: comprised of N. alanium. Group 6: comprised of N. lnnnannl because its peaks occurred greater than 3 times with at least 3 species. DISCUSSION It must be pointed out that species like N. a. anrn: duslnus (black and red abdomen) and N. sunlnnl where enough sample were available to be injected had obvious advantages over others with less sample. Efforts were made however, to scrutinize the chromatograms produced very well before leouan. use enema one HoOHucocu mo omnusOOuom "duodenum ad .a Avon. and .duddddud 9H .uquHflddH ta Hdfldddudfl ta .2 udduuduuu qz «sanded «d an ududfladu ea «udduuu ea .aduuadu oz needy and uaaqsau .a .uquauuuua .u qa auudduud ea Axocabv_udu nuanced .a .z udauuauuu .z ”duodenum .u an In udddddfluudu an .aduuddu qz «adduuu ea ududdndu ea «uddduud 1! Axosanv.uqu Acosocns uoaoa udduuAddu qz aduuddu qz udddddquudu ea ”unmanned qa ea con. aqua adduced .d ea ududdadu ea udddadu .d «undead ea nuduuuua.duuuua qz udduudddfl .2 uddddud ed 22 ududdaqu an .couv and «uddduud qz uddqdqduudu .a ddddfidu 2a nauduuuua 1a 1! Hudduuu ea .uandadu qz Acosocno nozoa duudauud ea Roman. «dad adduced ad ea aduudda ea .udddadu ed dudduau qz nuauuuua.«uuuua .z on.” em-.. a.-.» canoe. none no: confiscated guns; on newcomm .conusOOucmn uoasoauso Hausa no axoom one ambiance“ mo museuusooo mo ooumoc co comes aauzsm mo uoaoomm a mo unam50uu .HIHHd manna 267 mama .z Hosanna .M a 0 ea . . «nudged .a Go: and... 20.3. and nausea sanded ”squad naufluddé gas—«ad «3.3.3 .a dung .a Hg .1. $3..“ a . . «gag .z 89: and 33:2 Saga 53.3.3.2 add adequaé as: .a Andaman .a 3.63.: «am a «dang .z 6...: and... «and: .d .z ”savanna .a .a gun .a Headed .z an .2 I. dung .a a-.. euaam oeuao ounces «one no; confinedsoo exams can Hmouucocfi mo omsu:00uom scans on meaoomm A.ucoov .HIHH< Onnufi 268 making judgment. If not, features of chromatograms of spe- cies like N. Minus and D. slmllis with scanty samples would not have been so clearly presented. It must be empha- sized that lack of identification of the peaks and absence of enough samples in most species presented a problem not only in running the test but also in interpreting the re- sults. It is suspected that the 13 peaks which were common to all the species were mostly n-alkanes in homologous series. The n-alkanes are known to be derived directly from diet (Blomquist and Jackson, 1973) and since all the species feed on pine shoots, the n-alkanes in their cuticular hydro- carbon are likely to be similar. By the same reasoning, the peaks listed in Figure AII-3 would be mostly branched hydro- carbons. The results so far presented pointed out clearly that cuticular hydrocarbon could be considered for taxonomic use in pine sawflies. There were similarities with tax- onomic groupings of this six grouping and those Of Ross (1955) and Knerer and Atwood (1973). The group 6 in this study, N..lnunannl interestingly had peaks occurring about 3 times with 3 species. It was about the only species with such characteristics in this study and that suggested that N..lnnnatn1 might actually have some genetic connections with other species. This suggestion would be in line with the lecontei and sertifer group of Ross (1955). It was also of interest that N. dunlnsus_and N..1lnglnlnaus fell together into one group. The view of earlier workers on taxonomy of N.:1lnulalnaus, 269 complex (Ross, 1955: Becker et a1. 1966) supported this grouping. However, the Obvious quantitative difference in GLC peaks 12 and 13 is enough to separate them into species. Apart from these fine qualitative differences it*was evident as earlier pointed out that quantitative differences also exist between genera Dianna and Nnndlpnlnn and among species of sawflies. If this classification based on cuti- cular hydrocarbon is considered in conjunction with larval characters, host plant oviposition pattern, life history and male courtship behavior it will go a long way'at solving the taxonomy of pine sawflies. There is no doubt that cuticular hydrocarbon pattern has a role to play in chemotaxonomy of sawflies as demonstrated in this study. REFERENCES Armold, M. T. and F. E. Reguier. 1975. Stimulation of hydrocarbon biosynthesis by ecdysterone in the fleshfly Snrnnnhnnn aullnnn. J. Insect Physiol., 21: 1581-86. Becker, G. C., R. C. Wilkinson and D. M. Benjamin. 1966. The taxonomy of Neudiarina ruulfraas and N. duninsus (HymenOptera Tenthredinoidea: Diprionidae). Ann. Ent. Soc. Amer., 59 (1): 173-178. Blomquist, G. J. and L. L. Jackson. 1979. Chemistry and biochemistry of insect waxes. Prugr. Lipid. Res., 17: 319-345. Howard, R. N. 1982. Chemical ecology and biochemistry of insect hydrocarbons. Ann. Rev. Entomol., 27: 149-172. Jackson, L. L. 1972. Cuticular lipids of insects--IV. Hydrocarbons of the cockroach, Rnnnlnnntn jnanalnn and W nmnnlnnnn compared to other cockroach hydrocarbons. Comp. Biochem. Physiol., 41b: 331-342. Jackson, L. L. and G. J. Blomquist. 1976. Insect waxes. In Chemistry and Biology of Insects, P. E. Kolattukudy (ed.) pp. 201-233. Elsevier Scientific Publishing Co., Amsterdam. Jackson, L. L. and M. T. Armold. 1977. Insect lipid analysis. In Analytical Biochemistry of Insects. R. B. Turner (ed.), pp. 171-206. Elsevier Scientific Publishing Co., Amsterdam. Jacob, J. 1978. Sex dependent composition of cuticular lipids from beetle, Rhngnaynnn fulzn. Hoppe-Seyler's Z. Physiol. Chem., 359: 653-656. Knevev, G. and C. E. Atwood. 1973. Diprionid Sawflies: polymorphism and speciation. Science, 179: 1090-1099. Kolattukudy, P. E. .1976. Introduction to natural waxes. In Chemistry and biochemistry of natural waxes, P. E. Kolattukudy (edJ, lst edition, Elsevier, Amsterdam, pp. l-15. 270 271 Lockey, K. H. 1976. Cuticular hydrocarbon Of Locusta, thlsnnsnnnn and Ennlplnnnnn and their role in waterproofing. Insect Biochem., 6:457-472. Lockey, K. H. 1980. Insect cuticular hydrocarbons. Comp. Biochem. Physiol., 65B: 457-462. Nelson, D. R. and D. R. Sukkestad. 1975. Normal and branched alkanes from cast skins and grasshopper Ennisnnsnnsn,ungn (Suddar). J. Lipid. Res., 16: 12-18. Ross, H. H. 1955. The taxonomy and evolution Of the sawfly genus Nnnnlpnlna. Forest Science, 1: 196-209. Wigglesworth, V. B. 1976. Distribution of lipid in the cuticle of Rhodnius. In Insect Integument, H. R. Hepburn (add, lst edition, Elsevier, Amsterdam, pp. BIBLIOGRAPHY BIBLIOGRAPHY Armold, M. T. and F. E. Reguier. 1975. Stimulation of hydrocarbon biosynthesis by ecdysterone in the fleshfly Snnrnnahnun nullntn. J. Insect Physiol., 21: 1581- 1586. Atwood, C. E. and O. Peck. 1943. Some native sawflies of the genus Nnndlpzlna attacking pines in eastern Canada. Can. J. Res., 21: Sec. D. (5): 109-144. Atwood, C. E. 1961. 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