THESIS LIBRARY Michigan State University This is to certify that the thesis entitled PREDICTION OF INDUCTION AND TERMINATION 0F DIAPAUSE IN THE COOLING MOTH (Cydia pomonella (L.), Lepidoptera: Tortricidae) presented by Carlos Garcia Salazar has been accepted towards fulfillment of the requirements for MASTER OF SCIENCE degreein ENTOMOLOGY dmu / Major professor Date g/z/g# 0-7639 MSU is an Affirmative Action/Equai Opportunity Institution MSU LIBRARIES RETURNING MATERIALS: Place in book drop to remove this checkout from .—L—— your record. FINES will be charged if book is returned after the date stamped below. _.________'-—-———-'.__# a... l“ *7? 7mm I ~...da'~2-7~U1'3 FHKEINKTTKDBICH?ID“)LK:TFDDIIUNE)TTHKNUIHAJTCHW OF DIAPAUSE IN THE COOLING MOTH (Cydia pomonella (L), Lepidoptera: Tortricidae) by Carlos GarciaSalazar ATHESIS Submitted to Michigan State University in partial fulfillment of the requirement for the degree of MASTER OF SCIENCE Department of Entomology 198‘ ABSTRACT PREDICTION OF INDUCTION AND TERMINATION OF DIAPAUSE IN THE CODLING MOTH (Cydia pomonella (L), Lepidoptera: Tortricidae) by Carlos Garcia Salazar The induction and termination of diapause in the Michigan strain of the codling moth, deia pomonella (1..) showed that diapause was induced in the first to the fifth larval instars. Diapause induction could be eliminated 100% in the first two larval instars and a little more than 5096 in the third instar by modifying the temperature and photoperiod. The onset of diapause occurred when the daylength was 15 h at 13:58 h photophase, 5096 of the overwintering generation diapaused. A temperature change of 5° C during the larval rearing period shifted the onset of diapause to a shorter or longer photoperiod by l h. Diapause was terminated in all the specimens by a chilling period of 65 days at 2° C. Diapause termination occurred in 6296 unchilled larvae by the action of long-day photoperiod (16L:8D at 27° C). The length of time the larvae spent in diapause was also very important. Larvae in diapause for more than 50 days terminated diapause faster than those in diapause for less than 50 days. ACKNOWLEDGMENTS My many thanks go to my major professor, Dr. Mark E. Whalon, who graciously assisted me throughout my master's program and provided a laboratory well-supplied with equipment and able staff. I also thank Dr. James E. Bath, chairman of the Department of Entomology, who created a climate conducive to creative thought and study. I thank Dr. Haggai Podoler for his guidance and help with establishing the codling moth culture. I thank the members of my guidance committee: Drs. Dean Haynes, Stanley Wellso, and James Flore for their input. I thank Terry Davis for his technical support and help with using the computer and working out the details of thesis preparation. I thank my fellow graduate student, Bob Kriegel, for his help with computers, statistics, and the codling moth model. I also thank my colleagues Carlos Esmerelda, Steven Ray, and Zhang Zeng-Min for their kind assistance and support in writing the codling moth model. I thank, too, Kenneth Dimoff for his able and ever-ready assistance in statistical matters and computer graphics. My deepest appreciation goes to the Instituto Nacional de Investigaciones Agricolas, who provided some funding for my program and who granted me a leave of absence to continue my education, and the Consejo Nacional de Ciencia y Tecnologia, who provided the majority of the funding for my master's work. Finally, I thank my wife, Maria-Luisa Kiwen, and my two darling daughters, Rocio and Isis, for their support and understanding during this long, and at times, difficult period of study and research. 11 TABLE OF CONTENTS Bag; List of Tables ................................................. vii List of Figures ............................................... xii Introduction .................................................. 1 Objectives ................................................... 4 Literature Review ............................................. 5 Nature of Diapause ........................................ 5 Function of Diapause ...................................... 6 Endocrine Control of Diapause .............................. 6 Hormones Involved in Diapause .............................. 7 Physiological and Morphological Characteristics of Diapausing Insects ........................................ 8 Suspended Growth and Development ...................... 8 Reduced Metabolic and Respiration Rate ................. 9 Increased Storage of Reserves ........................... 9 Atrophied Gonads ..................................... 10 Factors Governing Diapause in the Codling Moth ............... 10 Photoperiod in the Diapause Process ..................... ll Diapause Induction by Photoperiod in the Codling Moth ................................................ l l Diapause Termination by Photoperiod in the Codling Moth ......................................... 12 Temperature in Diapause ............... . ............... 13 Sensitive Stages for Diapause Induction in the Codling Moth ......................................... 14 iii Table of Contents (cont.) Hypotheses ................................................... Materials and Methods ......................................... Codling Moth Culture .................................. Oviposition .......................................... Egg Sanitation ....................................... Larval Development ................................... Experiment I: The Sensitive State for Diapause Induction ................................................ Experiment 2: Photoperiodic Influence on Diapause Induction ................................................. Experiment 2.1: The Induction of Diapause under Field Conditions ............................................... Experiment 3: Effect of Temperature on Diapause Induction ................................................ Experiment 4: Termination of Diapause under Field Conditions ............................................... Experiment 5: Influence of the Low Temperature and Chilling Requirements of Termination Diapause ............... Results and Discussion ........................................ Experiment I: The Sensitive Stage for Diapause Induction in the Codling Moth (Cydia pomonella L.) ............ Diapause Induction .............. . ..................... Diapause Aversion ..................................... Discussion ............................................ Experiment 2: Induction of Diapause in the Michigan Strain of Codling Moth under different Photoperiods at Constant Temperature ..................................... iv Page 16 17 17 18 20 23 25 25 28 28 32 Table of Contents (cont.) Page Experiment 2.1: The Induction of Diapause under Field Conditions ............................................... 41 Experiment 3: Effect of Temperature on Diapause Induction ................................................ 52 Experiment 4: Termination of Diapause under Field Conditions ............................................... 59 Experiment 5: Effect of Low Temperature and Chilling Requirements to Terminate Diapause ........................ 64 Complimentary Experiment: Low Temperature and Chilling Requirements Affecting Diapause Termination in a Mexican Strain of Codling Moth ......................... 78 General Discussion and Conclusions ............................. 81 Diapause Induction .................................... 81 Photoperiodic Induction of Diapause ..................... 81 Effect of Temperature on Diapause Induction ............. 32 Diapause Temperature ................................. 83 Termination of Diapause by Photoperiod ................. 83 Effect of Low Temperature on Diapause Termination ......................................... 84 Complimentary Experiment ............................ 85 Diapause Termination under Field Conditions .............. 85 Conclusions .................................................. 36 Appendix I .................................................. 88 Appendix 2 .................................................. 89 Predictive Codling Moth Diapause Model ......................... 89 Components of the Model .............................. 91 Equations and Subroutines ............................. 91 Table of Contents (cont.) gage Corrected Photoperiod by Temperature ................. 92 Diapause Induction ................................... 93 Diapause Termination ................................ 94 System Design ....................................... 94 Diapause Termination Diagram ......................... 96 Literature Cited ............................................. 112 vi LIST OF TABLES Ingredients contained in the codling moth diet (Bio-Mix # 9370). ........ . ........................................ Sensitive stage for induction and aversion of diapause in the codling moth, Cydia pomonella (L): summary of treatments. ............................................ Photoperiodic induction of diapause in a Michigan strain of the codling moth, Cydia pomonella (L) reared on an artificial diet under 16L:8D at 27°C and transferred in the third larval instar to different daylengths t 22°C for diapause induction ....................................... Effect of 16 h of light daily at 27°C on reversing the diapause process in different larval instars of codling moth larvae reared under diapause inducing conditions (121.:120 at 22°C). ...................................... .. Effect of 12 h of light daily at 22°C on the diapause induction process in different larval instars of codling moth larvae reared under nondiapause inducing condi- tions (16L:8D at 27°C). .............. , ................... Comparison of the photoperiodic induction of diapause in three strains of the codling moth with the induction in a Michigan strain from Douglas, MI. ......................... Seasonal dynamics of diapause induction in the Michigan strain of the codling moth, Cydia pomonella (L) under field conditions. ........................................ vii 19 21 38 27 30 36 10. ll. 12. I3. 14. 15. Field observations on the dynamics of codling moth diapausing larvae in an unsprayed apple orchard at East Lansing, MI (1983) ....................................... Field observations on the dynamics of codling moth diapausing larvae in infested apples into a screened cage at E. Lansing, MI (1983). .............................. . . . . Field observations on the dynamics of codling moth diapausing larvae in an unsprayed apple orchard at Walker City, MI (1983). .................................. Field observations on the dynamics of codling moth diapausing larvae in an unsprayed apple orchard at East Lansing, MI (1984). ...................................... Field observations on the dynamics of codling moth diapausing larvae in infested apples into a screened cage at E. Lansing, MI (1984). . . ............................... Field observations on the dynamics of codling moth diapausing larvae in an unsprayed apple orchard at Douglas, MI (1984). ..................................... Some statistics for the dynamics of diapause induction in the Michigan strain of the codling moth under field conditions. ............................................ Influence of the prediapause temperature on diapause induction in codling moth larvae reared under l6L:8D at 27°C and transferred in the third instar to diapause induction at 17, 22, 27 and 30°C in different daylengths. ..... viii Page 43 44 45 46 47 48 50 16. 17. 18. 19. 20. 21. Influence of the prediapause temperature on diapause induction in codling moth larvae reared under l6L:8D at 27°C and transferred in the third instar to diapause induction at 17, 22, 27, and 30°C in different daylengths. Termination of diapause in a‘ population of codling moth larvae collected in the field during the winter and placed in different photophases to resume development at 22°C.* ........ ................................... Statistics for the regression photoperiod vs 96 diapause termination in codling moth larvae collected in the field and placed in different photoperiods at 22°C to resume development. . . . . . ........................................ Effect of low temperature (2°C) and chilling requirements to terminate diapause in the Michigan strain of the codling moth in function of the time that the larvae remained in diapause (resting period). .............. Selected statistics from summary output from the regression percentage diapause termination in function of a chilling period and the time that larvae remained in diapause; the resting period (Early Generation). ................ Selected statistics from summary output from the regression percentage diapause termination in function of a chilling period and the time that larvae remained in diapause; the resting period (Late Generation). ................ ix Page 56 6O 66 69 22. 23. Selected statistics from the summary output from the regression percentage diapause termination in function of chilling period and the time that larvae remained in diapause; the resting period (Early and Late Generation5)o......e.....-...o.............oo....... Effect of low temperature on diapause termination and chilling requirements to terminate diapause in a Mexican strain of the the codling moth, Cydia pomonella (L) from Chihuahua, Mex.* . . . . . . ........... . ..................... 75 79 2. LIST OF FIGURES Effect of 12 hours of light per day on inducing diapause in different larval instars of codling moth larvae *control (complete cycle at 16 hours of light per day). . . . Effect of 16 hours of light per day on reversing diapause in different larval instars of codling moth larvae. *Control (complete cycle at 12 hours of light per day). Photoperiodic reaction of the Michigan strain of codling moth transferred in the 3rd larval instar to several daylengths at 22°C for diapause induction. ............. Seasonal dynamics of diapause in codling moth larvae at East Lansing, MI. under field conditions: Years 1983 (A), and 1984 (B). ............................................. Seasonal dynamics of diapause in codling moth larvae at Walker City, and Douglas, MI under field conditions: Years 1983 (A), and 1984 (B). ................................ Dynamics of diapause in codling moth larvae from infested apples held into a screened cage at East Lansing, MI: Years 1983 (A), 1984 (B). ..... . ...... . .......... Photoperiodic response of the Michigan strain of codling moth transferred in the 3rd larval instar to several daylengths at 22°C for diapause induction. ................... Influence of the prediapause temperature on diapause induction in codling moth larvae transferred in the 3rd xi Page 26 29 33 35 49 49 39 10. ll. 12. 13. 14. 15. 16. instar to diapause induction at 17, 22, 27, and 30°C in several daylengths. .................... . ......... . Influence of the pre-diapause temperature on the onset of diapause in the codling moth (Cydia pomonella; L.). ,,,,,,,, Effect of low temperature and chilling requirements to terminate diapause in two populations of the Michigan strain of codling moth with different resting period "early" (more than 50 days of rest), "late" (less than 50 days of rest). . . ......................................... Termination of diapause in the "early" population of codling moth larvae by the interaction of the chilling and resting period. .......................................... Termination of diapause in the "late" population of codling moth larvae by the interaction of the chilling and resting period. ......................................... Termination of diapause in the "early" and "late" population of codling moth larvae by the interaction of the chilling and resting period. ....... . . . . . . . . . . .......... Block diagram presenting factors controlling diapause in the codling moth. ..... . . . . . . ............................ Systems graph for the codling moth diapause induction SYStem.OOO ..... OOOOOOOOICOOOOOOCOOOOOO ......... O ...... Systems graph for the codling moth diapause termination System. ............................................... xii Page 55 58 7O 73 76 9O 95 97 INTRODUCTION The codling moth, Cydia pomonella (L.), is a worldwide pest. Very few other species have such a capacity for ecological adaptation and migration. Having adapted to different climatic conditions, it has become almost universally a pest of major importance in apples, pears, walnuts, and plums (Shel'Deshova 1967). Introduced into eastern North America from Europe around 1750, the codling moth reached California in 1873, initially attacking apples and pears; however, 40 years later, it was found attacking walnuts, and in 1963, it became an economic pest on plums in Fresno and Tulare counties (Phillips and Barnes 1975). A similar progression from host to host occurred in the East and Midwest, but much earlier in the 20th century. Diapause is one of the most important mechanisms responsible for the adaptiveness of the codling moth. Diapause is the annual synchronizing mechanism that allows the codling moth to adapt to the phenology of its hosts (i.e., the race of codling moth that attaCks plum enters diapause in June, the walnut race enters diapause in mid-July, and the apple race does so in late July in the same locality) (Phillips and Barnes 1975). Diapause also allows the codling moth to adapt to different latitudes in both northern and southern hemispheres (Danilevskii 1970). To breed successfully at different latitudes, the codling moth adjusts its number of generations during the growing season to synchronize with fruit development, avoiding the continuous development where adverse environmental conditions would not allow survival (Shel'Deshova 1967). In geographic zones where the climatic conditions are conducive to the development of the codling moth, the number of generations during the season is two or more. In North American climatic regions, the percentage of damage in commercial orchards increases dramatically the further south one goes, from an average 596 in Michigan, USA, to 2096 in Chihauhau, Mexico, where the benign winter, the early spring, and the long growing season permit more than two generations per year (Anon. 1982, INIA-SARH 1982). Research in the last sixty years on the biology of the codling moth and its relationship to environmental factors has permitted the development of a better approach to managing this insect using the phenological models. Forecasting models have become an important part in managing this pest, due to the ease in determining when a particular life stage of the codling moth occurs in its diverse habitats. The first phenological models were designed in 1978: the Computerized Codling Moth Model developed at the University of California (Falcon et al. 1976), and the MOTHMDL developed at Michigan State University (Riedl and Croft 1978). In 1978, the PETE model (Predictive Extension Timing Estimator) developed in a multi-species pest complex, became a powerful predictive tool in a multi-species pest complex, including the codling moth (Welch et a1. 1978). Since the PETE model is a multi-species phenological model, it is useful in managing an apple pest complex sudi as occurs in Michigan and other states in the USA. In Chihauhau, Mexico, a phenological model based on the ecological relationship between the codling moth and the phenology of the apple tree was developed and tested in 1980 (Garcia 1980) These models mainly base their predictions on information (temperature in degree-days); they do not consider such factors as photoperiod and food availability. However, the most important adaptive dimension of the codling moth life cycle, diapause, is missing. The accumulation of heat units (degree- days) governs the phenology of the codling moth only after the termination of diapause. Therefore, it is not the only ecological factor with which to synchronize the PETE model and other phenological models to the seasonal phenology of this pest. Therefore, although these models are useful in-season, such as PETE, their lack of predictive adjustment does not allow them to be able to follow the seasonal phenology of the codling moth over several years. Moreover, since the codling moth responds to ecological factors relative to the latitude, the PETE model loses its accuracy when outside of Michigan because the onset of diapause occurs in accordance with characteristic ecological factors of each latitude, i.e., temperature and photoperiod (Shel'Deshova 1967). OBJECTIVES The objectives of this research on diapause of the Michigan strain of codling moth were (1) To obtain the necessary biological information to predict the induction and termination under the ecological conditions of Michigan, USA, (2) to gain a better understanding of the effects of the interaction of temperature and photoperiod, (3) to find the sensitive stage in which diapause can be induced or averted by the interaction of temperature and photoperiod, and (4) to investigate the effects of cold temperatures on diapause termination and to establish the Chilling requirement necessary to terminate diapause. LITERATURE REVIEW NATURE OF DIAPAUSE Diapause has been defined as a state of developmental arrest that persists even when environmental conditions are favorable for growth and development (Harvey 1962). In that state, the insect survives unfavorable environmental conditions due to physiological and metabolic changes, such as low metabolic rate, greatly diminished activity, and practical cessation of development (Dickson 1949). The causes of arrested development are physiological rather than the concurrently unfavorable environmental conditions (Beck 1965); however, in some insects, the arrest is facultative, i.e., environmental stimuli signal the organism either to continue or to terminate development. In other insects, the developmental arrest is obligatory: when reared under varied environmental conditions all individuals diapause regardless of these conditions (Les 1955). The codling moth, Cydia pomonella (L.), in North America is a bivoltine insect with considerable yearly variability in the second generation emergence due to the percentage of diapause that occurs in the first-generation larvae (Riedl and Croft 1978). Diapause in this insect has been characterized by Sieber and Benz (1980), as either obligated, i.e. dependent of ambiental factors, or facultative, i.e. primarily induced by ambiental factors such as photoperiod, temperature, and food availability. FUNCTION OF DIAPAUSE The function of diapause in the insect's life cycle is "to synchronize its phenology with the natural rhythm of food and climate suitable to growth and development" (Danilevskii 1970). Regular variation in environmental conditions during the year, which occur especially in temperate climates, are the principal reason for the seasonal rhythm of development. In these regions the temperature drops during the winter below that level necessary for development and reproduction of plants and poikilothermic animals. Thus, the seasonal activity of phytophagous insects must be synchronized with the appropriate time of the year if they are to survive. So, reproductive activity must occur during the warm growing season, and diapause usually occurs during winter when the adverse environmental conditions endanger the survival of insects and their host plants (Adkisson 1965). ENDOCRINE CONTROL OF DIAPAUSE Diapause in insects occurs in several developmental stages, i.e. during the embryonic, juvenile, and adult stages. This arrest is mediated by different glands of the endocrine system (Harvey 1962). The brain-thoracic glandular system controls larval and pupal diapause, which is arrested molting. Williams (1952) describes pupal diapause as a state of endocrine deficiency resulting from a temporary failure of the brain to secrete its trophic hormone following pupation. To continue development, the pupal tissue requires the prothoracic glands' growth and differentiation hormone which is available only after the brain recovers its secretory ability and triggers the function of the prothoracic glands. These glands degenerate in most adults, which consequently do not molt. In adult females, diapause inhibits egg maturation. Here, diapause is associated with failure of the corpus allatum function (Harvey 1962). Embryonic diapause is observed in some insects such as _B_gr_n_lgi_x_ 31931 L. where diapausing eggs are produced from the interaction between the brain and the subesophageal ganglion, which secretes the egg-diapause hormone. The brain either inhibits the secretion of the hormone in insects destined to lay non-diapausing eggs or causes its release in diapausing insects (Lees 1955). The different types of diapause are controlled and associated with the action of the brain, prothoracic glands, corpus allatum, and/or subesophageal ganglion. HORMONES INVOLVED IN DIAPAUSE Some of the secretions produced by the endocrine glandular system of insects that act in bringing about diapause are the brain hormone, juvenile hormone (JH), and molting hormone (ecdyson). For example, the JH titer of the last larval instar of the codling moth determines pupation or diapause, and larval diapause is maintained by a general state of hormonal inactivity (Sieber and Benz 1980). The brain hormone is produced by the neurosecretory cells, a group of specialized neurons that occur as a major group in the pars cerebralis (Lees 1955). JH is produced in the corpora allata. These glands are derived embryologically from buds of ectoderm between the mandibular and maxillary segments. The corpora allata secrete JH, which may vary in chemical structure in different insects or even in the same species at different times (Mordue et al. 1980). The molting hormone is secreted by the prothoracic glands. In pterygota insects, the prothoracic glands secrete ecdyson in larvae: in adult insects these glands degenerate shortly after the imaginal molt (Harvey 1962). PHYSIOLOGICAL AND MORPHOLOGICAL CHARACTERISTICS OF DIAPAUSING INSECTS Insects entering diapause may differ markedly from those in continuous development both in their physiological and morphological characteristics. Among those differences noted between diapausing and non-diapausing individuals are the retardation of the gonadal development, flight muscles, digestive tract degeneration, respiration rate change, fat metabolism, water relations and cocoon structure (Hansen and Harwood 1968). Wellso (1966) presents a better classification of these differences. He points out that most of the differences can be listed in four categories: (1) suspended growth and development, (2) reduced metabolic and respiration rate, (3) increased storage of reserves, and (4) atrophied gonads in some mature larvae and diapausing adults. Suspended Growth and Development. Suspended growth and development is the most typical characteristic of insects entering diapause; however, the stage in which arrested growth occurs varies within the species. Growth may be halted in eggs when the embryo is still rudimentary, half grown or young larva fully formed but still unhatched. Some insects with a facultative larval diapause growth may be suspended in more than one larval instar (Lees 1955). Some examples of this type of diapause are the codling moth (Cydia pomonella (L.)) and the Oriental fruit moth (Grapholithg molesta Busck.). Mature larvae of these moths become dormant at the end of the summer and resume growth and development the next year during the spring (Dickson 1949). The same type of suspended growth occurs in the stalk borer (911119 spp.) aestivation—diapause and in Diatraea grandiosella Dyar. whose mature larvae enter diapause during the dry season under short-day conditions, resuming development in the following spring (Chippendale and Yin 1973). Reduced Metabolic and Respiration Rate. The arresting of growth that occurs during diapause is always associated with a marked reduction in the insects' metabolic and respiratory rate, thus larvae and adult locomotor activity and feeding declines (Lees 1955). The reduced metabolism is closely related with oxygen consumption of diapausing individuals. In the bollworm Heliothis ;e_a_ (Boddie) male pupae in diapause consume one-tenth the oxygen of non—diapausing males; females consume approximately one-fifth of that for non-diapausing females (Wellso 1966). Also, in Diatraea gandiosella Dyar. diapausing larvae have lower oxygen consumption than non-diapausing individuals. Newly diapaused larvae have an oxygen consumption of approximately 0.85 ul per h/mg weight in non—diapausing larvae (Chippendale and Yin 1973). The same phenomenon occurs in the codling moth. Rates of oxygen consumption of larvae reared under long and short photoperiods are similar during the early fifth instar: however, an oxygen consumption rate of 1/2 that of mature non-diapausing larvae is associated with mature diapausing larvae (Hansen and Harwood 1968). Increased Storage of Reserves. Insects accumulate reserves during prediapause. These reserves are stored as either lipid or protein in the haemolymph. In the codling moth, the fat content of the larvae rises from 1196 in June to 18% of the dry weight at the beginning of diapause, and the water content decreases from 7296 to 58% (Lees 1955). However, there is no significant difference in the fat content of either diapausing or non-diapausing 10 larvae (Hansen and Harwood 1968). A soluble protein accumulates in the codling moth larvae haemolymph (Brown 1980). This protein accumulates during the last larval instar and remains constant throughout diapause. Atrophied Gonads. The comparison of non—diapausing stages with diapausing ones often involves gonadal development. For example, _C_Z_h_i_lg spp. diapausing larvae and a testicular volume of approximately 0.06 mm**3, while in non—diapausing individuals the volume is about 0.20 mm**3 (Scheltes 1978). Likewise, the testes of diapausing codling moth larvae are half the size of developing larvae, and there is a cessation of gametogenesis and degeneration of spermatids (Hansen and Harwood 1968). FACT ORS GOVERNING DIAPAUSE IN THE CODLING MOTH Diapause is one of the most interesting phenomena in the life of insects. Attempts to ascertain what factors govern diapause have pointed to photoperiod and temperature as the principal environmental factors governing the diapause process. The diapause process is comprised principally of two aspects: induction and termination. Both phenomena are usually regulated by the interaction of photoperiod and temperature. 4 The codling moth, like other insects from temperate climate zones, diapauses in response to daylength and temperature. Since diapause in the codling moth occurs after the summer solstice, the codling moth larvae stop their development as a response to the short-day photoperiod, resuming development when photoperiod and temperature conditions are suitable for growth and development. ll Photoperiod in the Diapause Process. Many have described the action of photoperiod in the physiology of immature stages of diapausing insects from temperate climates (Williams 1947, 1952, Van der Kloot 1955, Cloutier 1962, cited by Adkisson 1964). Further research on the codling moth demonstrated that the presence of JH during the fifth larval instar not only induced diapause, but also caused the preparatory behavior leading to great metabolic reserves vital for diapausing insects. Photoperiod is the controlling factor in the "switching on" of this hormone. Larvae reared under short—days have a relatively high titer of JH during the fifth larval instar and, therefore, enter diapause; larvae reared under long days have no JH during the last larval instar and, therefore, pupate (Sieber and Benz I978). Diapause Induction by Photoperiod in the Codling Moth. In the codling moth, the change in daylength appears to be the principal environmental factor inducing diapause. Dickson (1949), Geier (1963), Peterson and Hamner (1968), and Hamner (1969) examined factors involved in codling moth diapause induction. These studies were conducted at different latitudes, in the USA and Australia, and also in the laboratory and the field. The results show that the codling moth diapauses when the daily photophase is equal to 12 h light. Under these conditions, 100% of the larvae diapause. As the photophase increases to more than 13 h, the percentage of diapausing larvae drops to zero, and remains at zero during further increases in daylength. Hamner (1969) pointed out that 30 min of extra light per day beyond 14 h permits normal development in the codling moth; 30 min of less light each day induces diapause, a fact which confirms that the codling moth enters diapause at a precise time each year in response to reliable Changes in the daylength. 12 Field and laboratory studies show that diapause is induced when the daily photophase is from 12 to 14 h; only a small percentage diapause at 16 and 17 h of light. However, studies conducted in the USSR involving strains of codling moth from different latitudes (Leningrad 60 N to Samarkand 39 N), revealed that, under the climatic condition of Asia, the length of day needed to induce diapause varied from 18 to 13 h (Shel'Deshova 1967). This photoperiodic difference reflects the great genetic variability possessed by the codling moth to adapt to latitudes with very diverse daylengths. Diapause Termination by Photoperiod in the Codling Moth. The influence of the daily photoperiod in terminating diapause may act as a "switch" in the diapause clock. This seasonal response of insects to changes in the daily photoperiod was studied in Pectinomora gossypiella (Saunders) and Antheraea my L. (Adkisson 1964, 1965, Williams and Adkisson 1964), and the codling moth (Peterson and Hamner 1968, Hamner 1969, Riedl and Croft 1978). Results of this research show a strong relationship between diapause termination and daily photoperiod. Fourteen hours of light daily is the transition point between induction and termination of diapause. Thus, the same photoperiod inducing diapause also terminates it. Likewise, the codling moth can recognize photophases differing by only 15 min. Therefore, insects use a biological clock to determine when to terminate diapause. The biological clock measures the components of the photocycle (light and darkness) and separates the light period from the dark period (Adkisson 1964). 13 Temperature in Diapause. Temperature appears to be the second factor governing diapause. Temperature can affect the setting of the photoperiodic clock of diapause. Constant temperatures may modify the responsiveness of insects to the critical daylength. Likewise, low or high temperatures may completely reverse the photoperiodic effect, both during diapause induction and during diapause termination (Saunders 1976). Thus, daily temperature has two principal effects: diapause induction or diapause aversion. In general, temperatures greater than 24° C avert or reverse diapause induction in most insects from temperate climate zones. Temperatures lower than the developmental threshold do not permit the complete development of diapause because the larvae enter quiescence; however, low temperatures are required for some insects to break diapause. For example, codling moth larvae require a specific chilling period during diapause to resume growth and development. Diapause induction is modified by temperatures higher than 24° C or less than 12° C (Dickson 1949, Sunders 1976, Masaki and Kikukawa 1981, Tauber et al. 1982). For the aphid Megoura M L., the critical photoperiod decreases by about 15 min for every 5° C increment (Saunders 1976). However, in some lepidoptera, such as Acronycta rumicis L., the critical daylength to enter diapause Changes by about 1.5 h with a fall of 5° C. This change occurs in nature over a period of approximately two or three weeks (Danilevskii 1965). Thus, an overall reduction of the diapause response caused by elevated temperatures could shorten the critical photoperiod value without necessarily affecting the diapause clock. Townsend (1926) found that a chilling period was necessary for the codling moth to terminate diapause. He observed that larvae need to spend some time 14 at temperatures below 10° C to terminate diapause, concluding that the preparation process for breaking diapause may be explained by an autolytic enzyme in the tissue which is activated at low temperature. Low temperature averts the effect of photoperiod. The photoperiodic reaction of §y_di§ pomonella L., Grapholitha funebrana Tr. and Chilocorus species, disappears completely after sufficient chilling, and development is renewed at any daylength (Danilevskii 1970). Peterson and Hamner (1968) hypothesized that a chilling period could influence the termination of diapause without the intervention of photoperiod. Their findings demonstrated that larvae in diapause treated for a specific period at 4.50 C broke diapause in relation to the number of days at this temperature. Two groups of larvae chilled for 130 days under short-day conditions terminated diapause in 8:16 LD and 16:8 LD at the same time and in the same proportion when transferred to 26° C under both photoperiods; therefore, Peterson and Hamner concluded that even a diapause-inducing photoperiod, such as 8:16 LD, could not restrain diapausing larvae from further development, once they had been chilled sufficiently. Sensitive Stages for Diapause induction in the Codling Moth. Sensitive stages for the induction and reversion of the diapause process have been studied in several insects. In Lepidoptera, the most common sensitive stage for diapause induction is the larval stage; however, the sensitivity in different larval 11133313 varies. For example, Carposina tmonensis (Walshingham) does not have a critical larval instar for diapause induction, and photoperiod is operative during most of the larval period (Toshima et al. 1961). Similar results were observed in 15 Grapholitha molesta (Busck) (Dickson 1949). Sometimes more than one larval instar is sensitive to diapause induction and reversion. In Crambus spp. diapause induction occurs from the 2nd to the 5th larval instars (this insect passes through 8 larval instars), but other insects have a precisely sensitive stage (Kamm I970). The most sensitive stage (3153 Lag L. is the fourth larval instar (Barker et al. 1963), and for the stalk borer (91113 spp.) it is the fifth larval instar (Scheltes 1978). Sometimes diapause induction is determined by the photoperiod in which the parents have been reared, such as the pink bollworm, Pectinophora gossypiella (Saunders). The progeny of pink bollworms reared under long-day photoperiods enter diapause when transferred to short-days. Progeny of adults reared in short-day photoperiods tend to continue development even under short- days (Lukefahr et al. 1964). In addition, diapause induced in early larval instars by exposing the larvae to short-days can be reversed for the most part by the subsequent exposure of the last larval instar to long-days (Bell and Adkisson 1964). The sensitive stages for induction and reversion of diapause of the codling moth have been little studied, and are mentioned only broadly in the literature. For example, Sieber and Benz (1980a, 1980b) surmised that diapause is induced in early larval instars by short-days, and exposing larvae to these conditions during the first four larval instars induced facultative diapause in the mature larvae. 2.1 3.1 3.2 HYPOTHESES Codling moth larvae are sensitive to diapause induction from the first to fifth larval instar. Since the solar photoperiod is the only environmental factor that changes with mathematical precision, diapause begins after the longest day under decreasing photophase, and less than 15 h light daily induces diapause in the Michigan strain of codling moth. The termination of diapause in the codling moth occurs in the spring when the daily photoperiod increases to more than 12 h light daily. Daily temperatures modify the effect of photoperiod on the induction and termination of codling moth diapause. High or low temperatures, above or below the appropriate range of the temperature that induces diapause, avert or accelerate the diapause induction in the codling moth larvae. After diapause induction, a determined number of chilling hours at temperatures lower than 4° C are required to terminate diapause in the codling moth larvae. 16 MATERIALS AND METHODS Codling Moth Culture. Codling moth larvae were collected during the summer (1983) at the Trevor Nichols Research Complex at Douglas, MI (42° 44" N latitude). These larvae were used to initiate a culture of codling moth in the laboratory. Apples infested with codling moth larvae were brought into a greenhouse at the Pesticide Research Center, MSU, and kept at 16L:8D at 24- 270 C. Toward the end of larval development, corrugated strips of cardboard were provided as a pupation site. The pupae were transferred to a growth chamber at a constant 27°C, 16L:8D, and 80-90% R.H. Oviposition. After emergence, adults were placed in 8 x 25 cm cylindrical plastic cages to oviposit. The inner walls of the oviposition cages were lined with Parafilm to provide a suitable ovipositional surface. Both ends of the cages were closed with petri dish bottoms. Circles of filter paper were placed in the bottom petri dish to prevent oviposition on it, while the center of the top petri dish was cut out and covered with a plastic grid for ventilation. Adults were nourished with a 5% sugar solution applied onto cotton dental rolls at the bottom of the oviposition cage. The oviposition cages were cooled for 3 min in the refrigerator and the inactive adults were transferred to a new cage. The Parafilm with the eggs were removed every other day and replaced with new ones. 17 18 Egg Sanitation. To combat the presence of fungi and bacteria in the culture, the eggs were washed in a 0.0596 solution of sodium hypoclorite for three minutes, and rinsed with distilled water. The eggs were counted and placed in 1 oz plastic cups for incubation. Larval Development. After hatching, larvae were transferred by brush to l oz plastic cups and placed on artificial diet. To improve the larval establishment, the surface of the diet was scratched. The cups were closed with plastic lids and placed in growth chambers. The artificial diet for rearing codling moth larvae was supplied by BIO-SERV Inc. The Bio-Mix #9370 diet (Table 1) gave a more uniform population of larvae than green apples with ‘ respect to larval size and age (Garcia, unpublished). Cups and lids were held under a UV light before each use. To avoid contamination of the culture with fungi and bacteria, the surface of the diet was held under a UV light for 30 min before placing larvae on the diet. The trays used for stocking and storing cups were sterilized with a solution of methylparaben in ethanol 90% (4 gm/250 ml). Experiment 1: The Sensitive Stage for Diapause Induction To determine the sensitive stage for diapause induction in the codling moth, two experiments were conducted: one to investigate codling moth diapause induction sensitivity in different stages or larval instars, and the second to identify which stage or larval instar is most sensitive to diapause process reversal. In the first test adults, eggs, and larvae after each molt were exposed to inducing diapause conditions (12L:IZD at 22°c) (Dickson 1949). The number 19 Table 1. Ingredients contained in the codling moth diet: BIO-MIX #9370* Ingredients Amount (gm/l) Lima bean, finely ground 142.7 Brewer's yeast 29.0 Sucrose 19.0 Methylparaben 3.0 Ascorbic acid 3.0 Linseed oil 6.4 ground, raw, wheat germ 19.0 formaldehyde . 1.0 auger 14.3 * Bio-Serv Inc., Frenchtown, NH 08825 20 of individuals diapausing in each stage was recorded. Adults, eggs, and larvae reared in diapause-inducing conditions were transferred to non-diapause-inducing conditions (16L:80 at 27°C) (Peterson and Hamner 1968) to provoke the aversion of diapause induced in previous stages. With the exception of the fourth larval instar, all instars were tested. See Table 2 for a summary of these treatments. All mature larvae that failed to pupate after one month were considered to be in diapause. The stage or larval instar in which diapause was induced or averted in 100% of the individuals was defined as the sensitive stages for diapause induction, while the stage or larval instar in which 5096 of diapause induced in previous stages was averted was termed the critical stage for diapause induction. Experiment 2: Photoperiodic Influence on Diapause Induction Codling moth larvae from a culture held under non-diapause-inducing conditions were transferred in the critical stage (see results from Experiment 1) for diapause induction to the following photocycles: 16L:80, l5L:9D, 14.5L:9.5D, 14L:10D, 13L:11D, and 12L:12D at 22°C constant temperature. The number of diapausing individuals in each treatment was recorded. The photoPeriod at which the onset of diapause occurred and the critical photoperiod for the Michigan strain of codling moth were calculated and compared with that observed in other geographic strains of codling moth (California, USA, and Canberra, Australia). Experiment 2.1: The Induction of Diapause wider Field Conditions To investigate the field-induction of diapause in the codling moth, cardboard traps were fastened to the trunks of apple trees in unsprayed orchards 21 Table 2. Sensitive stage for induction and aversion of diapause in the codling moth, Cydia pomonella (L) : Summary of treatments. Trearment Stage maintained at Stage transferred to 16L:8D at 27 °c 12L:12D at 22 °C 8 Adults Eggs,....Pupa ‘g Adults, Eggs (AB) Larva 1,...Pupa E A E, Larva 1 Larva 2,...Pupa g A E, Ll, Larva 2 Larva 3,...Pupa .§. A E, L1,...Larva 4 Larva 5,...Pupa G Control (A E, Pupa) Treatment Stage maintained at Stage transferred to 12L:12D at 22°C 16L:8D at 27 °c .§ Adults Eggs,...Pupa g Adults, Eggs (AE) Larva 1,...Pupa 3 A E, Larva 1 Larva 2,...Pupa g A E, Larva 2 Larva 3,...Pupa g A E, L1,...Larva 4 Larva 5,...Pupa Control (A E, ... Pupa) _‘ Larval instars = L1, L2, L3,..... 22 at Douglas, Walker City, and East Lansing, MI. The traps were placed during late June and checked twice a week at E. Lansing, and weekly at Walker City and Douglas. Each cocoon found was marked and dated to record the date of pupation or diapause. The number of pupae and diapausing larvae per date were recorded and graphed, and the curve of diapause induction for each generation of larvae was obtained separately. In addition, apples infested with codling moth larvae were collected weekly and placed into cardboard strips in field-cages to observe the dynamics of diapausing larvae. Thus, it was possible to gain a better estimate of the onset of diapause and its progress without the interference of predators in the field. Experiment 3: Effect of Temperature on Diapause Induction To elucidate the effect of daily temperatures on diapause induction and to more fully understand its interaction with photoperiod, codling moth larvae reared under non-diapause inducing conditions were transferred to 16L:8D, 15L:9D, 14.5L:9.5D, 14L:10D, l3L:llD, and 12L:12D photoperiods during their critical stage (see results of Experiment 2) for diapause induction under three constant temperatures: 17, 27, and 30°C. These temperatures were above and below 22°C used in the Experiment 2 to induce diapause at the same selected photoperiods. The number of larvae entering diapause per treatment were recorded. The effect of the different temperatures on the onset of diapause and on the critical photoperiod were analyzed and compared with the results of Experiment 2. 23 Experiment 4: Termination of Diapause under Field Conditions The termination of diapause was studied under field conditions and under controlled conditions in the laboratory. Field collections of diapausing larvae were made during the winter of 1983 and 1984 at Walker City, Douglas, and East Lansing, MI. The larvae collected were placed into a screened cage at the MSU- Horticulture weather station and into laboratory growth chambers under 13L:11D, 14L:10D, 14.5L:9.5D, and 15L:9D at 22°C and 80-90% R.H. After 15 days the cocoons were opened and the number of pupae (non-diapausing) and diapausing larvae were recorded for each photoperiod. The results were analyzed to find out if there was a relationship between the percentage of individuals terminating diapause and the length of the photophase. Experiment 5: Influence of the Low Temperature and Chilling Requirements to Terminate Diapause The influence of low temperature and chilling requirements to terminate diapause were investigated by placing diapausing larvae in a refrigerator at 2°C for known periods of time (20 to 65 days). A strain of codling moth was obtained from Chihauhau, Mexico (240 larvae) to compare its diapause-terminating chilling requirement with those of the Michigan strain. Treatments. Three hundred and eighty diapausing larvae were separated into two groups according to larval age or resting period (i.e., the time they remained in diapause before entering the low temperature treatment). The first group, call the "Early Population," consisted of larvae having more than 50 days of rest. The second group, "Late Population," consisted of larvae with less than 24 50 days of rest. The chilling treatments assigned to both groups were: 0, 20, 30, 35, 45, 55, and 65 days at 2°C. After completing the chilling period, each treatment was transferred to 16L:8D at 27°C to terminate diapause and resume morphogenesis and development. To avoid the varying influence of humidity in terminating diapause, the relative humidity in the growth chambers was maintained between 80-90%. Each treatment was checked after 20 days to determine the number of larvae terminating diapause. The cocoons were Opened and the number of pupae and diapausing larvae per treatment were recorded. Complimentary Experiment. Two hundred and forty diapausing larvae collected in Chihuahua, Mexico at the end of September were divided into three groups and placed in the refrigerator at 2°C for 20, 30, and 56 days, respectively to compare their chilling requirements with Michigan larvae. After completing the chilling treatment, the larvae were transferred to diapause terminating conditions (16L:8D at 27°C). The treatments were checked after 15 days, and the cocoons opened and the number of pupae and diapausing larvae were recorded. RESULTS AND DISCUSSION Experiment 1: The Sensitive Stage for Diapause Indiction in the Codling Moth (Cydia pomonella L.). Diapause Induction. The results of this experiment showed that the codling moth has five larval instars and remains sensitive to diapause induction during its entire larval stage. The sensitivity observed diminishes as the larva approaches the last instar. All larvae (n = 100) reared in non-diapause-inducing conditions (16L:8D at 27°C) enter diapause when transferred to 12L:12D at 22°C (diapause- inducing conditions) during the first, second, and third larval instars. Only 15% of the larval population (3 reps, n = 40) entered diapause when the larvae were transferred to diapause-inducing conditions in the early fifth larval instar (Figure 1) (Table 4). The fourth larval instar was not included in the experiments; therefore, no data was available. These results are similar to those obtained by Dickson (1949) who found that diapause-inducing photoperiod had some effect on Oriental fruit moth larvae, if applied at any time during their feeding period. However, there did not appear to be a critical stage for diapause induction. All the early larval instars were sensitive, and diapause was averted or induced in 10096 of the larval population. The codling moth sensitivity observed in those experiments also is similar to that found in Qaflosina niponensis Walshingham. Although there was no specific stage for diapause induction, the diapause-inducing photoperiod was operative during most of the larval period (Toshima et al. 1961). 25 26 i 100 .... 1210 g0 - E 80. i g eo1 1:. O E 40. m o m if 20. 15 04 Q0 I l T ' F isthotar 2mm adhstor 4mm 5thhstar Contol' Figure 1. Effect of 12 hours of light per day on inducing diapause in different larval instars of codling moth larvae *Control (complete cycle at 16 hours of light per day). 27 Table 4. Effect of 16 h of light daily at 27°C on reversing the diapause process in different larval instars of codling moth larvae reared under diapause inducing conditions (12L:12D at 220C). Replicates Treatment 1 2 3 4 Total Per cent Instar L P L P L P L P Population Diapause A E, lst. 00 17 00 8 - — - - 25 00.00 A E,..2nd. 1 5 2 l4 4 20 4 14 64 17.18 A E,..3rd. ll 22 6 5 6 9 3 13 75 34.66 A E,..5th. 4 7 9 6 8 2 - - 36 58.33 Control 16 00 8 00 14 00 - - 38 100.00 A E = Adults & Eggs. L = Diapausing larvae. P = Pupae. 28 Diapause Aversion. The reversion of the diapause process was also studied. Like induction, there is a gradient from the first to the fifth larval instar with respect to sensitivity. Thus, when first instar larvae reared under 12L:12D at 22°C were transferred to non-diapause-inducing conditions, all larvae (2 reps, n = 25) developed without diapausing, but 82.8% reversion occurred when the second instar was transferred (4 reps, n = 64). The third larval instar seems to be the critical stage, since a little more than 50% of a larval population (4 reps, n = 75) maintained in diapause-inducing conditions resumed development when transferred to 16L:80 at 27°C during the third instar (Figure 2). Surprisingly, even diapause in the fifth larval instar can be averted when the larvae are placed in non-diapause-inducing conditions (41.6796) (3 reps, n = 36) of early 5th instar larvae raised in 12L:12D at 22°C continued development after being transferred to non-diapause-inducing conditions) (Table 5). These results are similar to those observed in Pectinophora gossygiella (Saunders) in which diapause induced in early larval instars was averted mostly by exposing the last larval instar to non-diapause-inducing conditions (Adkisson 1964). In Pieris rape L., diapause induced from the first to the fourth larval instar was almost totally averted when larvae were exposed to non-diapause- inducing conditions. However, only 66% of diapause is averted if larvae are exposed during the fifth larval instar (Barker et al. 1963). This is quite similar to the codling moth except that in the later diapause induced in early instars is stronger than that in Pieris rape L. (see Figure 2). Discussion. The sensitivity of the codling moth to photoperiod is different from other Lepidopterans, but corresponds to insects from temperate climate 29 100., Z 9 (D E 80 > ‘ 1.1.1 a: 1.1.1 (’0 3 a 60. S C U.- O 11.1 (D 40., < .— 2 1.1.1 0 5 20 a - 0.. 0.0 , 1 I r I h I ire lnstar 2nd instar 3rd instar 4th instar 5th instar Control' Figure 2. Effect of 16 hours of light per day on reversing diapause in different larval instars of codling moth larvae. *Control (complete cycle at 12 hours of light per day). 30 Table 5. Effect of 12 h of light daily at 22°C on the diapause induction process in different larval instars of codling moth larvae reared under nondiapause inducing conditions (16L:8D at 27°C). Replicates Treatment 1 2 3 Total Per cent Instar L P L P L P Population Diapause A E, lst. 13 00 10 00 00 00 23 100.00 A E,..2nd. 13 OO 20 00 13 00 46 100.00 A E,..3rd. 11 00 13 OO 7 00 31 100.00 A E,..5th. 2 8 1 7 3 19 40 15.00 Control 00 22 00 15 00 17 54 00.00 A E = Adults & Eggs. L = Diapausing larvae. P = Pupae. 31 zones, such as Q. molesta and Carposina niponensis. All the larval instars of the A codling moth are somewhat sensitive to the induction or aversion of diapause. These results suggest that the codling moth has a facultative diapause. However, the first larval instars are most sensitive to diapause induction or aversion. Thus, exposure of early larval instars to shorter photophases and 22°C temperature directs further larval development toward diapause. Likewise, the decreasing sensitivity in the subsequent larval instars ensures that there will always be a proportion of the population in diapause. That is, if diapause were averted in the first two larval instars by environmental conditions (principally temperature), 100% of the larval population might enter diapause if the conditions were suitable during the third larval instar, and even 15% of the fifth instar population could diapause. Conversely, if codling moth larvae, reared in diapause-inducing conditions during the three earlier larval instars, are transported to a geographic area where the environmental conditions are suitable for continuing development, these larvae would resume development and diapause would be averted in at least 65% of the population. Even if those larvae were transported in the early last larval instar, 41% of the population could resume development (Figure 2). The responsiveness of codling moth larvae to changes in daylength is advantageous because the codling moth can escape adverse conditions at the end of the larval period by diapausing or by continuing development. This variability in larval sensitivity to induction or aversion of diapause could be considered an additional adaptive mechanism that might predispose either early or late instars to become responsive to diapause-inducing photOperiod (Riedl and Croft 1978). 32 Experiment 2: Induction of Diapause in the Michigan Strain of Codling Moth under Different Photoperiods at Constant Temperature (22°C) The responsiveness of the Michigan strain of codling moth to different photocycles under constant temperature was evaluated. The results demonstrate that this strain of Codling moth diapauses in photocycles that varied from 15L:9D, 14.51.9515, 141.:100, 131.:1113, to 12L:12D at 22°C (Figure 1). However, exposure to a 16L:8D photocycle at 22°C during their critical stage (see results of Experiment 1) did not induce diapause. Even larvae reared during their entire larval period under these conditions did not diapause. A photophase of 15 h of light daily induced diapause in 26% of a larval population (2 reps, = 43). The critical photoperiod was around 14 h light per day (13.58 h calculated). A 14L:10D caused 51% diapause in larvae (2 reps, n = 49) reared under 16L:8D at 27°C, when transferred to this photocycle during their critical stage (the third instar). Likewise, 13L:11D elicited 71% diapause in larvae (6 reps, n = 106) transferred to this photocycle during the critical stage. Finally, larvae (3 reps, n = 44) diapaused when larvae were exposed to a 12L:12D photocycle (Figure 3). A linear regression analysis evaluated the length of the photophase as the independent variable predictor of the percentage of diapause observed in eaCh treatment. The observed percentage of larvae diapausing was transformed using arcsin (p/100)0'5 to linearize the curve of diapause induction (Figure 3) (a standard transformation used for 96 data). The resulting equation was: E(Y)= Bo - B (X) E(Y)= 335.40 - 20.76 (X) 0.97 1' t 11.98 33 100 q! z 9 80 _ y. o a o . E 3,‘ so . a < a S a ‘5 40 d m a < ... f. o 20 .. m 1.1.1 a ° it is 15 14.5 14 13 12 PHOTOPERIOD (Hours of Light per Day) Figure 3. Photoperiodic reaction of the Michigan strain of codling moth transferred in the 3rd larval instar to several daylengths at 220C for diapause induction. 34 where: E(Y)= Expected percentage of larvae diapausing X = Length of the observed photophase. The coefficient of determination yielded by this equation (r = 0.97), explains that 9796 of the variation in predicting the percentage of diapause observed is a result of the length of the photophase. Also, the coefficient of correlation value (B) was highly significant when tested by means of the "t" test (t; a = 0.05). (See Table 3 for a summary of statistics of regression.) Therefore, a strong positive relationship exists between induction of diapause and decreasing photoperiod between 16 and 12 h of light per day. The transformed data (Figure 4) show a constant positive effect with decreasing photoperiod. Discussion. The photoperiodic reaction of the Michigan strain of codling moth is no different from that observed in the other two strains of codling moth from the USA. Using the critical photoperiod (CPh 50) as a parameter of comparison, photoperiod the critical photoperiod of the Michigan strain is 13:58 h, the CPh 50 for the Californian strains is from 13:47 to 13:45h (from Riverside, CA (34 N latitude) and Tulare, CA (36 N latitude, respectively) (Dickson 1949, Cisneros 1971). The Michigan strain was at 42.114" N latitude. However, the strain of codling moth investigated by Peterson and Hamner (1968), also from California, showed a CPh 50 of 13:30 h. Comparing the Michigan strain to an Australian strain (CPh of 14:22 h) (Shel'Deshova 1967) shows a 24 min difference. Testing for differences among strains was done by transforming data (Dickson 1949, Shel'Deshova 1967, Cisneros 1971) using arcsin (p/100)0'5 and a regression analysis on each data set (Table 6). Confidence limits (a = 0.05) were established for the regression line of the Michigan strain; confidence limits for 35 A g - 3 2° - Diapause Onset ' End tst Generation ‘ d a - a . I --“‘~ 3 - - = ....I’IIII’B g o -- WV o—O/. . < I V 1 1' r T t fl ' V l *1 r '1 fir g .. m '1 End tst Generation ° 20 ‘ iii ‘ Dis ease Onset g I o .. '/ I r 1 ' I V I 1 200 2 10 220 230 240 250 260 JULIAN DATES Figure 4. Seasonal dynamics of diapause in codling moth larvae at East Lansing, MI under field conditions: Years 1983 (A), and 1984 (B). 36 Table¢5, Comparison of the photoperiodic induction of Diapause in three strains of the codling moth with the induction in a Michigan strain from Douglas, MI. Regression coefficients Regression statistics Confidence limits for Intercept Slope r2 t (a = 0.05) B0 B1 (1) 335.76 — 20.76 0.97 11.89 266.67 - 15.91 404.12 - 25.60 (2) 390.52 - 25.04 0.90 (3) 446.97 - 28.84 0.86 (4) 328.97 - 19.75 0.90 1) Michigan strain (Garcia, 1984); 3) Riverside, CA. strain (Dickson, Shel'Deshova, 1967). 2) Tulare, CA. strain (Cisneros, 1971); 1949); 4) Canberra, Aus. strain ( 37 the coefficients of correlation (Bo and Bi) also were calculated. The regression lines and the Bo and Bi coefficients were compared for all strains (Table 3). This analysis showed that, with the exception of the regression line for the Dickson's data, the regression lines were within the confidence limits for the Michigan strain, and therefore were not different (Figure 7). Likewise, the comparison of the intercept (Bo) and the slepe (Bi) of the regression lines for the Shel'Deshov's and Cisneros' data were not different from those calculated for the Michigan strain. Several possible reasons for differences between Dickson's and the other data may include a (1) limited data base, (2) genetic difference between Dickson's strain and the others, (3) different environmental rearing conditions, and (4) nutritional effects due to Dickson's diet. Another parameter observed was the onset of diapause. In Michigan, the codling moth responds to photophases shorter than 16 h of light daily; longer photophases elicit continuous development. In the onset of diapause, the Michigan strain was similar to the Tulare, CA strain. The onset of diapause in the Canberra strain (Shel'Deshova, 1967) occurred at longer photophase than the Michigan strain: 16 h of light daily induced diapause. However, the strain investigated by Dickson 91949) responded to photophases shorter than 15 h of light daily; this photoperiod does not induce diapause in that strain, whereas one hour less (14 h light per day) causes 75% diapause. With the exception of the onset of diapause, the photoperiodic reaction of the codling moth from Michigan is not much different from that observed in other strains. However, the question remains, why does the Michigan strain have a similar CPh 50 as the Californian strains? The latitudinal difference is quite wide, almost 10, which, according to Shel'Deshova (1967) and Riedl and Croft 38 Table 3. Photoperiodic induction of diapause in a Michigan strain of the codling moth, Cydia pomonella (L) reared on an artificial diet under 16L:8D at 27°C and transferred in the third larval instar to different daylengths at 22°C for diapause induction. Z diapause Z diapause Photophase observed are sin Z 16 0 0 15 25.58 30.40 14.5 31.22 33.96 14 51.02 45.63 13 71.69 57.86 12 100.00 90.00 Statistics for the regression photOphase Vs arc sin Z diapause. Regression coefficient B1 = — 20.76 Intercept B0 = + 335.40 t for B1 = 11.89** Critical Photoperiod (CPh 50)‘ = 13:58 h ** Highly significative at a = 0.05 level. 39 100 . z . _ 100 9 E(Y). 335.40 - 20.78 (X) 680 .— 80 . . 07 § F3: 0.97 2 w 3 a so . _ 15 a i - 50 u. o 40 . I a g 20. - 12 < 0 .. 0.57 0 1y T5 14" 1'4 1'3 1'2 PHOTOPERIOD (Hours of Light per Day) Figure 7. Photoperiodic response of the Michigan strain of codling moth transferred in the 3rd larval instar to several daylengths at 22°C for diapause induction. PERCENTAGE OF DWAUSE NOUC'flON 40 (1978), should shift the critical photoperiod of the Michigan strain toward a long photoperiod by about 1.5 h in relation to that observed in the Californian strains. The onset of diapause does occur, however, at different photoperiods even among strains from the same geographical area. Cisneros (1971) reported the onset of diapause in the Tulare strain at 15 h of light daily, but Dickson (1949) reported 14 h of light per day in the Riverside strain. The latitudinal difference between these places is 2. Between Tulare, CA (36 N latitude and Douglas, MI (42.44" N latitude) there is a difference of almost 7, but the strains of codling moth display similar photoperiodic reaction. Altitude might account for this similarity: Tulare's altitude is 450 ft, while Douglas' altitude is less than 50 ft. Another point is that the CPh 50 for the Michigan strain differs with that published by Riedl and Croft (1978). These authors reported a CPh 50 of 15:36 h, while in this study, under laboratory conditions, 13:58 h was the CPh. This large discrepancy is probably because Riedl and Croft made their observations in the field rather than in the more precise laboratory regimen reported herein. Additionally, error in Riedl and Croft's observations could have come from the measurement of photoperiod. Under controlled conditions in the laboratory there is no twilight and the insect's response is to the absolute duration of the photophase, while in field observation, twilight may be considered part of the photophase. Twilight adds approximately one hour or more to the photophase depending on the season of the year. Another possible source of error is daily temperature, which can alter the expression of the photoperiodic reaction, moving the CPh 50 toward long days in a cold year or toward short days under warm years. This will be discussed later. 41 Experiment 2.1: The Induction of Diapause under Field Conditions. The Michigan strain of codling moth enters diapause after the summer solstice (June 22 - 23) under decreasing photoperiod. Observations conducted at three different localities in Michigan (E. Lansing, Douglas, and Walker City) demonstrated that the onset of diapause occurred around the second half of July (202 Julian date + 7) that corresponds to July 22, when the photophase was 14:37 h of light per day (Tables 7-13). Another parameter observed in the dynamics of diapause was the displacement of the critical photoperiod (CPh 50) around 13:58 h of light per day. Field observations showed that 50% of the larval population entering diapause occurred when the daily phot0period was + 20 min of the CPh 50 (see Table 3, Experiment 2). Furthermore, two generations of diapausing larvae occur every year; however, the size of the first generation varies depending on the date of emergence of the overwintering generation and on the prevalent temperatures during the summer. But the most important generation is the second one, since it comprises 76% + 6.36 (SD) of the overwintering population every year (see Figures 4, 5, 6). An analysis of variance was performed on the date of diapause, photoperiod at onset of diapause and the critical photOperiod. This showed that the time variability in the development of diapause in the sites sampled and between the selected localities from year to year was small (Table 14). For example, the variability observed in the onset of diapause was 3.7% when Julian Date was used as a predictor. However, this variation decreased when the photoperiod at the onset of diapause was used as the predictor. Here, the variation observed in the onset of diapause as a function of photoperiod was 1.896. In addition, the 42 Table 73 Seasonal dynamics of diapause induction in the Michigan strain of the codling moth, Cydia pomonella (L) under field conditions. Onset of diapause Onset of diapause CPh 50* Date PhotOperiod Locality 1983 1984 1983 1984 1983 1984 E. Lansing 7/24 7/12 14:33 14:54 13:21 13:47 walker - Douglas 8/5 7/16 14:09 14:46 13:16 13:28 Screened cages MSU-Horticulture 7/24 7/17 14:33 14:46 13:00 13:54 * Critical Photoperiod in hours of light daily. 43 Table 8. Field observations on the dynamics of codling moth diapausing larvae in an unsprayed apple orchard at East Lansing, MI (1983). Date Number Percentage Photoperiod 7/22/83 0 0 - 7/24/83 1 3.0 14.56 8/1/83 2 9.0 14.29 8/3/83 1 12.12 14.22 8/5/83 2 18.18 14.15 8/15/83 2 24.24 13.81 8/29/83 3 33.33 13.33 8/31/83 1 36.36 13.26 9/2/83 1 39.39 13.20 9/12/83 14 81.81 12.82 9/28/83 100.00 12.30 44 Table 9, Field observations on the dynamics of codling moth diapausing larvae in infested apples into a screened cage at E. Lansing, MI. (1983). Date Number Percentage Photoperiod 7/24/83 1 1.72 14.56 8/1/83 1 3.44 14.29 8/3/83 2 6.89 14.22 8/8/83 1 8.62 14.02 8/10/83 1 10.34 13.89 8/17/83 2 13.79 13.74 8/23/83 1 15.51 13.54 8/29/83 6 25.86 13.33 8/31/83 1 27.58 13.26 9/2/83 1 29.31 13.20 9/16/83 6 44.48 12.72 9/22/83 19 77.58 12.54 9/28/83 13 100.00 12.30 Table 10, Field observations on the dynamics of codling moth diapausing 45 larvae in an unsprayed apple orchard at Walker City, MI. (1983). Date Number Percentage Photoperiod 8/5/83 2 8.33 14.15 8/10/83 3 20.83 13.89 8/18/83 0 13.71 8/30/83 4 37.75 13.23 9/7/83 4 54.16 13.02 9/13/83 5 75.00 12.82 9/21/83 2 83.33 12.54 9/29/83 4 100.00 12.27 Table 11- 46 Field.observations on the dynamics of codling moth diapausing larvae in an unsprayed apple orchard at East Lansing, MI. (1984). Date Number Percentage Photoperiod 7/12/84 2 1.60 14.90 7/17/84 6 6.40 14.73 7/19/84 1 7.20 14.63 7/24/84 3 9.60 14.49 7/28/84 8 16.00 14.36 7/31/84 0 8/6/84 8 22.40 14.02 8/10/84 24 41.60 13.91 8/18/84 8 48.00 13.64 8/26/84 24 67.20 13.37 9/4/84 26 88.00 13.06 9/9/84 15 100.00 12.89 47 Table 12. Field observations on the dynamics of codling moth diapausing larvae in infested apples into a screened cage at E. Lansing, MI. (1984). Date Number Percentage Photoperiod 7/17/84 3 5.88 14.77 7/19/84 2 9.80 14.70 7/24/84 1 11.79 14.53 7/26/84 2 15.68 14.39 7/28/84 1 17.64 14.36 7/31/84 4 25.49 14.29 8/6/84 3 31.37 14.02 8/11/84 19 68.62 13.91 8/20/84 7 82.35 13.55 8/26/84 7 96.07 13.30 9/2/84 100.00 12.99 48 Table 13, Field observations on the dynamics of codling moth diapausing larvae in an unsprayed apple orchard at Douglas, MI. (1984). Date Number Percentage Photoperiod 7/18/84 9 0.57 14.70 7/23/84 7 1.15 14.53 7/28/84 6 1.71 14.39 7/30/84 15 3.42 14.36 8/2/84 7 6.84 14.19 8/7/84 14 13.11 14.01 8/10/84 .15 20.00 13.91 8/17/84 19 30.78 13.67 8/22/84 4 33.06 13.50 8/30/84 21 45.03 13.23 9/6/84 54 75.81 12.99 9/10/84 40 100.00 12.85 49 A 20 " End 1st Generation u: " Diapause Onset ( C > d c s ' - ‘- O 0 cl ”\0—0/ 7" g T f r r I ' f r r T T r r fi 3 - g : 3 0 4o - IL 0 1 c d g - 01898080 0080' End 1st Generation 5 2° 1 d .’. '1 / O 1 ea—-——""" 1 ' I r I 7 .I 1 I : l ' T 1 200 2 1 O 220 230 240 250 260 JULIAN ones ' Figure 5. Seasonal dynamics of diapause in codling moth larvae at Walker City, and Douglas, MI under field conditions: Years 1983 (A), and 1984 (B). A “<‘ 20 . > 1 5 Diana e Onset End 1st Generation 2; .1 1 - z -i m -‘ .N a o_ o—-—O’.—-\/ g n v 1 W T f I i 1 ‘ 1 r I 1 a u 0 _ E 20.... Diapause Onset End 1st Generation 8 '3 q i, ._ J -———. 0—4 _/\o \e -_ r 1 T * fl ' 1 , ' I - ' r f T l 200 2 10 220 ~ 230 240' 250 260 JULIAN DATES Figure 6. Dynamics of diapause in codling moth larvae from infested apples held into a screened cage at East Lansing, MI: Years: 1983 (A), and 1984 (B). 50 Table lflw Some statistics for the dynamics of diapause induction in the Michigan strain of the codling moth under field conditions. Variable Mean S. Dev Coefficient of Var. Onset of diapause 202 (J. D.)* 7 3.68 Photoperiod at onset of diapause 14:37 h 0:15 h 1.81 Critical Photoperiod 13:28 h 0:20 h 2.49 * Julian Date. 51 variation in photoperiod in which 50% of the diapausing population occurred was 2.5%. Discussion. Diapause induction in the Michigan strain of codling moth under field conditions agrees with the results of Experiment 2. The codling moth in this particular geographic area responds to photoperiods at about 15 h of light daily. This measure of light corresponds to the period of light from sunrise to sunset during the second half of July. At this time of the year diapause began with a variance of + 7 days during the two years of study. Under optimal conditions of food and temperature, the onset of diapause in the Michigan strain of codling moth occurs at 15 h of light daily (see results Experiment 2). However, in the wild populations of codling moth, the onset of diapause occurs 15 to 30 min later (14.37 h). This variation represents three to six days under field conditions, a small difference considering the complexity of uncontrolled environmental factors that potentially could affect the beginning of diapause. In mid-August (l3 - 22) 5096 of the larval population is in diapause in the field when the daylength is equal to 13:28 h. Under laboratory conditions, Experiment 2 showed that 5096 diapause occurred, in the same strain, when the length of the photophase was 13:58 h (20 minutes less than under field conditions). The variation between laboratory and field observation data are close, and the CPh 50 calculated in Experiment 2 lies within one standard deviation of the field observations (Table 14). The differences between data from laboratory and field observations may be explained by the effect of temperature during the rearing period of the 52 larvae. Temperature influences development from the emergence of the overwintering generation through the onset of diapause. Under field conditions, a cold spring and a temperate summer might delay the onset of diapause toward a short-day photoperiod because of slow larval development. Under these conditions, the expected number of generations of diapausing larvae would be one. However, a combination of a cold spring and a warm summer also might have the same effect because the high temperatures suppress the photoperiodic reaction of the codling moth and the onset of diapause is delayed (see Figure 4) toward short-day photoperiods (see results Experiment 3). Finally, a temperate spring and temperate summer enhance the development of diapause in wild populations of codling moth by shifting the onset of diapause toward a long-day photoperiod. For example, in 1984 the onset of diapause occurred at almost 15 h photoperiod: 14:54 h for E. Lansing and 14:46 h for Douglas (Table 3, and Figure 4). Under these conditions, there would be two overwintering generations. Experiment 3: Effect of Temperature on Diapause Induction The effect of temperature on codling moth diapause induction and its interaction with photoperiod showed a strong influence of rearing temperature on the photoperiodic reaction of the codling moth. Diapause had a similar development in codling moth larvae held at 220 and 27°C constant temperature. The onset of diapause occurred in both treatments when the daylength was 15 h, while their critical photoperiods differed by only 5 min. However, high or low temperatures above and below this range altered completely the expression of the codling moth photoperiodic reaction. A 5°C decrease in temperature shifted 53 the onset of diapause toward long-day photoperiod by 1 h. Also, the critical photoperiod was moved from 13:0 hrs at 30°C to 15:07 at 17°C (i.e., one hour to a short-day photoperiod or one hour to a long-day photoperiod from the intermediate critical photoperiod (13:58 - 14:03 hrs) observed at 220 and 27°C). The expected effect of temperature on diapause was observed in both situations. Under a long-day photoperiod at low (17°C) temperature, 5096 of the larval population entered diapause when the length of the photophase was 15:07 h, one hour earlier than that observed at 22° and 27°C (Table 15). Conversely, less than 3096 of the larval population exposed to short-day photoperiod (12 and 11 h light per day) at 30°C entered diapause. The CPh 50 was delayed one hour. Since the maximum percentage of larvae entering diapause was 2796 (2 reps, n = 67) at 30°C, the CPh 50 was chosen to be the photoperiod in which the onset of diapause occurred (13 h light per day). See Figure 8 and Table 16 for a summary of results. Discussion. Temperature exerts a strong influence on the diapause induction process, confirming similar studies of other species. Diapause for Oriental fruit moth resides only within the range 20° to 27°C, and diapause is avoided when exposed to both high (30°C) or low (12°C) temperatures (Dickson 1949). The flesh-fly, Sarcophaga argyostoma Saunders, diapauses when exposed to a short-day photoperiod (101:140) at 15° to 20°C. Above 25°C, however, the photoperiodic response is eliminated (Saunders 1976). Another example of the effect of temperature on diapause is found in the cabbage whitefly, Aleyrodes brassica Wlk., which enters reproductive diapause under photoperiods ranging from 16L:8D to 12L:12D at 15° and 20°C, but temperatures higher than 25°C suppress the photoperiodic induction of diapause (Uche Iheagwam 1977). 54 Table 15. Influence of the prediapause temperature on diapause induction in codling moth larvae reared under 16L:8D at 27°C and transferred in the third instar to diapause induction at 17, 22, 27, and 30°C in different daylengths. Treatment Temperature Total Per cent Photophase oC Replicates L P Population Diapause 16 30 2 0 100 100 00.00 15. 30 4 0 91 91 00.00 14.5 30 3 0 91 91 00.00 14 30 2 O 47 47 00.00 13 30 3 25 69 94 26.59 12 30 2 18 49 67 27.00 11 30 3 18 47 65 24.61 16 27 2 0 , 50 50 00.00 15 27 2 5 83 78 6.41 14.5 27 4 17 77 94 18.08 14 27 3 41 18 59 69.50 13 27 3 54 5 59 91.51 12 27 3 66 2 68 97.00 16 22 2 O 47 47 00.00 15 22 2 5 73 78 25.58 14.5 22 3 10 22 32 31.25 14 22 2 25 24 49 51.02 13 22 6 76 30 106 71.69 12 22 3 44 _ 0 44 100.00 16 17 2 6 30 36 16.16 15 17 2 24 25 49 49.00 14 17 2 4O 0 40 100.00 13 17 2 39 0 39 100.00 1 44 0 44 100.00 12 17 L = Diapausing larvae. P = Pupae. 55 ‘00 11°C 22°C 1 27% 0 . O u: is 004 3 . 3 40 f E. sot . 0 0’ \0 E? 20‘ a e ’//r e . 0 .1 o—O—O to 16 14.5 14 '13 12 11 momma (Hm or Liam oar Day) Figure 8. Influence of the prediapause temperature on diapause induction in codling moth larvae transferred in the 3rd instar to diapause induction at 17, 22, 27, and 300C in several daylengths. 56 Tablelfiu Influence of the prediapause temperature on diapause induction in codling moth larvae reared under 16L:8D at 27°C and transferred in the third instar to diapause induction at 17, 22, 27, and 30 0C in different daylengths. 0 Temperature C Photocycle 17 22 27 30 16L:8D 16.66 * 00.00 00.00 00.00 15L:9D 49.00 25.58 6.41 00.00 14.5L:9.5D - 31.25 18.08 00.00 14L:10D 100.00 51.02 69.50 00.00 13L:11D 100.00 71.69 91.50 26.59 12L:12D 100.00 100.00 97.00 27.00 11L:l3D - - - 24.61 CPh 50** ‘ 15:07 h 13:58 h 14:03 h 13:00 h * Values for incidence of diapause are given in percentage. ** CPh 50 = Critical Photoperiod. 57 For the Michigan strain of the codling moth, low temperatures (17°C) enhance diapause induction, while high temperatures (30°C) avert or diminish the photoperiodic response. Thus, the codling moth enters diapause at temperatures less than the Oriental fruit moth. The codling moth response to the interaction of temperature with photoperiod is similar to the flesh-fly and cabbage whitefly, where high temperature elicits continuous development even under a diapause- inducing photoperiod (10L:14D). The onset of diapause and the critical photoperiod of the codling moth present the same response that was observed in other insects when exposed to a diverse regimen of temperatures. A 5°C decrease in temperature moved the onset of diapause and the CPh 50 l h to a longer photophase (see Figure 9). For Acronycta m L., diapause onset and the CPh 50 increase 1:30 h for each 5°C decrease in temperature (Danilevskii 1965). The foregoing research shows that the diapause mechanism in the codling moth is strongly associated with the daily temperature at which larvae are exposed during the feeding period. This feature reflects the great adaptiveness of this insect to environmental changes. In so doing, the codling moth synchronizes its number of generations and its seasonal phenology to the rhythms of warm seasons and cold weather by moving forward or backward the critical photoperiod and onset of diapause as a function of temperature. Also, it keeps a reasonable margin of 5°C (22° to 27°C) in which diapause presides "normally" but with the option of entering diapause or continuing development, if the environmental conditions are conducive. 58 3 ‘mq d O 3 a i 'w. .1 3 0 5 O 5 M- i t r 02- 13.: O 12 4 w T T? 10 15 2O 25 30 TEMPERArunetti Figure 9. Influence of the pre-diapause temperature on the onset of diapause in the codling moth (Cydia pomonella L.). 59 Experiment 4: Termination of Diapause under Field Conditions Field observations in 1983, in the area of East Lansing, field populations of codling moth terminated diapause during mid-May. Larvae kept under conditions were checked on May 5 and 13, 1983, to observe the initiation of diapause termination. Three larvae selected at random were checked and the cocoons opened. None of the larvae examined on May 5th had initiated development. These larvae were taken to a growth chamber at 16L:8D and 25°C. Four days later, the larvae had pupated; requiring 60 DD (10°C Base) to develop to the pupal stage. New pupae were in cocoons opened on May 13. The emergence of the first moths under outdoor conditions was recorded on June 4, 1983—22 days after the first pupae were observed. The Michigan strain of the codling moth terminates diapause in the winter, long before the mild temperatures during May contribute to the resumption of growth and development. Experiments were conducted with diapausing larvae collected during the winter of 1984. Diapausing larvae held under a 13L:11D at 22°C terminated diapause in almost the same proportion as another group placed at 15L:9D and 22°C terminated diapause in almost the same proportion as another group placed at 15L:9D and 22°C (this photocycle and temperature induced diapause in 2596 of the p0pulation of the same strain). After 15 days, 7196 of the individuals (2 reps, n=28) in the 13 h of light per day treatment terminated diapause, while in the 15 h of light per day 8196 (2 reps, n=21) terminated diapause (see Table 17). . 4 Under controlled conditions, 14L:10D induced diapause in 5096 of the codling moths at 22°C (see Experiment 2), but when diapausing larvae (more than 150 days old) were placed under these conditions 8296 (2 reps, n=28) of the larvae terminated diapause, even though these were diapause-inducing conditions. 60 Tableljh Termination of diapause in a population of codling moth larvae collected in the field during the winter an placed in different photophases o to resume develOpment at 22 C .* Photophase Days at 22 C Population Z diapause termination 13 15 28 71.42 14 14 28 82.14 14.5 15 29 72.41 15 14 21 81.00 * Larvae collected at Douglas and East Lansing, MI. (1984). 61 A simple linear regression analysis was performed to demonstrate the relationship between the percent of larvae terminating diapause and the photocycle to which the larvae were exposed. The equation obtained was: E(Y) = 28.37 (X), in which: E(Y) = Percentage of diapause termination expected. X = Length of the photophase. The coefficient of correlation was r = 0.51 r=0.26 The analysis showed a relation between the length of the photophase and the percentage of individuals terminating diapause. However, when the slope (31) was tested for significance using the "t" test (a = 0.05) 31 was not significantly different from 0 (see Table 18). Thus, the termination of diapause in those larvae could not be attributed to photoperiod. Since the larvae terminated diapause regardless of the photoperiod, diapause termination was probably due to freezing temperatures in the field. Once the larvae were placed in a temperature suitable for growth and development, postdiapause morphogenesis began. Discussion. The assumption that the Michigan strain of the codling moth terminates diapause in spring is incorrect. The data presented herein show that diapause ends during the winter, while the insect is subjected to shortday periods. At the beginning of the spring, the larvae are ready to resume morphogenesis and growth. These results are confirmed by (Peterson and Hamner (1968) who showed that diapausing larvae chilled for 130 days at 4.5°c 62 Table 18- Statistics for the regression on Photoperiod Vs ‘7. Diapause termination in codling moth larvae collected in the field and placed in different photoperiods at 22°C to resume development. Coefficients Regression Intercept r t for B1 (a = 0.05) 3.54 + 26.00 0.27 1.49 NS 63 at 8L:16D also terminated diapause at 8L:16D when the temperature was raised to 25°C. The percentage of individuals terminating diapause in their sample was similar to another group of diapausing larvae chilled under the same conditions but transferred to a 16L:8D photocycle. They concluded that low temperatures affect the sensitivity of the codling moth larvae to the short—day photoperiod; therefore, diapause is terminated by either long-day photOperiods or a period of chilling. A European strain terminated diapause either when exposed to long—day photoperiods or a period of chilling, and that under short-day photoperiods, diapause termination was influenced only by low temperature (Shel'Deshova 1967). Shel'Deshova considered that a chilling treatment at temperatures between 00 and 10°C was the most effective to terminate diapause in the codling moth. This study affirms that the termination of arrested development in field populations of codling moth is entirely due to the reactivating effect of the cold temperatures prevalent during winter. Danilevskii et al. (1970) also discussed the effect of low temperature on terminating diapause in the codling moth. They believe that the photoperiodic reaction disappears completely after a prolonged period of chilling, thus morphogenesis and development may occur at any daylength. The long and cold winter that normally occurs in Michigan and in the northern USA and Canada more than fulfills the number of days or hours at low temperature required to terminate diapause in the codling moth. This explains why larvae collected in the field during the winter and placed under 13L:11D, l4L:lOD, 1441:9513, and 151:90 photocycle at 22°C pupated irrespective of the photophase. 64 Experiment 5. Effect of Low Temperature and Chilling Requirements to Terminate Diapause Diapause termination by the codling moth, Michigan strain, was studied in the laboratory. The data show that the termination of diapause is not only a function of the amount of chilling, but also the time the larvae spend under diapause-inducing conditions—from the initiation of diapause up to the time of transfer to low temperatures. The low temperature treatment had a double effect on the termination of diapause depending on the length of the treatment. A short period of chilling (less than 30 days, at 2°C) delayed the termination of diapause, whereas a longer treatment accelerated the termination of diapause directly proportional to the length of the chilling period (Figure 10). The intensity of the response to the low temperatue treatment in the early and late populations differed markedly. The early population (more than 50 days of rest) reacted to the low temperature in relation to the length of the chilling period. Conversely, the late population is (less than 50 days of rest) responsiveness to the chilling treatments was poor, and at most chilling periods tested, less than 2096 of the individuals exposed terminated diapause (Table 19). The effect of this treatment in the late population was similar to that observed in the early one, except that in the late, the percentage of individuals terminating diapause was only 1196 (n=17) after being placed in 16 h of light per day at 27°C (Figure 10). The necessity of exposing the codling moth larvae to a period of low temperature to terminate diapause is unclear. Results of this research showed that larvae induced into diapause at 22°C in a short-day photoperiod (12L:12D) 65 80 z 1 Q ... ( 5 c 604‘ Ill .- W O {D 3 E s 40 4 O I“ 0 < 'i w 204 \ 0 C Ill 0. 20A/:5\. .$:.L./. O-t 0 so as as uuuaen or one AT 2 “c Figure 10. Effect of low temperature and chilling requirements to terminate diapause in two populations of the Michigan strain nf codling moth with different resting period "early" (more than 50 days of rest), "late" (less than 50 days of rest). 66 Table 19. Effect of low temperature (2°C) and chilling requirements to terminate diapause in the Michigan strain of the codling moth in function of the time that the larvae remained in diapause (resting period). Generation Resting period Chilling period Total Per cent (Days) (Days) Pop. Diapause Early 100 20 27 33.33 Late 48 20 18 00.00 Early 56 30 17 17.64 Late 40 30 17 11.76 Early 73 35 20 45.00 Late 44 35 20 00.00 Early 73 45 20 75.00 Late 44 45 20 15.00 Early 61 45 18 27.27 Late 24 45 23 4.34 Early 71 55 22 84.36 Late 44 55 20 5.00 Early 56 65 20 100.00 Late 49 65 48 18.75 Early 91 00 38 62.52 Late 42 00 33 27.27 Late 47 00 9 20.00 Early Generation = Larvae in diapause for more than 50 days. Late Generation - Larvae in diapause for less than 50 days. 67 when transferred to diapause terminating conditions, without a previous chilling treatment, terminated diapause in a higher preportion than that observed in larvae chilled for 20 to 45 days. After 25 days in long-day photoperiod at 27°C, 62% of larvae (n=38) in the early population and 2796 (n=33) in the later terminated diapause; after 65 days, 18% of larvae (n=48) from the late population terminated diapause. When larvae from the early population were transferred to diapause-terminating conditions without previous chilling treatment the percentage of individuals terminating diapause, after 25 days under these conditions, was higher than that observed in treatments after 35 or 45 days of chilling, and even higher than the percentage obtained from the 65 days chilling treatment for the late population (Figure 10). One factor previously not considered to be important in the termination of diapause was the age of the larvae. During these experiments, however, the resting period contributed a great deal to the responsiveness of the larvae to the chilling treatment. The individuals in the diverse chilling treatments were separated in two groups early and late population. Each population responded differently to the periods of chilling, and the termination of diapause was altered significantly (Figure 10). Larvae with less than 50 days of diapause were deep in diapause and even 65 days of chilling were unable to provoke an appreciable percentage of larvae to terminate diapause. Only 1896 (n=48) terminated diapause. Conversely, larvae held more than 50 days in diapause before the low temperature treatment were sensitive to the chilling treatment and yielded a percentage of individuals ending diapause proportional to the length of the low temperature period to which they were exposed. Even in the case of unchilled larvae, the percentage terminating diapause was a function of the resting period. 68 To obtain a more comprehensive understanding of the interaction of chilling and the resting period, a multiple regression analysis was performed for each population and for both together, using these variables as predictors of the percentage of larvae terminating diapause. For the early population, the equation that predicts the end of diapause is a second order polynomial: E(Y) = Bo +82 X1+ 32 X2 + 311 X1 + B22 X2 + 512 X1X2 where: E(Y) = Expected percentage of larvae terminating diapause. Bo = 35.29 81 =-7.84 82 = 2.82 511 = 0.072 B22 = 0.019 812 = 0.057 X1 = Chilling period (days). X2 = Resting period (days). The equation explains 9496 of variability in the percentage of larvae terminating diapause as a result of the effect of the variables involved and their interaction (see Table 20 and Figure 11). The same model was used for the late population, and the equation and its values are as follows: E(Y) = Bo +31 X1 +32 X2 +B11X1+ 1322 X2 +812 X1X2 where: E(Y) = Expected percentage of larvae terminating diapause. Bo = 52.0 B1 = -2.65 32 = 0.19 69 Tablez20. Selected statistics from summary output from the regression percentage diapause termination in function of a chilling period and the time that larvae remained in diapause; the resting period.(Ear1y Generation). Variables Multiple R R Square R Square Change Significance Square Chilling 0.70838 0.50180 0.50180 0.049 Chilling 0.85854 0.73709 0.23529 0.035 Rest x Chilling 0.94353 0.89026 0.15317 0.022 Square Rest 0.96786 0.93675 0.04649 0.038 Rest 0.96893 0.93882 0.00207 0.146 * Square terms and interactions are dummy variables created to improve the 'fitness of the polynomial regression. 70 . .moauma mswumwu mam wzwfiafizu wnu mo cowuwmhmuca wzu >£ mm>hma :uoE usaawoo mo scaumasmoa :haumw: wzu SH mwsmamfim mo :owumcHEuoh .HH unawah 35$ 851 3E. BSfiHMOJOIDWWmQELX 71 B“ = 0.016 B22 = -0.036 312 = 0.035 X1 Chilling period (days). X2 = Resting period (days). This equation explains 80% of the variation in the percentage of individuals terminating diapause by the effect of the chilling and resting period and their interaction on the physiological mechanism of the diapausing larvae (Table 21 and Figure 12). A third equation was developed which included both populations. This equation improved the prediction for the late population; however, it decreased the variation explained in the early population. The new equation developed and model were as follows: E(Y) = 30 + 51M+ B2 X2 + B11x1+ B122 X1 + B211><2 X1 + 312 X1 X2 where: E(Y) = Expected percentage of larvae terminating diapause. Bo =-11.90 Bl _ 5.105 32 = 0.78 311 = -0.098 3122 = 0.00068 8211: 0.0024 312 = -O.16 X1 = Chilling period (days). X2 = Resting period (days). This equation yields a r = 0.86. Therefore, the length of the low temperature treatment and the length of the resting period as well as their EH 72 Table 21. Selected statistics from summary output from the regression percentage diapause termination in function of a chilling period and the time that larvae remained in diapause; the resting period (Late Generation). Variables Multiple R R Square R Square Change Significance Chilling 0.35863 0.12861 0.12861 0.343 Square Chilling 0.79672 0.63476 0.50615 0.049 Square Rest 0.80472 0.64757 0.01281 0.130 Rest x Chilling 0.89113 0.79411 0.14653 0.110 Rest 0.89195 0.79557 0.00146 0.258 * Square terms and interactions are dummy variables created to improve the fitness of the polynomial regression. 73 .noauoa wcfiumon new mzwaafisu oSu mo cowuumuwucfi w:u >n mm>uma :uoE mafiawou mo :Ofiumasnoa :wumH: ozu :H mmDMQMHw mo coauMCfieumH .NH wu:uwm 0. DDD§I\ 74 interaction are the factors responsible for the variance on the percentage of larvae terminating diapause, and 8696 of this variation can be attributed to these factors (Table 22 and Figure 13). Discussion. The codling moth terminates diapause with or without a previous chilling treatment. Thus, larvae need not be exposed to a period of low temperature to terminate diapause. A low temperature treatment for less than 35 days instead of triggering the end of diapause, reinforces its depth (Figure 10). However, if the chilling treatment is longer than 35 days, the cold treatment satisfies the physiological requirements of diapause and termination is accelerated in relation to the length of the treatment. These data Peterson and Hamner (1968). Shel'Deshova (1967) found the same response in the Moldavia strain of the codling moth, as did Sieber and Benz (1980) who studied a Switzerland strain. Sieber and Benz defined the chilling period to which the larvae were exposed as "reactivating incubation." Some researchers have alluded to the importance of how long the larva remains in diapause before it becomes sensitive to the factors that cause diapause termination (Geier 1963, Cisneros 1971), but only Sieber and Benz (1980) documented it. Geier (1963) identified three factors as determiners of the mean emergence date: (1) the conditioning regimen, (2) the chilling regimen, and (3) the dormancy-ending regimes. Geier observed that when the conditioning and chilling regimen were insufficient, the mean date of emergence of the codling moth overwintering generation was delayed. The "conditioning regimen" meant the period of time that the larvae was in diapause-inducing conditions before the chilling treatment. Cisneros (1971) observed that 61 to 8496 of a California 75 Table 22. Selected statistics from summary output from the regression percentage diapause termination in function of a chilling period and the time that larvae remained in diapause; the resting period (Early and Late Generations). Variables Multiple R R Square R Square Change Significance Chilling x Restz 0.63059 0.39764 0.39764 0.007 Rest 0.69599 0.48440 0.08676 0.010 Rest x Chillingz 0.78061 0.60935 0.12496 0.005 Rest x Chilling 0.87157 0.75964 0.15028 0.001 Chillingz 0.89181 0.79533 0.03569 0.002 Chilling 0.92788 0.86095 0.06593 0.001 * Square terms and interactions are dummy variables created to improve the fitness of the polynomila regression. 76 .moauwa mcfiumwu mam waHHHfizu mcu mo cowuomnoucw mnu %n mm>uma :uoe mnwacoo no sowumasmon :mumH: mam :haumm: ecu :H mmstMHm mo cowumsfiaume ass 868 .50. IiNHWflleKflNMNEfl.8 .nH whamfim 77 strain reared on apples terminated diapause in a long-day photoperiod, depending on the age of the larvae. Also, when chilled larvae were placed in dormancy— ending conditions, the percentage of larvae terminating diapause was a function of the age of the larvae (the resting period). He concluded that the older the diapausing larvae the higher the percent of larvae termining diapause with or without chilling. Sieber and Benz (1980) said that the termination of diapause in the codling moth was a function of: (l) the rearing temperature during the larval period, (2) the duration of the preincubation period, (3) the period for which the larvae are chilled (the reactivating period), and (4) the complimentary incubation (the terminating diapause conditions). They reported that after a 90 day preincubation period, all adults emerged between 16 and 36 days when diapausing larvae were transferred from short-days at 19°C to long-days at 26°C. Conversely, only 32% of the population emerged after 30 days when the preincubation period was 30 days. Under natural conditions, diapausing larvae remain in diapause-inducing conditions from the onset of diapause in mid-July to the beginning of the spring. This period varies for different regions. In the northern USA and Canada, diapausing larvae remain in diapause until mid-spring or May. The time that the larvae remain in diapause-inducing conditions exceeds the resting time required for the larvae to become sensitive to the factors that promote the termination of diapause. In addition, the long winter ensures sufficient chilling to cause the termination of diapause long before the daily temperatures reach the developmental threshold required by the codling moth to resume growth and development. 78 Complimentary Experiment: Low temperature and Chilling Requirements Affecting Diapause Termination in a Mexican Strain The exposure of diapausing larvae from the Chihuahua strain of codling moth to low temperature treatment resulted in a rapid termination of diapause after the transference to 16L:8D at 27°C. There was no difference in the percentage of larvae terminating diapause among the chilling periods to which the larvae were exposed. After completing the chilling treatment, 14 days at 16L:80, and 27°C was great enough to terminate diapause in all larvae, regardless of the length of the chilling period to which they were exposed. Discussion. These results demonstrate a large difference between the Mexican and Michigan strains. The Mexican strain was more sensitive to the effect of low temperatures than the Michigan strain, and its chilling requirements to terminate diapause were much less in comparison with the requirements in the Michigan strain. The Mexican strain of the codling moth required only 20 days of chilling at 2°C to terminate diapause in 100% of the larval population, and further increments in the period of time at low temperature did not change the termination of diapause (Table 23). The same period of chilling in the Michigan strain, however enhances the termination of diapause in only 33% of the population. Also, the exposure of this strain to short periods of chilling (20 to 35 days) reinforces rather than triggers the termination of diapause (see Figure 10). In comparison, the Michigan strain required a chilling period of 65 days to terminate diapause in 100% of the larval population. In spite of these findings, there is the possibility that the Mexican strain of the codling moth does not require low temperature to terminate diapause, and 79 Table 23. Effect of low temperature on diapause termination and chilling requirements to terminate diapause in a Mexican strain of the codling moth, Cydia pomonella (L) from Chihuahua, Mex. * Chilling period Population Z Diapause termination 20 Days ' 44 ’ 100 35 Days 40 100 44 Days 20 100 * Larvae chilled at 2°C and transferred to 16L:8D at 27°C to resume development. 80 diapause termination in this strain would occur only by the effect of photoperiod; however, no supporting data are available. This experiment relates to the theory that geographic areas are important to diapause termination. The short period of chilling that the Mexican strain requires agrees with the short winter of northern Mexico, the apple growing area, where the number of days with freezing temperatures is usually less than 40. In Michigan, the number of days with freezing temperatures exceeds 100; thus the Michigan strain requires longer periods of chilling to ensure that no reactivation will occur in autumn before winter occurs. GENERAL DISCUSSION AND CONCLUSIONS Diapause Induction Sensitive State for Diapause Induction an Reversion. To evaluate diapause induction in the codling moth, it was necessary to answer: (1) what is the sensitive stage for diapause induction, and (2) can diapause be induced or averted? The answers provide basic biological information for a predictive model on diapause induction and termination under field conditions. In this study the codling moth remains sensitive to photoperiodic induction of diapause and reversion throughout its larval period. The first larval instar is the sensitive stage in which 100% diapause is induced or may be averted, while the third instar is the critical stage in which a little more than 50% diapause can be reversed by the action of photoperiod and temperature. Also, the early fifth larval instar is susceptible to induction or reversion of diapause, but with much diminished sensitivity. Photoperiodic Induction of Diapause. In nature, the last variable environmental factor signaling the end of the growth seasons is photoperiod. Every year the earth passes through the Summer Solstice, the longest day at all latitudes; with astronomical precision. In the Northern Hemisphere, this day corresponds to June 22-23. After the longest day, photophase decreases steadily until the shortest day at the Winter Solstice. Photoperiod plays a major role in the adaptation of insects to diverse ecological environments and in the synchronization of insects with their food source. 81 82 Diapause in insects may be induced by the action of photoperiod. This has been established as a general law of facultative diapause in insects (Lees 1955, Adkisson 1964, Danilevskii 1967, Saunders 1976). In the codling moth, photoperiod is the principal stimulus that initiates diapause (Dickson 1949, Peterson and Hamner 1968, Hamner I969). Codling moth larvae in Michigan are induced into diapause when the daylength is 15 h of light per day, around July 22. The length of the photophase is measured from sunrise to sunset, not including twilight. The responsiveness of the Michigan strain of codling moth to the period of light between these limits does not mean that the twilight period is not important in other biological functions of this insect. However, the field observations and the laboratory data herein discussed are in close agreement with this statement. Effect of Temperature on Diapause Inchction. The effect of temperature on diapause has been well documented (Lees 1963, Danilevskii 1967, Saunders 1976). Most research has been conducted in controlled conditions, to separate the effect of temperature from photoperiod. Temperature effects on diapause induction for the codling moth have been little studied (Shel'Deshova I967, Sieber and Benz 1980). The research presented in this thesis shows that temperature strongly influences diapause induction in the codling moth. In the Michigan strain, diapause in the laboratory occurred in the range of 220 to 27°C. However, 30°C suppressed the photoperiodic induction of diapause, and less than 30% of the larval population diapaused, even under a 11-12 H photoperiod that usually induced 100% diapause. Conversely, low temperature enhanced diapause induction, and 17°C caused the onset of diapause to occur one hour earlier in long-day photoperiod. 83 My research showed that a 5°C decrease in temperature shifted the onset of diapause and the response to the critical photoperiod toward a long-day photoperiod by one hour. Whereas, a 5°C increase in temperature shifted the onset of diapause and the CPh 50 toward a short-day photoperiod by one hour. This research has shown the following: 1. The codling moth is sensitive to photoperiodic induction of diapause from the first to fifth larval instars, and diapause in the Michigan strain of the codling moth is facultative throughout the larval period. 2. The Michigan strain of the codling moth responds to daylength, and diapause is induced by photophase less than 16 h of light daily. Half of the overwintering generation diapauses when the daily photophase is about 13:58 h. 3. Photoperiodic induction of diapause in the Michigan strain of the codling moth is altered by the effect of temperature, and the onset of diapause and the CPh 50 could be shifted backward and forward to shorter or longer photoperiods, by about-12 minutes per 1°C increase or decrease in the temperature during diapause induction. Diapause Termination. Photoperiod and low temperature are the principal environmental factors provoking diapause termination under natural conditions (Beck 1968, Tauber and Tauber 1976, Danilevskii 1970). Other factors as available moisture, relative humidity, and similar factors are also important. Termination of Diapause by Photoperiod. The photoperiodic termination of diapause may occur in insects under controlled conditions, but the time required to resume morphogenesis and development is usually longer than that observed in 84 insects chilled (Beck 1968). However, some studies suggest that field populations often terminate diapause sometime during the winter, but no one specific stimulus was found to actively end diapause (Tauber and Tauber 1976). In the codling moth, studies conducted with different geographic strains have shown that termination of diapause only by photoperiod does occur (Peterson and Hamner 1968, Shel'Deshova 1968, Sieber and Benz 1980). The Michigan strain showed a similar response. It also showed that diapause could be terminated by the influence of photoperiod without a previous chilling treatment. Effect of Low Temperature on Diapause Termination. Insects from temperate climate regions that enter diapause in late summer resume development only after the photoperiodic induction of diapause has been suppressed by the low temperatures that occur in winter (Lees 1955, Beck 1968, Danilevskii et al. 1970). The effect of low temperature on the codling moth diapause has been studied in different geographic strains from Europe and America. Results confirm that a chilling treatment enhances diapause termination (Peterson and Hamner 1968, Shel'Deshova 1967, Sieber and Benz 1980). The Michigan strain of the codling moth shows a response to low temperature similar to that observed in other geographic strains. A chilling period of 65 days at 2°C terminated diapause in 100% of a larval population after 20 days in diapause-terminating conditions. Only 62% of unchilled larvae terminated diapause after 20 days in the same conditions. However, exposing diapausing larvae to a period of chilling ranging from 20 to 35 days, the intensity 85 of diapause increased. A period of 30 days of chilling at 2°C terminated diapause in less than 20% of the population after 20 days in diapause-terminating conditions. An unexpectedly strong relationship between the termination of diapause and the time that the larvae remained in diapause before the cold treatment was discovered. Individuals in diapause for more than 50 days responded to chilling period treatments positively, and diapause termination was proportional to the length of the chilling period. However, larvae less than 50 days old usually remained in diapause. The longest chilling period (65 days) elicited diapause termination in less than 30% of the larvae treated. This response was also observed in unchilled larvae. After 20 days of long-day photoperiod at 27°C, diapause was terminated in 62% of larvae 98 days old, but only 27% diapause termination was observed in larvae less than 50 days old. Complimentary Experiment. The chilling requirements of the Michigan strain were higher than those from the Mexican strain of the codling moth. In the Mexican strain, 20 days of chilling at 20C terminated diapause in 100% of the larval population, whereas 65 days of chilling, at the same temperature, were required to terminate diapause in the Michigan strain. This wide difference in chilling requirement for diapause termination reflects the genetic differences between the two populations that come from very different environments. Diapause Termination Under Field Conditions. The end of diapause in wild populations of codling moth in the Lower Peninsula of Michigan occurs during the winter, long before the mild temperatures of the spring reactive morphogenesis 86 and development. At the end of the winter, codling moth larvae are ready to resume development if the temperatures are above the developmental threshold (10°C) even under a diapause-inducing photoperiod. Low temperatures during the winter suppress the photoperiodic reaction of the codling moth, and arrested development is maintained by the cold temperatures in early spring. CONCLUSIONS 1. Termination of diapause in the Michigan strain of the codling moth is affected by both photoperiod and low temperature. a) The Michigan strain does not require a low temperature treatment to terminate diapause. Reactivation may occur by transferring diapausing larvae to long-day photoperiods without a previous chilling treatment. b) Larvae terminate diapause even under diapause-inducing photoperiod once they have been sufficiently chilled, and a chilling treatment elicits a higher rate of diapause termination in treated larvae than in untreated ones. 2. The effect of low temperature and chilling requirements to terminate diapause in the Michigan strain differ from those of the Mexican strain. a) The Mexican strain requires 20 days of chilling to terminate diapause, while the Michigan strain requires 65 days of chilling at 2°C. 3. Diapause termination a function of larvae age (resting period). Diapause termination occurs faster in larvae older than 50 days than in younger larvae. Since this occurs in both chilled and unchilled larvae, some sort of physiological process must be completed before the larva becomes sensitive to diapause terminating factors. 87 4. Termination of diapause in field populations occurs during the winter, while codling moth are still under a short-day photoperiod. This is possible due to the cold temperature and/or the long resting period during the overwintering season. 88a APPENDIX 1 Record of Deposition of Voucher Specimens* The specimens listed on the following sheet(s) have been deposited in the named museum(s) as samples of those species or other taxa which were used in this research. Voucher recognition labels bearing the Voucher No. have been attached or included in fluid-preserved specimens. Voucher No.: 1934-4 Title of thesis or dissertation (or other research projects): PREDICTION OF INDUCTION AND TERMINATION OF DIAPAUSE IN THE CODLING MOTH (Cydia pomonella (L.) Lepidoptera: Tortricidae) Museum(s) where deposited and abbreviations for table on following sheets: X’ Entomology Museum, Michigan State University (MSU) Other Museums: Investigator's Name (3) (typed) Carlos Garcia Salazar Date 11/8/84 *Reference: Yoshimoto, C. M. 1978. Voucher Specimens for Entomology in North America. Bull. Entomol. Soc. Amer. 24:141-42. Deposit as follows: Original: Include as Appendix 1 in ribbon copy of thesis or dissertation. Copies: Included as Appendix 1 in copies of thesis or dissertation. Museum(s) files. Research project files. This form is available from and the Voucher No. is assigned by the Curator, Michigan State University Entomology Museum. 88b APPENDIX 1.1 Voucher Specimen Data Page 1 of Pages ._l__ puma unflnymw. vm\m\HH mama 86.83 88% as? a: xonOEOucm muwmuo>ficb oumum cmwficufiz ecu :« ufimonmv pom mamEHumam woumfia m>onm msu pm>wmomm Vlvmmm .oz umnoso> Apoamuv Hmumwmm cannon mONHmo Amvmsmz m.uoumwfiumm>cH Akummmoooc ma mucosa HmEOfiuwpvm omav Mm.umm.&mwmszms..mas Mozummzmm mmmwowHuHOE "mumuQOpwmun mm: v v V mm m :mmwnuHE .mmwmzon «.9» wwwmcosom mwmmu 460+ pmuwmonop can pom: mo pmuumaaoo coxmu Honuo no mmaumam m e r r m m e .m % mcwEHooam you pump Henna erOdEIIBPWS S e D.e .n u u D. m .5 u.n e t t .d .d u v. a as M w.d.1 Au .5 1n D. N .L “n "mo ponasz APPENDIX 2 PREDICTIVE CODLING MOTH DIAPAUSE MODEL The codling moth seasonal phenology can be predicted with more accuracy by incorporating a diapause model in the predictive models actually used in pest management. Since diapause is the natural synchronizing mechanism coordinating the phenology of the codling moth with its host's phenology, induction and termination of diapause are key events to initialize a predictive model. Thus, the actual prediction (based on the accumulation of heat units) can be improved a great deal, and crop damage can be predicted more accurately. The predictive Codling Moth Diapause Model (CMDM) was developed from biological information available in the literature and from our research. We first identified the factors governing diapause in the codling moth. Next, we established the causal relationship of these factors with the biology of the codling moth, and determined how they interacted to induce and terminate diapause in natural conditions. Biotic and abiotic factors governing diapause in the codling moth are presented in Fig. 14. The principal factors governing diapause of insects as genetics, food availability and quality, population density, photoperiod, and temperature. Some of these factors only influence a specific phenological stage. These factors are dynamic, and the intensity of their influence varies with time and other environmental factors. In addition, the insect's physiological and morpohological changes modifies their response to environmental stimuli; very often their response is a function of physiological age. Thus, a continuous quantification of the insect's response to these stimuli throughout its life cycle is required if an accurate predictive model is to be built. 89 90 “mm” ran" , 6 r Larval period In. to 5th. DIEM] Figure 14. Block diagram presenting factors controlling diapause in the codling moth. 91 The model developed herein predicts the induction and termination of diapause in wild codling moth populations in Michigan. The prediction is based principally the knowledge of the specific interaction between the two main factors governing diapause: photOperiod and temperature. Components of the model. The components of the CMDM are (1) a table of values for the longest and shortest day at latitudes from 30 to 43 N latitude used for calculating the daily photoperiod, (2) the PETE model life table used to predict the codling moth phenology and the number of larval generations, (3) a subroutine used to calculate degree days and chilling units (Welsh et al. 1978, Cage and Haynes 1977), and (4) a diapause table that calculates percentages of fifth instar larvae entering diapause as a function of the daily photoperiod and its interaction with temperature. Equations and Subroutines. In calculating the daily photoperiod, the following equation were used: 52 = (175-JD) * $10pe + longest ph for JD < 175 $2 = (JD-175) * slope + longest ph for 175 < JD < 358 $2 = (365-JD+174) * slope + longest ph for JD < 358 where; 52 = Daily observed photoperiod slope = (shortest photoperiod - longest photoperiod)/ 174. where shortest and longest photoperiod are given by the chosen latitude. JD = Julian Date. longest ph = longest photoperiod. 92 These equations calculate the daily photoperiod, which is used to estimate percentages of diapausing larvae entering diapause or terminating diapause as a function of daylength. The estimated values of photoperiod are used to find the percentage of diapause induction or termination only where the daily average temperature is from 22° to 27°C. Otherwise, the daily photoperiod is over— compensated for by the effect of temperature. The modified photoperiod was used to predict the percentage of diapause induction as a function of photoperiod and its interaction with temperature. Corrected Photoperiod by Temperature. An increase or decrease in the prediapause temperature above or below the range of 22° to 27°C modifies the photoperiodic reaction of the codling moth larvae, and the onset of diapause and the percentage of larvae entering diapause are shifted toward short or long days by 1 hour for every 5°C change. This modification is expressed by the following equations: 1) Corrected Photoperiod = 52 + Ph where: 52 = Observed photoperiod Ph = Compensation by temperature 2) Ph=(To-T1* 11 where To = Temperature thresholds (22° and 27°C) T1 = Daily average temperature (maximum 4» minimum)/2 u = Constant 93 The values for Ph can be positive, negative, or zero depending on the average temperature. 50 that: -Ph for T1 F To = 22°C +Ph for T1 > 1' = 27°C 00 for T1 = To < 27°> 22°C These equations estimate the photoperiod compensated by the effect of temperature used to predict the appropriate percentage of diapause induction as a function of photoperiod and its interaction with temperature. Diapause Induction. The prediction of the beginning of diapause and the percentage of larvae entering diapause was obtained in Experiment 2. From that, it has been shown previously that some Michigan codling moth diapause when the daylength is equal to or less than 15 h of light per day with 100% diapause occurring at 12 h of light per day. Also, as was shown in Experiment 3, diapause occurred within those limits when the rearing temperature was between 220 to 27°C. From this information, a table with the percentages of fifth instar larvae diapausing between 15 and 12 h of light per day was calculated on a daily basis. This table and the equations to estimate the daily photoperiod and its compensations by temperature were used to calculate diapause induction. Predictions were made by matching the daylength observed or compensated by temperature with their corresponding value for diapause induction in the table, where: Percent Diapause Induction = 0.0 for $2 > 12, Percent Diapause Induction = Table for 12 < 52 > 15. 94 Diapause Termination. The termination of diapause was predicted by the model in two ways: (1) by the action of the accumulation of chilling units or (2) by the effect of the daily photoperiod (increasing daylength). The use of chilling units accumulated during the autumn, winter, and spring is a good estimator of the end of diapause in wild codling moth populations from Michigan, USA. Results of Experiments 4 and 5 showed that codling moth larvae terminate diapause during the winter due the effect of the low temperatures, and that exposure to more than 55 days at 2°C terminate diapause in codling moth larvae. Thus, after 800 h at temperatures lower than 4°C, the model predicts the end of diapause, and post-diapause development is predicted by the degree days accumulation above 10°C, the developmental threshold of the codling moth (Riedl and Croft 1978). When the chilling requirements were not fulfilled, the termination of diapause, as observed in Experiment 5, occurred by the action of photOperiod by reversing the short-day diapause inducing photoperiod to long-day diapause- ending photoperiod. Therefore, the percentage of larvae terminating diapause was calculated by using the inverse of the diapause induction equations, where: Percentage of Diapause Termination = 0.0 for 52 < 12, Percentage of Diapause Termination = 100.0 for $2 > 15, Percentage of Diapause Termination = Table for 12 > 52 < 15. System Design. The CMDM has two basic designs: (1) the diapause induction diagram and (2) the termination diapause diagram. Inputs for the diapause induction diagram (Fig. 15), are the age (x) of the larval population, the photoperiod observed at time (t), and the temperature (maximum and minimum) 95 (51) 5th 1nmr__,L larvae. ($3) Taper-attire— r—sml IO (52) PMWPIPIO‘ . o ' E —> “zit-U. .11 a" H— a,(e-I) ‘ L______1 Larvae in R1 Quiescence 01 apauafng n2 Larvae in .1 Dave! ell-ant 3 Figure 15. Systems graph for the codling moth diapause induction system. 96‘ observed at time (t). Depending on the interaction of photoperiod with temperature, there are three possible outputs generated in this system: (1) % of larvae in quiescence (R1) that occurs when the daily average temperature is lower or equal to the developmental threshold, (2) % of larvae pupating (R2) that occurs when the daily average temperature is higher than 30°C, and (3) the percentage of larvae entering diapause R3) that occurs when the daily photoperiod and temperature are within the range that induces diapause. Considering the larval population of age (X) as S 1, the observed photoperiod as $2, and the temperature as 53, the input-output equation representing the model of diapause induction is: S1+SZ + 53 + 52(33) = R1 + R2 + R3 Diapause Termination Diagram. A diagram representing the termination of diapause in codling moth larvae requires four inputs (Fig. 16): (I) the diapausing larval population coming from the first and second larval generations, (2) the daily photoperiod at the chosen latitude, (3) the current temperature transformed into chilling and heat units, and (4) the relative humidity which provides the necessary water to restore the water balance indispensable to reactivating the metabolism of the larva. These mentioned stimuli interact to generate a single output: diapause termination. The equation that describes the system is: Y (t) = L (t-l) + NH) + C (t-l) + H(t-1), 97 Relative "rid“: Diapausing Population ’ O I Photoperiod 1(M) ,___J, Di aoause Tami nation OCH" P riod ' . (Barron-.W‘ ' ‘5‘” Figure 16. Systems graph for the codling moth diapause termination system. 98 where: Y = Percentage of larvae terminating diapause at time (t) L = Population of diapausing larvae at time (t-l). P = Observed photoperiod at time (t-l). C = Temperature (maximum and minimum) at time (t-l). H = Relative humidity at time (t-l). 99 Program SYSB43: Var Dtable : arraytl..65,1..2]of real: EggTable,LarvaeTable,FifthTable : arraytl..180Jof real: PupaeTable,RdultTable : arrayfl..180]of real: lndexTable : arraytl..180]of integer; Short,Long : arrayt30..433of real; Php,Maxtemp,Mintemp : arraytl..365]of real: newpop,pop,dgen1,dgen2,first,year,gen,h,i,J,JJ,k,21,za,23 I integer; perbroke,broke,chillday,accch1,accch2,induce,fifth,totper : real: accdd,d1,d2,d3,degday,ph,temp,perdiap : real; ovip,eggsl,hatch,larvae1,pupael,emerge,preov,aging,adult1 : real; Latitude,eggs2,larvae2,pupae2,adult2 : integer; come,go,UTHR_STRING : string: lfile,lfn,local : text: answer : char: begl,bega : boolean: Function DDiMax, {maximum temperature for the day} Min, (minimum temperature for the day) Base, {lower threshold for the calculation} Upper, (upper horizon threshold} Uppera areal) {upper vertical threshold} : Real: {THIS FUNCTION CRLCULRTES THE DEGREE-DAYS BETNEEN MAX 9ND MIN BY THE B-E METHOD. CURVE 15 TEMP I G*COS(B*TIME) + C HHERE A ' 9MPLITUDE = (max-n1~)/2 B I FRED B 2*91124 C 8 RVG I (MRX+MIN)/2 } Const DI-3.141593 Var 9,8,6, {parameters of the above curve) L1,L2,L3: real: {intersections of these thresholds) Function Acosicosine : real):rea1: {calculates the arc-cosine of a value} Var Acostmp: real: (in radians} Begin If cosine - 0 then acostmp:=PI/2.0 Else if ABSicosine) > 1.0 then acostmp:-0 {test if legal} Else acostmp:=atan(SGRT(1-SGR(cosine))lcosine): If acostmp ( 0 then acosxe acostmp + 91 Else acosr-acostmp: End: (function acostmp) Function CurvintiS, {starting value) Fzreal) (finishing value} :real: (function integtes the curve between the starting and ending values} Begin Curvint:8(A/B*(sin(B*F)-sin(8*5))+(C-base)!(F-S)1/12.03 100 End: (function curvint) Function Cnstintis, (starting value) Fireal) (finishing value) :real: (function integrates a constant between the starting and ending values) Begin Cnstint:IiUpper-Base)*(F-S)/la.0: End; (function cnstint) Function CurvinviTnpcreal) :real; (calculates the inverse of the curve to find the time a temperature was) Begin Curvinv:-acos((TMP-C)/A)/B: End; (function curvinv) Begin (function DD) (test for special conditions) If Max (- Base then 00:80.0 Else if (Upper? ) 0) and (Min )8 Upperal and (Max )- UpperE) then DD:=0.0 Else (ok to calculate) begin (get curve parameters) fls- (Min-Max)/2.0: 88' 91712.0: C:- (Min+Max)/2.0: (find the intersection points of the thresholds) If Min ( Base then Ll :- Curvinv(Base) Else L18-0: If (Upper ) 0) and (Max ) Upper) then begin If n i) 0 then begin If Min ( Upper then L2:= CurvinviUpper) Else L2:-0: end Else L2:-0: end Else L2:-12.0: If (Uppera ) 0) and (Max ) UpperZ) then L3:= CurvinviUpperE) Else L3:-12.0: (calculate the degree-days) DD:=Curvint(Ll,L2) + Cnstint(L2,L3); end: End: (function DD) Function Chilling(max,min : real) : real: Const pie-3.14: Var amp,sum,x,y : real: Begin 101 (a This procedure will calculate the number of a} (s chilling units on the given day using the a) (* max and min temperature to make a sine curve 5) (i and integrate over the curve with the upper s} {a boundary of 4C and lower boundary of 0C. 9) maxa-(max - 32) O (5.0 9.0); mini-(min - 32) e (5.0 I 9.0)1 ampxemax-min: sums-0.0; xi-0.0: ifimin ( 4)then begin repeat y:=amp e sin(x) + min: if(y ) 0)then begin if(y ( 4)then begin yx-(y + (amp 8 sin(x + 0.05) + min))/2: sum:-sum + ((4 - y) a 0.05); end: end else begin sums-sum + 0.2: end; xa-x + 0.05: until(x )- pie): end: Chilling:-sumg End: (a Procedure Chilling *) Procedure lnit_Fifth: Begin (* This procedure will read the important data from *) (* the data table generated by the PETE model. a) for Ji-l to 10 do begin . EggTableIJJ:-0.0: AdultTabletJJ:-0.0g lndexTableEJJ:-(J - 1) a 20: if(J ( 6)then ' begin LarvaeTableEglxsl00.0: PupaeTableEJJ:-0.0: end: end: ‘ LarvaeTableIBJ:3Bl.5: LarvaeTableE7J:-68.4: LarvaeTableEBJ:-S7.3: LarvaeTablet91:-52.4g LarvaeTablet10J:-47.B: PupaeTableIGJ:-18.5: PupaeTableE7ls-31.6: PupaeTableEBJ:-42.6: 102 PupaeTableESJaI47.4; PupaeTableEl0J:I51.7; JxIll; reset(lfile,’fl5:cm.text'l; read(lfile,k); if(k I llthen begin read(lfile,i,ovip,eggsl,hatch,larvael,fifth,pupael,emerge); readln(lfile,preov,aging,adultl); end else begin read(1file,ovip,eggsl,hatch,larvae1,fifth,pupae1,emerge); readln(lfile,preov,aging,adultl); end; - EggTableEJ18I-l.0; LIerIleletJll'-l.$$ PupaeTableIJliI-l.0; RdultTabletJJ:I-1.0; FifthTableIglxI-1.0; repeat {s Read the degree day value into IndexTable. a) ifik I 1)then begin lndexTableEJJ:Ii; end; (s Read the fifth instar abundance into FifthTable. *) ifik I 2)then begin FifthTabletglfoifth; end; {a Read the egg abundance into EggTable. *)- if((i )I 220)and(i ( 1040)and(k I 2))then begin EggTabletglsIeggsl; end else begin ((i )I l040)and(i ( l440)and(k I 3))then begin EggTableEJJ:Ieggsl; end else begin ~ if((i )I 1440)and(k I 8))then begin EggTabletglrIeggsl; end else begin EggTableEJJ:I0.0; end; end; . end; (a Read the larvae abundance into LarvaeTable. *) O if((i (I 450)and(k I 1))then begin LarvaeTabletglxIlarvaei; end else 103 begin if((i ) 460landii (I 1420)and(k I 2))then begin LarvaeTabletjllearvael; end else begin if((i ) l420)and(i (I 1540)and(k I 1))then begin LarvaeTabletgliIlarvael; end else begin if((i ) 1540)and(k I 2))then begin LarvaeTableEJJIIlarvael; end; end; end; end; (s Read the pupae abundance into PupaeTable. *) if((i (I 860land(k I 1))then begin PupaeTableEJJ:Ipupae1; end else begin if((i ) 860)and(i (I l420)and(k I 2))then begin PupaeTabletglzIpupael; end else begin ' if((i ) l420)and(i (I 1940)and(k I l))then begin PupaeTableEJJ:Ipupae1; end else begin if((i ) 1940)and(k I 2))then begin PupaeTabletjlxIpupael; end; end; end; end; (I Read the adult abundance into AdultTable. *) if((i (I 1120)and(k I l))then begin AdultTableCalxIadultl; end else begin if((i ) 1120)and(i (I l420)and(k I 2))then begin RdultTabletJl:Iadultl; end else begin - if((i ) l420)and(i (I 2220)and(k I 1))then begin AdultTableIJJIIadultl; end else begin if((i ) 2220)and(k I 2))then begin AdultTableCJJ:Iadultl; end; end; end; 104 end; if((EggTable[JJ () -l.0)and(LarvaeTabletJJ () -l.0)and(PupaeTableIJJ () -1.0)and(RdultTable[JJ () -l.0)and(FifthTabletJJ () -l.0))then begin J"J*1i EggTableIJJII-l.0; LareTableEJJ:I-1.0; FifthTableIJJiI-l.0; PupaeTableIJlaI-l.0; AdultTableIJJ:I-l.0; end; (e Do the actual input from data file. a) readilfile,k); if(k I l)then begin read(lfile,i,ovip,eggsl,hatch,larvael,fifth,pupae1,emerge); readln(lfile,preov,aging,adultl); end else begin read(lfile,ovip,eggsl,hatch,larvael,fifth,pupae1,emerge); readln(lfile,preov,aging,adultl); end; ' 'until(eof(lfile)); close(lfi1e,lock); End; (I Procedure Init_Fifth s) Procedure Init_Photo; Var JJ : integer; Longest,Shortest : real; Function Photoperiod( daynum : integer; Longest,Shortest : real ) : real; Var slope : real; - (i This function will return the value of the photoperiod. t) (I All values are in hours even decimal values. *) Begin slope:I(Shortest - Longest)/l74; if(daynum ) l74)then begin if(daynum ) 357)then begin . Photoperiod:=((539 - daynum) e (slope)) + Longest; end else begin Photoperiod:I((daynum - 175) I (slope)) + Longest; end; end else 105 begin Photoperiod:I((l75 - daynum) I (slope)) +.Longest; end; End; (I Function Photoperiod I) ShortLBBJaI9.54; Shortf3938I9.46; Shortt40J:I9.37; LongE3BJ:I14.B; LongI391:I14.9; Long£40J:I15.0; SHORTE4138I9.23; LONGL413:I15.13; SHORTL423:I9.11; L0NBE42]:I15.25; SHORTE43]:I9.0 LONGE43]:I15.37; repeat Begin (I Initialize the different shortest and longest I) (I daylengths for the latitudes between 30.8 40. I) Short130J:Il0.2; Longt30lxIl4.12; Shortt3llaI10.12; LongCSllzI14.2; . Shortt323:I10.04; Long[323:Il4.28; Shortt33]:I9.96; Longt33J:I14.36; Shortt343:I9.BB; Longt343:Il4.45; Shortt3SJxI9.B; Longt353:I14.54; Shortl363:I9.7l; Longt363:I14.62; ShortE37J:I9.63; Longt37J:I14.7l; write(’Hhat is the degree of latitude? (Between 30 and 43): ’); readln(Latitude); until((Latitude ) 29)and(Latitude ( 44)); Shortest:IShort[Latitude]; Longest:ILong£LatitudeJ; for JJ:I1 to 365 do PhpIJJJ:IPhotoperiod(JJ,Longest,Shortest); End; (I Procedure lnit_Photo I) Procedure Init-Dtable; Var lfile : text; m : integer; (I Input file for X diapause vs. photoper. I) (I Loop index. I) (I This procedure will initialize the Diapause table for I) (I the percentage diapause related to photoper. I) Begin reset(lfile,’05:results.text’); for m:I1 to 62 do begin readlnilfile,DtableEm,lJ,Dtab1e[m,23); end; End; (I Prodecure Init-Dtable I) Procedure Induction; 106 (I This procedure will attempt to simulate the induction I) (I of a x of the codling moth population into the state I) (I of diapause. a} Begin IF TEMP ( 12.0 THEN perdiapaI0.0 ELSE begin IF TEMP ( 22.0 THEN pthph I ((temp - 22.0) I 0.20) ELSE IF TEMP ) 27.0 THEN phaIph + ((temp - 27.0) I 0.20); if(ph ) 15.0)then perdiap:I0.0 else begin if(ph ( 12.0)then begin perdiapII100.0; end else begin k;I0; J8'1i repeat if((ph ( Dtabletg,13)and(k I 0))then begin perdiap:IDtableLJ,23; krIl; end; J3'J+18 until((J ) 61)or(k () 0)); and; end; end; End; (I Procedure Diapause I) Procedure Breaking; Var JJ,kk : integer; xx 8 real; Begin (I This procedure will keep track of the breaking of I) (I diapause by the overwintering larvae. I) if(temp ( 0.0)then begin xxaI0.0; end else begin phinh I ((temp - 4.0) I 0.05); if(ph 15.0)then begin xx:Il00.0; end 107 else begin if(ph ) 9.0)then begin ph:I(15.0 - ph) + 12.0; JJl'li kk:I0; repeat if((ph ( DtableLJJ,lJ)and(kk I 0))then begin xinDtabletJJ,2J; kk:Il; end; JJI'JJ+1( until((JJ ) 62)or(kk () 0)); end; ' end; end; if(xx ) broke)then broke:Ixx; End; (I Procedure Breaking I) (: . I) Begin (I Main Program SYSB43 I) writeln(’How many trees are in the orchard? IPlease no more than 30000'); read1n(newpop); rewrite(local,’05:output.text'); writeln(local,’ ':2S,'INITIRL POPULRTIDN I ',newpop:6); writeln(local); (I Initialize the diapause vs. photoperiod table, I) (I daily photoperiods, and PETE data information. I) Init_Dtable; lnit_Photo; lnit_Fifth; firsts-0; broker-0.0; accch11I0.0; accch2:I0.0; (I Set up loop for a threei3) year period. I) for yeaeri to 1 do begin (I Connect the proper weather file for the year. I) NRITE(’Enter name of weather file: ’); READLN( NTHR_STRING ); RESET( LFN, NTHR_STRING ); NRITELN( LOCAL, ’Heather file I ’, HTHR_STRING ); poprInewpop; 103 writeln(local); write(local,’DRY’:5,’DD’:5,’EGBS':B,’LARVAE':8,'PUPAE':B); writelnilocal,'ADULTS’:B,’PHOT0’:B,'TEMP ':B,' DIAPQUSED 1BROKEN'); write(local,’ ’); writeln(local,' '); (I Read in the daily weather information. I) for ixIl to 365 do begin readln(lfn,Maxtemp[iJ,Mintemptil); end; accdd:I0.0; totper:I0.0; beg1:Itrue; beg2:Itrue; (I Set up the loop for the daily calculations. I) for i: to 365 do begin temp:I0.0; perdiaprI0.0; degday;-DD(Maxtemp[iJ,Mintempti],50.0,88.0,0.0); chilldainChilling(MaxtempIiJ,MintemptiJ); (I Accummulate the chilling units for each generation.I) if(beg1)then accchl:Iaccch1 + chillday; if(beg2)then accch2:Iaccch2 I chillday; (I Bccummulate the degree days each day. I) accdd:Iaccdd + degday; (I Take the average daily temperature and convert to I) (I celcius degrees. I) temp:I(MaxtempEiJ I MintempIiJ)/2; temp:I(temp - 32.0) I (5.0 I 9.0); induce:I0.0; ph:IPhptiJ; (I If date is before June 22 then determine % broken.I) if((i ( 165)and(broke ( l00.0))then Breaking; (I If degree days are ) 1420 then first generation I) (I is terminated, according'to PETE predictions. I) if((accdd ) 1420)and(first I 0))then begin 109 (I Calculate numbers and write out results. I) firsthl; . dgen1:Itrunc((totper / 100.0) I pop); writeln(local); d2:I(totper I 100.0) I pop; write(local,‘TOTAL DIAPAUSE IN GENERATION 1 I ',dgen1:5); writeln(local,’ LARVAE.'); writeln(local,totper:4:2,'x OF POPULATION DIAPAUSED.'); writeln(local); writeln(totper:4:2,'$ OF POPULATION DIAPAUSED.‘); writeln; writeln(local,’CHILLING DAYS IN BREAKING DIAPAUSE I ',accch2:6:2); writeln(local); writeln(local); totper:I0.0; popxIpop - dgenl; end; (I If date is after June 22 then calculate induction. I) if(i ) 165)then begin ph:IPhp[iJ; Induction; if(perdiap ) 0.0)then begin haIl; repeat (I Search through IexTable for proper fifth instar I) (I abundance value. I) if(trunc(accdd) ( IndexTableEhJ)then begin d2:IFifthTable[hJ-FifthTableEh-ll; if(d2 ( 0.0)then begin d2:I0.0; end; h:I185; induce:=(perpiap I d2)/100.0; end; h:=h+1; until(h ) 180); totper:Itotper I induce; end; end; (I Set the index value "h" for abundance values. I) 110" h:=0; repeat h:=h+1; if((trunc(accdd) ( IndexTableIhJ)or(IndexTab1eEhJ I 2500))then begin d3:=-l.0; end; until(d3 ( 0); (I Calculate the number of entries in each life stage I) (I for daily output. I) eggs2:=trunc((EggTableEhJ / 100.0) I pop); larvae2:Itrunc((LarvaeTableIhJ I 100.0) I pop); pupae2:=trunc((PupaeTableEhJ / 100.0) I pop); adult2:Itrunc((AdultTableIhJ / 100.0) I pop); d2:=(totper / 100.0) I newpop; write(local,i:5,accdd:5:2,eggse:8,larvae2:8,pupae2:8); writeln(loca1,adu1t2:e,PhpEi]:B:2,temp:8:2,d2:7:2,broke:7:2); write(i:5,accdd:5:2,egg52:8,larvae2:8,pupae2:B,adu1t2:B); writeln(PhpEi]:6:2,temp:6:2,d2:10:2,broke:10:2); (I If induction starts then start accummulating I) (I chilling units for each of the generations. I) if((d2 ) 0.0)and(not(begl)))then begin beg1:Itrue; accchl:=0.0; end; if((d2 ) 0.0)and(not(beg2))and(accdd ) 1420))then begin beg2:=true; accch2:=0.0; end; if((accdd ) 500)and(accdd ( 1500)and(beg1)and(bega))then begin beg1:Ifalse; beg2:=false; end; end; (I Daily loop 1-365 I) (I At the end of each year calculate the number I) (I entries in the diapause state. I) dgen2:=trunc((totper / 100.0) I newpop); d2:=(dgen2 / (eggsE + larvae2 + pupae2 I adult2 + dgen2)) I 100.0; writeln(local); write(local,’TOTAL DIAPAUSE IN GENERATION 2 I ’,dgen2:5); writeln(local,’ LARVAE.’); writeln(local,d2:4:2,'% OF POPULATION DIAPAUSED.'); writeln(local); writeln(d2:4:2,'$ OF POPULATION DIAPAUSED.’); writeln; writeln(local); write(local,'FIRST GENERATION HAS ACCUMMULATED ’,accch1:6:2); writeln(local,‘ CHILLING UNITS.‘); writeln(local); write('FIRST GENERATION HAS ACCUMMULATED ’,accch1:6:2); writeln(‘ CHILLING UNITS.'); writeln; write(local,’SECOND GENERATION HAS ACCUMMULATED ’,accch2:5:2); 111 writeln(local,’ CHILLING UNITS.'); writeln(local); write(’SECOND GENERATION HAS ACCUMMULATED ’,accch2:6:2); writeln(' CHILLING UNITS.'); broke:I0.0; first:=0; (I Reset the population for the next year. I) (I 40% mortality for overwintering population. I) newpop:=trunc((dgenl I dgen2 I pupae2) I 0.60); Closeilfn,lock); end; (I Yearly loop 1-3 I) Closeilocal,lock); End. 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