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Development, diapause and seasonal ecology of the insect parasite, apanteles rubecula (hymenoptera; braconidae) Nealis, Vincent Graham 1983

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DEVELOPMENT, DIAPAUSE AND SEASONAL ECOLOGY OF THE INSECT PARASITE, APANTELES RUBECULA (HYMENOPTERA; BRACONIDAE >. by VINCENT GRAHAM NEALIS B.Sc. (Honours), Carleton University, 1974 M.Sc, Carleton University 1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Plant Science and Institute of Animal Resource Ecology) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1983 (E> . ^V i n c e n t Graham Nealis, 1983 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date DE-6 (3/81) i i ABSTRACT Apanteles rubecula i s a s o l i t a r y insect parasite of Pier i s  rapae (small cabbage white b u t t e r f l y , imported cabbageworm). The parasite has been successfully introduced to Aust r a l i a and Vancouver, Canada but has f a i l e d to become established at other North American release s i t e s . This p r a c t i c a l problem i l l u s t r a t e s a fundamental aspect of insect ecology. The seasonal biology of insects i s interpreted here as an interaction of responses to ambient conditions. Emphasis centers on the rates at which l i f e history phenomena occur and the importance of the insect's b i o l o g i c a l chronometers on the outcome of i t s ecological relationships with i t s host and i t s l o c a l climate. Comparisons are made between Canberra, Australia and Vancouver, Canada. The parasite's developmental response to temperature i s similar in Canberra and Vancouver but the host response d i f f e r s . Canberra A. rubecula have a longer generation time r e l a t i v e to the host at low temperatures, but shorter generation times at higher, midseason temperatures. Vancouver parasites always have faster generation times than their hosts but the season i s truncated in August by a diapause response to daylengths shorter than I5h. The beginning of the season i s delayed u n t i l late May by the high thermal requirement to terminate diapause. These l o c a l responses to temperature and photoperiod result in d i f f e r e n t phenologies which, while appropriate l o c a l l y , are disastrous elsewhere. The f a i l u r e of North American attempts to esta b l i s h Vancouver A. rubecula i s attributed to the diapause c h a r a c t e r i s t i c s of the released insects. They entered diapause while ambient temperatures remained warm enough for morphogenesis and were unable to survive the obligatory period to diapause termination. Manipulation of the diapause response i s one technique in ecological pest management. A methodology for a breeding program and i t s analysis i s presented. P r a c t i c a l suggestion for b i o l o g i c a l control e f f o r t s are made and the role of individual physiological responses in insect seasonal ecology are discussed. .iv TABLE OF CONTENTS Abstract i i L i s t of tables v i L i s t of figures . v i i i Acknowledgements x Chapter 1 INTRODUCTION 1 The Insects 3 P i e r i s rapae 3 Apanteles rubecula 4 Description of the Study Areas and General Methods 8 Chapter 2 GROWTH AND DEVELOPMENT - RESPONSE TO TEMPERATURE 14 Introduction 14 Temperature-dependent time 15 Methods 16 Results 18 Discussion 31 Chapter 3 INDUCTION OF DIAPAUSE - RESPONSE TO PHOTOPERIOD AND TEMPERATURE 33 Introduction 33 Methods 36 Results 39 Discussion 51 V Chapter 4 MANIPULATION OF THE DIAPAUSE RESPONSE 53 Introduction .. 53 Methods 55° Analysis 57 Results 59 Discussion 73 Chapter 5 DIAPAUSE TERMINATION AND POST-DIAPAUSE DEVELOPMENT 75 Introduction 75 Methods 77 Results 80 Diapause termination 87 Post-diapause pupal development 94 Discussion 101 Chapter 6 PHENOLOGY AND HOST-PARASITE ECOLOGY 104 Phenology 104 Host-parasite synchrony 108 Diapause ...110 Bi o l o g i c a l Control 112 LITERATURE CITED 115 v i LIST OF TABLES TABLE 1.. Thresholds (t±SE) and degree-day requirement (K±SE) for development of the immature stages of P. rapae and A. rubecula in VANCouver and CANBerra. a) from Jones and Ives T 1 9 7 9 ) ; b) from N. Gilber t unpublished 19 TABLE 2. Chronological and physiological development periods for the p a r a s i t i c stages (egg to emergence) of A. rubecula reared on plants at the Plant Science F i e l d Station, UBC, Vancouver. 23 TABLE 3. Estimated generation times for P. rapae and A. rubecula at three average temperatures in Vancouver, Canada and Canberra, A u s t r a l i a . 25 TABLE 4. Diapause response in A. rubecula reared under variable temperature/photoperiod combinations. a T constant temperatures b) alternating temperatures (square wave) c) fluctuating temperatures (sine wave) 40 TABLE 5. Summary of diapause incidence in field - r e a r e d parasites, Vancouver (UBC) 1981. Daylengths from 1981 Canadian Almanac and Directory corrected to c i v i l daylengths after Beck (1980). Daylength for median date of i n t e r v a l , average temperature over entire i n t e r v a l . 47 TABLE 6. Summary of diapause incidence in fie l d - r e a r e d parasites from two locations (UBC and BBY) near Vancouver 1982. Daylengths from 1982 Canadian Almanac and Directory corrected to c i v i l daylengths after Beck (1980). Daylength for median date of i n t e r v a l , average temperature over entire i n t e r v a l . 48 TABLE 7. Diapause in A. rubecula larvae c o l l e c t e d at BBY on September 16-17, 1981 and transferred to laboratory conditions which promote continuous development (22°C 16L:8D). 50 TABLE 8. Summary of l o g i t analysis of experiments to test the effect of maternal age on the diapause response of the progeny. Development time in days at 20°C 15.5L:8.5D. 60 TABLE 9. Summary of l o g i t analysis of experiments to test the effect of maternal diapause history on the diapause response of the progeny. Development time in days at 20°C 15.5L:8.5D. 60 v i i TABLE 10. Mean development time at 20°C of progeny of young and old females. Analysis of variance used Log (dev time + 1). 65 TABLE 11. Mean development time at 20°C of progeny of females which had, or had not experienced a previous diapause. Analysis of variance used Log (dev time + 1 ) . 65 TABLE 12. F i t t e d models for l o g i t analysis of diapause response of d i f f e r e n t parental groups (dev=development time in days at 20°C). D-diapause, N-nondiapause; m-male, f-female. 67 TABLE 13. Mean development time of progeny of each parental group. Analysis of variance used Log (dev time + 1). 70 TABLE 14. Contrasts among l o g i t s at mean development time of 16 days at 20°C. D-diapause, N-nondiapause; m-male parent, f-female parent. 71 TABLE 15. Mean cocoon weights and time to develop to adult for male and female A. rubecula after three or four months storage at 0° or 10°C. 81 TABLE 16. Mean time in days to adult emergence of diapause cocoons taken to the f i e l d on January 20, 1982, returned to the laboratory on indicated dates and held at 22°C, 16L:8D. 83 TABLE 17 Descriptive measures of time required for male and female A. rubecula to terminate diapause at several temperatures following s p e c i f i e d storage conditions. N-number of observations, mean termination time ± standard deviation and the range are given. 88 TABLE 18. Descriptive measures of time required for male and female A. rubecula to terminate diapause at several temperatures following storage in the f i e l d for varying periods. N-number of observations, mean termination time ± standard deviation and the range are given. 89 TABLE 19. Estimated thermal constants for post-diapause pupal development in female and male A. rubecula. 95 v i i i LIST OF FIGURES FIGURE 1. Temperature-dependent rate of development for P. rapae larvae. Open c i r c l e s are unparasitised larvae, closed c i r c l e s are larvae parasitised by A. rubecula during the second inst a r . Bars are 95% confidence i n t e r v a l s , a) t h i r d instar rate b) fourth instar rate. 21 FIGURE 2. Temperature-dependent f i n a l cocoon weight of A. rubecula. Open symbols are females, closed symbols are males. Parasitisms began in f i r s t instar hosts ( c i r c l e s ) , second instar hosts (triangles) or t h i r d instar hosts (squares). Bars are 95% confidence i n t e r v a l s . 28 FIGURE 3. Correlation between between adult female fresh weight and the number of eggs and oocytes in their ovaries. Standard variables are plotted. Open c i r c l e s are unmated females, closed c i r c l e s are mated females. . . 30 FIGURE 4. Photoperiodic response curve under d i f f e r e n t controlled temperatures for Vancouver A. rubecula. 42 FIGURE 5. Per cent diapause in Vancouver A. rubecula at 16L:8D photoperiod. Open c i r c l e s are constant temperature conditions, closed c i r c l e s are fluctuating temperature conditions plotted at their mean d a i l y value. Bars are 95% confidence i n t e r v a l s . 44 FIGURE 6. Development period in days at 20°C, 15.5L:8.5D, for the p a r a s i t i c stages of the offspring of old and young female A. rubecula. Black portions are diapause i n d i v i d u a l s . 62 FIGURE 7. Development period in days at 20°C, 15.5L:8.5D, for the p a r a s i t i c stages of the off s p r i n g of female A. rubecula with d i f f e r e n t diapause h i s t o r i e s . Black portions are diapause individuals. 64 FIGURE 8. Diapause pro b a b i l i t y of the offspring of d i f f e r e n t parental groups. Bars are 95% confidence i n t e r v a l s . Estimates are from models A and B in Table 12. The proportion of males in the associated groups i s shown below the abscissa. 69 ix FIGURE 9. Spring emergence of A. rubecula overwintered in the f i e l d in Vancouver (1982). Diapause date i s the date for the beginning of f i e l d storage of diapause in d i v i d u a l s . S o l i d bars are males, open bars are females. . 86 FIGURE 10. Julian dates of diapause termination in Vancouver A. rubecula overwintered in the f i e l d , 1982-83. So l i d bars are males, open bars are females. Pm and Pf are the mean termination dates predicted from laboratory data for males and females respectively. Minimum and maximum f i e l d temperatures and the thermal accumulation above the threshold t, are given. 93 FIGURE 11. Physiological time (degree-days) required from diapause termination to adult emergence in same individuals as in F i g . 10. Pm and Pf are predictions from laboratory estimates of males and females respectively. 98 FIGURE 12. Relationship between thermal requirements to complete development to adult (Kp) and the thermal requirements for; a) diapause termination (Kdt) b) pupal morphogenesis (Km). Numbers are frequencies. Squares indicate 10 or more points per c e l l . 100 X ACKNOWLEDGEMENTS This thesis i s a di r e c t descendant of my happy collaboration with N e i l G i l b e r t and Rhondda Jones in their work with P i e r i s rapae. I thank them for their gentle but persistent prodding and continued support. Bryan Frazer opened many doors for me and Dr. W.G. Wellington i n s t i n c t i v e l y made sure I went through. Each influenced my research by example but as the best of teachers, encouraged me to do i t my own way. Debbie A l l a i n , Helene Contant, Sheryl McFarlane and Lewis Wilson were not only competent assistants but laughed in a l l the right places. I thank Dr. A.John Petkau, Institute of Applied Mathematics, UBC, for helping with the analysis in Chapter 4 . Thanks to those who helped in so many ways; C-K. Chan, M. Gross, L. Hansen, J. Heraty, D.L. Johnson, D.A. Raworth and A.T.S. Wilkinson. Thanks are due to the H i l l & Sons Market Garden, P i a l l i g o , A u s t r a l i a and a l l the gardeners in Burnaby, Canada. I would l i k e to thank the i n s t i t u t i o n s whose h o s p i t a l i t y made t h i s research possible and p a r t i c u l a r l y the individuals who grace them; Division of Entomology, CSIRO, Canberra (Dr. R.D. Hughes); Department of Zoology, James Cook University, Queensland (Dr. R.E. Jones); the Vancouver Research Station, Agriculture Canada (Drs. A.R. Forbes and B.D. Frazer); Plant Science F i e l d Station, UBC (D. Pierce and A. Neighbour). This research was generously funded by a NSERC Postgraduate Scholarship. Thanks to the insect participants seems irreverent. Rather, an apology from Fred Cogswell's "Butterfly"; Bedraggled now and crushed, f r a i l soul of f l u t t e r i n g things, forgive my hands that brushed the sun dust from your wings; VINCENT NEALIS Vancouver, B.C. July, 1983 1 CHAPTER 1 INTRODUCTION Time i s the most elusive of physical quantities. Changing concepts of time mark revolutions in modern s c i e n t i f i c thought. Newton limited time to an instant and so described a universe in which relationships change with a continuous and constant flow of time. His methods s t i l l dominate applied sciences but Newton's cosmology has been replaced by Einstein's r e l a t i v i t y and time intervals have come to depend upon your point of view. The universe behaves in a surprising fashion at the l e v e l of the quanta. Natural philosophers knew the earth and i t s organisms changed. L i f e grew, reproduced and died in apparent harmony with i t s environment. Darwin understood change and pondered the variants and the exceptions. Darwin's revolution showed where these variations could lead, i f only given enough time. Ecologists seek to explain the d i s t r i b u t i o n and abundance of organisms. An evolutionary approach must explain how organisms "cope" with the deluge of changes encountered in a single, precarious l i f e t i m e . We are reminded that the size and importance of changes depends on your point of view. In insect ecology, i t is f i r s t of a l l necessary to gain the insect's point of view. 2 This thesis examines one l e v e l of time and seasonal rhythm in a host-parasite relationship. It proposes that the dynamic nature of ecological relationships between insects can only be understood against a background of daily and seasonal responses to ambient weather and photoperiod. Parasites have a clear objective. They must have a l i f e history which ensures a period of reproductive a c t i v i t y concurrent with suitable hosts -- they must be in the same place at the same time. Their intimate relationship with the host make parasites a good s t a r t i n g point for the comparative dynamics of interacting populations. The remainder of this chapter introduces the host and parasite and my experimental approach. Chapter 2 describes continuous growth and development as temperature-dependent rates. Chapters 3 to 5 examine the biology of diapause in the parasite. The la s t chapter discusses the parasite's phenology and diapause and compares the success of i t s l i f e history strategy under di f f e r e n t ecological situations and relevance to b i o l o g i c a l c o n t r o l . 3 THE INSECTS PIERIS RAPAE (Lep., Pieridae) Pier i s rapae L., the small cabbage white b u t t e r f l y , i s a native of Europe where i t s larvae are minor pests of cu l t i v a t e d c r u c i f e r s . Richards (1940) i s the best source of information for the biology of P. rapae in B r i t a i n . Recent studies by Dempster (1967, 1968) provide a l i f e table approach and analysis of mortality factors in England. The small cabbage white's rapid dispersal through the countries of introduction i s documented by Beirne (1971) CANADA, Muggeridge (1942) NEW ZEALAND and Peters (1970) AUSTRALIA. In North America, P. rapae is also known as the imported cabbageworm. What has been unfortunate for cabbage growers has been a boon to entomologists interested in the comparative population dynamics of insects. The imported cabbageworm is readily sampled and eas i l y reared so that extensive l i f e table s t a t i s t i c s have been compiled from a d i v e r s i t y of locations (Harcourt 1962, 1966; Ito et a l . 1960, 1975; Parker 1970; Parker et a l . 1971, 1972; Pimental 1961). P. rapae has been the subject in studies on behaviour (Jones 1977a,b; Jones and Ives 1979), dispersal (Jones et a l . 1980) and growth and development (Jones et a l . 1982). Female b u t t e r f l i e s lay their eggs singly on the underside of the outside leaves of wild and cu l t i v a t e d c r u c i f e r s . The five l a r v a l instars and the pupae develop within a temperature range of 10° to 30°C (Richards 1940; Jones and Ives 1979). At 4 at an average temperature of 17°C, a complete generation requires fiv e to six weeks . Development i s continuous u n t i l a combination of photoperiod and low temperatures, acting on late l a r v a l stadia, induces diapause in the pupa. Richards (1940) c i t e d three and a p a r t i a l fourth generation per year in England but Dempster (1967) claims that only in exceptionally warm years would three generations be completed. There are three generations in Ontario (Harcourt 1963) but only two in Vancouver (Jones and Ives 1979). There may be as many as six generations in the southern United States (Parker et a l . 1971) and in Canberra, Au s t r a l i a (Jones and Ives 1979). APANTELES RUBECULA (Hym., Braconidae) ' Apanteles rubecula Marshall i s a braconid parasite of the larvae of P.rapae. It i s native to Europe and B r i t a i n but has been introduced to A u s t r a l i a (Wilson 1960) as a b i o l o g i c a l control agent of the cabbageworm. A. rubecula has not become established throughout i t s host's range in A u s t r a l i a ; i t has not been recovered in Tasmania or Queensland. The Vancouver population was introduced accidently (Wilkinson 1966) and I have co l l e c t e d the parasite in the Okanagan Valley at Summerland. Several attempts to es t a b l i s h A. rubecula in North America outside B r i t i s h Columbia have f a i l e d (Oatman and Platner 1969, 1972; Puttier et a l . 1970; D.G. Harcourt and J . Heraty, personal communication). Its potential for*control of the imported cabbageworm has been demonstrated (Parker and 5 P i n n e l l 1972), but releases must be renewed for reasons which w i l l be discussed later in t h i s thesis. A. rubecula had apparently become established in China before biocontrol workers were able to make releases of Canadian material (Hu et a l . 1981).. The o r i g i n a l A. rubecula was described ex P. rapae (Marshall 1885) but Schenefelt (1972) catalogues six other hosts from various sources. In his review of the B r i t i s h Apanteles, Wilkinson (1945) was convinced that most of the alternate host records were due to either a m i s c l a s s i f i c a t i o n of the host or of the parasite. Moss (1933) was unable to rear A. rubecula from the cl o s e l y related Pier i s brassicae (L.) and Hamilton (in Richards 1940) f a i l e d to get oviposition in either P. brassicae or Pier i s napi (L.). Krombien et a l . (1980) l i s t Pier i s protodice Boisduval and LeConte as an alternate North American host but a search of their references c i t e d does not support t h i s claim. Modern workers, with the exception of cataloguers, consider A. rubecula to be an obligate parasite of P.rapae. 6 Biology The genus Apanteles i s a large group of temperate-zone braconids u t i l i z i n g lepidopterous larvae as hosts. Our knowledge of their biology i s fragmentary (Matthews 1974). Gautier and R i e l (1921) published the f i r s t notes of the biology of A. rubecula F r e e - l i v i n g adults probably feed on nectar and pollen. In c a p t i v i t y , they feed readily on honey droplets and do not seem to require protein supplements to sustain egg production for several days. Adult behaviour shows that odour plays the p r i n c i p l e role in host location, as has been found in other braconids (Read et a l . 1970; Sato 1979; Vinson 1976). Visual cues, and chemoreceptors in or near the ovipositor, may be necessary for f i n a l acceptance of the host by the parasite (Fisher 1971). Apanteles wasps attack by thrusting the abdomen between the legs and jabbing with the ovipositor. In A. rubecula, which lays a single egg, oviposition requires less than a second and i s followed by a change in wasp behaviour; the female moves out of the immediate v i c i n i t y and grooms extensively. Delucchi (1950) provides morphological descriptions and figures of the l a r v a l instars of A. rubecula. The single e l l i p t i c a l egg i s injected into the host haemolymph and i s i n i t i a l l y nourished by the trophamnion. The caudate I-instar parasite can often be seen within the trophamnionic membrane. The II - i n s t a r i s vesiculate (proctodaeum evaginated) and the 7 las t instar t y p i c a l l y hymenopteriform (mandibulate, apodous, white c y l i n d r i c a l body). Schisler (1981) reports that A. rubecula remains a haemolymph feeder during the f i r s t two instars but feeds on host tissues during the l a s t i n s t a r . The mandibles of the last instar are c e r t a i n l y functional as i t emerges from the host by b i t i n g through the c a t e r p i l l a r ' s body wall between the t h i r d and fourth abdominal segments. It then spins a cream-coloured cocoon. The insect either diapauses at th i s prepupal or eonymph stage, or develops to an exarate pupal form and f i n a l l y to an adult, which bites a hatch and pushes i t s way out of the cocoon. Mating i s similar to that described for Apanteles  glomeratus (L.) (Tagawa and Hidaka 1982; Tagawa and Kitano 1981). Males approach females from downwind. A s p e c i f i c sequence of behaviour appears to be necessary before females permit copulation. The male approaches the female from the rear, wings arched and vibrating and antennae held above the female's thorax. Copulation can l a s t t h i r t y seconds to fi v e minutes. The female remains motionless after the male leaves and i f she i s immediately approached by another male, he too w i l l copulate, uncontested. Once female a c t i v i t y resumes, she appears to be no longer a t t r a c t i v e to males. Rahman (1966) estimated female fecundity at about t h i r t y eggs, but I have obtained that many successful ovipositions from a female in one day. Parker and Pinn e l l (1970) record longevity under mass-rearing conditions as 25.1 days for females and 21.8 days for males. Parker and Pinn e l l (1972) estimated that adults 8 survived six weeks during a Missouri autumn. A. rubecula is attacked by hyperparasites in both A u s t r a l i a and Canada. In Canberra, the pteromalid Eupteromalus  braconophagus (Cameron) is an infrequent hyperparasite but in Vancouver, the eulophid Tetrastichus galactopus (Ratz.) accounts for a high mortality toward the end of the season (Nealis 1983) and there are additional hyperparasitisms by various Gelis spp. DESCRIPTION OF THE STUDY AREAS AND GENERAL METHODS Par a s i t i c insects present special problems for f i e l d sampling. The adult i s a small, active predator whose prey are more or less aggregated in p a r t i c u l a r areas. There may be opportunities to observe wild females but there are no r e l i a b l e methods for quantitative sampling of populations. Most parasite studies use, as the base observation, the proportion of hosts p a r a s i t i s e d . When there is a r e l i a b l e sampling method for the host population and the proportion par a s i t i s e d consistent, the immature parasite population may be reasonably sampled. But the unique nature of the host-parasite rel a t i o n s h i p can cause d i f f i c u l t i e s in the extrapolation from the sampled host population to the parasite's ecology. For example, there are e f f i c i e n t sampling designs for the imported cabbageworm in 9 commercial plots (Harcourt 1962) but c a t e r p i l l a r s in commercial plots are only a small proportion of the broader population which serve as hosts to A. rubecula. An explanation by way of describing host-parasite ecology may help to c l a r i f y the sampling d i f f i c u l t i e s . The cabbage white is a vagrant species; the bu t t e r f l y finds and oviposits on host plants wherever they occur within i t s range. The d i s t r i b u t i o n of host plants i s an important factor in the species' population dynamics. Large patches of host plants, such as commercial cole plots, have a r e l a t i v e l y low egg density due to but t e r f l y f l i g h t and oviposition behaviour (Jones 1977a) and poor l a r v a l survival due to in s e c t i c i d e s . But l a r v a l survival may be very good on many small, undisturbed patches of host plant. In both North America and A u s t r a l i a , P. rapae i s the scourge of backyard gardens. In Canberra, R.Jones and I have made l i f e tables for several cohorts of c a t e r p i l l a r s in nine home gardens. They a l l received large numbers of eggs. Survival of c a t e r p i l l a r s varied widely but there was substantial damage in a l l gardens even though we removed c a t e r p i l l a r s after they entered the f i n a l instar. Raising cabbages in Canberra requires constant vigilance or in s e c t i c i d e s . Yet, A. rubecula was absent from most s i t e s and a minor mortality factor in the remainder. A larger and less disturbed habitat for the cabbage white i s represented by a picnic s i t e at Hut Crossing on the Murrumbidgee River near Canberra. This shaded, well-watered area has an annual population of wild mustard (Hirshfeldia) which serves as host 10 for a small but persistent population of P. rapae. The proportion of c a t e r p i l l a r s p a r a s i t i s e d by A. rubecula at this s i t e i s consistently high (>50%) from late January u n t i l the end of the season in A p r i l . But A. rubecula can also exploit large and newly available habitats in p a r t i c u l a r circumstances. The H i l l & Sons Market Garden in P i a l l i g o , near Canberra has been a consistent source of b u t t e r f l i e s for several years. Chemical control of cabbage aphid and diamond-back moth make i t an area of poor cabbageworm s u r v i v a l . On February 5, 6 and 13, 1981, heavy rains flooded one p l o t , making the large cabbages unsaleable. No further i n s e c t i c i d e s were applied. C a t e r p i l l a r c o l l e c t i o n s on February 17, 20 and March 4, 11 and 20 estimated parasitism rates exceeding 80%. By March 20, parasite cocoons were more numerous than' host larvae. A. rubecula had thoroughly p a r a s i t i s e d the host population. Similar rates in experimental plots are reported by Parker and P i n n e l l (1972). In Vancouver, A. rubecula can be commonly found p a r a s i t i s i n g cabbageworms in home gardens but i s most consistently c o l l e c t e d in areas such as the Burnaby Rental Plots where there i s a moderately large population of c u l t i v a t e d c r u c i f e r s in small, individual p l o t s . . In view of the above habitat associations of the host and parasite, my f i e l d samples were opportunistic. Rather than sampling intensively under some schedule, I chose to c o l l e c t host larvae and parasite cocoons at several s i t e s throughout 11 the main growing season, concentrating my e f f o r t s at key times of the year (e.g., onset of diapause). At each s i t e I would search entire host plants for c a t e r p i l l a r s and parasite cocoons u n t i l I had enough (50-200) c a t e r p i l l a r s to provide an adequate estimate of the rate of parasitism or u n t i l i t was evident that I must either s e t t l e for less or c o l l e c t every larva in the host population. I kept a record of the search time (1-2 hours) but I do not consider my counts accurate measures of host density. This scheme s a t i s f i e d the necessities of obtaining f i e l d data and experimental material when they were most needed with minimum disturbance to a sometimes sparse population. My treatment of f i e l d - c o l l e c t e d insects was as follows. C a t e r p i l l a r s were brought back to the lab and sorted by instar. Depending on the objectives of the c o l l e c t i o n , each instar was reared separately on potted c r u c i f e r s under controlled or natural conditions, or dissected under saline so that parasite eggs could be counted. The proportion, per stadium, of pa r a s i t i s e d hosts was always obtained. The time to emergence in s p e c i f i c conditions could be used to estimate the approximate age of the parasite on the c o l l e c t i o n date. The parasite's age and possible superparasitism were observed d i r e c t l y by dissection of the host. The sex-ratio and hyperparasitism were also recorded. These data are the background for subsequent experiments. Interpretation of these f i e l d data requires a good deal of q u a l i f i c a t i o n and a large part of t h i s thesis i s devoted to experimental investigation of hypotheses suggested by the f i e l d notes. 1 2 Other f i e l d data are experimental. A known number of hosts of various instars and/or female wasps were seeded into an area according to a pa r t i c u l a r experimental design. In some cases, hosts were parasitised in the laboratory and then taken to the f i e l d . These methods and those of the laboratory w i l l be discussed with the experiments. Insect culture , Standardized laboratory conditions cause unavoidable selection in insect stock. It i s impossible to anticipate the consequences for laboratory material even when we know the intensity and di r e c t i o n of the selection. In a l l experiments, I used insects which, i f not freshly c o l l e c t e d from the f i e l d , were no more than three generations removed from i t . P. rapae pupae and A. rubecula cocoons can be stored three weeks at 10°C without increased mortality and the adult wasps l i v e up to six weeks under these same conditions i f supplied with honey and water. B u t t e r f l i e s were fed a 10% honey solution absorbed on cotton. Mating in both the b u t t e r f l i e s and wasps i s inhibited by a r t i f i c i a l l i g h t , so newly emerged adults were kept outside or near a window. Recently develped broad-spectrum fluorescent lamps (Vita-Lite ) seem to be an improvement but thorough comparisons have not been made. 13 The imported cabbageworm can be reared on a variety of c r u c i f e r s but the experiments involving A. rubecula u t i l i z e d Brussels sprouts (Jade Cross) occasionally supplemented by kale (Maris Kestrel) or cabbage (Early B a l l ) . A l l experiments started with plants six to ten weeks old. The temperature of each controlled environment f a c i l i t y was recorded continuously and these records were calibr a t e d with mercury thermometers, which were in turn calib r a t e d monthly in ice and b o i l i n g water. Temperatures were almost always within ± 1°C of the stated mean. A t r u l y constant humidity i s d i f f i c u l t to maintain when there are potted plants in the growth chambers. A r e l a t i v e l y constant humidity (approximately 75%) was achieved by keeping the s o i l in the pots moist and preventing standing water from accumulating in the chambers. Photoperiod was provided by two to six cool, white fluorescent lamps supplemented by two broad-spectrum lamps. 14 CHAPTER 2 GROWTH AND DEVELOPMENT - RESPONSE TO TEMPERATURE INTRODUCTION The time arthropods require to grow and develop during their most active season i s largely determined by temperature. As poikilotherms, insects are dependent on ambient temperature to f u l f i l l the thermal requirements for growth, maturation and reproduction. Each process may have d i f f e r e n t responses to temperature so the biology of the in d i v i d u a l , and of the population , i s an interaction of a l l processes in a thermally fluctuating environment. T y p i c a l l y , insects have adapted splendidly and occupy the entire range of climates offered by t e r r e s t r i a l habitats, at a l l hours of the day. This relationship between temperature and rate of development means we must c a l i b r a t e an insect's chronometer with our thermometer. This chapter discusses physiological time in the host-parasite relationship. It describes temperature-dependent rates of growth and development during the major portion of the active season in Vancouver, Canada and Canberra, A u s t r a l i a . These rates may be modified by other factors which are also considered here. 15 Temperature-dependent time There are several functions which describe the rate of development of arthropods over a wide range of experimental temperatures (Logan et a l . 1976; Stinner et a l . 1974). Campbell et a l . (1974) argue that f i e l d temperatures during the period of greatest insect a c t i v i t y are mostly in the range over which the rate of development i s d i r e c t l y proportional to ambient temperature. I f i t rate-temperature linear regressions to estimate a threshold t, the temperature at which the rate of development w i l l be zero, and a "physiological time" K, which estimates the number of degree-days above the threshold required for development to be completed. When average d a i l y temperatures remain close to the threshold, degree-days underestimate the rates and an expression such as Logan et a l . ' s (1976) may provide a better prediction (J. Regniere, personal communication). Any nonlinear response at low temperatures i s not i n t r i n s i c to the insect but results from variation in the threshold within the population. Observation of rates at chronically low temperatures are biased by d i f f e r e n t i a l temperature selection for individuals with lower thresholds, making the estimate inaccurate (Gilbert et a l . 1976). The choice of any p a r t i c u l a r function should be pragmatic. In t h i s case, the linear model provided an adequate f i t over most of the season (Table 2) and allowed simple comparisons with the host. Campbell et a l . (1974) describe the empirical estimation of t and K and their standard errors, and I use their methods here. 16 METHODS A l l parasites and hosts were f i r s t or second generation laboratory insect, the parental generation was raised on potted cabbage, kale or Brussels sprouts at 22°C 16L:8D. In i n i t i a l t r i a l s with Australian parasites, l a r v a l hosts of d i f f e r e n t stadia were caged with adult wasps for one to two hours at 23°C and then randomly assigned to temperature treatments. In a l l other t r i a l s , host larvae were removed from the parasite cage as soon as an attack was observed, to minimize superparasitism and to avoid using unparasitised individuals. Host larvae were checked d a i l y at the lowest temperatures and twice or three times d a i l y at intermediate and higher temperatures, to determine the survival and duration of host instars and the times when parasite larvae emerged from the host. Once dry (1/2 to 1 day), pupal.cocoons were weighed and then returned to the same temperatures u n t i l adults emerged. Other A. rubecula were raised at one temperature (25°C) and the cocoons d i s t r i b u t e d among di f f e r e n t temperatures to check whether the conditions experienced by the larvae affected the thermal requirements of the pupae. As there was no e f f e c t , pupae with d i f f e r e n t l a r v a l rearing conditions have not been distinguished further. Time to imago and the sex of each individual were recorded. Temperature treatments ranged between 13.9° and 30°C. In Vancouver, the a b i l i t y of my estimated t and K values to predict generation times of A. rubecula in the f i e l d was checked by rearing cohorts of p a r a s i t i s e d hosts in large screen 17 cages (3m X 3m X 1m high) at the Plant Science F i e l d Station at the University of B r i t i s h Columbia (UBC). Daily maximum and minimum temperatures, recorded at an adjacent meteorological station, were used to compute degree-day accumulation in the f i e l d , using the algorithm provided by Frazer and Gil b e r t (1976). Fecundity of A. rubecula Preliminary dissections of adult female A. rubecula showed that the number of eggs in the swollen, proximal portion of the common oviduct roughly doubled over the f i r s t two days after eclosion, and then remained constant for at least three weeks. To avoid confounding weight with d i f f e r e n t i a l egg maturation rates, I used females which were two weeks old and of similar, limited foraging history. The adults were anaesthetized, weighed and immediately dissected under sa l i n e . A l l eggs and oocytes were counted. Although t h i s count may underestimate absolute fecundity, i t is the comparison for individuals of di f f e r e n t weights which i s of int e r e s t . 18 RESULTS Table 1 l i s t s the thermal requirements for the immature stages of P. rapae and A. rubecula in Canberra and Vancouver. The thermal constants for P. rapae are from Jones and Ives (197S). Neil G i l b e r t provided estimates for Canadian pupae and the development rates of unparasitised larvae shown in F i g . 1. The egg and l a r v a l development rate in P. rapae i s i d e n t i c a l in both countries, but Canadian pupae have a subst a n t i a l l y higher threshold than Australian pupae. This difference was confirmed by N. Gilbert for pupae held at 10°C in both places; the Australian pupae eventually produced adults whereas none of the Canadian pupae showed any development. At the same f i e l d temperatures, Canadian individuals w i l l develop more slowly than Australian. Parasitism by A. rubecula retards development of the host larva (Fig. 1). Note that the early stages of parasitism do not aff e c t host development at high temperatures but later stages slow host development at a l l temperatures. In contrast, the host size had l i t t l e e f f e c t on the parasite's threshold or i t s rate of development. Male and female A. rubecula developed synchronously through the p a r a s i t i c stage so the thermal requirements for egg and l a r v a l A. rubecula in Table 1 include both sexes from a l l host sizes. The threshold for these p a r a s i t i c stages of A. rubecula i s higher than, but not s i g n i f i c a n t l y d i f f e r e n t from that of i t s host in either l o c a l i t y . 19 STAGE LOCATION t(SE) K(SE) P. RAPAE egg,larva VANC 10.0 (0.8)a 216.8 (6.5)a CANB 9.8 (0.7)a 221.0 (4.4)a pupa VANC 10.4 (0.9)b 107.0 (3.0)b CANB 6.7 a 134.0 a A. RUBECULA egg,larva (parasitic) VANC 10.7 (0.3) CANB 11.6 (0.9) 119.0 (1.4) 122.0 (6.0) pupa VANC 11.4 (0.2) CANB 11.7 (1.0) 79.4 (1.3) 97.1 (4.7) TABLE 1. Thresholds (t±SE) and degree-day requirement (K±SE) for development of the immature stages of P. rapae and A. rubecula in VANCouver and CANBerra. a) from Jones and Ives (1979); b) from N. Gil b e r t unpublished. 20 FIGURE 1. Temperature-dependent rate of development for P. rapae larvae. Open c i r c l e s are unparasitised larvae, closed c i r c l e s are larvae p a r a s i t i s e d by A. rubecula during the second ins t a r . Bars are 95% confidence i n t e r v a l s , a) t h i r d instar rate b) fourth instar rate. 60i 50 a . Ill—instar Pierls rapae T 401 30 I-Z LU 2 Q. *~ 201 O * —1 a LL! ° > 5 10 m © D ' o Li. ~ O « 50| LU 5 < ^ 40| b. I V - i n s t a r Pieris rapae 301 4> 201 10 15 20 25 T E M P E R A T U R E ( ° C ) 30 22 There was no corr e l a t i o n between the degree-day requirement of the p a r a s i t i c l a r v a l stages and that of the f r e e - l i v i n g pupal stage. Male A. rubecula pupae develop faster than female (F=25.65 df=1,205 p<0.0l). The thresholds were i d e n t i c a l but males required f i v e degree-days less than females to complete pupal development, about one day less in the f i e l d . I do not consider th i s difference here because i t does not result in major differences in t o t a l generation time. There was a s l i g h t additional effect of weight so that smaller male adults emerged e a r l i e s t in a cohort. Canadian A. rubecula pupae required fewer degree-days to complete development than did Australian (t=3.33 df=106 p<0.01). Both Vancouver and Canberra parasite populations had a much lower heat requirement than their hosts. The net eff e c t i s that host generation times are shorter in Au s t r a l i a and parasite generation times are shorter in Canada. How useful are the thermal constants in Table 1 for f i e l d prediction? Table 2 l i s t s the chronological and physiological developmental period for Vancouver cohorts of A. rubecula larvae reared from c a t e r p i l l a r s on cabbage. Four of the five observed values are very close to that estimated in the laboratory. The exceptions are parasites reared during A p r i l and May when mean temperatures are close to the threshold. The linear model underestimates the real heat accumulated by the insect in the f i e l d . In Chapter 5 I w i l l show that spring 23 CALENDAR TIME PHYSIOLOGICAL TIME N MEAN (SE) August 10-30, 1981 A p r i l 28 - June 6, 1982 July 16 - August 3, 1982 July 31 - August 23, 1982 August 6-31, 1982 LAB ESTIMATE (TABLE 1) 119.5 (0.82) 30 56.2 (3.02) 10 114.4 (0.60) 18 118.3 (0.54) 47 120.8 (1.08) 37 119.0 (1.40) 300 TABLE 2. Chronological and physiological development periods for the p a r a s i t i c stages (egg to emergence) of A. rubecula reared on plants at the Plant Science F i e l d Station, UBC, Vancouver. • 24 emergence of wasps in Vancouver does not occur much sooner than June 1 so that, in practice, I w i l l not have to consider early-season predictions. Table 3 compares host and parasite generation times at various average temperatures for Canadian and Australian populations. In A u s t r a l i a , A. rubecula w i l l have longer generation times than their hosts at lower temperatures but faster times at higher temperatures. In Vancouver, even at temperatures as low as 15°C, the parasite generation i s always shorter than the host. This difference i s due to the faster rate of development in Canadian parasite pupae and the extended generation time of the host due to i t s higher pupal threshold. Although oviposition in hosts of d i f f e r e n t ages had a ne g l i g i b l e effect on parasite development rate, the parasites emerging from older, larger hosts were c e r t a i n l y larger than those from younger, smaller hosts (Fig. 2) so r e l a t i v e growth rates must be affected by host age. As in many insects, large females had more eggs and unmated females had as many eggs as mated females of the same size (Fig. 3). Females must grow r e l a t i v e l y faster than males since both sexes develop synchronously through the p a r a s i t i c stage but females are consistently 10% larger than their male sibs. Male weights ranged from 3.60 to 7.83 mg and females from 4.36 to 8.76 mg so there i s considerable overlap. Within the range of temperatures favourable to development 25 GENERATION TIME (DAYS) . VANCOUVER CANBERRA 15° 20° 25° 15° 20° 25° P. RAPAE 66.7 32.8 21.7 58.6 31.8 21.8 A. RUBECULA 49.1 21.9 14.4 64.7 26.1 16.4 TABLE 3. Estimated generation times for P. rapae and A. rubecula at three average temperatures in Vancouver, Canada and Canberra, A u s t r a l i a . 26 (15° to 28°C), parasites reared at higher temperatures were smaller than those reared at lower temperatures (Fig. 2). Oviposition in older hosts produced larger parasites at a l l temperatures. Parker and Pinnell (1973) made t h i s same observation using a laboratory culture of A. rubecula from Vancouver stock. There i s no apparent interaction between temperature, host size at oviposition and sex of the parasite; the rel a t i o n s h i p between weight and these three variables can be expressed as; WT = 7.49 - 0.109 TEMP + 0.25 HOST - 0.65 SEX R2=0.29, df=295, p<0.0l where WT i s in mg, TEMP in °C, HOST size is 1, 2 or 4 for instar, males are 1 and females are 0. This equation i s based on Vancouver data only. Australian data for these same variables are not so complete; the magnitude and dire c t i o n of the trends i s the same but host size appears to contribute less to f i n a l parasite weight. 27 FIGURE 2. Temperature-dependent f i n a l cocoon weight of A. rubecula. Open symbols are females, closed symbols are males. Parasitisms began in f i r s t instar hosts ( c i r c l e s ) , second instar hosts (triangles) or t h i r d instar hosts (squares). Bars are 95% confidence i n t e r v a l s . 28 7.0 cr. E C D 111 6.01 O O o O 5*| o 4.01 0 •I o 0 1 II + Apanteles rubecula 1 I I i 15 18 21 24 27 R E A R I N G T E M P E R A T U R E ( ° C ) 29 FIGURE 3. C o r r e l a t i o n between between adult female f r e s h weight and the number of eggs and oocytes i n t h e i r o v a r i e s . Standard v a r i a b l e s are p l o t t e d . Open c i r c l e s are unmated females, closed c i r c l e s are mated females. A c t u a l number of eggs and oocytes i n A. rubecula ranged from 35 to 128. 30 r=0.49 d f =55 C O o (D HI o • • oo # o o o -1 -2 -2 -1 0 1 2 3 A D U L T F E M A L E W E I G H T ( m g ) 31 DISCUSSION The parasite and host thermal requirements shown in Table 1 can be compared with those of aphids and their parasites (Campbell et a l . 1974, Table 1). The threshold of A. rubecula i s only s l i g h t l y higher than that of i t s host in comparison with the large differences between the thresholds of aphids and their parasites; but the aphid hosts have much lower thresholds than does P. rapae. A more important difference here is that, unlike the aphid parasites, A. rubecula has a much lower degree-day requirement than i t s host. In A u s t r a l i a , generation time in A. rubecula i s longer, r e l a t i v e to the host, at average temperatures below 17°C but shorter than the host's at higher temperatures. Since 17°C i s approximately the mean seasonal temperature for Canberra, parasite generations there are shorter than the host's for half of the season but longer than the host's during the other h a l f . Frazer and G i l b e r t (1976) have already shown the importance of alternating temperature-driven development advantages in a predator-prey relationship. In Vancouver, A. rubecula completes a generation faster than i t s host at a l l times of the year. The small number of generations per year in Canada may outweigh any p o s s i b i l i t y of over-running the host population in Vancouver. Population growth i s a function of development time but i t is helpful to view the growth of individuals as a cl o s e l y associated but d i s t i n c t process (Gilbert 1983). Size, and therefore growth rate, can vary enormously in parasites being 32 dependent on sex, temperature and host size among other variables. But even though highly variable in size, most parasites take about the same length of time to complete development because the thermal constants do not vary as widely. A heterogeneous environment does not disrupt the developmental synchrony in parasites; i t merely determines their s i z e . This implies high l o c a l v a r i a b i l i t y in fecundity compared to timing and i s consistent with Cole's (1954) argument that a population's rate of increase i s more sensitive to variations in generation time than to equivalent variations in fecundity. 33 CHAPTER 3 INDUCTION OF DIAPAUSE - RESPONSE TO PHOTOPERIOD AND TEMPERATURE INTRODUCTION Diapause i s a peculiar physiological state characterized in arthropods by cessation or drastic modification of some metabolic processes leading to a marked reduction in the rate of morphogenesis. Diapause can occur at any stage in the insect l i f e cycle but for a pa r t i c u l a r species, there i s usually only one stage capable of prolonged dormancy. Once i n i t i a t e d , development through the diapause stage requires a p a r t i c u l a r sequence of events to be completed. Diapause i s an adaptive phenomenon enabling insects to l i v e in areas when environmental conditions are unsuitable for a c t i v i t y . It thus serves to synchronize active stages in the l i f e cycle with seasonal periods favourable to growth and development. Preparation for diapause requires a response under conditions favourable to insect a c t i v i t i e s and must therefore be i n i t i a t e d by environmental cues which f o r e t e l l periods unsuitable for continuous development. Several possible cues are recognized; declining food q u a l i t y and low temperatures were among the f i r s t factors investigated, but i t i s now recognized that, in temperate regions at least, diapause i s commonly a response to natural photoperiods (Danilevskii 1965). Photoperiod has the virtue of being a r e l a t i v e l y invariant 34 harbinger of seasonal changes but i s not in i t s e l f an adverse condition. Moreover the e f f e c t s of photoperiod on temperature, of far more immediate importance to insects, are delayed, permitting insects to make the necessary preparations for dormancy. The exact location of the insect photoreceptors involved in the induction of diapause are not known. Eyes and o c e l l i are not necessary. Control of diapause i s d e f i n i t e l y neuro-endocrine (Novak 1975) so l i g h t may act d i r e c t l y on neurosecretory c e l l s in the cerebral ganglia (Lees 1964). The s p e c i f i c mechanisms of the clocks measuring the l i g h t and dark phases and their modification by other variables are fascinatingly complex (Beck 1980; Saunders 1976, 1981). Diapause in parasites may be influenced by the host (Salt 1941; Vinogradova and Zinovjeva 1972) but in braconids, an independent photoperiodic response has been demonstrated (Dansilevskii 1965; Maslennikova 1959; Rabb and Thurston 1969). Schisler (1981) interpreted f a i l u r e of A. rubecula to diapause under short days when reared in hosts lig a t e d at the neck as indicative of parasite reliance on the host endocrine system. But the same study showed an e f f e c t of parasitism on biosynthesis of juvenile hormone in host larvae: who i s c o n t r o l l i n g whom? I have reared A. rubecula under conditions where approximately half the parasites entered diapause (Chapter 4) and never observed diapause in the unparasitised c a t e r p i l l a r s developing side by side with the parasites. In 35 Vancouver, A. rubecula diapause begins two to three weeks before i t s host, so some degree of independence on the part of the parasite must be recognized. The adaptive role that diapause plays in synchronizing the insect population with i t s environment i s of primary interest. The important ecological questions pertain to the relationship between seasonal p e r i o d i c i t y in photoperiod and temperature, and the induction and duration of diapause. The photoperiodic response curve expresses the diapause response of a population to a series of stationary photoperiods. Typical of such curves i s the c r i t i c a l photoperiod, a period which marks the abrupt change from diapause induction to continuous development (Beck 1980; Danilevskii 1965; Saunders 1976). In the experiments reported below, I added constant, alternating (square wave) and fluctuating (sine wave) temperatures as factors to investigate the interaction between photoperiod and temperature. The interpretation was validated with experimental evidence from the f i e l d . I also i d e n t i f i e d the developmental stages of A. rubecula which are sensitive to photoperiod. 3 6 METHODS Laboratory: General Host and parasite laboratory stocks were taken from Vancouver populations and reared under conditions promoting continuous development (22°C 16L:8D) for one generation before the experiments started. Late-II and e a r l y - I l l instar c a t e r p i l l a r s were exposed singly to female wasps. After an attack, the c a t e r p i l l a r s were transferred to Brussels sprouts (Jade Cross) in 15 cm pots and then randomly assigned to a par t i c u l a r temperature-photoperiod combination. A l l laboratory experiments were carried out in controlled environment f a c i l i t i e s at the Vancouver Research Station, Agriculture Canada and the University of B r i t i s h Columbia. Every e f f o r t was made to standardize the hosts' food. The inevitable changes in plant condition through the experiment, p a r t i c u l a r l y evident at low temperatures, could have been somewhat ameliorated by replacement of plants at regular intervals but thi s would have necessitated handling the insects during transfer. Fortunately, c a t e r p i l l a r s p a r a s i t i s e d by A. rubecula eat very l i t t l e during the l a t t e r stages of parasitism, so food qu a l i t y then would have a reduced e f f e c t . G i l b e r t (1983) shows that declining food qu a l i t y affects weight gain but not development time in P. rapae. Moreover, there i s l i t t l e ambiguity regarding the diapause response at short 37 (<15h) photophases. The cocoons of the emerged parasites were coll e c t e d d a i l y , recorded by emergence date and, in some cases, weighed on a Mi t t l e r ME-30 electronic balance. They were then held at 22°C 16L:8D. Under such conditions, A. rubecula which are not in diapause eclose in about one week (Chapter 2). After 15 days, individuals could be scored as either diapause (D) or nondiapause (N). C r i t i c a l Photoperiod During 1981, experiments were designed to determine the c r i t i c a l photoperiod for the diapause response of A. rubecula at d i f f e r e n t constant temperature-photoperiod combinations. The large number of combinations necessitated s p l i t t i n g the experimental stock into two groups, one for current t r i a l s and a second for continuous rearing for the next set of t r i a l s . The las t t r i a l s used third-generation laboratory adults but there were no anomalous responses which could be attributed to a r t i f i c i a l selection for or against the diapause response. During these t r i a l s , I included two runs at alternating temperatures and 16L:8D. Experiments in 1982 were designed to see i f the photoperiodic response was greatly modified by temperatures which fluctuated d a i l y over a range t y p i c a l of late summer in Vancouver (15°-25°C). These t r i a l s were conducted in i d e n t i c a l 38 controlled environment chambers equipped to permit temperatures to fluctuate as a sine wave with the maximum temperature occurring at the mid-point of the photophase. Photoperiods used were 14L:10D, 14.75L:9.25D, 15.5L:8.5D. A fourth cabinet operated as a control at a constant average temperature of 20°C and a 15.5L:8.5D photoperiod. F i e l d data F i e l d data pertaining to diapause are from two sources. Hosts from general f i e l d c o l l e c t i o n s were reared on plants in outdoor cages when c o l l e c t i o n s were made during the estimated c r i t i c a l period (July 21 to August 30 in Vancouver, February 15 to March 31 in Canberra). Host c a t e r p i l l a r s c o l l e c t e d in Burnaby (BBY) on September 16, 17 1981 were caged on potted plants in the laboratory at 22.5°C 16L:8D. Time to parasite emergence was recorded and converted to physiological time. Assuming an average thermal requirement of 120 degree-days above a threshold of 11°C to complete l a r v a l development (Chapter 2), I estimated the age at which the parasites, were transferred from diapause ( f i e l d ) to nondiapause (laboratory) conditions. These data were used to estimate the photosensitive stage of A. rubecula and examine the p o s s i b i l i t y of disrupting the diapause response. The second source of f i e l d data was the cohorts of pa r a s i t i s e d hosts reared on plants in large screen cages (3m X 3m X 1m high) at the Plant Science F i e l d Station (UBC) and, to 39 a lesser extent, on the adjacent grounds of the Vancouver Research Station. Late-II and e a r l y - I l l instar c a t e r p i l l a r s were exposed to adult parasites and, after an attack, placed on potted plants and permitted to s e t t l e overnight at 22°C. Plants with p a r a s i t i s e d c a t e r p i l l a r s were then placed in the f i e l d . When parasites emerged, the cocoons were c o l l e c t e d d a i l y but kept in a Stevenson screen for two additional days to insure ample opportunity to pupate under ambient conditions. Once they were returned to the lab, diapause was determined by the same c r i t e r i a as for lab experiments described previously. Insects in diapause were stored in gelatin caps overwinter in the Stevenson screen to determine su r v i v a l and the timing of spring emergence (Chapter 5). RESULTS C r i t i c a l photoperiod Table 4 includes the results of a l l t r i a l s to determine the c r i t i c a l photoperiod under controlled conditions. Figs. 4 and 5 i l l u s t r a t e two aspects of the analysis. Figure 4 shows data from constant and fluctuating temperatures and demonstrates the clear photoperiodic response. The c r i t i c a l photoperiod l i e s between 15h and 16h and appears to be s l i g h t l y modified by temperature. At constant temperatures less than 19°C, a proportion of parasites entered diapause with a 16h photophase (Fig. 5). The closed c i r c l e s in F i g . 5 are alternating temperatures plotted at their average d a i l y value. TEMPERATURE PHOTOPERIOD N DIAPAUSE a) 15° 16° 17° 16L:8D 16L:8D 18° 20° 1 0L 1 2L 1 3L 1 4L 1 5L 1 6L 17L 14D 1 2D 1 1D 1 0D 9D 8D 7D 22' 16L:8D 12L:1 2D 1 3L: 1 1D 14L:1OD 15L:8D 15.5L:8.5D 16L:8D 14L:1OD 15L:9D 16L:8D 60 23 32 48 33 25 64 58 26 95 64 63 41 54 119 61 88 30 72 120 45 1 5 32 48 33 25 64 27 1 22 64 63 41 48 53 39 0 30 65 0 75% 65% 100% 1 00% 100% 100% 100% 46% 4% 23% 1 00% 100% 100% 89% 44% 64% 100% 90% b) 12°-20° 1 i °-l9° c) 20±5° constant 20' 16L:8D 16L:8D 14L:10D 14.75L:9.25D 15.5L:8.5D 15.5::8.5D 60 50 73 68 68 68 0 0 73 68 0 36 100% 1 00% 53% TABLE 4. Diapause response in A. rubecula reared under variable temperature/photoperiod combinations. a) constant temperatures b) alternating temperatures (square wave) c) fluctuating temperatures (sine wave) F I G U R E 4. P h o t o p e r I o d i c r e sponse cu r ve under d i f f e r e n t c o n t r o l l e d t empera tu res f o r Vancouver A . r u b e c u l a . 42 GSnBdBjQ % FIGURE 5. Per cen t d i a p a u s e 1n Vancouver A. r u b e c u l a at 16L:8D p h o t o p e r i o d . Open c i r c l e s a r e c o n s t a n t t empera tu re c o n d i t i o n s , c l o s e d c i r c l e s a r e f l u c t u a t i n g t empera tu re c o n d i t i o n s p l o t t e d at t h e i r mean d a l l y v a l u e . Bars a r e 95% c o n f i d e n c e i n t e r v a l s . 100 0 CO ZJ CO Q . CO 50 H 0 1 0 6 15 16 17 18 19 T 20 Temperature 4». 45 With alternating temperatures, low average temperatures did not induce diapause, provided the maximum was at least 19°C, although the development time was close to what would be expected at the average temperatures. This maximum temperature is regularly reached during the Vancouver season, even after diapause has already been induced in f i e l d populations of A. rubecula (Tables 5 and 6). Hence, temperature, although demonstrably operative under experimental conditions, may play a minor role in diapause induction in nature. The experiments u t i l i z i n g fluctuating temperatures and a variable photoperiod demonstrate the predominance of the photoperiodic response and limited interaction between photoperiod and temperature in determining the diapause response in A. rubecula (Table 4c). The t r a n s i t i o n between diapause and nondiapause induction at t y p i c a l Vancouver summer temperatures i s sharp. At less than 15h photophase, diapause i s induced in a l l individuals in a cohort. Within the neighborhood of the c r i t i c a l photoperiod (I5h-16h) temperature has some moderating e f f e c t ; in pa r t i c u l a r a high d a i l y maximum temperature leads to continuous development even though the same constant average temperature can lead to a substantial proportion of the insects entering diapause. An additional observation was made from these data. The portion of a cohort which entered diapause consisted of the las t individuals to emerge from their hosts i . e . , those with the slowest development rates. This association between 46 development time and the diapause response i s not shown here because i t would be comparing individuals reared under d i f f e r e n t conditions. The relationship i s examined in Chapter 4. F i e l d data Tables 5 and 6 are weekly summaries of diapause incidence in f i e l d c o l l e c t i o n s of A. rubecula from BBY and cohorts reared at UBC during 1981 and 1982. Temperatures and c i v i l daylengths at Vancouver are given. In the absence of information regarding the spectral s e n s i t i v i t y of A. rubecula, I assume the natural photoperiod includes a large portion of the c i v i l twilight periods (see Beck 1980 and Saunders 1975 for a discussion). C i v i l daylength i s , in any case, the period which environmental chambers most closely mimic and i t i s these data which are compared. The f i e l d data confirm laboratory experiments and show that diapause i s f i r s t induced in the f i e l d at photoperiods between 15.5h and 16h photophase. At I5h, almost every individual enters diapause. In 1981, the f i r s t individuals observed to enter diapause emerged from their hosts on August 17; in 1982 on August 7. Most individuals emerging to spin cocoons during the l a s t week of August in both 1981 and 1982 entered diapause despite t h i s being a warmer period during 1982. Diapause incidence was intermediate during the second and t h i r d weeks of August. There 47 EMERGENCE PERIOD TEMPERATURE range mean ( °c ) DAYLENGTH c i v i l h min N DIAPAUSE % July 27 - Aug 8 1 2-25 17.3 1 5 30 52 0 -August 27-30 1 0-20 16.1 1 4 40 17 17 1 00% September 5-8 1 2-28 18.1 1 4 1 2 16 1 6 1 00% September 10-19 1 0-26 16.6 13 44 21 21 1 00% after Sept 20 - - <13h 16 16 1 00% TABLE 5. Summary of diapause incidence in field - r e a r e d parasites, Vancouver (UBC) 1981. Daylengths from 1981 Canadian Almanac and Directory corrected to c i v i l daylengths after Beck (1980). Daylength for median date of i n t e r v a l , average temperature over entire i n t e r v a l . 48 EMERGENCE PERIOD TEMPERATURE DAYLENGTH N DIAPAUSE % range mean c i v i l (°C) h min July 11-22 (UBC) 10-24 15.4 17 12 85 0 -July 23-31 (UBC) 10-28 18.3 16 36 39 0 -August 1-7 (UBC) 1 2-25 16.1 16 14 53 2 4% (BBY) 13 0 — Aug 8-15 (UBC) 9-22 15.2 15 45 29 3 10% (BBY) 37 1 4 38% Aug 16-23 (UBC) 1 2-22 16.6 1 5 25 87 87 100% (BBY) 18 9 50% Aug 24-31 (UBC) 12-25 17.4 1 4 54 45 45 100% (BBY) 63 45 71% Sept 1-8 (UBC) 1 3-24 17.4 1 4 1 2 58 58 100% (BBY) 17 16 94% Sept 9-18 (UBC) 8-23 14.5 13 45 73 73 1 00% Sept 19-25 (UBC) 12-22 15.3 1 3 10 25 25 100% TABLE 6. Summary of diapause incidence in fiel d - r e a r e d parasites from two locations (UBC and BBY) near Vancouver 1982. Daylengths from 1982 Canadian Almanac and Directory corrected to c i v i l daylengths after Beck (1980). Daylength for median date of i n t e r v a l , average temperature over entire i n t e r v a l . 49 were a few individuals c o l l e c t e d at BBY which did not enter diapause after emerging the l a s t week of August, 1982. This may have been due to temperature; either because d a i l y temperatures at BBY are, on average, warmer than those measured at UBC or because para s i t i s e d larvae c o l l e c t e d at BBY were reared in small wooden cages which absorbed far more heat than did the large screen cages used for UBC cohorts. But the f i n a l demonstration of the predominance of the photoperiodic response over that of temperature was fortuitous. A cohort i n i t i a t e d on August 15, 1982 began emerging and spinning cocoons on August 30. Seventeen A. rubecula were co l l e c t e d between August 30 and September 9 and were, as expected, a l l in diapause. On September 8 the cage was relocated behind a greenhouse only 30m away. Greenhouse l i g h t s extended the daylength in the v i c i n i t y to close to I8h. Only one of the ten parasites emerging one week after t h i s move entered diapause, despite average temperatures during that period which were the coolest since the preceding spring. When paras i t i s e d hosts were transferred from short-day conditions in the f i e l d to long-day conditions in the laboratory, the diapause response of the population decreased with the time spent under nondiapause conditions (Table 7). Unlike Schisler (1981) I found that an appreciable disruption of the diapause response required over 40 degree-days, or about one-third of the l a r v a l parasite's lifespan be spent under long-day conditions. These data agree with Schisler that 50 DEGREE-DAYS ABOVE 11° REQUIRED TO COMPLETE PARASITE DEVELOPMENT IN THE LABORATORY ESTIMATED PROPORTION OF PARASITE DEVELOPMENT COMPLETED AT TIME OF FIELD COLLECTION N DIAPAUSE % 23 0.81 10 10 1 00% 34 0.71 6 5 83% 46 0.62 15 8 53% 58 0.52 23 5 22% 69 0.42 1 6 1 6% 80 0.33 5 0 -92 0.23 2 1 50% 115 0.04 1 0 -TABLE 7. Diapause in A. rubecula larvae c o l l e c t e d at BBY on September 16-17, 1981 and transferred to laboratory conditions which promote continuous development (22°C 16L:8D). 51 A. rubecula larvae mid-way through the developmental period are sensitive to diapause-inducing photoperiods but neither set of data permits d i s t i n c t i o n between photosensitivity to diapause induction and disruption. It i s possible that a l l immature stages are photosensitive but require a minimum number of short photoperiods before diapause i s induced. Diapause would be averted when transfer to long-day conditions occurred early in the developmental period, as Schisler and I have observed. How early such a disruption would occur might depend on temperature, since low temperatures increase developmental time and lead to more photoperiodic cycles per l i f e stage. DISCUSSION Vancouver A. rubecula show a photoperiodic response t y p i c a l of "long-day" insects . Long daylengths (I6h) and high temperatures (>19°C) promote.continuous development while shorter daylengths and lower temperatures lead to diapause. It is the p a r a s i t i c l a r v a l stage which responds to the photoperiod, although s e n s i t i v i t y may be extended to the prepupal (eonymph) stage (Schisler 1981). The c r i t i c a l photoperiod for diapause induction of Vancouver parasites l i e s between 15h and 16h of l i g h t per day. The consistency of the response depends to some extent on temperature. Unusually cold conditions could lead to premature diapause in some individuals but the predominant and abrupt photoperiodic response ensures that the t r a n s i t i o n from 52 continuous development to diapause i s l i k e l y to occur over a very short period of time in the f i e l d . There i s good evidence that absolute daylength i s measured because no diapause has been observed when daylengths in the f i e l d are decreasing but s t i l l above the c r i t i c a l daylength (July in Vancouver; January, February and March in Canberra). In Vancouver, diapause i s noted as early as the second week of August and includes the entire population by the end of the month but in Canberra, diapause is not observed u n t i l after the autumnal equinox. This cannot be explained by the r e l a t i v e l y warmer temperatures in Canberra. The small moderating effect of temperature on the photoperiodic response established in my experiments was confirmed by Parker and Pi n n e l l (1972) who noted that Vancouver A. rubecula released in Missouri entered diapause in early September, despite the warmer temperatures in their new environment. This early diapause in the Vancouver population may be part of the problem with establishing A. rubecula in warmer local e s . I return to th i s question in the f i n a l chapter. 53 CHAPTER 4 MANIPULATION OF THE DIAPAUSE RESPONSE INTRODUCTION Manipulation of the diapause response in insects by selective breeding of both pests and predators i s an intriguing p o s s i b i l i t y for pest management (Chippendale 1982; Hoy 1976, 1978). The general h e r i t a b i l i t y of the diapause response i s amply demonstrated by the rapid selection for nondiapause in laboratory strains (Harvey 1957; House 1967; Hoy 1975, 1978). Hybridization of laboratory strains or natural populations d i f f e r i n g in their diapause response often y i e l d s ' o f f s p r i n g with a diapause response which i s intermediate to that of the parents; an indication of polygenic control (Danilevskii 1965; Hoy 1975, 1978; Tauber and Tauber 1978). There may be additional nongenetic maternal e f f e c t s on diapause. Diapause increases with maternal age in some insect parasites (Saunders 1965; Simmonds 1948) and i t i s the photoperiodic response of the mother which determines the diapause response in the offspring (Ryan 1965; Saunders 1965, 1966). Henrich and Denlinger (1982) showed that the diapause history of the female f l e s h f l y , Sarcophaga bullata Parker, af f e c t s diapause in her progeny. Interestingly, the effect was determined, not by diapause i t s e l f , but by the diapause-inducing photoperiod experienced by the larva of the maternal 54 generation. The l a s t chapter showed that the photoperiodic responses of Vancouver and Canberra populations d i f f e r e d markedly. This difference could be due to differences in the o r i g i n a l native stock or to divergence following i t s introduction to Canada and A u s t r a l i a . Either si t u a t i o n implies within-species v a r i a b i l i t y of the diapause response. I suggested that the high c r i t i c a l photoperiod of the Vancouver population has contributed to the d i f f i c u l t i e s encountered in attempting to e s t a b l i s h t h i s population in other parts of North America. This chapter develops a methodology for a breeding program to modify the diapause response of Vancouver A. rubecula . Considering the p o s s i b i l i t y of maternal e f f e c t s , experiments to check for effects of maternal age and diapause history were considered prerequisites of any breeding program and are included. The preliminary data i l l u s t r a t e new features of the biology of diapause in A. rubecula and suggest a p r a c t i c a l approach for future releases of the parasite in North America. 55 METHODS Experiments in Chapter 3 indicated that i f A. rubecula larvae were reared at 20°C 15.5L:8.5D, only a portion of the cohort would enter diapause. These conditions were chosen as the standard for a l l experiments described in t h i s chapter. A kale-based diet modified from Wilkinson et a l . (1972) was used as a plug at one end of a 7-dram v i a l and held in place by a rubber "0" ring. Three para s i t i s e d host larvae (late-II or early-III instar) were placed on the diet and the v i a l placed upside down on a tray in the incubator. Survival rates of host larvae were near 67%, less than that of unparasitised larvae. The time required from oviposition to emergence of the parasite from the host was generally one day longer at 20°C on the diet than i t was on potted plants. A similar lag in development time has been noticed in unparasitised P. rapae and i s thought by N. Gi l b e r t to be due to an i n i t i a l reluctance of the c a t e r p i l l a r s to feed on the di e t . The f i r s t experiment compared the diapause response of progeny from old and young female parents. Old females were six weeks old and had oviposited many times. They had been stored at 10°C, then moved to 16°C for three days before being used in the experiment. Young females had eclosed only two to four days e a r l i e r and were held, with males, at 22°C. The second experiments compared the diapause response of 56 progeny of females which had entered diapause under the short-day conditions of previous experiments (Chapter 3 ) , with the progeny of females which had never experienced-a diapause stage. Individuals which entered diapause in the experiments on diapause history became the diapause group (D) and were stored at 0°C. Those which continued to develop to adult were the nondiapause group (N) and were mass-mated and their o f fspring reared at 2 0 ° C 1 2 L : 1 2 D . A l l of these progeny entered diapause and were stored with the D-group. So both D and N parents experienced a diapause stage. Total storage time for the diapause group was four months and for the nondiapause group, about three weeks le s s . These formed the following parental groups: D male X D female N male X D female D male X N female N male X N female plus unmated D and N females. Differences in maternal age were avoided. A l l females used in these experiments had been caged with males for two weeks and had limited foraging history. 57 Analysis Experiments to detect maternal and genetic e f f e c t s on the diapause response of the offspring were analyzed in the same way. I was interested in the effects of maternal condition (old vs young or post-diapause vs no diapause) or parental group on the diapause response. I recorded development time because I suspected i t would also a f f e c t the diapause response (Chapter 3). This mix of categorical and continuous explanatory variables with a binary response variable (diapause or nondiapause) was analyzed with l o g i t models (Feinberg 1980). These log-linear models employ a weighted regression of transformed variates. Logit analysis extends the log-linear model to allow estimation of parameters and prediction of the binary outcome. Structure of the model, and to some extent i t s interpretation, is analogous to standard regression analysis (Neter and Wasserman 1974). The transformed p r o b a b i l i t y , p, of an individual entering diapause under sp e c i f i e d conditions i s given by; l o g i t p = log[p/d-p)] The l o g i t regression model assumes that the dependence of this 58 transformed probability of entering diapause on the various explanatory variables can be expressed as a linear relationship such as; l o g i t p = B0 + B, X/ + BtXi + ... + B/ X/ + ... + BflXtf where X^'is an explanatory variable such as parental group. As in multiple regression, powers of variables and interactions between variables can be included. The models are f i t t e d by the method of maximum l i k e l i h o o d . Alternative models are compared and tested for goodness-of-fit by the method of l i k l i h o o d r a t i o . Sokal and Rohlf (1981) discuss the merits of the lo g - l i k e l i h o o d test s t a t i s t i c . The analysis of nongenetic maternal effects used a s t a t i s t i c a l package (BMD:PLR Engelman 1981) but the analysis of the parental groups was extended by Dr. A.John Petkau, S t a t i s t i c a l Consulting Service, UBC, to consider contrasts among l o g i t s corresponding to the d i f f e r e n t parental groups. I am indebted to Dr. Petkau for introducing me to l o g i t analysis and for the several hours he spent in analysis and consultation on t h i s problem. Analysis of parental effects on offspring development rates under constant conditions employed analysis of variance with orthogonal contrasts (Steel and Torrie 1982). The experiments on maternal age and diapause history consisted of three and six t r i a l s respectively. These t r i a l s 59 represent d i f f e r e n t days of set-up and therefore s l i g h t l y d i f f e r e n t diet q u a l i t i e s . Different t r i a l s proved to have a s i g n i f i c a n t e f f e c t of the outcome in both experiments (Tables 8 and 9 ) . In the experiments with parental crosses, a large number of individuals were set up each day so that the number of t r i a l s was limited to two. Developmental times between t r i a l s were similar and the ef f e c t of d i f f e r e n t diets on the diapause response was v i r t u a l l y n i l in this experiment. Data from d i f f e r e n t t r i a l s in these genetic experiments were pooled for the f i n a l analysis. RESULTS Effect of maternal age and diapause history The results of the stepwise l o g i t regression analysis are in Tables 8 and 9 . Development time always had the largest effect on the diapause response. Diapause individuals were among the slowest to develop in a cohort (Figs. 6 and 7 ) . In one case, there was an additional quadratic ef f e c t of development time (Table 9 ) . Including t r i a l (day of set up) as a variate improved the f i t in both cases, indicating the diapause response was sensitive to differences in the d i e t , perhaps v i a i t s effect on development rates. The extreme differences in maternal age had a s i g n i f i c a n t effect on the diapause response in the progeny but th i s may have been due to a high c o r r e l a t i o n between maternal age and IMPROVEMENT GOODNESS-OF-FIT STEP TERM df CHI-SQUARE p-va lue CHI-SQUARE p-va lue 1 Cons tan t 39 163. 199 <0. 001 2 Development t ime 38 1 14 .562 <0 001 48. 637 0. 1 16 3 T r i a l 36 13 . 146 0. .001 35. .490 0. .493 4 Materna l age 35 3 .310 0. ,069 32 . 180 0. 605 5 Dev X Age 34 6 .855 0. .009 25 . 325 0. 859 TABLE 8. Summary of l o g l t a n a l y s i s o f expe r imen t s to t e s t the e f f e c t o f mate rna l age on the d i a p a u s e r esponse of the p rogeny . Development t ime i n days a t 2 0 ' C 1 5 . 5 L : 8 . 5 D . IMPROVEMENT GOODNESS-OF-FIT STEP TERM df CHI-SQUARE p-va lue CHI-SQUARE p-va lue 1 Cons tan t 69 274. .329 <0. .001 2 Development t ime 68 170, ,516 <0. .001 103. .812 0. .003 3 Dev X Dev 67 12 .465 <0, .001 91 . 347 0. .026 4 T r i a l 62 22 .698 <0. 001 68 . 649 0. . 262 TABLE 9. Summary o f l o g l t a n a l y s i s o f expe r imen t s to t e s t the e f f e c t o f mate rna l d i a p a u s e h i s t o r y on the d i a p a u s e r esponse of the p rogeny . Development t ime i n days a t 20' C 1 5 . 5 L : 8 . 5 D . 61 FIGURE 6. Development period in days at 20°C, 15.5L:8.5D, for the p a r a s i t i c stages of the offspring of old and young female A. rubecula. Black portions are diapause individuals. 62 30 o c CD D D" 0) III 40 30 20 10 y o u n g 11 12 13 14 15 16 17 18 19 20 Development period (days at 20°C) 63 FIGURE 7. Development period in days at 20°C, 15.5L:8.5D, for the p a r a s i t i c stages of the offspring of female A. rubecula with d i f f e r e n t diapause h i s t o r i e s . Black portions are diapause individuals. 64 50 40 30 20 O 1 0 C 0 o CJ 60 50 40 30 20 10 p o s t d i a p a u s e n o d i a p a u s e 12 13 14 15 16 17 18 19 20 Development period (days at 20°C) 65 MATERNAL AGE PROGENY DEVELOPMENT TIME (SE) N (days at 20°C) YOUNG 131 14.08 (0.17) OLD 114 15.40 (0.18) F=28.52 df=1,243 P<0.001 TABLE 10. Mean development time at 20°C of progeny of young and old females. Analysis of variance used Log (dev time + 1). MATERNAL DIAPAUSE HISTORY PROGENY DEVELOPMENT TIME (SE) (days at 20°C) N NO DIAPAUSE 242 15.20 (0.12) POST DIAPAUSE 145 15.08 (0.15) F=0.467 df=1,385 p=0.50 TABLE 11. Mean development time at 20°C of progeny of females which had, or had not experienced a previous diapause. Analysis of variance used Log (dev time + 1). 6 6 developmental period (r=0.56, df=34, p<0.05). The o f f s p r i n g of o l d females showed an increased diapause response and a l s o took longer to develop than those of young females (Table 10). There was no e f f e c t of maternal diapause h i s t o r y e i t h e r before or a f t e r the e f f e c t s of development time and t r i a l were included.. Nor were development times of the progeny d i f f e r e n t (Table 11). In the g e n e t i c a l experiments, the p r o b a b i l i t y of diapause again increased with development time; i n the a l t e r n a t e models i n Table 12, a l l slopes are greater than 0 (p<0.05). There was an a d d i t i o n a l p a r e n t a l e f f e c t ( F i g . 8). Of the a l t e r n a t e models i n Table 12 and F i g . 8, model A i s the simplest and provides an adequate f i t but the a d d i t i o n of a term for the i n t e r a c t i o n of par e n t a l group and development time under model B s i g n i f i c a n t l y improves the f i t . The nature of the i n t e r a c t i o n i s not c l e a r ; there were no d i r e c t p a r e n t a l e f f e c t s on mean development time of the o f f s p r i n g (Table 13). By i n c l u d i n g the i n t e r a c t i o n , d i f f e r e n c e s between p a r e n t a l groups depend on the developmental per i o d at which the comparisons are made. At mean development time (16 days), e i t h e r model i n d i c a t e s an unusual g e n e t i c a l s i t u a t i o n ; the o f f s p r i n g of crosses have a lower p r o b a b i l i t y of ent e r i n g diapause than e i t h e r inbred l i n e , mated or not ( F i g . 8 ) . Normally, one expects crosses to be intermediate t o , or l e a s t i n d i s t i n g u i s h a b l e from, the inbred l i n e s . 67 l o g i t (p) PARENTAL GROUP MODEL A MODEL B Dm X Df 0 .377 + 1 .141 (dev- 16) 0. 503 + 1 .321 (dev- 16) Nm X Nf 0 .805 + 1 . 141 (dev- 16) 0. 564 + 0 .856 (dev- 16) Nm X Df -o .146 + 1 . 141 (dev- 16) -o. 171 + 0 .956 (dev- 16) Dm X Nf -0 .427 + 1 . 141 (dev- 16) -0. 446 + 0 .989 (dev- 16) Nf 0 .799 + 1 .141 (dev- 16) 2. 033 + 2 .206 (dev- 16) Df 1 .937 + 1 .141 (dev- 16) 2. 256 + 1 .406 (dev- 16) GOODNESS-OF-FIT G 2 57.46 df=62 43.37 df=57 TABLE 12. F i t t e d models for l o g i t analysis of diapause response of d i f f e r e n t parental groups (dev=development time in days at 20°C). D-diapause, N-nond.iapause; m-male, f-female. oo FIGURE 8 . D i apause p r o b a b i l i t y o f the o f f s p r i n g of d i f f e r e n t p a r e n t a l g r o u p s . Ba rs a r e 95% c o n f i d e n c e i n t e r v a l s . E s t i m a t e s a re from models A and B i n T a b l e 12. The p r o p o r t i o n of males i n the a s s o c i a t e d groups 1s shown below the a b s c i s s a . 1 0 1 O) a CO .75 co n o CD co 3 CO a co • mmm a ,50 .25 Df Nf DmDf N m N f NmDf DmNf 1.0 1.0 .51 .89 proportion males ,51 A B .41 ON to 70 PARENTAL GROUP N PROGENY DEVELOPMENT TIME (SE) (days at 20°C) Dm X Df Nm X Nf Nm X Df Dm X Nf Nf Df 181 1 54 121 1 16 97 87 15.8 (0.19) 16.3 (0.21) 16.1 (0.18) 15.5 (0.21) 15.6 (0.27) 16.0 (0.25) F=2.08 df=5,750 p>0.20 TABLE 13. Mean development time of progeny of each parental group. Analysis of variance used Log (dev time + 1). MODEL A MODEL B CONTRAST ESTIMATE SE p-va lue ESTIMATE SE p-va lue DmDf • - NmNf -0. .43 0. .30 0. , 16 -0 .06 0. 35 >0 . 20 DmDf • - NmDf -0. 28 0. .37 >0 . 20 -0. .28 0. 35 >o . 20 Nf • - Df -1 .  14 0. 39 0, .004 -0. . 22 0. 83 >0. . 20 DmDf • - Df -1 . ,56 0. 36 <0, 001 -1 . 75 0. 58 0. .003 NmDf • - Df -2. 08 0. 38 <0. .001 -2 . ,43 0. 57 <0. 001 DmNf • - Nf -1 . 23 0. 40 0. .002 -2 . 48 0. 70 0. .001 NmNf • - Nf 0. .01 0. 34 >0. .200 - 1 . ,47 0. 69 0. .030 TABLE 14. C o n t r a s t s among l o g i t s at mean deve lopment t ime of 16 days at 20'C. D-d l apause , N-nondlapause ; m-male p a r e n t , f - f ema l e p a r e n t . 72 The progeny of unmated females show a s i g n i f i c a n t l y higher diapause response than any of the other l i n e s (Table 14, F i g . 8 - model B). Their offspring are a l l males and thi s provides a clue to a possible explanation of the re s u l t s . F i g . 8 includes, with each parental l i n e , the proportion of male progeny. The proba b i l i t y of diapause increases with the proportion of males in the experimental cohort. The r e l a t i v e magnitude of t h i s proportion can also explain the apparent parental effects but the data do not permit separation of the two possible covariates. The data could be explained as purely due to sex or genetics or some combination of the two. But i f the effect of sex were genuine, and there remains a s i g n i f i c a n t interaction, then there should be an interaction between sex and development time within parental groups. There i s not; at least not for nondiapause individuals from a l l groups. Chapter 2 showed we should not expect any relationship between sex and development time in A. rubecula larvae, and these data confirm t h i s . Moreover, in the experiments using old and young wasps, the offspring of older females had an increased diapause response even though the proportion of males was much lower in that group (0.24) than in the young mother group (0.50). So the effe c t of sex was then in the opposite d i r e c t i o n . In the absence of independent data to distinguish between the e f f e c t s of sex and parental group, inclusion of either term i s , at best, p r o v i s i o n a l . Given the importance of the interaction between parental group and development time under model B, i t i s best to consider the model which also includes 73 the parental group as a main e f f e c t . In t h i s model (B), the ef f e c t s of inbreeding are s l i g h t and dependent on where in the developmental period the comparisons are made. DISCUSSION Under temperature and photoperiods where only a portion of the parasites of any parental o r i g i n entered diapause, the larvae which emerged from the host f i r s t were less l i k e l y to enter diapause than those which emerged l a t e r . This relationship has also been noted in the o r i e n t a l f r u i t moth (Dickson 1949). Its s i m i l a r i t y to the ageing effect on the photoperiodic response of maternal Nasonia v i t r i p e n n i s (Walk.) (Saunders 1965) suggests a common mechanism. Saunders (1976) subsequently showed that photoperiodism required not only measurement of the duration of the photoperiod but also summation of successive days' information; i . e . a clock and a counter (Saunders 1981). A. rubecula which require longer development times experience more photoperiods and are more l i k e l y to enter diapause. In A. rubecula males and females develop at the same rate so there w i l l be no sexual selection via development time, but the p o s s i b i l i t y of a dir e c t maternal influence on sex r a t i o which in turn a f f e c t s the proportion of progeny entering diapause cannot be ignored. After a l l , the mother determines the sex of her offspring. The lack of influence of maternal physiology is not surprising since the larva i s the stage which 74 i s sensitive to diapause-inducing conditions (Chapter 3). Analysis of results of the single selection for diapause is inconclusive. The disadvantages of selecting for a binary character such as diapause (we have no idea how 'far away' are parental means) which requires a three-month obligatory incubation period between generations makes selection on the diapause response impractical. But the results suggest an a l t e r n a t i v e . It should be possible to select, under nondiapause conditions, fast and slow developers. Development rate i s a good quantitative character in A. rubecula ranging from 11.5 to 22.0 days at 20°C. Since h e r i t a b i l i t y of development time has been demonstrated in insects (Goldschmidt 1980), i t would be of more than merely p r a c t i c a l interest i f selection for development rate also modified the diapause response as both are e c o l o g i c a l l y related in the control of seasonal development. There i s l i t t l e reason to doubt that modification of the diapause response in A. rubecula should be any more d i f f i c u l t than in other braconid species. But given the existence of the Australian population which does not enter diapause u n t i l daylength i s less than 12h, the most sensible advice for b i o l o g i c a l control programs in areas where cabbages are a late-summer or autumn crop i s to introduce stock from the Australian, rather than the Vancouver source. 75 CHAPTER 5 DIAPAUSE TERMINATION AND POST-DIAPAUSE DEVELOPMENT INTRODUCTION The conditions and time required to terminate diapause set the clock for the resumption of a c t i v i t y in overwintering populations. Termination of diapause marks the s t a r t i n g point for the phenological model. This chapter takes us back to the f i r s t of the season. It considers the effects of storage conditions on diapause termination rates and the developmental response to temperature following diapause. By corroboration with f i e l d data, I discuss winter and spring ecology of the parasite A. rubecula. Andrewartha. (1952) was one of the f i r s t to recognize that diapause was an active state; physio- and morphogenesis were suppressed but not arrested. He proposed the term "diapause development" to emphasize that diapause was an alte r n a t i v e developmental strategy with s p e c i f i c requirements for i t s completion. The confusion and controversy regarding diapause terminology (Danilevskii 1965; Mansingh 1971; Sheldon and Macleod 1974; Thiele 1973) r e f l e c t s the necessity for better understanding of the physiology of diapause and the resumption of normal development. Yin and Chippendale (1973) have shown that diapause maintenance can be under neuro-endocrine control and, as in 76 diapause induction, some measurement of time i s involved. The termination of diapause may be a response to s p e c i f i c stimuli such as c h i l l i n g (Schneiderman and Horwitz 1958) or the insect may show a decreasing response to diapause-inducing stimuli (Tauber and Tauber 1976) or simply no response to any stimuli u n t i l a certain refractory period has passed (Mansingh 1971). If diapause is an alternative developmental strategy, i t should be possible to characterize i t s progress and completion as a response to ambient conditions. Photoperiod i s the most important factor for induction of diapause but termination and post-diapause development are often temperature responses; photoperiodic s e n s i t i v i t y decreasing after the onset of diapause (Danilevsky et a l . 1970). The same lag in the daylength-temperature rela t i o n s h i p that produced r e l a t i v e l y warm but short days in late summer results in long spring days when temperatures are d i s t i n c t i v e l y cool. Even when diapause termination i s a s p e c i f i c response to a c r i t i c a l photoperiod, that photoperiod i s usually surpassed in nature while temperatures are too low for appeciable morphogenesis. The insect remains in torpor u n t i l temperatures exceed a developmental threshold (Tauber and Tauber 1978). There i s no evidence that diapause termination in A. rubecula i s a photoperiodic response. A l l diapause eonymphs from the experiments reported in Chapters 3 and 4 were transferred from short to long-day conditions immediately after spinning a cocoon and development did not resume before three 77 months cold storage. There was no diapause when eonymphs were transferred from a 16h to 12h photophase at several temperatures so f r e e - l i v i n g stadia may not show a photoperiodic response at any time. If that i s the case, parasite larvae l i t e r a l l y moult their photoperiodic s e n s i t i v i t y . The experiments reported here investigate diapause termination and post-diapause devlopmental rates in A. rubecula. There i s a series of laboratory experiments in which cocoons were incubated under variable storage conditions and then brought to temperatures which promote continuous development. Other cocoons were stored in the f i e l d and then brought to controlled conditions in the laboratory and s t i l l others were l e f t in the f i e l d to determine the dates of diapause termination and adult emergence under natural conditions. METHODS Experiments in 1982 used A. rubecula cocoons from the c r i t i c a l photoperiod experiments of Chapter 3. E a r l i e r t r i a l s had shown sporadic adult emergence and high mortality when diapause eonymphs were stored at a moderate temperature (23°C) or for less than three months at low temperatures (0°or 10°C). It was also noted that although photoperiod experienced by the larva did not affe c t post-incubation development time, there was a s l i g h t effect of l a r v a l rearing temperatures, probably via the effects of temperature on f i n a l weight (Chapter 2). A l l 78 experiments which analyze developmental time used cocoons of larvae reared at 20°C. To determine weight loss during incubation, weighed cocoons were stored for three or four months at 0°C, then re-weighed and brought to 23°C. Weight after incubation, time to adult emergence and the sex of each individual were recorded. A second series was s i m i l a r l y divided into two storage times, three or four months, and further subdivided into two storage temperatures, 0° and 10°C. These cocoons were not weighed u n t i l after they were removed from storage. They were held under the same conditions as the previous t r i a l s (23°C). Time to adult emergence and the sex of each individual was recorded. The factors were coded with dummy variates and analyzed by multiple regression (Gilbert 1973). A p a r a l l e l series was incubated under natural conditions in a Stevenson screen at UBC on January 20, 1982. Fifteen cocoons were brought to 23°C on March 29, A p r i l 23 and May 15. Ten more cocoons were l e f t in the Stevenson screen to determine f i e l d emergence dates. One attempt was made in 1982 to determine post-incubation development time at temperatures between 15° and 28°C and a long day; optimal conditions for development. None of the insects at 15°C terminated diapause and over 25% of the insects at 17°C were s t i l l in diapause aft e r sixty days. The experiments in 1983 were more extensive. I 79 distinguished diapause termination and post-diapause development times at 17°,19°,23° and 28°C. When diapausing eonymphs were transferred to these warmer temperatures after cold storage for at least three months, morphogenesis did not begin immediately. The eonymph is the mature parasite larva; therefore the midgut i s blind and does not open into the proctodaeum. At the f i n a l moult, the guts join and the feces and l a r v a l c u t i c l e are cast together (the meconium). When illuminated, these post-diapause individuals can be i d e n t i f i e d by this dark meconium at the posterior of the cocoon. At thi s point, I consider diapause i s terminated and subsequent development i s that of the pupa. It should be noted that t h i s d e f i n i t i o n of diapause termination i s s t r i c t l y operational as active development must occur before the appearance of the meconium. 80 RESULTS Although diapause cocoons stored for four months at 0°C lost s l i g h t l y more weight than those stored for three months, the proportion of weight l o s t in either group did not d i f f e r s i g n i f i c a n t l y (F=3.52 df=1,71 0.05<p<0.10). Males and females lost an equal proportion (about 16%) of their weight under these storage conditions. Thus the relationship between sex and weight remained the same after diapause, with females heavier than males. As in continuous development, males develop to adult more rapidly than females (F=27.78 df=1,71 p<0.00l). Weight may be an additional determinant of development time since smaller individuals tend to develop more rapidly, but thi s trend was not as convincing here as i t was in the experiments measuring continuous development (Chapter 2). There was a marginal effect of storage time on the developmental period with those insects stored for four months taking less time than those stored for three months at 0°C (F=5.45 df=1,71 0.0l<p<0.025). The second experiments confirmed that after three or four months storage at temperatures below the developmental threshold, females were, on average, heavier than males and required more time to develop to adult (Table 15). Once the sex of the parasite was considered, there were no additional e f f e c t s of weight, rearing conditions, or storage time on the time to adult emergence. 81 SEX N WEIGHT (SE) TIME TO ADULT (SE) mg (days at 23°C) FEMALE 78 5.54 (0.0635) 17.90 (0.202) MALE 103 4.93 (0.0552) 15.66 (0.143) TABLE 15. Mean cocoon weights and time to develop to adult for male and female A. rubecula after three or four months storage at 0° or 10°C. 82 Adding storage temperature did s i g n i f i c a n t l y improve the f i t of the regression leading to the descriptive model; DEVELOPMENT TIME = 17.484 + 0.799 ST.TEMP - 2.258 SEX (days at 23°C) R2=0.37, df=178, p<0.0l where ST.TEMP=0 for 0°C, 1 for 10°C; SEX=0 for females, 1 for males. Thus females take longer to develop to adult than males and a lower storage temperature decreases post-incubation development time. F i e l d data from 1982 showed a d e f i n i t e effect of storage time on post-diapause development, with those stored for longer periods taking less time to develop to adult (Table 16). Note that the largest difference occurs between those removed from the f i e l d on March 29 and A p r i l 23 despite the fact that the period of greatest temperature accumulation was between A p r i l 23 and May 15. The main conclusions of the 1981 experiments were that, given a s u f f i c i e n t storage period, i . e . , at least three months below the developmental threshold of 11°C and temperatures greater than 15° to 17°C, the most important determinant of subsequent time to adult emergence was the sex of the insect; males emerged before females. This same emergence pattern was evident in the experiments using conditions promoting continuous development (Chapter 2) indicating winter diapause 8 3 FIELD STORAGE DATE RETURNED TO N MEAN DEVELOPMENT TIME DURATION LABORATORY (days at 22°C) 10 weeks March 29 14 25.2 (0.59) 14 weeks A p r i l 23 12 19.9 (0.57) 17 weeks May 15 13 19.1 (0.49) TABLE 16. Mean time in days to adult emergence of diapause cocoons taken to the f i e l d on January 20, 1982, returned to the laboratory on indicated dates and held at 22°C, 16L:8D. 84 does not disrupt what appears to be a fundamental difference in male and female developmental biology. This difference i s not due to differences in weight, although females are consistently heavier than males. The difference i s confirmed under natural conditions where individuals stored outside in a Stevenson screen emerged over a r e l a t i v e l y short period of time but males s t i l l emerged f i r s t (Fig. 9 ) . Larval rearing conditions appeared to have no e f f e c t on development aft e r diapause. In addition, storage conditions had l i t t l e e f fect although the pa r t i c u l a r treatments used, especially with respect to duration were a r t i f i c i a l l y short, so comparisons may not be e c o l o g i c a l l y relevant. There were consistent indications that increased storage time decreased the subsequent developmental period. This i s apparent in the small data set from natural storage conditions (Fig. 9 ) . Individuals not taken to the f i e l d u n t i l winter emerged later the following spring than did those which had been stored for the entire autumn-winter period. oo in FIGURE 9. S p r i n g emergence of A. r u b e c u l a o v e r w i n t e r e d 1n the f i e l d a t Vancouver (1982 ) . D i apause d a t e 1s the da te f o r the b e g i n n i n g of f i e l d s t o r a g e of d i a p a u s e i n d i v i d u a l s . S o l i d b a r s a r e ma les , open b a r s a r e f e m a l e s . June 15 Julyi July is EMERGENCE DATE 87 Distinguishing diapause termination from post-diapause pupal development was a s i g n i f i c a n t improvement in the design of the 1983 experiments. I was able to follow Morris and Fulton (1970) and divide the t o t a l heat requirement for post-storage development to adult into two parts, Kp = Kdt + Km, where dt represents diapause termination and m represents pupal morphogenesis. I w i l l discuss each process separately. Diapause termination Tables 17 and 18 give descriptive measures of the time required to terminate diapause at d i f f e r e n t temperatures following variable storage h i s t o r i e s for male and female A. rubecula. Males completed diapause development sooner than females. Cocoons stored in the laboratory for four months at 10°C took the longest to terminate diapause at any temperature and th i s group suffered the highest mortality. A series stored for twelve months at 10°C terminated diapause in three to ten days at 23°C but less than a t h i r d of these survived beyond diapause termination. Cocoons stored in the f i e l d for the winter required less time to terminate diapause than those stored for a comparable period at 10°C but more time than those stored for longer periods at lower temperatures. F i e l d temperatures at UBC during th i s period (November to March) were mostly between 0° and 12°C at a mean of 6.4°C and so were intermediate to the controlled storage temperatures. N MEAN TERMINATION TIME (DAYS) + SD (RANGE) STORAGE MONTHS TEMP MORTALITY SEX 17' 19' 23' 4 10 15% m 10 30 .8 ± 3.12 (25-36) 55 23 .8 ± 5.14 ( 17-37) 38 14.6 ± 3.84 ( 10-26) 31 11.4 ± 1.12 (10-14) f 2 3 6 . 0 + 2.83 (34-36) 16 26 .6 ± 6.63 ( 17-37) 1 1 14.9 + 4.04 ( 1 1-26) 9 11.1 ± 0 .93 (10-13) 6 0 11% m f 15 8.5 ± 1.60 (6-13) 10 9 .0 ± 1.56 (7-12) 12 4 .8 ± 1.03 (3-6) 5 6 .6 + 1.52 (5-9) 17 3.1 ± 0 .33 (3-4) 8 3.6 ± 0.52 (3-4) 7 o m f 19 4 .9 + 1.13 (4-7) 9 4 .6 + 1 0 1 (4-7) 8 0 10% m 6 11.5 + 4 .23 (5-16) 15 6 .6 + 2.53 (4-13) 18 4 .8 ± 1.31 (3-7) 1 1 3.1 ± 0 .30 (3-4) f 4 11.2 + 0 . 5 0 ( 11-12) 6 7.3 + 1.21 (5-8) 5 7.0 + 1.87 (5-10) 8 3.8 ± 0 .46 (3-4) TABLE 17 D e s c r i p t i v e measures o f t ime r e q u i r e d f o r male and f ema le A. r u b e c u l a to t e r m i n a t e d i a p a u s e at s e v e r a l t empera tu res f o l l o w i n g s p e c i f i e d s t o r a g e c o n d i t i o n s . N-number of o b s e r v a t i o n s , mean t e r m i n a t i o n t ime ± s t a n d a r d d e v i a t i o n and the range a re g i v e n . 00 00 N MEAN TERMINATION TIME (DAYS) ± SD (RANGE) FIELD STORAGE MORTALITY SEX 19' 23' 28' 4 months 6% 14 13 14 m 1 6 . 1 + 3 . 3 0 9 . 8 + 1 . 9 9 7 . 4 + 0 . 7 6 (14-27) (8-15) (7-9) 9 6 6 f 19.1 ± 3.33 11.3 ± 1.86 7.5 + 0 .55 (14-24) (8-13) (7-8) 5 months 8% 12 m 5.2 ± 0 .58 (5-7) 9 f 6 .3 + 1.00 (5-7) 6 months 12% 7 m 7.4 + 0 .53 (7-8) 9 f 9 . 0 + 1 . 1 2 (8-11) TABLE 18. D e s c r i p t i v e measures o f t ime r e q u i r e d f o r male and fema le A. r u b e c u l a t o t e r m i n a t e d i apause at s e v e r a l t empera tu res f o l l o w i n g s t o r a g e in the f i e l d f o r v a r y i n g p e r i o d s . N-number o f o b s e r v a t i o n s , mean t e r m i n a t i o n t ime ± s t a n d a r d d e v i a t i o n and the range a r e g i v e n . oo 90 The cocoons stored six, seven or eight months at 0°C had comparable termination rates and were pooled to obtain the following regressions on temperature: TERMINATION RATE (MALE) = -0.219 + 0.0191 TEMP R2=0.63, df=112, p<0.0l TERMINATION RATE (FEMALES) = -0.188 + 0.0164 TEMP R2=0.74, df=57, p<0.0l Thermal constants estimated by these regressions show that males and females have the same threshold for diapause termination (11.4°C) but females have a higher heat requirement (61 degree-days compared to 52 degree-days for males). Although development may proceed slowly at temperatures between t h i s threshold and 15° - 16°C, i t appears the actual moult to a pupa requires warmer conditions. On two separate occasions A. rubecula has shown no sign of diapause termination after sixty days at 15° - 16°C, even after several months storage. Desiccation then becomes a major mortality factor. It should be stressed that t h i s thermal requirement i s peculiar to diapause eonymphs because when eonymphs emerging from the host at 22°C were placed in the same 16°C conditions, they were a l l able to moult within three days. A nonlinear model would, under these circumstances, provide a more r e a l i s t i c estimate of thermal requirements for the-population (see Chapter 2) but would require development times from a wide range of fluctuating temperatures. 91 Nevertheless, the estimates from the linear regressions work very well in a situation where temperatures do fluctuate — in nature. F i g . 10 i l l u s t r a t e s data obtained in the f i e l d . It shows the actual Julian date of diapause termination for Vancouver A. rubecula in a Stevenson screen at UBC in 1983. The f i r s t to terminate were males from groups stored six or eight months at 0°C and then taken to the f i e l d on A p r i l 1, 1983. Only 1 degree-day had accumulated by that date. The rest of the groups are homogeneous despite the differences in the date of diapause induction the previous summer. Using d a i l y temperatures at UBC (shown in the fi g u r e ) , the linear temperature summation model agrees quite well with the Julian dates for diapause termination. The date of 50% emergence may be a few days e a r l i e r , depending on the actual d i s t r i b u t i o n of termination times. Another interesting point to note in F i g . 10 i s the role ambient temperature plays in the pattern of termination. During colder periods few individuals terminate diapause and the pattern appears sporadic; but warming periods accelerate rates and "compress" the apparent termination times so that the population terminates diapause over a f a i r l y short period of calendar time (May 15 to 27, .1 983). 92 FIGURE 10. Julian dates of diapause termination in Vancouver A. rubecula overwintered in the f i e l d , 1982-83. Solid bars are males, open bars are females. Pm and Pf are the mean termination dates predicted from laboratory data for males and females respectively. Minimum and maximum f i e l d temperatures and the thermal accumulation above the threshold t, are given. 94 Post-diapause pupal development There was no correlation between the time required to complete diapause termination and the developmental period of the pupal stage at any temperature. Nor were there any e f f e c t s of the d i f f e r e n t storage temperatures or durations on post-diapause pupal development. Combining data from a l l laboratory experiments, post-diapause pupal development rate can also be expressed as a li n e a r function of temperature: PUPAL DEVELOPMENT RATE (MALE) = -0.126 + 0.0112 TEMP R2=0.83, df=l82, p<0.0l PUPAL DEVELOPMENT RATE (FEMALE) = -0.107 + 0.0100 TEMP R2=0.84, df=66, p<0.0l The calculated thermal constants from these regressions are given in Table 19. The threshold i s similar for males and females and i s indistinguishable from the independent estimate in Chapter 2 for pupal development without diapause. The thermal requirement for post-diapause pupal development of both sexes i s higher than in nondiapause generations. The real heat requirement must be higher as measurements of post-diapause pupal development time do not include the period from spinning the cocoon to casting the meconium which can take up to two days at 20°C (approximately 20 degree-days). Employing these estimated parameters in temperature summation follwing diapause termination at UBC in the spring of SEX N t (SE) K (SE) FEMALE 68 10.7 (0.85) 99.7 (5.42) MALE 184 11.2 (0.47) 89.2 (3.011) TABLE 19. Estimated thermal constants for post-diapause pupal development in female and male A. rubecula. 96 1983 predicts a J u l i a n period of adult emergence in the f i e l d which i s later than actually observed (Fig. 11). It may be just coincidental that t h i s observed d i s t r i b u t i o n of emergence is accurately predicted i f the thermal requirements from Table 1 are used. Males c l e a r l y emerge before females but once again the emergence of most individuals i s compressed into a few calendar days. On the physiological scale, v a r i a t i o n in the t o t a l time required for adult emergence is cl o s e l y associated to variation in the diapause termination period rather than that of pupal morphogenesis (Fig. 12). This i s consistent with.the suggestion of Danilevskii (1965) and Morris and Fulton (1970) that the thermal requirements for active development show l i t t l e i n t r a s p e c i f i c v a r i a t i o n , whereas processes associated with diapause are subject to rapid selection and consequently, geographic v a r i a t i o n . V3 •~1 FIGURE 11. P h y s i o l o g i c a l t ime (degree-days ) r i n same i n d i v i d u a l s as in F i g . 10. l a b o r a t o r y e s t i m a t e s of males and e q u i r e d f rom d i a p a u s e t e r m i n a t i o n to a d u l t emergence Pm and Pf a r e p r e d i c t i o n s f rom fema les r e s p e c t i v e l y . 4 + C 2J 1 J n >55 58 63 68 73 78 Physiological May 29 May 31 m 1 83 88 time 93 >95 June 5 99 FIGURE 1 2 . Relationship between thermal requirements to complete development to adult (Kp) and the thermal requirements for; a) diapause termination (Kdt) b) pupal morphogenesis (Km). Numbers are frequencies. Squares indicate 10 or more points per c e l l . 100 355' 295 H 2351 175H 115H 3 4 2 1 2 2 1 1 9 2 1 1 1 8 33 2 • 2 7 2 5 3 J 2 1 1 5 1 1 1 11 4 3 1 3 1 1 2 1 2 1 2 3 3 2 2 1 1 3 1 2 1 B 1 1 1 5 2 2 4 3 1 1 295 — i — 55 i 115 175 K d t 235 355 A 295-235-175H 55 i 3 3 5 2 2 4 2 . "J 2 1 3 4 1 3 2 2 1 1 5 2 2 5 3 2 1 1 1 1 3 8 1 1 1 2 4 2 2 1 1 1 1 1 • 1 8 1 • 2 3 5 7 2 1 3 1— 115 K 175 101 DISCUSSION After the eonymph enters diapause within the cocoon, there is a period of at least three months when individuals are incapable of continuing development to adult. The longer the incubation period, the more rapid i s the resumption of morphogenesis. But t h i s i s only true when incubation temperatures are below the threshold for morphogenesis. A. rubecula appears to be similar to the f a l l webworm, Hyphantria cunea Drury, where the post-diapause pupal heat requirement decreased to a minimum after six to eight months cold storage and then increased with extended storage (Morris and Fulton 1970). The period of c h i l l i n g in the f i e l d i s roughly seven to eight months for both A. rubecula and H. cunea. There may be an inverse relationship between temperature and diapause development such that diapause terminates most quickly after an extended period at lower temperatures. Evidence for th i s i s the observation that cocoons stored at 0°C required less time to terminate diapause than did those at 10°C. Insects which overwintered in the f i e l d at a mean temperature of 6.4°C required an intermediate period of time to terminate diapause. This same effect of low temperatures on the rate of diapause development has been observed in several insect parasites (Nechols et a l . 1980; Schneiderman and Horwitz 1958; Wylie 1977). C h i l l i n g may be a s p e c i f i c cue to terminate diapause but 102 in A. rubecula i t i s more l i k e l y that low temperatures simply preserve the insect during i t s extended dormancy. A few diapause cocoons stored at 20° to 23°C s t i l l emerge after ten to twelve weeks but their emergence i s sporadic and mortality very high. After a s u f f i c i e n t storage period (four to eight months) at low temperatures, diapause termination i s well synchronized within any one group and mortality low. Diapause then terminates most rapidly at higher temperatures. Variation in the heat requirement for adult emergence i s largely due to variation in the heat required to terminate diapause. This i s also similar to that found in the f a l l webworm (Morris and Fulton 1970). However, in A. rubecula the high heat requirement for the post-diapause pupal moult means that diapause termination w i l l not occur u n t i l temperatures are consistently high. This f a c i l i t a t e s the synchronized termination of diapause and ensures post-diapause pupal development w i l l not commence u n t i l d a i l y thermal accumulation rates are high. From a chronological point of view, the i n t r i n s i c v a r i a b i l i t y in diapause termination times would be minimized in the f i e l d . This i s exactly what we observe in nature. Storage at low temperatures for variable periods in the laboratory did not r e a l l y change the date of diapause termination whether these insects went to the f i e l d in late summer (August 19, 1982) or early spring (April 1, 1983). The f i r s t insects to terminate diapause are usually males but the relationship between termination rate and sex i s not overwhelming. 103 Post-diapause pupal development i s unaffected by l a r v a l rearing conditions or storage history and not correlated with diapause termination rates. The thermal requirements are similar to those described for temperature-dependent pupal development in.Chapter 2 except the t o t a l heat requirement for adult emergence appears to be higher following diapause. This helps delay spring emergence. Males emerge f i r s t but most of the population becomes active within a short period of calendar time so mating success i s as good as i t can be under the circumstances. 104 CHAPTER 6 PHENOLOGY AND HOST"PARASITE ECOLOGY To everything there i s a season, and a time to every purpose under heaven a time to keep silence, and a time to speak Ecclesiastes 111; 1 ,7 This f i n a l chapter reviews the experimental data and describes the phenology of A. rubecula in Vancouver, Canada and Canberra, A u s t r a l i a . I discuss some general aspects of seasonality in host-parasite ecology and conclude with a discussion of the relevance of seasonal models to our understanding of insect ecology and b i o l o g i c a l c o n t r o l . PHENOLOGY Phenology i s the study of the v i s i b l e response of organisms to seasonal changes in their environment. The recurrence of common b i o l o g i c a l events (flowering, migration, hibernation) at pa r t i c u l a r times of the year give r i s e to familiar seasonal cycles and the fundamental ecological tenet that the d i s t r i b u t i o n of organisms i s the result of their b i o l o g i c a l interaction with their environment. Descriptions of the seasonal biology of organisms are as old as the f i r s t observations in natural history. The modern concept of phenology i s well-developed in plant sciences. Coupling precise 105 meteorological data with b i o l o g i c a l processes has resulted in "phenological networks" which attempt to map the progress of plant communities throughout the year (Lieth 1974). The importance of climate and weather in entomology has been stressed in several c l a s s i c works (Andrewartha and Birch 1954; Uvarov 1931). Local weather impinges on a l l aspects of the population biology of insects and an appreciation of t h i s interaction between insects and th e i r abiotic environment i s necessary to gain the insects' point of view (Wellington 1977). The conversion to a temperature-dependent time scale, that is from calendar time to physiological time, i s a f i r s t step. There are several analytic models that make t h i s conversion. Nearly a l l employ the concepts of a threshold below which development i s imperceptible and a t o t a l heat requirement to complete development. Throughout t h i s thesis I have used linear regression to estimate.these thermal constants. The u n r e l i a b i l i t y of the technique to accurately predict development rates when temperatures are low was a minor disadvantage in timing the spring emergence of overwintering A. rubecula. Over the rest of the season, the regressions worked very well. Their greatest a t t r i b u t e i s s i m p l i c i t y as the estimation of standard errors i s straightforward (Campbell et a l . 1974) and comparisons r e l a t i v e l y unambiguous. My interest is not in how accurate can a prediction be made under a l l possible conditions but in what are the differences and s i m i l a r i t i e s between interacting insect populations and how i s 106 their seasonal biology tuned to broad weather patterns. The season for A. rubecula begins in the spring with the end of diapause and pupation of the parasite within i t s cocoon. The insect i s capable of resuming pupal development after a few months dormancy but in Vancouver, diapause does not end u n t i l r e l a t i v e l y late (May), almost nine months after i t was induced. Part of t h i s delay i s due to the high heat requirement to terminate diapause. At least a few days with temperatures sustained over 15°C are necessary for the f i n a l moult. The date w i l l , of course, depend on the weather. In Vancouver in 1983, diapause termination occurred over the last two weeks of May; in 1982 i t was two weeks l a t e r . But termination i s synchronous at one location in any year, at least with experimental material. The high heat requirement for diapause termination ensures pupal a c t i v i t y w i l l be delayed u n t i l average temperatures are better than just marginal for development. The estimated thresholds for post-diapause and nondiapause pupal development were similar but post-diapause pupae had a higher heat requirement. Males had the lowest heat requirement and so were the f i r s t adults to emerge. Males w i l l attempt to copulate with females immediately. Newly emerged females are ready to oviposit but require a few days before residual fats are depleted and oocyte production complete. In 1.982, females were attacking hosts by June 1. The spring of 1983 was warmer and parasites were active in Burnaby by the la s t week of May. Temperature summation for both 1981 and 1982 shows there 107 could be three generations in Vancouver. The second-generation parasites mature before the c r i t i c a l photoperiod i s reached although a late spring emergence and cool summer temperatures could r e s t r i c t populations in some areas to two generations per year. The adults of t h i s generation remained active into September. The last generation parasites emerge from the host from the end of August through September while temperatures are s t i l l warm enough for normal development but daylength is shorter than the c r i t i c a l photoperiod. The season ends with diapause in the entire t h i r d generation. There were small differences in the thermal constants between populations from Vancouver, Canada and Canberra, A u s t r a l i a . Australian populations had a higher heat requirement but the most important difference for phenology was the c r i t i c a l photoperiod for the induction of winter diapause. In Vancouver, the c r i t i c a l photoperiod was between f i f t e e n and sixteen hours of l i g h t d a i l y so the season was truncated in late summer. In Canberra, a c t i v i t y continued well into autumn; only a small portion of cocoons co l l e c t e d from March 20-23, 1981 (autumnal equinox) entered diapause so there must have been at least one autumn generation. I have not had the opportunity to follow A. rubecula for an entire season in Canberra, but I know i t was not common at Pt. Hut u n t i l January in 1980 or 1981. Using calculated thermal constants and meteorological data for Canberra in 1981, there would have been three to four generations between January 1 and March 23, 1981. If there were, in addition, a spring generation during 108 December, there would have been a t o t a l of fiv e to six generations of A. rubecula during the active season in Canberra. Host-parasite synchrony As in several insect parasites, the developmental threshold of A. rubecula i s higher than that of i t s host. The similar range of the threshold of so many insect species suggests the threshold i s primarily a physiological constraint although the difference between host and parasite parameters could be a mechanism promoting host-parasite synchrony (Campbell et a l . 1974). Of more interest to t h i s study was the r e l a t i v e l y low heat requirement to complete one generation for the parasite. During the main portion of the growing season, the parasite w i l l complete more generations in a given period of time than w i l l the host. There is a theore t i c a l certainty that the parasite population w i l l o u t s t r i p that of the host. However, r e a l i t y prevails when we consider the entire seasonal picture. In Vancouver, where P. rapae has only two generations per year, a high c r i t i c a l photoperiod for diapause induction in A. rubecula r e s t r i c t s the parasite to three generations per year at the most. In Canberra, the c r i t i c a l photoperiod i s lower and the parasite population could p o t e n t i a l l y achieve more generations than the host. But the lower thermal requirements of the Australian hosts results in comparatively 109 short generation periods for the host at the lower temperatures encountered at the beginning and end of the season. Consequently, both host and parasite have f i v e or six generations per year in Canberra. In both l o c a l i t i e s , the f i r s t parasite adults of the season lag behind the appearance of the f i r s t b u t t e r f l i e s by several weeks. This i s due to the extended dormancy of A. rubecula resulting from the high thermal requirement to terminate diapause. The asynchrony in spring emergence enables the host population to get a head start so that when parasites f i n a l l y emerge, there i s a good chance there w i l l be susceptible host larvae available for parasitism. A recurrent observation in th i s study was the synchrony in development times of individual parasites. There i s l i t t l e v a r i a b i l i t y in the thermal requirements at any stage so that adult emergence for a cohort at any time of the season occurs over a short period of time, although males consistently emerge before females. This synchrony in developmental pattern may be s i g n i f i c a n t in parasite ecology. Price (1980) has developed a theory of non-equilibria in which parasite populations are characterized by their frequent extinction in l o c a l patches but rapid recolonization and growth in other patches. Parasites, by necessity, occupy ephemeral and heterogeneous habitats. Such a precarious l i f e would be well served by developmental synchrony as i t would increase the p r o b a b i l i t y of mating in sparse 110 populations and insure that parasites in a l l patches experience the same seasonal pattern irrespective of minor l o c a l variations in weather. Diapause The c h a r a c t e r i s t i c s of diapause in A. rubecula were examined in d e t a i l in Chapters 3 to 5 and need not be repeated. The importance of diapause to phenology has also been stressed. But the difference in diapause response between Vancouver and Canberra populations deserves comment. The f i r s t A. rubecula released in Aust r a l i a were from Switzerland (Wilson 1960) and l a t e r material came from I t a l y , near the southern end of the parasite's range (Delucchi 1950). But we do not know where the Vancouver population originated (Wilkinson 1966) so i t i s not possible to determine whether these differences in the photoperiodic response r e f l e c t differences in the colonizing stock or changes afte r introduction to new habitats. It would be simple enough to see i f the c r i t i c a l photoperiods in the introduced populations f a l l within the range of v a r i a b i l i t y for European populations. Whatever their o r i g i n , the diapause response in Vancouver and Canberra i s remarkedly appropriate for l o c a l circumstances. In Canberra, where the host i s active at least seven months of the year, the parasite also has a r e l a t i v e l y long season and as many generations as i t s host. In Vancouver, the high c r i t i c a l photoperiod ensures a short season. Both strategies permit 111 A. rubecula to make the most of l o c a l host populations and survive l o c a l climates. At f i r s t , Vancouver parasites appear to overcompensate, entering diapause well before the end of the summer while b u t t e r f l i e s are s t i l l laying eggs. However, there are at least two additional risks to late season Vancouver parasite populations. The f i r s t i s weather. Autumn temperatures f a l l more prec i p i t o u s l y at higher l a t i t u d e s . Although l a r v a l A. rubecula can develop at temperatures down to 11°C, i f conditions are cool and moist when they emerge, the s i l k cocoon, the insect's primary protection, i s not properly formed. Cocoons spun during October in Vancouver were misshapen and thin even when the insect was reared from hosts on young plants under laboratory conditions before being taken to the f i e l d . None of these insects survived to pupate. In Canberra, where autumn days are r e l a t i v e l y clear and dry and often warm for at least a few hours of the day, thi s may be less of a problem. The second late-season risk to Vancouver A. rubecula i s predation. Hyperparasitism by T. galactopus i s substantial in Vancouver during September (Nealis 1983). T. galactopus i s a true hyperparasite; i t oviposits d i r e c t l y into the primary parasite through the body wall of the primary host. It i s incapable of hyperparasitism once A. rubecula has formed a cocoon. Hence i t i s the p a r a s i t i c stages and not diapause eonymphs which are at r i s k . In contrast, hyperparasitism in 1 12 Canberra i s p r a c t i c a l l y nonexistent. My c o l l e c t i o n s of E. braconophagus were the f i r s t records for Au s t r a l i a (I. Naumann, personal communication). Thus, diapause may serve several ends. After years of watching insect a c t i v i t y , I am f i n a l l y impressed by the importance of doing nothing. BIOLOGICAL CONTROL One of the few benefits of the pesticide treadmill was the belated r e a l i z a t i o n that pest management i s an ecological problem. At times, i t may seem that t h e o r e t i c a l zeal d i s t r a c t s the p r a c t i c a l perspective (Wellington 1977) but taken together, theory and practice in insect ecology contribute to our understanding of natural systems. And even i f , as Holling (1978) and others suggest, understanding natural systems i s not necessary for their management, the more complete is our knowledge of basic ecological relationships, the more able w i l l we be to respond to new situations when things go wrong. As an ecological approach to pest management, b i o l o g i c a l control demands not only that the basic biology of the participants be understood but that we formulate some hypotheses about the nature of interactions between trophic l e v e l s . Studies in b i o l o g i c a l control can make a virtue of necessity and tr u l y become large-scale f i e l d experiments on natural populations (Myers 1978). 1 13 This study began with the observation that despite the wide d i s t r i b u t i o n of a pest species, i t s s p e c i f i c parasite seemed far less adaptable and consequently, more r e s t r i c t e d . Several attempts to introduce the parasite using innovative methods demonstrated the effectiveness of A. rubecula to control the imported cabbageworm but establishment was unsuccessful (Parker and P i n n e l l 1972; Puttier et a l . 1970). The clue for these f a i l u r e s was actually contained in Parker and P i n n e l l (1972) who observed that in Missouri, A. rubecula obtained from Vancouver entered diapause in early September even though the host was f u l l y active for several more months. It i s now clear why t h i s must be so. The long obligatory dormant period for Vancouver parasites meant they would remain in diapause while ambient average temperatures were greater than 15°C, conditions which we can now recognize as l e t h a l . To restate my proposed solution, the Vancouver stock should remain in Vancouver. Parasites from A u s t r a l i a are more promising candidates for the southern United States. The importance of finding natural enemies with the appropriate climatic adaptations for a b i o l o g i c a l control program has been demonstrated many times (Messenger and van den Bosch 1971). These workers combined entomological knowledge with simple experiments to i n i t i a t e the integrated pest management approach (Huffaker 1980). Their methods have been updated, although not always improved, by current attempts to quantify a l l the processes and model the entire system 1 14 (Gutierrez et a l . 1980). One such class of models, and perhaps the simplest, are phenological models. They require precise biometeorological data and good measurements of environmental physiology and the recognition that there may be several explanations for a phenomenon, depending on your point of view. It i s the ecologist's task to i d e n t i f y which point of view offers the more sati s f a c t o r y explanation (Gilbert et a l . 1976). A descriptive model, such as outlined here, can offer considerable insight and a simulation model can provide an opportunity to investigate t h e o r e t i c a l consequences. But no one; entomologist, ecologist, modeller or insect can predict tomorrow's weather with certainty. It i s t h i s uncertainty which makes insect ecology complex and nature so fascinating. 1 15 LITERATURE CITED Andrewartha, H.G. 1952 Diapause in re l a t i o n to the ecology of insects. BIOL REV 27:50,107 Andrewartha, H.G. and L.C. Birch 1954 THE DISTRIBUTION AND ABUNDANCE OF ANIMALS. University of Chicago Press, Chicago, xv + 782p. 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