(Lepidoptera : Geometridae). by Olive Wall, B.Sc

The biology and egg development of two species of Chesias Treitschk (Lepidoptera : Geometridae).

Olive Wall, B.Sc. (Loud.), A.R.C.S.

Thesis submitted for the Degree of Doctor of Philosophy.

July 1970 Imperial College of Science and Technology, Silwood Park, Sunninghill, ASCOT, Berkshire. -1-

ABSTRACT

The biology, and in particular the embryonic development, of two species of Chesias (Lepidoptera: Geometridae) are described and compared. Some aspects of the general biology of these species are examined, and these include the time of occurrence of the different stages of the life cycle, the behaviour (particularly during oviposition) of the adults, and the parasites attacking the larvae. The morphology of the developing embryo is described in detail, and comparisons between the two species are made. Morphogenesis is divided into a number of arbitrary stages, and the relative duration of the different stages is compared. The temperature relations of the developing embryo are examined in detail in both species. In particular, the changing temperature requirements of the embryo of C. leqatella, which diapauses at an early stag- ,are determined by the examination of large samples of eggs killed at different times during embryonic development. The existence of parental effects on the embryonic development of the progeny is also investigated, and certain aspects are discussed. -2-

TABLE OF CONTENTS Page ABSTRACT 1 TABLE OF CONTENTS 2 GENERAL INTRODUCTION 6 GENERAL MATERIALS AND METHODS 7 (i) Collecting 7 (ii) Rearing 10 1. BIOLOGY 16 (i) Introduction and Review of Literature 16 (ii) Habitat and Distribution 16 (iii) Life Histories 17 (a) Life History of C. legatella 18 (i) Adults 18 (ii) Oviposition 23 (iii) Egs 27 (iv) Larvae 28 (v) Pupae 31 (vi) Parasites 33 (b) Life History of C. rufata 37 (i) Adults 37 (ii) Oviposition 42 (iii) Eggs 43 (iv) Larvae -3-

Page

(v) Pupae 46

(vi) Parasites 46

(iv) Discussion 50

(v) Summary 53

(a) C. legatella 53

(b) C. rufata 54

2. EGG DEVELOPMENT 56

(i) Introduction and Review of Literature 56

(ii) Experimental Methods 74

(a) Handling of Eggs 74

(b) Sterilisation of Eggs 74

(d) Incubators 78

(e) Fixation, Preparation and Examination 81 of eggs

(iii) The External Morphogenesis of the Etbryos 86 of C. legatella and C. rufata (a) The Stages of -Embryonic Development 86 in C. legatella (b) The Stages of Embryonic Development 101 in C. rufata

(iv) The Egg Development of C. rufata 116

(a) The Duration of Development in an 116 Outdoor Insectary

(b) The Duration of Development at Two 121 Constant Temperatures

(0) Tmbryogenesis at 20°C. 124 (i) Timetable of Embryonic Development 124 Page (ii) Variability in Rate Development 130 (d) The Effect of Temperature on 138 Embryonic Development (i) The Effect of Temperature on 138 the Rate of Development (ii) The Effect of Temperature on 140 Variability in Developmental Rate (iii) The Effect of Temperature on 141 the Proportion of Eggs Hatching (e) Parental Effects on Embryonic 141 Development (i) The Effect of Maternal Age on 143 the Rate of Embryonic Development (ii) The Effect of Maternal Age when 145 mated on the Proportion of Infertile Eggs produced (iii) The Effect of Maternal Age on 145 the Proportion of Eggs Hatching (f) Summary 147 (v) The Egg Development of C. legatella 149 (a) Duration of Development under 149 Natural Conditions (b) Duration of Development in an 152 Outdoor Insectary (c) Etbryogenesis in an Outdoor Insectary 154 Summary of (a), (b) and (c) 165 (d) 'embryonic Development under 165 Controlled Conditions (i) The Effects of Three Constant 187 Temperatures on Egg Development (ii) The Effect of a Low Temperature 192 Regimeon Subsequent Development at 20C. -5--

Page (iii) The Effect of High Initial 193 Temperatures on Embryonic Development (iv) The Effects of 20°C. and 9°C./3°C. 198 on Embryonic Development after Blastokinesis (v) The effects of Dif7erent 199 Temperature RegiEes with a common mean of 6 C. (e) Further Aspects of Embryonic Develop- 203 ment under Natural and Insectary Conditions (i) The Effect of Temperature early 203 in Development on the Dispersion of the Emergence Curve (ii) The Effect of Temperature on 210 Eclosion

(f) Parental Effects on Egg Development 215 (i) Parental Effect on Duration of 215 Development (ii) Parental Effect on Egg weight 218 (iii) The Effect of Maternal Age on 219 Egg Viability (iv) The Influence of Egg Weight and 223 Time of Oviposition on the Duration of Development (g) Summary 225 (vi) Discussion 229

SUMMARY 256 ACKNOWLEDGEMENTS 258 REFERENCES 260 STATISTICAL APPENDICES 272 -6-

GENERAL INTRODUCTION

The genus Chesias Treitschke is represented in this country by two species, Chesias legatella Schiffermiiller and Chesias rufata Fabricius. These species are of no economic importance, but are of interest because their larvae feed on the same host plant, Sarothamnus scoparius (L.) Wimmer, and yet they differ in many features of their biology. This thesis seeks to provide information on the biology of C. legatella and C. rufata and of their parasites (mainly Braconidae in the genera Ap.nteles nrster and Microplitis nrster), and provides a detailed comparative account of the embryonic development of both species. The effects of various factors on the development of the embryo are examined in several ways. A detailed investigation has been made of the morphogenesis of both species, and of the variation in its rate under different conditions. Studies of this kind, involving the use of large samples of eggs, previously have been confined almost entirely to Orthopterat and this is probably the first attempt of its kind within the Lepidoptera. GENERAL kATERIALS AND THODS

(i) Collecting The abundant broom at Silwood Park provided a moderate supply of C. lesatella. The overwintering eggs, which are laid on the younger stems, were collected by the close examination of branches gathered from the field. They can be recognised by their bright orange colour, even though many are hidden in the stem axils. Larvae were obtained at all developmental stages by beating broom bushes onto a white boating tray; the larger ones were picked up individually, whilst the younger instars were taken up in an aspirator. The adults of this species we-'e collected by beating bushes of broom, and in two light traps. The beating method sometimes yielded reasonable numbers of both sexes, particularly during the latter part of a warm sunny afternoon; the moths would fly when disturbed, and could be caught in a net. The light traps were placed in an area of broom bushes; trap No.i (Plate 1 0 fig. a) was a standard Rothamsted trap fitted with a 200 w. tungsten lamp Oilliams, 1948); trap No.2 was a portable version with a 125 w. Phillips U.V. lamp, collapsible legs, and "Perspex" panels replacing the glass. In both traps the conventional killing jar was replaced by a large polythene bag filled

with egg packing, and attached at the top by a strong

elastic band; moths entering the bag settled quietly

under the packing, and the whole bag could be removed the following morning and its contents sorted in the laboratory. The traps were operated by a time switch, which switched them on one hour after sunset and off one hour before sunrise. Large numbers of C. legatella were caught with these traps, but females were in the

minority. Considerable difficulty was experienced in locating a plentiful supply of C. rufata. This moth is only present in very small numbers at Silwood Park. Areas of broom were beaten in the following localities at various times during the summer of 1967, in the hope of disturbing

adult moths: National Locality Grid Reference

Disused Gravel Pit, Easthampstead, Berks. ... SU 873659

Bisley Camp, Surrey. ... SU 94.4579 Blackwater, Hants. ... SU 846597

Ascot Heath, Berks. ... SU 924.693 Farnham By—Pass, Surrey. ... SU 833460

Romsey, Hants. ... SU 343201 This proved unsuccessful, and it was not until A.L. Goodson a. :iothamsteM. Light . trap b. Sleeved broom plant

c. Mating Cage

Plate 1. Collecting and Rearing Apparatus. -10—

(personal communication) recommended Barnes Common,

London, 8.7.13 (Nat, Grid. Ref. TQ 225759), that larvae

were located in large numbers, and collected in September

of that year.

Subsequent collecting during the adult season showed

that, even when present in large numbers, this moth is

very reluctant to fly when disturbed during the daytime.

Consequently this species may be present at some of the

localities mentioned above; however, a plentiful supply

from Barnes Common made the verification of this unnecessary.

At dusk, moths of this species suddenly become very active,

and may then be collected with the aid of a net and torch

as they fly between the broom bushes. During this time

females can be seen vibrating their wings on the top of

broom bushes, and may be collected in specimen tubes.

The small size of the eggs, and the dense foliage of

the broom plants, made the collection of eggs of this

species quite impractical.

(ii) Rearing

Rearing for experimental purposes took place in an

outdoor insectary, thus providing all stages of the life

cycle of each species with conditions comparable with

those in the field. The result of this was that only one generation was reared in each species in any one year, -11—

since both have an obligate diapause in each generation.

When any one stage in the life cycle was subjected to experimental conditions, neither the later stages of that generation nor subsequent generations bred from that material were used in further experiments.

(a) Larvae were reared in a variety of ways.

When individual rearing was necessary, each larva

was kept in a 7.6 x 2.5 ems. flat bottomed glass

specimen tube. A 1:1 mixture of moist sand and

peat was placed in the bottom of the tube to

provide a medium for pupation, and a piece of

broom was provided for food (Pig.la). If the cut

end of the broom twig was inserted into the sand

and peat, the foliage usually remained fresh for

several days or until it was eaten. The tube was

closed by fine terylene net (2L meshes per cm.)

stretched across a bored cork.

Larvae were reared collectively either in

sleeves made of fine terylene net, which were placed

over all or part of potted broom plants (Plate 1,

fig. b), or in transparent plastic cages 10.5 ems. in

diameter and L1..5 ems. high, in the lid of which there

was a ventilation hole 2.5 ems. in diameter covered

with terylene net (11 meshes per cm.) (Fig.lb).

-12-

a cork terylene net

broom twig

flat bottomed specimen tube

5 cms. sand and peat

transparent plastic cage

IIIIIIIIIIIIIII!IIIIIIIIIIIIIIIII

cotton wool roll

polythene cap dried broom twig

Fig. 1 Rearing Cages -13-

A potted broom plant two to three years old and approximately 75 ems. high will support twenty larvae for their entire larval life; however, mortality increases considerably if too much overcrowding occurs.

Larvae reared in this way were taken from the sleeve on reaching the prepupal stage, and alloed to pupate in the sand/peat mixture. Those larvae kept in plastic cages were fed on broom twigs kept fresh by inserting the cut ends into a moist cotton-wool roll, of the type used in the dental profession. This had one big advantage over the sand/peat mixture used in individual rearing, in that the bottom of the cage and the larval frass remained dry; this helped to prevent the spread of fungal infection. At first the prepupae were removed as above, but later it was found that both species pupated readily on the bottom of the cage; these pupae were removed at a later date, sexed and then placed in the moist sand/peat mixture. It is interesting that the cocoons of Apanteles spp., bred from the moth larvae, are particularly resistant to fungal attack, and. Ford (1943) has made a similar comment.

(b) Both the hibernating pupae of C. rufata and those of C. legatella were kept in aslightly moist -14- pupating mixture in glass tubes or plastic cages.

For the greater part of this stage the tubes or cages were packed into a large box filled with dry peat, which was placed in a closed part of the insectary; thus, extremes of temperature were avoided. Towards the time of emergence the tubes and cages were restored to the lighted part of the insectary, and dry broom twigs were inserted into the tubes. On emergence the adults would climb these twigs to expand their wings; however, many moths failed to expand their wings in the confined space of a tube. Moths emerging in cages did not experience this difficulty, and this was by far the more satisfactory method.

A piece of dried broom was placed in each cage, together with a supply of a 10r,i; w/w solution of honey (Fig.ib).

The latter was provided by soaking a cotton-wool roll with the solution and placing this in a small polythene cap. Cockayne (1936) states that geometrid moths do not require food, but must have water. However, both species of Chesias were attracted to and fed on the honey solution, and therefore this food was provided throughout the rearing and experimental work.. -15—

Mating was not readily achieved when males and females were confined in pairs. The most reliable way of ensuring that mating occurred was to place up to fifty virgin males in a large well ventilated cage

(Plate 1, fig. c) 90 cms. high x 45 ems. square, in which there was a potted broom plant and a supply of honey solution. This cage was fitted with a small sliding door enabling the introduction and removal of moths at will. The cage was kept in an outdoor insectary, and during the late evening two or three females were introduced at a time. The males immediately commenced to fly, and mating took place very shortly after each female had exposed her scent organs. Each copulating pair was then removed to a plastic cage, which was placed in the appropriate experimental conditions. The successful fertilisation of eggs was indicated by the large number laid on the first two nights after mating, and also by the changing colour of the eggs from green to orange in the first few days of development. -16-

1. BIOLOGY

(i) Introduction and Review of Literature Very little information has been found in the literature on the life histories of C. legatella and C. rufata and virtually nothing was known of the biology of these species before this study. The only information on the life histories has been confined to notes on the times of occurrence of the different stages of the life cycle in such publications as Kirby (1898), Stokoe (19118), South (1961) and Blesznski (1965). The different stages are described briefly by these authors, and these descriptions have been augmented by the detailed descriptions of the genitalia of both species by Pierce (1914) and of the eggs by Tonge (1933). Accounts of the life cycle of both species are given, together with certain aspects of their biology. These include the behaviour of the adults, particularly during mating and oviposition, the development of the immature stages (including information on the stages in which diapause occurs) and the insect parasites attacking the larvae of both species.

(ii) Habitat and Distribution The larvae of both species feed on the leaves, and sometimes the flawers, of Scotch Broom, S. scoparius, and -17-

pupate in the soil at the base of the plant. Kirby (1898) and BleszAlski (1965) state that larvae of C. rufata also feed on Genista Linnaeus. The adults use broom bushes as resting sites and the females deposit their eggs on the younger branches. Thus the whole life cycle of both species revolves around the food plant, S. scoparius, and their distribution patterns are consequently very similar and reflect the distribution of their food plant. In this country both species occur in most places where broom is well established (South, 1961). Abroad their ranges extend over most of Central and Southern Europe, from Spain eastwards to Poland. They are known to occur as far north as Southern Scandinavia and south to North Africa (Fletcher, personal communication).

(iii) Life Histories The descriptions of the life histories of C. legatella and C. rufata refer to populations associated with Scotch Broom at Silwood Park, Berks and Barnes Common, London, S.W. 13 respectively. It is interesting that, although the broom at Silwood Park maintains a moderate population of C. legatella, very few C. rufata were obtained from this locality. Conversely, there was a flourishing population of C. rufata at Barnes Common during the course of this work, but no C. legatella were located. -18-

The life histories of these two species are quite different (Fig. 2), but have a common feature in that the larvae feed on the broom during one of the main periods of growth, Scotch Broom has two main periods of growth during the year, one in spring just before flowering and the other, when most of the growth takes place, after pod formation in the summer. (Waloff, 1968). (a) Life History of C. leatella (i) Adults. The adults of C. legatella can be found in the field from mid September to mid Nbvember and are particularly abundant during the second half of October. Variations in this general pattern occur from year to year as indicated by light trap catches during the autumns of 1967 and 1968 (Fig. 3). These catches were dependent not only on the presence of adults but also on climatic conditions being favourable for flight activity. Thus the minor fluctuations in the numbers caught are undoubtedly a result of the latter factor. Nevertheless it is apparent that in 1967 the adults appeared a little earlier than in 1968. These results are based on the catches of male moths, since the number of females attracted to the traps was very small in both years. The evidence from light trap records is supported by the dates of emergence of, adults reared in an outdoor insectary. The emergence of adults commenced. on September YEAR 1 YEAR 2

Cs. leoatella

rialteZ v

Egg

Larva

Pupa *.,WW&X`VV*;404 ORagla ?WOW +VAMS`ik • .W41.*:,",,,O.V&\ j'AMANAWV C. rufata .'"War V4iK: ; Adult

II Jan. Mar. May Jul. Sep. Nov. Uan. Mar. May Jul. Sep. Nov

Fig. 2 The Life Histories of C. legatella and C. rufata -20-

23rd in 1967 and October 1st in 1968, and ceased on October 30th in both years. Fig. 4 clearly shows that the emergence of both males and females occurred earlier in 1967 than in 1968y and that in both years males preceded females. `Ile emergence of individual adult moths was observed to occu.2 almost entirely at or soon after dusk, although a few individuals did emerge at other times. The adults are nocturnal, and field and insectary observations have indicated that flight activity occurs during the first four to five hours after dusk. When not in flight the moths settle on broom twigs, usually with their wings folded round the twig in a very characteristic manner (Plato 2 fig. a) resembling a seed pod. South (1961) reports that this species has been recorded flying by day; certainly, if broom bushes containing adults are disturbed on a warm sunny afternoon, males in particular will fly but usually settle again at the earliest opportunity. Females tend to drop to the ground when disturbed during the day, spinning like open seed pods as they do so, and males will also do this on colder days. No attempt has been made to study the diurnal activity pattern of this species in any greater detail. Mating takes place at night during the period of general activity. When enclosed in a large cage with several actively flying males, a female will settle in a prominent

Fig 4Emergence Fig. 3LightTrapRecords1967,1968-SilwoodPork,Berks. % Emergence (Accumulative) Number Caught 100 80 - 60 40- 20- 0

,._ ,- _ _ • 22 2630481216 2024281

f 16 24282610141822263037111519 •') Sept. Sept. •

• ( , i

• of Adults-Insectary 1967,1968 •

• T • •

• o "T

, )0 Se r • 0 • Y

-P • • TT

I " 0 . • C, • •

V •

•

1 m • • ,00 , i

r) •

or' • ,00 •

,-, • Oct rp • TV Oct 0 i

V d

il o I

U Li -21- 0 i ••

o • %•• I

(C. legatella) Nov I • • • [ Females1968(N.91) • Moles 1968(N.93) Males 1967(N.66) Females 1967(N-61) (C. Nov. legate& • Males 1968 Males 1967 a. Resting Adult b. Mating Pair

c. Eggs laid in captivity on broom twig

Plate 2. C. legatella -23-

position (on top of a broom bush, if one is provided), open her wings and extend her genitalia. It is thought that a scent is produced, since the males become extremely excited until one of them is successful in mating with the female. The flight activity of the other males then ceases. Copulation was observed to last for one to two hours in most case-:c During copulation the pair usually remain stationary on a broom twig in a characteristic lepidopteran mating position (Plate 2, fig. b), the female's wings partly overlap- ping those of the male. If disturbed the female will crawl away dragging the male behind her, and may even attempt to fly whilst still remaining in cop. A spermatophore is produced, and this was used as an indication of successful mating (females being dissected on death). Mating was never observed unless fifteen to twenty males were present in the cage. However, moths were never observed in groups in the field during extensive collecting at night. The mating behaviour of this species, and of C. rufata (which proved to be very similar) warrants further study, but this is outside the scope of the present work.

(ii) Oviposition. Oviposition takes place at night. Adults kept in an outdoor insectary and also at 15°C. (10/24 hour light regime) did not oviposit at any other time. Observations were made at intervals of eight

-24-

hours and all the eggs laid in the insectary (over 2300) were deposited between 24.00 hours and 08.00 hours; likewise all 3700 eggs laid at 15°C. ware depooited during- the same period, the dark period commencing at 18.00 hours. Field samples have indicated that the eggs are deposited on broom twigs, either singly or in pairs, and are attached by a water soluble adhesive. The oviposition sites were classified according to the diameter of the twig (small, medium, large) and the position on the twig (stem groove, stem axil, leaf axil). There is a strong preference in this species for the stem axils of small and medium sized twigs as oviposition sites (Table 1).

Table 1. Choice of Ovi osition Site in the field 1W6fe711a)

Ovip. Site Stem Stem Leaf Total Twi Groove Axil Axil Dian.

small 3 56 1 60 medium - 37 - 37 large - 7 7 Total 3 100 1 104

These twigs are the result of the autumn and spring flushes of growth of the year in which the eggs are laid. -25-

Hardly any eggs were located in any other position. Makings (1956) showed that females of C. legatella Prefer twigs 1.5 mm. in diameter and shallow crevices 065 mm. wide as oviposi- sites. Twigs of this diameter are the youngest on a plant. Of 26 eggs collected in the field by liakings, 18 were found in twig axils and 12 in stem grooves. An experiment by the same author on choice of oviposition sites, using caged adults, indicated a gong preference for those two sites, stem grooves being preferred to stem axils. During the course of the present work it has become evident that when adult females of this species are kept in cages with a restricted supply of broom, their oviposition behaviour is quite different from that in the field. They will lay eggs in large groups down the stem grooves of the broom twigs provided (Plate 2, fig. c) and also on the muslin of the cage. This suggests that cage experiments on the choice of an oviposition site in species which normally lay their eggs in small groups over a wide area should be conducted with the greatest caution, and must be supported by field experiments or sampling. The longevity and fecundity of the adults were not investigated thoroughly, but most adults in captivity in the insectary lived for approximately one to two weeks; their longevity was extremely variable and was probably affected -26-

by temperature. A few adults placed at 2000. on emergence died within three to four days; clearly this constant temperature is close to the upper limit of the temperature range of this species. In many cases longevity could not be determined either because the moths were captured as adults or because eggs were collected from them over only a few days and no record was kept of the date of death. However, three mated females were kept in an outdoor insectary from emergence to death, and these lived for 11, 13 and 14 days. The fecundity of these three females was 151, 129 and 193 eggs respectively, giving a mean of 158 ± 19. Host eggs are usually laid on the first four or five nights (Table 2).

Table 2. Total Number of Eggs Laid blljr_Females - insectary 1568_0. 1Katel1a7"--

Night 1 2 3 4 5 6 No. of Eggs laid 35 199 82 9 40 20 22 Night 8 9 10 11 12 13 14 No. of Eggs laid 25 16 7 7 4 0

When eggs were collected from six females in the insectary for seven nights only, the mean number of eggs laid by each female was 165 :L 15. For most experiments enough eggs were collected in three nights. -27-

(iii) Eggs. The eggs of C. legatella resemble those of many Geometridae, being ovoid in shape with a slightly concave dorsal surface. The anterior end is rather flat, bears the micropyle at its centre, and it is through this end that the young larva emerges. At its posterior end the egg is more rounded and dorsoventrally flatter, No distinctive chorionic sculpturing is evident under a dissecting microscope. A sample of twenty eggs was measured soon after oviposition and the mean dimensions were found to be: Length 0.831 ± 0.011 mm. Width 0.510 ± 0.003 mm. When first laid the eggs are green in colour. Within a few days they change colour, if fertile, passing through pale green (formation of blastoderm) and pale yellow, to orange (formation of serosal pigment). Finally, a few days before hatching the orangecacuration disappears and the almost black larva becomes visible within the whitish semi- transparent chorion. Peterson (1962 a) refers to similar colour changes in the eggs of Lepidoptera, particularly within the Geometridae. He describes how the colour of the entire egg can change during development from green to a uniform orange, red, brown, or deep purplish-brown within the clear chorion. Gaumont (1950) describes the colour changes of Operophtera brumata (Linnaeus) from pale green soon after oviposition through beige to red, which remains -28-

until the larva is fully formed. The duration of the egg stage under different conditions will be discussed in detail in a later section. However, the embryo passes through a diapause (see later), and hatching occurs the following spring when the broom buds are bursting. Field samples have shown that hatching occurs from late March to early May, but this is dependent on weather conditions, and again this subject is discussed later. (iv) Larvae. The larvae of C. legatella are usually dark green in colour and can attain a maximum length of 3-4 cm. when fully grown. They have the usual characteristics of geometrid larvae, which include the possession of only two pairs of abdominal prolegs; as a result they proceed in the "looping" fashion normally associated with larvae of this family, and when at rest they resemble a small broom twig, being attached only by their prolegs with the rest of the body held straight and at an angle to the twig on which they are resting (Plate 3, fig. a). The larvae feed externally on the leaves, and sometimes the flowers, of S. scoparius. There is some variation in colour, and this is influenced by the type of food material; larvae feeding on very young leaves tend to have a paler colouration than those feeding on the older darker leaves, and when the bright yellow flowers are consumed the larvae may, to a certain extent, be coloured yellow. -29-

a. Larva in characteristic resting position

b. Larva shortly before pupation Plate 3. C. legatella -30-

The number of larval instars has been determined by the measurement of the width of the head capsules of samples of larvae from the field, and also by rearing individual larvae and recording the number of moults through which each larva passes. The measurement of head capsules indicated four distinct larval instars, and this was confirmed by the rearing method. Table 3 shows the mean head capsule widths of samples of 20 larvae of each instar.

Table 3. Mean Head Capsule Width of Each Larval Instar (C. legatella)

Head Capsule Width Instar (Mean ± S.E.* in mm.)

1 0.282 ± 0.004 2 0.564 ± 0.008 3 1.086 ±0.008 4 1.888 ± 0.020

*S.E. . Standard Error. Field collections of larvae in 1967 revealed that first instar larvae were present from the first two weeks of April. In 1968 egg samples taken from the field indicated tht hatching was occurring during the greater part of April, and this was confirmed in 1969 when 73 eggs, which had been laid on broom bushes the previous autumn, hatched between April llth and May 2nd. -31-

In both 1967 and 1968 collected larvae were reared in an outdoor insectary and the dates of pupation were observed. In 1967 the 92 larvae pupated between May 14th and June 5th, but 79;: of them pupated between May 23rd and May 26th. The following year pupation was later being between June 7th and June 27th (218 eggs), but almost all of these (90) pupated between June 7th and June 17th. The conclusions which can be drawn from these data are that the larvae hatch from late March to early April, and pupate by the end of June at the latest. Prior to pupation the larvae become sturdier and the body segments become very pronounced (Plate 3, fig. b); they make their way down to the ground and burrow to a depth of five to seven centimetres in order to pupate. The prepupal stage was observed to last two to three days in the insectary, but no accurate determination of the duration of this stage was made. (v) Pupae. The pupae are light brown, but the colour darkens as development proceeds. They are not enclosed in a cocoon. Pupal development takes approximately four months in the field, and, although no information was obtained on the exact duration of this stage in the field, accurate records were kept for pupal development in the outdoor insectary. Table 4 shows the mean duration of pupal development in the insectary in the summers of 1967 and 1968. -32-

Table 4. Duration of Pupal Development in Insectary 1967 and 1968 (C. lezptella)

Mean Devel. Time (Days) ± S.E. Year Males. Females.

1967 131.6 I 1.2 135.8 '1.- 1.2 1968 122.9 ± 0.8 129.6 ± 0.8

*S.E. . Standard Error. These developmental times clearly indicate that there must be an interruption of metmnorphosis despite the summer temperatures, which are normally favourable to insect development. Lees (1955) states that although diapause is usually confined to one stage in the life cycle of insects, there anafew exceptions. One of these exceptions is the winter moth, O. brumata. The life cycle of this species is very similar to that of C. legatella and Kozhanchikov (1950) states that there is an obligatory diapause in both the egg and the pupa. The egg overwinters in a manner not unlike that of C. leatona, and the pupa exists from approximately June to September (or even as late as February according to latitude). Kozhanchikov has clearly shown that the rate of pupal development increases with decrease in temperature, and also suggests that the adults emerge earlier in a cold autumn than in a warm one. -33-

The mean developmental times for male pupae shown in Table 4 are less than those for female pupae in both years. This difference was not significant in 1967 (.1 > p > .05) but was very highly significant in 1968 (p < .001). Incubation of pupae of both sexes under constant temperature conditions may well indicate significantly shorter developmental periods for male pupae. This sexual difference in pupal developmental rates could well explain the tendency for male adults to emerge earlier than female adults. There is, then a suggestion from the data obtained, that C. legatella diapauses in the pupal stage in addition to the diapause of the overwintering egg. The development of the pupa requires much closer examination, and that of the egg is dealt with in a later section. (vi) Parasites. During the course of this work several parasites were found in the larval stages of C. legatella. No evidence was obtained of any egg parasites in this species; a total of 200-300 eggs of C. legatella were collected at various times during the winters of 1967 and 1968, and not one of these was found to be parasitised. However this evidence is not conclusive, since many of the eggs were killed for examination of the embryonic development and these may have contained parasitic eggs which escaped notice. No data were acquired on the possible existence of pupal parasites, since pupae of C. legatella

-34--

were not collected from the field at any time. The following parasites were reared from larvae of C. legatella collected in the field: Primary Parasites Apanteles vitripennis Curtis (Hymenoptera: Braconidae) Apanteles fulvipes Halliday (Hymenoptera: Braconidae) Microplitis fordi sp. n. (Nixon, 1970) (Hymenoptera: Braconidae) Phoridae (Diptera) Hyperparasite Mesochorus sp. Gravenhorst (Hymenoptera: Ichneumonidae) 400 larvae of C. legatella (100 of each instar) were collected from the field during the early summer of 1967 and reared individually in an outdoor insectary. Details of the results of this rearing are shown in Table 5. Table 5. Results of Rearing Collected Larvae - summer 1967 (C. le atella)

Host Instar Collected Result 1 2 3 4 Live Pupae 30 57 53 57 Dead Larvae 52 23 4 6 Dead pupae/prepupae 11 12 14 32 A. vitripennis 6 8 25 - A. fulvipes - - 3 5 M. fordi - - 1 - Phoridae 1 - - - Total 100 100 100 100 -35-

A. vitripennis was by far the commonest parasite of C. legatella larvae (accounting for 80% of the parasitism), and although the larvae of this parasite were found in small numbers in first and second instar host larvae, they were much more numerous in third instar larvae and absent from the final instar. This suggests that A. vitripennis lays its eggs mainly in third instar larvae of C. legatella, but will attack the younger instars also. The small number of A. fulvipes were present in only third and fourth instar host larvae, and so the same may apply to this species. No conclusions can be made from the one record for M. fordi in a third instar host larva, but the occurrence of phorid - parasites in a first instar host larva is interesting, since the parasites did not pupate until the prepupal stage of the host was reached. All the larvae of A. fulvipes also pupated at the prepupal stage of the host, and the larva of M. fordi emerged from the fourth instar host larva for pupation. Larvae of A. vitripennis also emerge from the host larva before pupating, and the instars of the host larva in which this happened are indicated in Table 6. It is clear that almost all emergence of the larvae of A. vitripennis took place from the third or fourth instar larvae of the host, and emergence from the third instar host was the most common.

-36-

Table 6. Details of Emergence of A. vitripennis (C. legatella)

Host Instar Host Instar Collected from which parasite emerged. 1 2 3 4 Total

1 0 - - - 0 2 0 1 - - 1 3 6 7 12 - 25 4 0 0 13 0 13 Prepupa 0 0 0 0 0 Total 6 8 25 0 39

Only one larva, of A. vitripennis or M. fordi emerged from an individual host larva, but in both A. fulvipes and the phorid parasite several larvae emerged from a single host larva. The emergence of larvae of A. vitripennis and M. fordi _from late instar host larvae nearly. always occurred from the side of the sixth or seventh abdominal segment; in most cases the host lived for several hours after the emergence of the parasite, but always died eventually. Upon emergence, the larvae of these two parasites immediately spin a cocoon for pupation, and this is usually attached to a broom twig. The cocoons of A. vitripennis are white (typical of the genus Apanteles) and those of M. fordi are grey with slight longitudinal ribs. -37-

The larvae of both A. fulvipes and the phorid parasite emerged in the soil after the host had attained the prepupal stage. The larvae of A. fulvipes again spin white cocoons, but the phorid larvae pupate in the soil without any cocoon. The pupae of all the parasites reared from larvae of C. legatella developed without interruption, and emergence of the adults took place in the same summer. The average duration of pupal development in A. vitripennis was 12.9 ± 0.4 days, and all the other parasites had emerged by the end of June. One of the records shown in Table 5 for A. fulvipes did in fact result in the emergence of a hyperparasite (Mesochorus sp,). The cocoons of A. fulvipes were formed in the usual way, but the adults which emerged from them were Mesochorus sp. (one from each cocoon). (b) Life History of C. rufata (i) Adults. The adults of C. rufata can be found in the field from March to August. Bleszyliski (1965) comments that the adults are on the wing in Poland from April to June, and South (1961) states that in this country the moth emerges from May to July, but that its time of appearance is uncertain, and it may come up in early spring or not until early autumn. The small numbers of this species at Silwood Park during this study resulted in very poor light trap catches; the numbers caught were so low that Table 7 is merely a -38-

summary of the number of moths caught in each month.

Table 7. Number of Adults caught in Light Traps - 1967 and 1968 (C. rufata)

Month 1967 1968

March 1 0 April 3 1 May 7 2 June 0 1 July 21 4 August 8 0 Total 40 8

The only positive indication in these data is that the peak of emergence in 1967 appears to have been in July; however, the numbers are so low that the records show very little more than this. The dates of emergence of adults reared in an outdoor insectary from larvae collected in 1967 are shown in Fig. 5. 119 adults emerged between June 22nd and July 23rd 1968, and there is little indication in these data that males emerged earlier than females (cf. C. legatella). The adults which emerged at this time represent only 85% of the total emergence. The other 15% emerged the following year, from May 5th to June 5th. Table 8 gives details of the adult emergence 100 0 • 006400 ••••01r•• 90 • 00 o o • 80 0 •0 ) • ive t 70

la • 0

mu 60 • 0 Accu 0 ( 50 •o ence 40 0 erg

Em • Males (N.67) e 30 •o

tag Females (A1.52) n 20 • rce 000 Pe 10 ojo oo•

21 25 29 3 7 11 15 19 23 27

June July

Fig. 5 Emergence of Adults - Insectary 1968 (C. rufata) from pupae which overwintered for a second year.

Table 8. Emergence of adults from Two Year Old Pupae - 1969 (C. rufata)

No. Adults -Emerged Date Males Females Total May 5 1 1 n 7 1 1 " 11 1 1 2 " 15 1 1 " 16 1 1 " 22 1 1 2 " 23 2 2 " 24 3 3 " 26 1 1 2 " 27 1 1 " 29 1 1 " 31 1 1 June 3 1 1 I, 5 2 2

Total 10 11 21

Thus, the pupae which overwintered for two years in the insectary produced adults earlier in the summer than those which completed their development in one year. It seems likely that the sporadic appearance of adults of this species in the field from March onwards is the result of a small proportion of a population remaining in the pupal stage for --41-

two years. The dates of emergence of the main part of the population were confirmed in 1969, when 59 adults emerged from pupae reared from the previous year's eggs. These adults emerged between June 28th Rnd July 10th, 58% of them emerging during the last three days of June. Adults of C. rufata have a diurnal activity pattern quite similar to that of C. legatella. Thus, emergence from the pupa and general adult activity take place during the first few hours after dusk. Flight activity is more noticeable in the field in this species than in C. legatella, possibly because of the higher prevailing temperatures during the adult season. Adults were observed to commence their flight activity at dusk, approximately 45 minutes after sunset. When not in flight adults of this species settle on the broom bushes in a similar fashion to those of C. legatella, but during the daytime they are extremely reluctant to fly when disturbed. The mating behaviour of this species is extremely similar to that of C. legatella and need not be described separately. However, females were observed in the field situated in prominent positions on the top of broom bushes, fanning their wings and exposing their genitalia. Thus the behaviour of the female in the field is confirmed as being -42-

similar to that observed in the insectary. (ii) Oviposition. Oviposition usually takes place at night during the first few hours of darkness. For instance, all of 1483 eggs laid in the insectary by seven females were laid between 16.00 hours and 08.00 hours, and 66% of these were laid before midnight. Only 1.5% of 532 eggs laid at 15°C., and in 16/24 hour light regime, were laid in the light, and these were laid between 16.00 hours and 24.00 hours (dark period commencing 24.00 hours). However, at 20°C., and in a 16/24 hour light regime, 25% of the eggs laid by 24 females were laid between 16.00 hours and 24.00 hours, and 8% were laid between 08.00 hours and 16.00 hours, the total number laid being 4214. These results indicate that oviposition commences earlier in the night in this species than in C. legatella, and that although oviposition normally takes place in the dark, there is a certain amount of inherent periodicity of oviposition activity which results in the deposition of eggs between 16.00 hours and 24.00 hours even in the presence of light. The natural oviposition sites of this species were not investigated, since the examination of large quantities of thickly leaved broom would have been too time consuming at the height of the season. However, the behaviour of captive ovipositing females was very similar to that of females of -43-

C. legatella. The mean longevity of 19 adult mated females at 20°C., and in a 16/24 hour light regime, proved to be 14.5 ± 1.2 days. The mean fecundity of these same females was 176 t 13 eggs, and the mean fecundity of four females in the insectary was 221 ! 27 eggs. All these females had been reared from collected larvae. Most of the eggs are usually laid on the first few nights after mating, as in C. legatella (Table 9).

Table 9. Total Number of Eggs Laid by Three Females - insectary 1968 (C. rufata)

. Night 1 2 3 4 5 6 7 No. of Eggs Laid 342 102 26 52 47 63 15 Night 8 9 10 11 12 13 14 No. of Eggs Laid 15 20 26 4 11 0 8

(iii) Eggs. The eggs of C. rufata are very similar to those of C. legatella in shape, although they tend not to be quite so flattened, and the dorsal depression is not so conspicuous. However, the eggs of this species are considerably smaller than those of C. legatella. A sample of twenty eggs was measured soon after oviposition, and the mean dimensions were: Length 0.617 ± 0.004 mm. Width 0.397 ± 0.003 mm. Like those of C. legatella, freshly laid eggs of this species are green, but within a very few hours they turn to pale green (formation of blastoderm) and after approximately two days to pale yellow (segmentation). No deep orange pigment is laid down, and the eggs remain pale yellow until the pigmented fully formed larvae consume the serosa prior to hatching. The duration of the egg stage is discussed later, but it can be noted here that the eggs hatch in approximately 10-12 days under outdoor insectary conditions. (iv) Larvae. The larvae of C. rufata are very similar to those of C. legatella, but do not usually attain a length of more than approximately 3 ems. They move and rest in a similar way also, and feed externally on broom leaves. Although there is some variation in body colour amongst larvae of this species, this is not as noticeable as in C. legatella, partly because there are no flowers present on the broom when the larvae are feeding. The number of larval instars is four, as in C. legatella. This was determined by rearing collected larvae, and also larvae subsequently obtained from eggs, individually in the insectary to see how many moults each larva passed through. No measurements were made of head capsules because all the larval material was required for rearing purposes (to provide adults). -45-

The information obtained on the timing of the emergence of adults in the insectary suggests that first instar larvae might be found in the field from early July onwards, and that the peak of hatching occurs at the end of the month. Field collections of larvae in August and early September in 1967 yielded the larval instars shown in Table 10. The collected larvae pupated in the insectary between August 22nd and September 20th.

Table 10. Larval instars collected in 1967 (C. rufata)

Date of No. of each instar Total Collection 1 2 3 4 16.8.67 1 12 6 1 20 20.8.67 13 97 174 91 375 2.9.67 9 35 44

Total 14 109 189 127 439

It may be concluded that the larvae produced by the main population hatch from mid July probably through to mid August, and that pupation takes place from late August to late September. The fully grown larvae of C. rufata are very similar to the larvae of C. legatella in their behaviour prior to pupation. The body segments thicken and become very pronounced and the larva burrows into the soil, after which two to three -46-

days are spent in the prepupal stage before pupation actually takes place. (v) Pupae. The pupae of C. rufata are reddish- brown with bright green wing cases. As metamorphosis proceeds the green colouration disappears and the pupae become dark brown shortly before emergence of the adults. Once again no cocoon is formed. The duration of the pupal stage in the outdoor insectary was approximately ten months in those individuals which emerged the following year, and 21-22 months in those which emerged in the second year. There is evidence that the pupae of this species diapause, and that low temperatures are required to terminate diapause, since not one of 20 pupae kept at 20°C. continuously completed development. All remained a pale reddish-brown colour, and after ten months (the time taken for most pupae to develop in the field) they were all dead. The temperature relations of the pupae of this species were not investigated in any more detail. (vi) Parasites. The existence of several larval parasites of C. rufata was established during the course of this work. No data were obtained on the possible existence of egg or pupal parasites, since these stages of the life cycle were not collected in the field at any time. The following parasites were reared from larvae of -47-

C. rufata collected in the field: Primary Parasites A. vitripennis (Hymenoptera : Braconidae) M. fordi (Hymenoptera : Braconidae) Phoridae (Diptera) Hyperparasite Mesochorus sp. (Hymenoptera : Ichneumonidae) Thus, with the exception of A. fulvipes, the same larval parasites were found in both hosts. 441 larvae of C. rufata were collected from the field during late August and early September, 1967 and reared individually in an outdoor insectary. Details of the results of this rearing are shown in Table 11. Table 11. Results of Rearing Collected Larvae - summer 1907 (C. rufata77

Host instar collected Result 1 2 3 4

Live Pupae 36 26 38 42 Dead Larvae 57 46 11 16 Dead pupae/prepupae - 1 6 12 A. vitripennis - 18 29 3 M. fordi 7 7 15 27 Phoridae - - 1

Total 100 100 100 100 (n = 14)(n = 109)(n = 191)(n = 127) *Numbers refer to percentages. n = number of host larvae collected. -48-

In contrast to the population of C. legatella at Silwood Park in the same year, the larvae of C. rufata at Barnes Common were parasitised equally by A. vitripennis and M. fordi (almost 52% and 48% of the total parasitism respectively). Once again A. vitripennis appears to lay its eggs mainly in third instar larvae of the host, although second instar larvae were also attacked. M. fordi appears to attack rather later, ovipositing mainly in third and fourth instar larvae. Almost all the larvae of A. vitripennis emerged from third instar host larvae for pupation, as can be seen in Table 12.

Table 12. Details of Emergence of A. vitripennis (C. rufata)

Host Instar Host Instar Collected from which parasite emerged. 1 2 3 4 Total 1 _ - - _ - 2 - 0 - - 0 3 - 20 53 - 73 4 - 0 2 4 6 Prepupa - 0 0 0 0 Total 0 20 55 4 79 -49--

This evidence is substantiated by the fact that only 3% of 127 4th instar larvae collected from the field contained a larva of A. vitripennis. All 73 larvae of M. fordi emerged from the fourth instar of the host larva. The behaviour of these parasite larvae during emergence was identical to the behaviour of larvae of the same species during emergence from larvae of C. legatella. However, Nixon (1970) comments that the cocoons of the later generation of M. fordi (Lathose emerging from larvae of C. rufata) tend to be more pointed at each end, and to have more sharply emphasised whitish ribs, than those from the earlier generation (emerging from larvae of C. legatella). The larvae of the phorid parasite emerged from the pupal stage of C. rufata and pupated without any cocoons. Once again only one larva of A. vitripennis or M. fordi emerged from each host larva, but several larvae of the phorid parasite emerged from the one host larva parasitised by this species. The pupae of A. vitriPennis completed their development in a mean of 11.5 ± 0.3 days, after which time the adult parasites emerged. However, the cocoons of M. fordi and the pupae of the phorid overwintered and emergence of the adults did not take place until the spring. The larvae of M. fordi spun their cocoons, and presumably pupated, between August 22nd and September 20th; the adults emerged between April -50-

20th and May 6th, 79% of these emerging between April 20th and 22nd. The nine larvae of the phorid parasite pupated on September 1st and the adults emerged between March 30th and April 16th. Three of the cocoons of M. fordi were found to be parasitised by the hyperparasite, Mesochorus sp. These cocoons were formed in the noimal manner, but the following spring a single adult of the hyperparasite emerged from each one, the emergence dates being April 22nd and May 6th.

(iv) Discussion The present work has confirmed that the life histories of C. legatella and C. rufata are very different. The times of occurrence of the different stages in the life cycle, and the duration of those stages differ considerably and are summarised in Fig. 2. C. legatella overwinters in the egg stage, which lasts approximately six months, and also aestivates in the pupal stage for approximately four months. C. rufata overwinters in the pupal stage, in which it remains for approximately ten months. The relative durations of the larval and adult stages of C. legatella are similar to those of C. rufata although they occur at different times of the year. The adult behaviour and diurnal activity patterns have been shown to be basically similar in the two species. -51-

However, females of C. rufata oviposit much earlier in the night than females of C. legatella. The mating behaviour of the two species in captivity is very similar, and warrants much further investigation. The occurrence of the equivalent stages in the life cycles at different times of the year indicates that the temperature requirements of those stages would differ between the two species. This has been verified in the adult, the egg (discussed later) and the pupa. The larvae of both species occur in the slimmer months and probably have similar temperature requirements. Both species have been shown to diapause in the pupal stage, and a comparison of the pupal development of the two species would be very interesting, since one of the pupae hibernates (C. rufata) and the other aestivates (C. legatella). An investigation into the causes of the extended diapause in some pupae of C. rufata, and the fate of the adults which emerge early in the season as a result, would also be valu- able. This might throw some light on the significance of this phenomenon in Lepidoptera. The existence of two diapausing stages in the life cycle of C. legatella is an interesting and uncommon feature of this species. The differences in the life cycles of the two species under consideration suggest that both species would be able to survive in -52-

the same locality. However, within the limitations of this study, it appears that only one of the species can exist abundantly in one habitat. A thorough ecological study of these two species could well prove very rewarding and supply the reasons for this. In this context it is interesting that both species are attacked by the same parasites. One of these, M. fordi, has only been recorded from C. legatella, C. rufata and Thera juniperata Linnaeus (Nixon, 1970). If C. legatella and C. rufata co-existed in one habitat, then M. fordi could complete its annual life cycle using these two species as alternate hosts. However, when only one of these hosts is present there must be at least one other host species present. Nixon (1970) has suggested that Euclidimera (= Callistege) mi (Clerck) might be an alternative host for parasites bred from C. legatella. This is quite possible since the larvae of E. mi feed on clovers from July to September (South, 1961). Large numbers of larvae of Eupithecia castigata Httbner have been found between August and September, and this species may also be a host of M. fordi. No suggestion has yet been made as to which host species is parasitised by adults of M. fordi when they emerge from overwintering cocoons in areas where C. legatella is not present. Further work on the biology of M. fordi is needed. -53-

(v) Summary (a) C. legatella (i) The adults emerge between September and November and lay eggs which diapause and then hatch between late March and early May the following year. (ii) The general period of flight and mating activity of the adults is during the first few hours of darkness. (iii) The eggs are laid singly or in pairs on the stems of S. scoparius, mainly in the stem axils, during the second half of the night. They are ovoid in shape, with a flattened dorsal surface, and their mean dimensions are 0.831 ± 0.11 x 0.510 ± 0.003 mm. (iv) The larvae feed externally on the leaves and flowers of broom during the spring flush of growth. (v) There are four larval instars and pupation, which takes place in the ground, is completed by the end of June. (vi) The pupal stage passes the summer in a state of diapause. (vii) No egg parasites were found. (viii) The larvae were found to be attacked by several parasites, of which the most common was A. vitripennis (Hymenoptera : Braconidae). This parasite appears to attack mostly third instar host larvae. One fully grown parasite -54-

larva emerges from each host larva and pupates within a white cocoon. The adult parasite emerges approximately two weeks later. (b) C. rufata (i) The main adult emergence is between late June and late July, and these adults lay eggs which develop without interruption and hatch in approximately 10-12 days. (ii) The general period of adult activity is similar to that of C. legatella, but oviposition takes place earlier in the night. (iii) The mean longevitrof mated females at 20°C. was 14.5 ± 1.2 days, and their mean fecundity was 176 ± 13 eggs. (iv) The eggs have a similar shape to those of C. legatella, but are smaller, their mean dimensions being 0.617 ± 0.004 x 0.397 ± 0.003 mm. (v) The larvae feed on the leaves of broom during the late summer flush of growth following pod formation. (vi) As in C. legatella, theinaare four larval instars and pupation takes place in the ground; the latter is completed by late September. (vii) This species overwinters in the pupal stage which diapauses; adults emerge from most of the pupae the following summer (June/July), but a small proportion of the -55-

pupae (15%) overwinter for a second time and adults emerge from these earlier in the summer than the main emergence. (viii) With one omission, the parasites attacking the larvae of C. rufata were found to be the same as those attacking C. legatella. A. vitripennis and M. fordi (hymenoptera : Braconidae) were equally numerous. The former parasitises mainly the third instar host larvae, and the latter attacks third and fourth instars. All larvae of M. fordi emerged from fourth instar host larvae prior to pupation; this species overwinters in the cocoon and the adults emerge the following spring. The duration of the pupal stage of A. vitripennis when bred from C. rufata was similar to its duration when bred from C. legatella. -56- 2. EGG DEVELOPMENT

(i) Introduction and Review of Literature The eggs of C. legatella and C. rufata have been chosen for a comparative study as a result of their similarities in shape and ecological niche and their dissimilarities in time of occurrence and duration of development. The aspects of embryonic development studied include the external morphology of the developing embryo, the duration and variation of development and their relationships to temperature, the temperature requirements of the different embryonic stages with particular reference to the egg of C. legatella, and parental effects on embryonic development. Most embryological studies on Lepidoptera have been accomplished by sectioning eggs, and few published works cover the external morphology of a lepidopteran embryo adequately. One notable exception is the work by Anderson and Wood (1968) on the embryo of EiphYas,postvittana (Walker). These authors regarded their study as an exercise in func- tional morphology and were able to establish the morphological basis of the embryonic movements previously described in E. postvittana by Reed and Day (1966). Christensen(1953) described the embryonic development of Cochlidion limacodes Hufnagel (= Apoda avellana (Linnaeus)) and based his description on the examination of living dated eggs. However, both E. postvittana and C. limacodes are particularly -57-

easy to study, since their eggs are small, flattened and have a transparent chorion. Many lepidopteran eggs areSpha.ical or ovoid, contain a considerable quantity of yolk and possess a thick and often non-transparent chorion. These factors render examination o the embryo, even by sectioning, extremely difficult; nevertheless some authors have succeeded in describing the external morphology of the embryo with varying success. Eastham (1927 , 1930) gives a very full account of the embryology of Pieris rapae Linnaeus, and describes the changes in body fol.= from the end of gastrulation onwards with great clarity (Eastham, 1930). Johannsen (1929) describes the body form of the embryo of Diacrisia virginica (Fabricius), and illustrates the changes from the cup-shaped germ band up to the completion of segmentation. Grandori (1932) and Saito (1934) give excellent accounts of the development of the silkworm and the Tusser moth (Antheraea pernyi (Guerin-Meneville) respectively. Saito (1934) presents very detailed figures and divides development into three stages, although each stage covers a considerable span of morphogenetic development and is therefore of doubtful meaning. Grandori (1932) even succeeds in presenting some very clear photographic plates of eggs with the whole development of the embryo revealed. Presser and Putasky (1957) give an equally clear account of the development of -58-

Heliothis zea (Boddie), including details of the external morphology and movements of the embryo together with the exact timing of events. Christensen (1942) describes the early embryonic development of Orgyia antiqua (Linnaeus) up to Lhe completion of gastrulation in great detail, and 1.2.j.erates the morphology and movements of the embryo with the help of three dimensional diagrams. Although Lattenschlager (1932) describes the whole of the embryological development of Solenobia triquitella (Abner), he also only illustrates the body form of the very early stages. Other descriptions of the external morphology of developing lepidopteran embryos are those of Ephestia kuehniella Zeller (Sehl, 1931), Plodia interpunctella Hamer (Willer, 1938), and Mamestra configurata (Walker) (Hempel, 1951). The continuous process of insect embryonic development has been divided into a number of morphologically different stages by many authors. Such a system provides a means of cormaring development in different species and within a single species under different conditions. The latter is the reason why Gaumont (1950) describes the embryonic development of the winter moth, G. brnmata, in terms of eight morphological stages; he is then able to compare the rate of development through each stage at different temperatures. In a previous paper Chancogne, Gaumont and Grison (1949) made use of seven -59-

rather crude stages in their work on the effects of sodium dinitrocresylate on the eggs of the winter moth at different times during embryonic development. Lindsay (1954) divides the embryonic development of Agrotis orthogonia Morrison ito twelve stages equivalent to each of the days of develop- mcy)t at 30°C., but does not describe the morphology in any detai]. He then compares the relative time of occurrence of the stages at four other temperatures, and shows that tempqL'ature does not have a differential effect on the rate of development through the stages. It appears that these authors have, so far, been alone in dividing the development of a lepidopteran embryo into morphological stages, except for the rather poor attempt of Saito (1934). This system is adopted in the present work, but development has been examined in more detail. The description of embryonic development in terms of morphological stages has been confined almost entirely to the Orthoptera, find within this Order work has concentrated on the Acrididae. Some thirteen authors have contributed to this field in the Acrididae; four have used morphological stages which also correspond to daily intervals during development (Slifer, 1932; Salt, 1949; Salzen, 1960; Riegert, 1961), and the remainder have used various systems of arbitrary stages based sole' on morphological criteria (Steele, 1941; Jhjngran, 1947; Bodenheimeorand Shulov, 1951; Matthee, -60-

1951; Shulov and Pener, 1959, 1963; Kiciikeksi, 1964; Van Horn,. 1966 a; Chapman and Whitham, 1968). Chapman and Whitham review the work of the other authors, and suggest a system of nine major stages which could be applied to the Orthoptera _1, general, and they list the equivalent stages of the pre-

-vi(,us authors. Moore (1948) makes use of some of Slifer's (1932) embryonic stages in his work on variation in grasshopper eggs; the stages he describes partition the developmental period into ten equal parts. In addition Brookes (1952) and Rakshpal (1962 a) have made contributions to this field in the Gryllidae, and the latter author points out the close similarities in embryonic morphology and development between the Gryllidae and the Acrididae. The embryonic stage equivalent to the physiological state of diapause has often been determined. Lees (1955) gives an excellent review of the known embryonic diapausing stagminEsects up to that date. Previously, Umeya (1950) reviewed the field and classified diapausing and hibernating embryos into five main types according to the morphological stage at which diapause or hibernation occurs; Andrewartha (1952) and Andrewartha and Birch (1954) record three embryonic diapausing stages in their list of the known insect species diapausing at some stage in their life cycle. Lees (1955) states that diapause cannot occur prior to the formation of the blastoderm or during the differentiation -61-

following blastokinesis, and Howe (1967) states that diapause in eggs usually occurs at one of the following three stages: before segmentation is complete, near the close of anatrepsis, or when the embryo is fully formed. Among the Lepidoptera it has been found that embryonic dipause ensues most often early in development or in the definitive embryo, and it seems appropriate in this instance to restrict examples to this Order. The arrest takes place in. the newly formed germ band in O. brumata (Kozhanchikov, 1950), Dendrolimus undans excellens (Butler) and Archips rlosteana (Linnaeus) (Umeya, 1946), Notolophus (=Orgyia) thyellina (Butler)(Umeya, 1950), Papaipema nebris (Guen6e) (Decker, 1931) and Bombyx mori (Linnaeus) (Umeya, 1950). Archips cerasivorana (Fitch) is also reported by Andrewartha (1952) and Andrewartha and Birch (1954) to diapause before segmentation is complete, but in the original paper Baird (1918) merely states that very little development takes place in the overwintering eggs until the spring. Christensen (1937) records that the diapause of O. antiqua occurs towards the end of anatrepsis, but in the remainder of the examples found it is the fully formed embryo which diapauses, and the species are all in the family Lymantriidae. This type of diapause has been recorded in Lymantria dispar Linnaeus and Lymantria monacha Linnaeus (Tuleschkov, 1935), and -62-

Malacosoma neustria (Linnaeus) (Tuleschkov, 1935). There are cases in which embryonic diapause in insects is not necessarily accompanied by a complete arrest of development (Lees, 1955). The orthopteran examples of vetroicetes cruciata Saussure (Andrewartha , 1943) and

1).e.-;Lostaurus maroccanus (Thunberg) (Bodenheimer and Shulov, 19fel) are notable. Lees (1955) places two Lepidoptera in this category, namely N. thyellina and D. undans. This is presumably because the embryos of both these species develop slowly during the winter (Umeya, 1946). However Umeya suggests that diapause is, in fact, terminated by the end of November in both these species, at a time when the embryo is still in the early germ band stage. There is a close parallel here to the situation in C. legatella, as will be demonstrated. The duration of embryonic development has been investigated in a wide range of insects for many reasons. The time required for an insect egg to develop under any given set of environmental conditions is vital to the understanding of the processes of embryonic development, and is often econom- ically important. The relationship between developmental period and temperature can be calculated from data obtained at a series of constant temperatures, and predictions of the times of outbreaks of certain insect pests may be based on this type of information. -63-

Various methods have been employed in presenting data on developmental periods, such as the shortest developmental period, the mode, the median and the ffean.. The latter is the most commonly adopted statistic, but it can be affected by the presence of one or two abnormal individuals to a greater degree than the median. Howe (1952) states that in some ways the median is more satisfactory than the mean for this very reason, a view strongly supported by Messenger and Flitters (1958); but in a later paper Howe (1966) records that the mean is the statistic most often used for developmental periods of stored products beetles, and that the amount of variation between individuals is then represented by some derivative of the variance. The whole topic of the statistical representation of the developmental period has been critically reviewed by Howe (1967). Knowledge of the conditions which are most favourable for the egg stage (those conditions giving rise to the highest proportion hatching with the least variation and in the shortest time) is required for species which diapause during this part of the life cycle, as well as for non-diapausing species. The three criteria of proportion hatching, and the mean and variance of the developmental periods have often been used in the evaluation of a given set of conditions for the successful termination of diapause. Browning (1952 b) -64-

used all three criteria in his work on diapause in the eggs of Gryllulus (=Acheta) (=1912mallya) commodus Walker. Analyses of variance were performed on the results of all the treatments using each of the criteria. Suitable transforma- tions were first required in the cases of the proportions hatching and the variances. Nasaki (1956) also makes use of these three criteria in his work on L. dispar, but does not find it necessary to perform analyses of variance. He shows that both the standard deviation and the coefficient of variation of the hatching period are reduced by increase in exposure to low temperatures during diapause, thus illus- trating that the reduction in variation of the hatching time has biological , as well as statistical, significance. Stanley (1946) combines the proportion of eggs hatching and the duration of development by expressing them as a ratio of the former to the latter, thus creating an environmental index which is used to measure the suitability of different temperature treatments to the non-diapausing eggs of Tribolium confusim Duval.

The criteria mentioned so far are all characteristics of hatching and are therefore not ideal, since although diapause may be completed under a given set of conditions subsequent development may be abnormal and this may not become apparent until the larval stages or even later in the life cycle (Browning, 1952 b; Andrewartha and Birch, 1954). -65-

In this connection Browning suggests that better criteria would be the ability of young nymphs or larvae to feed and grow, the ability of the eggs to produce fecund adults, or measurements could be made of oxygen consumption (Bodine, 1932). However, as Browning points out, hatching is practic- able, and for this reason it has been used in the present work together with the timing of morphological events during development. The latter is achieved by constructing a timetable of development for any particular set of conditions from records of the morphological stages present in samples taken at regular intervals during development. Few authors have examined the distribution of stages within their samples in any detail (Slifer, 1932; Moore, 1948; Shulov and Pener, 1959, 1963; Salzen, 1960; Amaro, 1963; Van Horn, 1966 a,b), which is surprising since variation does occur and this method not only provides a means of constructing a timetable of development for a population of eggs but also of examining variation in developmental rates throughout development and the factors contributing to that variation in a population. Several methods have been applied to describe statistically samples of embryos classified into stages, some more appropriate than others. Brookes (1952) records the modal stage at regular intervals throughout development in -66--

G. commodus, and gives details of the proportion of eggs at the modal stage and behind or ahead of it. Salzen (1960) employs a similar procedure for the eggs of Locusta migratoria migratorioides (Reiche and Fairmaire). Shulov and Pener used the mean in their won': on the same species (Shulov and Pener, 1959) and Schistocerca gregaria (ForskR1) (Shulov and Pener, 1963). In these two papers they describe variation in terms of the range and standard deviation respectively. Slifer (1932) uses the mean stage for embryos of Melanoplus differentialis Uhler, but this is justified since each morphological stage represents one day in development. Variation is described in terms of days behind or ahead of the mean stage. Moore (1948) also uses the mean stage for the embryos of three grasshopper species, but ac;ain the stages represent equal proportions of the total developmental time, and the mean is expressed as the mean percentage development. Van Horn (1966 a) describes the embryonic of developmentAAulocara elliotti (Thomas) in terms of arbitrary morphological stages, and then shows that a linear relationship exists between embryonic stage and age, at least during pre-diapause development. She therefore makes use of the mean stage and standard deviation with justification. In the present work careful consideration has been given to the question of the appropriate statistics needed to describe and analyse the data collected. -67-

Amaro (1963)examines the variation in the time taken to develop to certain stages in the embryogenesis of Acarus siro Linnaeus, and uses, as his parameters, the median time and semi-interquartile range because these are not affected by a few individuals in a long tail. This approach is possible because he only examines three stages in development, one of which is hatching, and the other two stages can be recognised easily in live eggs. Thus the accurate timing of development through each of the three stages can be achieved simply by frequent observations. King (1959) investigated the differences between populations of Drosophila melanogaster Meigen in embryonic developmental rates by examining the time taken to develop to each of five stages in embryogenesis. He compared the median times taken to reach each stage, which were calculated by means of probit analysis, and also the variation which was described in terms of standard deviations of the developmental distributions. The various aspects of the temperature relations of insect eggs have been investigated by many authors, and it is practical to make reference to only a selection of the more relevant works. However, the subject is reviewed in detail up to the end of 1965 by Howe (1967). The temperature requirements of the embryo, as indicated by the duration of its development, its viability and the rate of development at different times during embryogenesis, were the aspects chosen for investigation in both the non-diapausing eggs of C. rufata and the diapausing eggs of C. legatella Many attempts have been made to construct, and describe mathematically, curves representing the effect of temperature on the duration of embryonic development (see reviews by Uvarov, 1931; Belehr6dek, 1935; Andrewartha and Birch, 1954; and Howe, 1967). Howe points out the dangers of doing this without adequate precise data. A curve has been constructed in order to show that the duration of the egg stage of C. rufata is influenced by temperature in a way characteristic of the non-diapausing stages of insects, to indicate the optimum temperature for development, and to make a comparison with the egg of C. legatella. Shrtage of material has prevented a more extensive examination of the range of temperatures favourable for embryonic deve3opmert in this species, and no attempt has been made to describe the temperature/duration curve mathematically. There is evidence which suggests that different embryonic stages may have slightly differing temperature requirements in insects. Browning (1952 a) shows that a logistic curve can be made to fit data on the embryonic development of G. commodus more closely if separate parts of development are considered rather than the entire development of the egg stage. The embryo of T. confusum is differentially sensitive E-69-

to brief exposures to high temperatures at different stages during its development (Stanley and Grundmann, 1966). The work of Richards (1956, 1959, 1964) has emphasised that development should be regarded as consisting of numerous processes, each with its own temperature relations. Work on this aspect of embryonic temperature requirements has been confined to the post-diapause embryonic development of C. legatella, since a detailed examination of the embryogonesis of C. rufata was made at only one constant temperature. In addition to an examination of the range of constant temperatures over which post-diapause development in C. legatella can proceed, the effects of alternating temperature regimes which have means approaching the developmental threshold have been investigated. This was stimulated by the interesting results of Richards (1956, 1959) concerning the embryonic development of Oncopltus fasciatus (Dallas) in these conditions, and also those of Lin et al (1954) for 0. fasciatus and T. confusum and Messenger and Flitters (1959) for three species of fruit fly. In both cases alternating or fluctuating temperatures near the constant temperature developmental threshold appear to have had a dramatic effect on embryonic development in comparison with the equivalent constant temperatures, but Messenger and Flitters give a good critical account of the difficulties encountered in the interpretation of such data. The determination of the constant temperature equivalent of an alternating or fluctuating temperature regime is very difficult and it is therefore equally difficult to detect whether the variable temperature has had an accelerating or inhibiting effects However Richards (1959) clearly shows that the proportion of eggs of 0. fasciatus which hatch, and the vitality of the larvae are greater when the eggs are incubated in alternating temperatures, and Lin et al report similar findings. The temperature relations of diapausing insects have been examined in great detail by many authors, and much of their work is mentioned in the reviews by Andrewartha and Birch (1954), Lees (1955) and Dnnilevskii (1965). The temperature requirements of the embryo of C. legatella during pre-diapause, diapause and post-diapause development have been investigated. The conditions favourable for the termination of diapause have been examined rather than the conditions needed to induce diapause (if any), since it was found that diapause is obligate in this species. Diapause enables insects to survive unfavourable dry, hot or more often cold conditions (Howe, 1967). In temperate regions the diapausing stage in insects is usually adapted to survival during the cold season, and some early workers therefore regarded diapause as the result of tae presence of an inhibitory substance which was destroyed by the action of low temperatures (Bodine, 1932; Salt, 1947). Andrewartha -71-

(1952) was quick to point out that different insect species have different temperature requirements during diapause, and these do not always include low temperatures. This author put forward the idea that diapause is a stage during the physiological development of the insect, often, but not always, accompanied by a cessation of morphological development, which requires certain temperature conditions. The range of temperatures favourable for diapause development may or may not overlap with the range favourable for morphogenesis, and this is dependent on the climatic conditions experienced by each species. Lees (1955) describes the temperature requirements of the insect species worked on up to that time, and discusses the subject further in a later paper (Lees, 1956). He states that many insects from temperate climates with moderately severe winters have been found to respond most readily to temperatures within the general range 0-12°C. (Lees, 1955), and many European Lepidoptera fall within this range, with optima for diapause being approximately 7-10°C. (Lees, 1956). More recent work has revealed further examples with these general requirements for diapause development: P. rapae and L. dispar are further representatives of the Lepidoptera (Masaki, 1955, 1956) and Leptohylemia coarctata Fallen (Diptera: Muscidae) also has similar requirements (Way, 1959). The latter author has shown that the temperature requirements can change during -72-

diapause, and concludes that there are two phases in the diapause of L. coarctata with different temperature optima. Danilevskii (1965) discusses the relationship between geographical distribution and the temperature requirements for diapause development. Most of the examples quoted are European Lepidoptera, and he concludes that photoperiodic and temperature reactions determining the onset of diapause, and also those temperature adaptations of the diapausing stages on which depend the duration of the resting stage and the rate of reactivation are subject to marked intra- specific geographical variation as a result of adaptation to zonalgeographical conditions. This is particularly evident in a close relative of the species studied, namely the winter moth, 0. brumata, which has a life cycle very similar to that of C. legatelia. This topic is elaborated in the discussion. Variations in embryonic development between populations of insects have been recorded by several authors (Moore, 1948; Kozhanchikov, 1950; Van Horn, 1966 a), and these can be largely attributed to genotypic differences (Speyer, 1938; King, 1959; Wylie, 1960). However, it has been established in several species that the embryonic development of the progeny is influenced by the physiological state of the parents (Roubail, 1928; Simmonds, 19118; Matthee, 1951; -73-

Kogure, 1933; Albrecht et al, 1958; Albrecht, 1959; Papillon, 1960; Rakshpal, 1962 b; Wellington, 1965). Van Horn (1966 b) states that there may be considerable influence from environmental factors and from maternal age on the expressivity of the genotype, and much recent work suggests an endocrino- logical basis for this, particularly that of Hasegawa (1963), Yamashita and Hasegawa (1964 a), Masner, S1 ma and panda (1968), and Wellington (1969). Certain aspects of the maternal control over egg development have been examined briefly, in particular the effects of maternal age. There is very little information concerning the relative times taken by different insect species to reach particular stages of development. The only known literature on this subject refers to the Acridoidea (Chapman and Whitham, 1968). A brief comparison between C. rufata and some other Lepidoptera is included in the discussion. -74-

(ii) Experimental Methods

(a) Handlinz of Eggs

Females of both species laid their eggs either along the ribs of the broom twig provided or on the sides

of the cage, Each egg was cemented to the substrate with a water soluble substance. Eggs, previously moistened with

a fine camel hair brush, could be dislodged with either the same brush or some blunt instrument such as a seeker. The thickness of the chorion prevented damage to the eggs when

they were handled in this way. When moist eggs were subsequently placed onto a surface, they usually became attached once more by means of the adhesive, and this, together with their flattened elongate shape, prevented their loss.

(b) Sterilisation of Eggs In one set of experiments eggs of CJegatella were kept at a high relative humidity for several months, and they were therefore washed in a solution of a wetting agent to remove fungal spores. This wetting agent is marketed under the trade name of Ilyamine 1622 (B.D.11.), and consists of Di-iso-butyl phenoxy ethoxy ethyl dimethyl benzyl ammonium chloride monohydrate. (c) Containers for Eggs

When no humidity control was needed, eggs were kept either in the cages in which they were laid, or on -75-

filter paper in petri dishes. These containers were used both outdoors and in constant temperature rooms, but not

when a strict control of temperature and humidity was essential. In all other instances eggs were kept in

containers which could be placed in constant temperature

chambers, water baths, or the outdoor insectary.

Each container consisted of a small polythene

cap 2 ems. in diameter, to one side of which was attached a small piece of adhesive plastic foam (Fig. 6 a,b). eggs were placed either on part of a cotton—wool roll inside the cap (Fig. 6 a), or on parachute nylon stretched across the top of the cap and attached to the polythene by the application of heat (Fig. 6 b). The latter method proved more successful, since nylon is very resistant to fungal attack. The eggs were not dislodged from the nylon because of their adhesive properties. Photographic slide labels were used for numbering the caps.

These egg containers were placed in a variety of tubes:—

(i) Containers kept in the outdoor insectary were placed in 7.5 x 2.3 ems. airtight flat bottomed tubes. Another polythene cap at the bottom of each tube contained the humidity controlling liquid, and the egg container was held approximately half way up the tube by its plastic foam pad (Fig. 6 a). -76—

a. b.

11111111111111111111111111111111111111111111 rubber bung

cotton wool test roll tube _11 polythene plastic 11111111111111111111111111111111111 cap foam

airtight flat bottomed tube parachute nylon

11111111111111111111111111111111111 .111111111111111111111111111111111

polythene cap (containing sat. NaCI. solution)

c. non - absorbent cotton wool

glass vial gelatin

lead 1 111111111m1111111111 shot

5 cms

Fig. 6 Tubes for Egg Containers _'77_

(ii) Egg containers kept in constant temperature water baths were placed in 15 x 2.5 cms. test tubes sealed with a rubber bung. Each test tube was weighted with lead shot covered by gelatin containing copper sulphate. The gelatin ensured that no poisonous fumes could be given off by the lead, and the copper sulphate prevented fungal growth. A tight padding of cotton wool supported a polythene cap containing humidity controlling liquid, and the egg container was held in the tube as above (Pig.6b). (iii) In one instance, when eggs were kept at a low temperature in a refrigerator, the containers were covered by small glass vials 2.4 ems. high and 2.3 ems, in diameter (1'ig.6c). (iv) In two constant temperature cabinets several containers were placed in a plastic box together with humidity controlling liquid. The design of the egg containers facilitated the transference of eggs from one temperature to another, and also from the insectary to a constant temperature bath. Two humidities were used, almost 100% R.H. given by distilled water, and 756 R.H. given by a saturated solution of sodium chloride. The relative humidity given -78-

by this salt varies only 1.56 over the range of temperatures

used (Winston and Bates, 1960).

Only in one set of experiments was cotton—

wool used as a substrate for the eggs, and than the wool

in each container was moistened with 20 drops of a saturated aqueous solution of an anti—fungal agent known as Nipagin L (a methyl ester of para hydroxy benzoic acid). No humidity control was used, but the eggs in contact with the damp cotton wool were subjected to an almost 100% relative humidity. It was in these experiments that the

eggs were washed with Hyamine 1622 before incubation. It was subsequently discovered that Nipagin M has a deleterious

effect on the egg development of these moths. It was not,

therefore, used again, and the eggs were kept on parachute nylon instead.

(d) Incubators

During the course of the experiments described below eight temperatures, ranging from 2°C. to 2000.,

were required. A combination of constant temperature water baths and cabinets was used to provide this wide range. A

10/24 hour light regime was adopted throughout the experi— ments with C. legatella (except where stated otherwise), and a 16/24 hour regime was used for C. rufata. The three higher temperatures (20°C., 15°C.,

900.) were provided by water baths. A B.T.L. "Unitemp" -79-

water bath, fitted with a light-proof lid containing two Phillips TLC 15 w./29 fluorescent tubes, was run at o 20 C. + 0.1 oC. A small aquarium tank was placed inside a well ventilated light box in a constant temperature room,

and this gave a temperature of 15°C. + 0.1 °C. The light box was illuminated by two Phillips TLAD 15 w./29 fluorescent tubes. Although the water was not stirred,

there was hardly any indication of a temperature gradient within the tank; this was probably the result of the constant air flow around the tank. Another aquarium tank

fitted with stirrer, 60 w. heater operated by a Gallenkamp mercury contact thermometer and Sunvic Electronic Relay,

and cooling coil, was run at 90C. + 0.1 0C. under a bank of four Phillips TLAP 20 w./29 fluorescent tubes. The coolant was supplied by a Frigidaire refrigerator, which contained several litres of an ethylene glycol/water mixture; the latter was circulated by a small centrifugal pump (Grant Instruments: No. 4.4378). When the system had stabilised the temperature of the coolant in the refrigerator was exactly 300. below that of the water bath. The tubes containing eggs which required a temperature of 6°C. were therefore placed in a small white container filled with water, which was partially submerged in the coolant. A "Perspex" lid, fitted to the refrigerator, -80—

allowed light from the bank of lights to fall upon these

eggs. The temperature fluctuation was only + 0.1 00.

TemperatUres of 200. and 3°C., which were not

required at the same time, were provided by aSternette

refrigerator which was converted into an incubation chamber.

A "Perspex" lid was fitted to facilitate illumination of

the eggs. A fan circulated air within the incubator, and

a Sunvic thermostat (Type TS 3 ii0)„ coupled to a hotwire

vacuum relay (Type F103/3), was fitted to control the

temperature. Egg containers were placed inside an empty

aquarium tank, which was closed by a glass lid; this reduced the temperature fluctuation inside the tank to

+ 0.2°C., but there was probably much less variation

inside the egg containers.

In any of the above incubators, the test tubes

containing eggs were supported in stainless steel or

"Perspex" racks.

Temperatures of 14.500. and 7.5°0. were obtained in two Gallenkamp cooled incubators. Two 6v. 0.1 amp. torch bulbs were placed inside each incubator to provide. light. Each bulb was fitted with a silver foil reflector and placed 15 cms, from a small plastic box containing the eggs. The current for the bulbs was supplied by a mains transformer operated by a time switch. The bulbs were of -81-

such low wattage that they had a minimal effect on the

temperature within the incubators, the fluctuation being

only + 0.2°C.

The intensity of the light received by the eggs

in all incubators was of the order of 10-20 foot candles.

The temperature and relative humidity in the

outdoor insectary were recorded on a thermo-hygrograph.

During one winter two artificial 'eggs' (Plate 4 , fig. a)

consisting of orange painted 26 s.w.g. copper-constantin

thermocouples, were placed on a broom bush in the field

(Plate 4 , fig.b ), one in an exposed sunny position and the other on the shaded side of the bush. These thermo-

couples provided a continuous temperature record to the nearest 0.25°C. for each position on a Rustrac twin- channel potcntiometric recorder. This method is a modification of that used by Way and Banks (19624). (e) Fixation Preparation and Examination of Eggs

Since a large number of eggs was fixed, a strongly penetrating fixative was used so that the eggs did not have to be pierced to facilitate the penetration of the fixative. Bouin's fixative ivo.2 was found to be satisfactory, and was used to fix all the eggs to be examined. At least 24 hours were allowed at room temperature for fixing. -8?

a. Close-up of Thermocouple tip on broom twig

b. Site of Recording Apparatus - (41, = broom bush) Plate 4. Apparatus for measuring Temperature in the Field. -83—

After fixation the eggs were washed in 900

alcohol, and then dechorionated with forceps. A method of dechorionation developed by Slifer (1945) for locust eggs, using a 3,A solution of sodium hypochIorite , was

tried and found to be satisfactory, but the timing of the

process was very critical, and the method was not used.

In the ovcrwintering eggs of C. IegateIla the

serosa forms a deep orange membrane which completely obscures the contents of the egg. However, a few weeks

storage in alcohol, or a few days in distilled water, removed this orange colour; the eggs could then be placed

in a suitable clearing agent for examination. The eggs

of C. rufata are also coloured orange, but the serosa is not completely opaque. Little difficulty was encountered

in the examination of these eggs. Lost of the usual clearing agents were tried without success, Either the eggs would not clear, or the embryo became as transparent as the yolk and could not be readily distinguished. A

1:1 mixture of 70,,e6 alcohol and glycerol (Hogan, 1959) was found to clear a large number of eggs. When they

were examined under a dissecting microscope against a black background with reflected light (the angle of the incident rays being very critical), the embryos showed up white and opaque in the otherwise transparent eggs. This -84—

was satisfactory when it worked, but in many eggs no

embryo could be seen.

iviost of the usual stains would not penetrate

the eggs of C. legatella, before any of these stains were

tried, the eggs were washed in a 10% solution of ammonium

acetate in 90% alcohol, in order to remove excess picrates.

Peulgen's solution was found to be satisfactory, and was

therefore used after the eggs had been hydrolysed in N HC1

at 60°C. for 8 minutes. The eggs were cleared in a mixture

of benzyl benzoate (62,5,M and 2 ethoxy ethanol (37.3%)

(Peters, 1961). The correct mixture was found by trial

and error.

Even the last method was not entirely satisfact-

ory. Some embryos of C. legatella were extremely difficult to see. It is interesting that in this species, the eggs

in any one group (i.e. all subjected to the same conditions) stained and cleared to different degrees.

Rubstc (1963) refers to a similar phenomenon in the eggs of a tortricid moth. He records that some embryos stain more weakly after they have been subjected to a period of frost.

It was subsequently found that staining is not necessary. Eggs which had been dechorionated were passed through absolute alcohol into the clearing mixture of benzyl benzoate and 2 ethoxy ethanol, in which they were -85— stored and later examined under a dissecting microscope with transmitted light. -86—

(iii) The External Hornhogenesis of the Embryos of C. legatella and C. rufata

The continuous process of embryonic development can be divided into a number of stages based on the external morphology of the embryo as seen in whole mounts of dechorionated eggs.

(a) The Stages of Embryonic Development in C. legatella

STAGE I — The development of the blastoderm prior to the formation of a germ band. The egg is pale green before and after the formation of the blastoderm. Infertile eggs cannot be distinguished from fertile ones at this stage; however, infertile eggs remain green, and eventually collapse after several weeks.

STAGE II — The formation of the germ band. Successive divisions in part of the blastoderm produce an area of small, closely packed cells called the germ band. This band lies on one side of the egg so that its lateral margins extend onto the dorsal and ventral surfaces, thus covering approximately one half of the total surface area of the egg

(Fig. 7a ). It is readily visible in live eggs as a whitish patch on a yellowish—green background. The larger, more flattened cells of the serosal rudiment cover the remainder of the yolk.

STAGE III — The germ band becomes more compact, covering approximately one quarter of the surface area of -87—

the egg, and during this process the serosa extends over

the surface of the germ band, thus enclosing it (Fig. 7b).

STAGE IV — The embryo becomes cup—shaped (Fig. 8a ),

resembling the pyriform stage in which several Lepidoptera

are known to enter diapause (ULEYA, 1950). The embryo

is surrounded by yolk, and the serosa becomes coloured

deep orange, thus obscuring the embryo from view in

live eggs.

STAGE V — Slight anteroposterior elongation of the

embryo takes place, but in other respects this stage is

similar to the previous one (Fig. 8b).

STAGE VI — The elongation of the embryo proceeds

further, together with the formation of two cup—shaped

anterior lobes, separated by a ventral longitudinal groove, and one large posterior pouch (Fig. 9a ). As the embryo elongates it becomes dorsoventrally curved

in a plane parallel to the surface on which the egg rests.

STAGE VII — The posterior, or caudal pouch, enlarges and the anterior, or cephalic lobes, become more prominent as the embryo increases in length and dorsoventral

curvature (Fig. 9b).

STAGE VIII — The commencement of segmentation. A transverse groove clearly separates the cephalic lobes from the rest of the embryo. The postoral segments of the head -88-

are delineated by transverse ventral grooves, and the

remainder of the embryo becomes narrower and continues to

elongate, thus forming a trough which is U-shaped in cross

section (Fig.10a).

STAGE IX - Segmentation of the thorax is complete, and

some of the anterior abdominal segments are also delineated

(Fig.10b ). The ventral surface of the embryo faces the

surface of the egg throughout these early stages in

embryogenesis. Consequently, as a result of the elongation

and curvature of the embryo, the cephalic lobes and caudal

pouch come to face each other. This becomes more evident

in the next stage.

STAGE X - segmentation is complete. All the body segments are clearly delineated and the embryo attains its greatest length prior to blastokinesis (Pig.11a ). The mandibular, maxillary and labial segments of the head, and the three thoracic segments all extend ventrolaterally to form a pair of appendages on each segment. Ten abdominal segments and a telson are clearly visible, and the sixth segment bears two ventrolateral swellings which later develop into prologs. A terminal invagination of the telson to form the proctodaeum can be seen in cleared specimens. -89—

STAGE XI — During the process of gastrulation the

embryo becomes shorter and thicker. The head appendages

begin to group around the mouth, and the thoracic

appendages increase in length posteriorly. The labral

and antennal lobes are visible in lateral view (Fig.11b).

STAGE XII — The dorsal closure of the body wall is

complete. The embryo becomes less coiled, as a result of

the reduction in length, and is ready to undergo blastokinesis. The gnathal appendages condense further and the labrum becomes prominent. The telson and tenth

abdominal segment can no longer be distinguished as separate structures (Fig.12a).

STAGE XIII — The embryo undergoes blastokinesis.

At the start of the revolution the posterior abdominal segments are turned ventrally (Pig.12b ). The thoracic appendages move to a ventral position and no longer lie in

contact with the body wall. The ninth abdominal segment fuses with the tenth segment and telson.

STAGE XIV — Elongation of the embryo results in an anteriorly directed movement of the last abdominal segments (Fig.13a ), so that they come to lie opposite the thoracic segments and between the thoracic appendages.

The head is temporarily prognathous. -90—

STAGE XV - On completion of the revolution the head

and last abdominal segments again lie in close proximity, but the dorsal surface of the embryo faces the surface of

the egg (Fig.13b Of Fig.11a ). The thoracic limbs enlarge and lie on each side of the last four abdominal segments, The prolegs on the sixth abdominal segment and

the claspers on the telson are clearly visible.

STAGE XVI - Further elongation results in the dorsal surface of the embryo coming into contact with the serosa, and also in a slight twisting of the embryo to accommodate the posterior segments of the abdomen next to the rapidly enlarging head capsule. The latter, which becomes hypognathous, occupies much of the anterior part of the egg, and the gnathal appendages begin to assume their final position and shape (Fig.14a).

STAGE XVII - The lateral ocelli are clearly visible as pigmented structures. The mouthparts develop further, and the mandibles are weakly sclerotised. The thoracic legs arc clearly segmented, and the spiracles are visible.

During these latter stages of embryogenesis the egg yolk is eaten by the embryo and can be seen clearly in the midgut of cleared specimens.

STAGE XVIII - The mouthparts are complete and more heavily sclerotised than in the last stage. The thoracic -91-

Abbreviations used in Figs. 7 - 15. A.P. = Anterior Pole abd. 1, 2.. = abdominal segment ant. = antenna ant. 1. = antennal lobe cl. = clasper c. 1. = cephalic lobes

c. p. = caudal pouch g. b. = germ band 1. = labium lbr. = labrum 1.s. = labial segment rand. = mandible mnd. s. = mandibular segment mx. = maxilla mx. s. = maxillary segment oc. = ocelli pr. = proleg p. inv. = proctodaeal invagination s. = serosa soc. - socket of developing seta sp. = spiracle s.r. = serosal rudiment st. inv. . stomodaeal invagination t. = telson th. 1. = thoracic limb th. 1, 2.. . thoracic segment y. = yolk

Figs. 7 - 15 are drawn to scale shown in Fig. 7. a.

s.r.

A.P.

0-5mm

Fig. 7 C. legatella a. Stage II embryo b. Stage III embryo _93_ a.

Fig. 8 C. lega tella a. Stage IV embryo

b. Stage V embryo -94-

c. I. c. p.

Fig. 9 C. legatella a. Stage VI embryo

b. Stage VII embryo a.

mnd. s.

Fig.10 C. legatella a. Stage VIII embryo

b. Stage IX embryo piny

abd 7

Fig. 11 C. legatella a. Stage X embryo

b. Stage XI embryo Dorsal Wall Complete a.

lbr ant. I. abd. st. inv 10+t. mnd.

-4A mx.

abd 6

abd. 9+10#t.

Fig. 12 C. legatella a. Stage XII embryo

b. Stage XIII embryo -98- a.

abd. 6

Fig. 13 C. lega tella a. Stage XI V embryo

b. Stage XV embryo a.

ant. lbr mnd. mx.

Fig. 14 C. legatella a. Stage XVI embryo

b. Stage XVII embryo -100- a. soc.

Fig. 15 C. legatella a. Stage XVIII embryo

b. Stage XIX embryo -101—

legs are complete with terminal claws, and the whole body is covered with developing setae (Fig.15a).

STAGE XIX — The fully formed embryo. Sclerotisation and pigmentation are complete. Further elongation of the body results in a twist of the abdomen, so that the posterior segments are turned back with their ventral surface adjacent to the ventral surface of the preceding abdominal segments (Fig.15b ). The thoracic legs are held forward. The embryo lies coiled up inside the transparent chorion, having ruptured and consumed the egg membranes.

STAGE XX — This is represented by the hatched larva.

In hatching the caterpillar eats its way out of the anterior end of the egg. There is no evidence that the empty shell is consumed by the young larva.

(b) The Stages of Embryonic Development in C. rufata

There are several differences between the embryo— genesis of this species and that of C. legatella. These differences are more pronounced during the early part of development.

STAGE I — The development of the blastoderm prior to the formation of a germ band. The egg is pale green at this stage as in C. legatella; however, in this species it remains green until segmentation is almost complete. -102-

STAGE II - The differentiation of the blastoderm into germ band and serosal rudiment is similar to that in

C. legatella, but the germ band occupies such a large proportion of the surface area of the egg (Fig.16a), that the serosal rudiment consists only of a narrow longitudinal strip on one side of the egg. The germ band is orientated in a similar way to that of C. IeRatella with its lateral portions extending over the dorsal and ventral surfaces of the egg.

STAGE III - The serosa spreads over the entire surface of the germ band, completely enclosing it. A very slight reduction in the extent of the germ band in the mid- lateral zone now takes place (Fig. 16b ) and the embryo occupies approximately three quarters of the surface area of the egg.

STAGE IV - The embryo, which remains in close contact with the serosa (cf. C. legatella Stage IV), occupies approximately two thirds of the surface area of the egg

(Fig.17a ). However, the anterior and posterior regions do not become reduced in size, and these are destined to form the cephalic lobes and caudal pouch respectively.

STAGE V - The embryo possesses recognisable cephalic lobes which are separated from each other by a ventral longitudinal groove. A large caudal pouch extends -103—

posteriorly from a point just behind the head region

(Fig. 17b ). The caudal pouch is open dorsally even at its posterior extremity.

STAGE VI — Morphologically this stage is very similar in both species. However, the embryo of

C. legatella is very compact and is enclosed in the yolk, whereas the embryo of C. rufata lies on the surface of the yolk and partially surrounds it. The head lobes become more pronounced and the middle region of the embryo is the narrowest part (Fig. 18a ).

STAGE VII — Elongation of the embryo takes place within the confined space of the egg, and results in a dorsoventral curvature similar to that found in the embryo of C. legatella. The cephalic region and caudal pouch occupy the anterior and posterior ends of the egg respectively (Fig. 18b ).

STAGE VIII — As in C. legatella, segmentation commences with the delineation of the postoral cephalic segments (Fig.19a ). The cephalic lobes become clearly separated from the rest of the embryo. Further elongation of the embryo causes the cephalic lobes to take up a position posterior to the mandibular segment, and the caudal pouch is reduced in size. -104-

STAGE IX - This is very similar to the Stage IX

embryo of C. legatella, with the cephalic and thoracic

segments delineated (Pig. 19b). However, the entire embryo is near the surface of the yolk and the egg remains

a greenish colour. The cephalic lobes and caudal pouch

lie in close proximity as a r: suit of the elongation process. Slight protuberances can be seen on the

postoral and thoracic segments, and are the origins of the appendages of those segments.

STAGE X - At this stage the embryo sinks into the yolk (Fig. 20a), and the egg becomes yellowish. The embryo, which can no longer be observed clearly in the live egg, becomes completely segmented and is extremely similar to the Stage X embryo of C. legatella.

STAGES XI to XV are so similar to those of C. legatella that no further description is necessary.

STAGES XVI to XIX - Since the egg of C. rufata is

considerably smaller than that of C. legatella, development of the embryo beyond Stage XV results in an enlargement of the egg. In this species the final

turning of the posterior abdominal segments commences at Stage XVI, and not at Stage XIX as in C. legatella

(Fig. 23a cf. Fig. 15b). Consequently elongation of the embryo is completed by Stage XVIII in C. rufata -105—

(cf. C. legatella). Stages XVIII and XIX in this species differ only in the degree of pigmentation and not morphologically. Stages XVI to XIX resemble their counterparts in C. legatella in all respects other than those mentioned above. -106-

Abbreviations used in Figs. 16 - 24.

A.P. = Anterior Pole abd. 1, 2. . = abdominal segment ant. = antenna ant. 1. = antennal lobe el. = clasper c. 1. = cephalic lobes c. p. = caudal pouch g. b. = germ band 1. = labium lbr. = labrum 1.s. = labial segment mnd. = mandible mnd. s. = mandibular segment mx. = maxilla mx. s. = maxillary segment oc. = ocelli pr. = proleg p. inv. = proctodaeal invagination s. = serosa soc. = socket of developing seta sp. = spiracle s.r. = serosal rudiment st. inv. = stomodaeal invagination t. . telson th. 1. = thoracic limb th. 1, 2.. = thoracic segment y. = yolk

Figs. 16 - 24 are drawn to scale shown in Fig. 16. a.

A.P.

0-5mm. b.

Fig. 16 C. rufata a. Stage II embryo b. Stage 111 embryo -108- a.

C. c. p.

Fig. 17 C. rufata a. Stage IV embryo b. Stage V embryo -109-

Fig. 18 C. rufata a. Stage VI embryo b. Stage VII embryo a. -110-

th. 1

Fig. 19 C. rufata a. Stage Vlll embryo b. Stage IX embryo -111-

p. inv. mnd.

mx.

Fig. 20 C. rufata a. Stage X embryo

b. Stage XI embryo -112-

Dorsal Wall Complete a. lbr ant. I. abd. 10+t. st inv.

abd. 6

abd. 9+10+t. th. 1. cl.

Fig. 21 C. rufata a. Stage XII embryo b. Stage XIII embryo -113- a.

abd. 6

Fig. 22 C. rufata a. Stage XIV embryo

b. Stage XV embryo -114-

sp.

mnd. 1. mx. Fig. 23 C. rufata a. Stage XVI embryo

b. Stage XVII embryo s. (Stage XVIII only)

Fig24 C. rufata Stages XVM & XIX -116—

(iv) The Egg Development of C. rufata

(a) The Duration of Development in an Outdoor Insectary

The eggs laid by five females in an outdoor

insectary were incubated in the insectary at very nearly

10C% relative humidity, and under temperature conditions similar to those in the field. (Thermograph records for

the duration of this work are available). The freshly

laid eggs were collected at intervals of eight hours, and the subsequent observations for hatching were made with the same frequency. The mean developmental time was 253.33 4, 1.05 houruond this was based on 887 eggs hatching from a total of 1312 laid. The frequency distribution for hatching is shown in Fig.25 . The bimodality of this distribution is shown by a plot of the accumulated percentage hatch on probability paper

(Harding, 1949). There was considerable variation in the developmental time of these eggs (the hatching period extending over 112 hours), and this can be attributed to a number of factors,

Details of the developmental time for the eggs of each female are set out in Table 13.

20 h tc Ha e

tag 10 en rc Pe

0 216 224 232 240 248 256 264 272 280 288 296 304 312 320 328

Developmental Time (Hours)

Fig. 25 Hatching Distribution - Insectary 1968 (C. rufata) -118—

Table13 — Duration of Development in an Outdoor Insectary (C. rufata)

r Po. of Eggs Mean Time (Hrs.) Coeff. ofl Female % Variation Laid Hatched Hatch +— S.E 4' 51

A 302 260 86.09 237.32 ± 1.11 7.51

B 201 153 76.12 235.29 ± 1.09 5.74

C 270 211 78.15 256.04 + 1.74 9.86

DRR 315 209 66.32 273.42 ± 1.46 7.69

mm s 224 54 24.11 293.19 + 2.02 5.06

TOTAL 1312 887 67.61 253.33 ± 1.05 12.35

3€ S.E. = Standard Error.

Collected (i.e. not reared from larva) A one way analysis of variance revealed that the developmental times of the eggs from individual females differed to a highly significant extent (p < .001). The females used in this experiment emerged on different days, and hence the mean temperature during incubation also differed. Fig. 26 represents the relationship 300 )

rs • (Hou 280 ime T

l • ta men 260 lop

e •

1•• Dev an

Me 240 • •

17 18 19 20 21

Mean Temperature (*C.)

Fig. 26 Relationship between Developmental Time and Temperature -

Insectary 1968 (C. rufata) -120—

between the mean developmental time of the eggs of each female and the mean temperature during incubation. Quite clearly there was an increase in the rate of embryonic development with rise in mean temperature over the range indicated and the fluctuations in temperature could well explain the bimodality of the data. Thus, in this experiment any intrinsic effects of the parents on the rate of development of their eggs cannot be separated from the ambient temperature effects.

The individual variation in developmental rate among the eggs of each female is represented by the coefficient of variation (Table 13), and this differs from one female to another. The value of this coefficient, which ranges from 5 - does not appear to be related to mean temperature, but may to some extent have been influenced by the number of eggs hatching.

It seems likely that variability in the development of the eggs was influenced by the parents in some way.

This has been found to be the case in the winter moth,

O. brumata (Speyer, 1938;Wiie, 1960a; Briggs, 1957).

The data also suggest that the proportion hatching increases with increase in temperature, but this is discussed in detail later (p.141). -121—

(b) The Duration of Development at Two Constant Temperatures The eggs laid by four females at 20°C. in a 16/24 hour light regime were incubated under these conditions and at very nearly 10c relative humidity. The mean developmental time was 228.57 ± 0.30 hours (Table 14. (i) and FigZW, based on 895 eggs hatching out of a total of 1200 laid. This compares with a mean developmental time at 15°C. in a 16/24 hour light regime of 435.92 ± 1.39 hours for 202 eggs hatching from 532 laid by three females at 15°0. in a 16/24 hour light regime (Table14 (ii) and Fig. 27b). -122-

Table14 - Duration of Development at Two Constant Temperatures (C. rufpta)

No. of Eggs bean Time (Hrs. Coeff. of Female 2(2 n, m Variation Laid Hatched Hatch + 0..6.

(i) 20°C.

A 225 118 52.114 230.72 ± 0.81 5.55

B 162. 141 87.04 232.40 + 0.7L1. 3.17

CRR 491 375 77.00 227.63 + 0.46 3.61

DKR 342 261 79.57 226.88 ± 0.55 3.23

TOTAL 1220 895 74.58 228.57 ± 0.30 3.86

(ii) 15°C.

ARR 172 72 41.86 429.11 + 2.26 3.85

B 235 106 45.11 /43/4.19 + 1.91 3.83

cxm 125 24 19.20 464.00 ± 4.02 3.74

TOTAL 532 202 37.97 435.92 + 1.39 4.52

S.E. - Standard Error

KRCollected (i.e. not reared from larva) -123-

a. 20°C.

200 208 216 224 232 240 248 256 264 272

Developmental Time (Hours)

b. 15°C.

I i---1 416 424 432 440 448 456 464 472 480 488 496 504 512

Developmental Time (Hours)

Fig 27 Hatching Distribution - C. rufa ta -124-

Howe (1966) states that for a reasonably accurate determination of the mean and variance, the observations should cover a minimum range of 10-20 periods. This order of accuracy has been achieved by making observations at eight hour intervals, the smallest range being ten periods at 20°C.

The hatching distributions at both temperatures are normal (as shown on probability paper), but positively skewed.

The variability of the individual eggs of each female, as represented by the coefficient of variation, is low, being only 3-5.5% over the two temperatures. (e) Embryogenesis at 20°C. (i) Timetable of Embryonic Development

Over 500 eggs were incubated at 20°C. in very nearly 100% relative humidity, and at least 50 eggs were killed and fixed each day during development. The eggs were obtained from five females, each female contributing ten eggs to each group fixed. The eggs were collected at eight hour intervals and developmental time was measured from the time of collection. The results are shown in

Table 15.

It would have been preferable to measure developmental time from the mid-point between two -125—

collections. An egg fixed on day "x" would then have been "x" days old + 4 hours. However, for practical reasons this was impossible; consequently this egg was "x" days old + 0-8 hours.

Almost all eggs reached the same stage of development after any given time; there were a few eggs which lagged behind, and even fewer which were more advanced. Slifer (1932) found an almost identical situation in M. differentialis. A timetable of develop— ment has been obtained, using the modal stage for each day of development (Table 16). -126-

Table.15 g Etab onj,c Stages reached during Development at `/C. (C. rufata).

Stage XX 5 27 XIX 52 18 XVIII 45 6 4 XVII 45 12 6 2 XVI 1 1 60 10 2 XV 47 4 1 1 1 XIV 3 XIII 4 XII 52 XI 51 X 2 50 1 IX 1 VIII 5 VII 38 VI 6 V IV III II I 3 2 1 1 1 1 2 4 17 0 1 2 3 4 5 6 8 9 10 Age in Days -127—

Table 16 — Timetable of Embryonic Develoment at 200C. (C. rufata)

Age of Embryo in Days Stage

0 I 'I VII 2 A 3 x1 4 x11 5 Xv 6 Xvi 7 XVII 8 XVIII 9 xix 10 XX (hatch)

It is at once apparent that the arbitrary stages chosen to describe the morphological development of these embryos do not represent a regular sequence with respect to time. Stages I — X, which represent the early development up to the point when segmentation is completed, are passed through rapidly in 48 hours. Likewise, the process of blastokinesis, which is represented by three stages (XIII — XV), is completed -128—

within 24. hours butween the fourth and fifth days. Subsequent work on eggs incubated at room temperature (approximately 2000.) revealed a timetable for the early stages of development (Table 17).

Table 17 — Timetable of Early Embryonic Development at 20°C. rufata)

Age of Embryo in Hours Stage 0 I 16 II 18 III 21 V 23 VI 24 VII 48 x

From the above information it has been possible to calculate the proportion of the total developmental time taken to reach each stage at 2000. (Pig. 28). The total developmental time has been taken as the mean developmental time obtained at 2000. (Table 14(i) ). Thus, approximately 10/0 of the developmental time is taken up by the formation of the germ band and its early development prior to segmentation, which is completed after 20;0 of the time. The dorsal body wall is formed after 42%, and the rapid process of X XIX XVIII XVII XVI XV

XIV Yin XIII

e XII • XI

Stag X • ic IX on VIII bry VII • Em VI ian

d V •

Me IV Ill • 11 • - •

0 20 40 60 80 100

Developmental Time (%)

Fig. 28 Relationship between Embryogenesis and Developmental Time -

20°C. (C. rufata) -130-

blastokinesis follows immediately, and is completed at the 52.514 level, The second half of embryogenesis consists of the continuous processes of differentiation and further elongation of the larva until, after 94.5% of the developmental time, it is fully pigmented and development is almost complete. (ii) Variability in Rate of Development The timetable of development described above was based on the evidence that most of the eggs developed at the same rate at 2000. However, some variation in rates of development did occur, and the nature of this was investigated by examining about 3,000 eggs incubated at 20°C., and very nearly 10056 relative humidity, which were fixed at different times during embryogenesis. Approximately 600-800 eggs were fixed on each of four days. Each group consisted of all the eggs laid by four or five females. The results are shown in Table18. The variation at different times during development cannot be compared unless the frequency distributions of stages take account of the variable duration of the different stages. Figs. 29-30 are frequency distributions indicating the variation in rate of development, the stages having been grouped where necessary so that each column represents the development -131-

Table 18 - Stage Frequency Distributions during Rmbryonic Development j9. rufata .

Stage XX 1 XIX 7 XVIII 284 XVII 1 352 XVI 4 557 46 XV 11 55 11 XIV 1 13 11 XIII 5 13 4 XII 2 815 27 11 XI 9 5 1 X 686 IX 2 VIII VII 1 VI 1 V IV III II I 80 37 65 64 2 4 6 8 Age in Days -1 3 2 - DAY 2 100-

80-

R.' 60 -

40 -

20-

Xl XII XVI XVII XVIII XIX XX

Stage

XI XII XIII► XVI XVII XVIII XIX XX

Stage

Fig. 29 Stage Distribution - 20°C. (C. rufata) -1 33- DAY 6 100

60 t..

I II. VIII. XI XII XVI XVII XVIII XIX XX

Stage

DAY 8 100

60 L. -Q 144 40

I II. VIII. XI XII XIII. XVI XVII XVIII XIX XX

Stage

Fig. 30 Stage Distribution - 20°C. (C. rufata) -1

taking place in 24 hours. Fig.29, for example, shows

that on day 2 almost all the eggs were at stages VIII -

X, but a small proportion were one day behind (i.e. stages II — VII), a slightly larger proportion were one day in advance (i.e. stage XI), and a few more were two and five days in advance. This permits a direct comparison between the distribution of stages at different times during development. However, the distributions, in their grouped form, do not have large enough ranges for satisfactory estimates of the variation to be made (Howe, 1966). A quantitative assessment of the change in variability during embryogenesis is not, therefore, of much value, but a few general points emerge. It is interesting that, although none of the stage distributions are normal, there is a trend towards normality during development (as shown on probability paper). There is very little variation in the develop— mental rate of eggs before blastolcinesis (Fig. 29). During the second half of embryogenesis variability increases (Fig. 30 ) . In fact, the frequency distribution for day 8 has a different mode (Stage XVII) from that of the smaller sample taken in the timetable experiment on the same day (Stage XVIII). -135-

The nature of the variation alters during development, the first two frequency distributions being virtually symmetrical, whereas the last two have a strong negative skew, The latter type of distribution indicates that more eggs were lagging behind the average stage than were developing more quickly. This is in agreement with the hatching distribution at the same temperature (Fig.2), which has a positive skew.. Similar results have been obtained by others working on grasshoppers (Slifer, 1 932. ; Riegert,1961). Fig.31 shows the hatching distribution at 2000. plotted as percentages of the total number of eggs which developed, and with the observations grouped into numbers hatching on each day. The histogram has been plotted in reverse, with the slowest developing eggs on the left, so that it can be compared directly with the stage distributions in the previous four figures. On the left hand side of the figure is a histogram showing the stages reached by the eggs which failed to hatch (also as percentages of the eggs which developed). Both have a negative skew. The eggs which lagged behind during the latter part of embryogenesis were equivalent to those which hatched late, and perhaps to those which failed to hatch. More detailed work is required to clarify this -136-

Stage Distribution of Hatching Distribution 100 Unhatched Eggs

60 (._ 4-41 '44 40

XVI XVII XVIII XIX i 12 11 10 9

Stage Age (Days)

Fig. 31 Incubation until Eclosion - 20°C. (C. rufata)

II. VIII. XI XII XIII. XVI XVII XVIII XIX XX

Stage

Fig. 32 Stage Distribution DAY 6 - Insectary 1968 (C. rufata) -137—

point. Amaro (1963) was fortunate enough to be able to

follow the embryonic development of A. siro by observing the change in external appearance of the eggs. He concludes that there is no constant rate of development at any one temperature, and that the rate of development of

individual eggs varies during incubation. But Howe

(1967) suggests that there may have been an unsuspected diapause early in development, which would account for the fact that all the variation occurred in the early stages.

Fig.32 shows the stage distribution for a group of eggs incubated for six days in the insectary.

Approximately 650 eggs from five females were incubated.

A comparison between this distribution and that obtained on day 6 at 20°C. (Fig.30) reveals that the median stage reached in the insectary is less than that at a constant temperature of 20°C. by an amount equivalent to one day's development at 20°C. This was presumably caused by the lower temperatures outdoors. There is less variation in the rate of development of the insectary eggs, but this may be the result of a more uniform parental age than in the experiments at 2000.

Examination of the stage frequencies for the eggs of each female at 20°C. (Appendix 1 ) shows that -138—

the eggs of some females had far more variable develop—

mental rates than others. There is a suggestion in these

data that this variation increases with the ago of the

female when mated.

(d) The Effect of Temperature on Embryonic Development

(i) The Effect of Temperature on the Rate of Development

The results of the incubation of eggs at

different temperatures have been used to construct a

curve indicating the relationship between temperature

and the rate of development in the eggs of C. rufata o over the range 15-20 C. (Fig. 33 ). The rate of develop— ment is expressed as the average percentage of develop—

ment taking place in one hour. The corresponding develop—

mental time curve is also shown. Each point represents

the mean rate of development (or mean developmental time)

for the eggs of one female. There are three such points

on each curve corresponding to 15°C., and four corres— ponding to 20°C. For the intermediate temperatures, the mean rate (or mean developmental time) for the eggs of each female in the insectary has been plotted against the mean temperature during the incubation period. The latter was calculated from daily maxima and minima.

The results show that between 15°C. and 20°C. ° 500 0.5 A vera 0 ge % D

Q.' 400 0.4 evel o pm ent a)

E per H °-o 300 0.3 our

Flo • 200 0.2 •

15 16 17 18 19 20

Mean Temperature (°C.)

Fig. 33 Relationship between Embryonic Development and Temperature (C. rufata) -140—

the rate of embryonic development increases with

increasing temperature, and that the optimum constant

temperature is probably in excess of 2000.

(ii) The Effect of Temperature on VariabiliLy in Developmental Rate

There is no evidence from the above data that

a relationship exists between temperature and variability

in the developmental rates of individual eggs between

1500. and 2000. This variation is expressed as the

coefficient of variation for the eggs of each female and for each treatment as a whole (Tables 13 and. 14).

Slight differences exist between the progeny of different females, but the coefficient of variation at the two constant temperatures is of the order of only 3-4 (with one exception of 5.5/0 at 2000.). However, in the fluctuating conditions outdoors the coefficient of variation 155..10%. Since the oviposition period for each female extended over several days, the individual eggs of each female developed under different temperature conditions. This probably accounts for the variation in developmental rates being greater than that at the two constant temperatures. -141—

(iii) The Effect of. Temperature on the Proportion ,of Eygs Hatching The effects of temperature and parental age on the proportion of eggs hatching, expressed as a percentage of the total number of eggs laid by each female, were investigated by means of a partial regression. The partial regression coefficient for the effect of parental age on the number of eggs hatching proved to be insignificantly different from zero. Pig. 34, therefore, represents the regression of number of eggs hatching against temperature. These data provide a rather poor estimate of the regression coefficient which is

8.52 ± 2.47 (Appendix 2 ). Nevertheless, a correlation coefficient of 0.74 was obtained; this would be much improved by a more accurate estimate of the regression coefficient. (e) Parental Effects on Embryonic Development

One way analyses of variance on mean develop— mental times show that there is a parental effect on the developmental time of the eggs of O. rufat.a (p < .001 at 2000. and .05 > p > .01 at 15°C. Appendix 3). This supports the evidence obtained under variable temperature conditions (p. 118). 100

Y. -93206 + 8 516X • •

80 •

• h

tc 60 Ha

e •

tag • • 40 rcen Pe

• 20 - •

0 ' 15 16 17 18 19 20

Mean Temperature ( °C)

Fig. 34 Relationship between Proportion of Eggs Hatching and Temperature -

(C. rufata ) -143—

(i) The Effect of fraternal Age on the Rate of Embryonic Development The difficulties encountered in persuading moths to copulate, resulted in females being mated at different ages (from one to eleven days old). Fig. 35 illustrates the median stage reached by the eggs of o individual females after different periods at 20 C. The median stage reached in each case has been plotted against the age of the maternal parent when she was mated. Two points emerge from this figure. Firstly, it again illustrates that there is very little variation in developmental rate before blastokinesis (i.e. in the first four days of development) and that variability increases with time in the second half of embryogenesis. (The variation being expressed here as differences in median stage reached by the progeny of different females). The second point indicated by the figure is the fall off in embryonic developmental rate with increase in maternal age at the time of mating. This becomes manifest during the second half of development. However, there is no more than an indication of this; the data do not warrant statistical treatment, and further work is needed to establish the validity of this idea. Thus, the evidence here and on p. 137 suggests that the rate of embryonic development becomes slower and more variable the older the mother is when she is mated, -144-

20°C. - C rufata a. Xi

X - • • • • • DAY 2

b. XI II

XII -• • . • DAY 4

tag XI S

c. ic XVI - on • • • bry

Em XV DAY 6 ian

d • Me XIV

d. XVIII - • •

XVII • • DAY 8

• XVI 2 4 6 8 10 12 Age of Female when Mated (Days)

Fig. 35 Relationship between Embryogenesis 8 Maternal Age -145—

(ii) The Effect of Maternal Age when mated on the Proportion of Infertile Eggs produced The proportion of infertile eggs laid (expressed as a percentage of the total number of eggs) is correlated with the age of the maternal parent when maced. Fig.36 shows the regression of these two variables at 20°C. (data taken from the experiment described on p. 130 ). The correlation coefficient of 0,7157 is very highly significant (p < .001 0 Appendix 4). The regression coefficient has a standard error of ± 0.2531. The regression line passes through the "x" axis at day 1; it is very likely that females in the field mate when one day old, since they emerge at night arid presumably mate the following night. (iii) The Effect of Maternal Age on the Proportion of Eggs Hatching At 20°C. and 150C. the proportion of the eggs laid which hatch does not decrease in the eggs laid late in the oviposition period. Only one female at each temperature oviposited for two full weeks. Table 19 shows the percentage hatch in the eggs laid during the first week of oviposition, and in those laid during the second week. Fig 36Relationshipbetween ProportionofInfertileandMaternal Age- Percentage Infer tile Eggs 25 35 20 30 15 10 5 0 20°C. (C.rufata ) • 1 23456789 101112 Y. -2.258 Age ofFemalewhen Mated(Days) + 2.188X I • • • • • I • I • • I I -147-

Table 19 The proportion of eggs hatOning when laid at different times drin the oviposition period C.

Oviposition Period: Temper- Female ature First Week Second deek No.eggs laid (A -Mite lo.eggs laid °,:k Hatch o 20 C. C 423 76 68 76 15°C. B 121 36 85 68

There was no decline in the percentage hatch at either temperature; in fact, at 15°C. the percentage hatch increased significantly in eggs laid in the second week.

(f) Summary (i) The eggs of C. rufata develop without interruption and hatch in approximately 9 days at 20°C. and 18 days at 1500. (ii) Egg viability, as shown by the proportion of eggs hatching, is positively correlated with temperature o b(-3tween 15 C. and 2000. (iii) Information on egg viability and developmental rates suggests that the optimum thermal conditions for egg development in this species are in excess of 20°C. (iv) In almost all eggs embryogenesis is a rigid process with morphologically recognisable stages

occurring at precise times during the course of develop—

Such variation as does occur consists mainly of a retardation of some eggs, and this only becomes evident after blastokinesis has been completed.

(v) The retardation of some eggs in a population is also illustrated by the positive skewness

of the hatching distribution at both 20°C. and 15°C.

(vi) Parental control of embryonic development has been indicated in two ways:

(a) There are indications that the age

of the maternal parent at the time

of mating influences the rate of

development of the progeny.

(b) There is a significant correlation

between the age of the maternal parent

at the time of mating and the number

of infertile eggs produced. -149—

(v) The Egg Development of C. lealt91.12

(a) Duration of Development under Natural Conditions Detailed work on the duration of the egg stage

under natural conditions was executed during the winter of 1968/69. However, some information was obtained

during the previous two winters from light trap records,

together with egg and larval collections. The light trap records ( p. 21 ) provided information on the occurrence of adult moths in the autumn, and hence the period during which oviposition was possible; the collection of eggs and young larvae in the spring provided an estimate of the time of hatching. Fig. 3 (p. 21 ) shows that the bulk of the adult population was on the wing during the last three weeks of October. The graph only shows the number of males caught in the light traps because comparatively few females were attracted; the peak of the female population probably occurred at some time during this period also, even though this species may be somewhat pr otandr ous. Eggs of this species, which were incubated in a greenhouse with open sides during the winter of 1966/67, -150—

did not hfftch until the following spring. Various collections of eggs were made at Silwood Park in late

January, 1967, and these confirmed that this species passes the winter in the egg stage. Collections of larvae revealed that late first instars were present during the first two weeks of April, from which it is estimated that in the field hatching took place in late March and early April.

During the spring of 1968 eggs were collected at fortnightly intervals from January 16th onwards. The main purpose of these collections was to study the development of the embryos, but information about hatching dates was also obtained. Not one of 20 eggs collected on April 9th had yet hatched, but 19 out of 20 eggs collected on April 23rd had hatched.

During the winter of 1968/69 two groups of eggs were kept on broom bushes in the field; one group had been laid by three females between October 12th and 18th, 1968, (group A), and the others had been laid between

October 30th and November 2nd, 1968, also by three females (group D). One object of this experiment was to establish the duration of development in both groups, and the other was to compare the hatching period in the field with that in a similar

-151—

experiment in an outdoor insectary. Both groups of eggs in the field hatched at approximately the same time

in the spring, between April 11th and 1,ay 2nd (Fig. 51,

p. 212 ). The mean duration of development in each

group of eggs is shown in Table 20.

Table 20 - Duration of Egg. Stage in the Field (C. legatella)

--r-' ho. of Dates of Dates of Lean Dev. Time Group Dggs Oviposition Hatching + S.E. (Days)

A 26 12th - 15th 11th April - 190.0 + 1.2 Oct. 1968 2nd May, 1569

B 147 30th Oct. — 11th Anril - 172.8 -1- 0.7 2nd Nov.1968 30th April, 1569

+B. 73 178.9 + 1.1

x S.E. = Standard Error

To summarise briefly, the eggs of C. legatella are laid in October and early November, and hatch in late

March, April, and early The duration of the egg stage, which varies from year to year according to the climatic conditions and time of oviposition, is -152—

approximately 180 days, with a range from 160-200 days.

(b) Duration of Development in an Outdoor Insectary

The temperature conditions in the outdoor

insectary approached those in the field, but certain

important differences were observed. The effect of some

of these differences is discussed later. (Thermograph

records for the duration of the work are available).

During the winter of 1966/67 109 eggs were

incubated to hatching on filter paper in petri dishes in

a greenhouse with open sides. These eggs were laid

between October 20th and 31st, 1966 and hatched between

February 5th and April 4th, 1967. record was kept

of the number of eggs laid on each day, so an approximate

estimate of the median duration of the egg stage under these conditions was calculated as the time in days between the mid—point of oviposition (i.e. October 25th ) and the day on which 50% of the total hatch occurred

(i.e. Larch 15th). Thus the figure of 141 days was obtain— ed for the median developmental time under these conditions.

In the winters of 1967/68 and 1968/69 more accurate determinations of the duration of the egg stage were obtained, and the results are shown in Table 21. -153—

The eggs were incubated in the outdoor insectary; in

'1967/68 they were kept on cotton wool moistened with an antifungal agent until early spring when they were transferred to petri dishes as before, and in 1968/69 the eggs were kept on the twigs on which they hod been laid in plastic cages. The freshly laid eggs were collected once a day, and observations on hatching were made with the same frequency. A record of the exact developmental time of each egg was obtained, and the mean duration of the egg stage was calculated for each year. -154—

Table 21 - Duration of Egg,Stage in Insectary (C. legatella)

No. of No.Eggs / Dates of Mean Dev.Time Year Females Laid Hatch Oviposition + S.E.N (Days)

1967/68 2 230 34.4 18-27 Oct. 180.3 + 4.0

1968/69 3 427 83.4 13-19 Oct. 165.1 + 1.7

1968/69 3 560 86.8 27 Oct. — 162.7 + 0.7 2 Nov.

1968/69 6 987 85.3 13 Oct. — 163.7 ± 1.2 (Pooled) 2 Nov.

S.E. = Standard Error An analysis of the possible causes of differences between field and insectary results, and between insectary results in different years is presented on pp. 203-215 . The extremely low proportion of eggs hatching in 1967/68 was probably caused by the antifungal agent (p. 78 ). (c) Embryogenesis in an Outdoor Insectary The embryonic development was studied throughout the winter months of 1967/68 and 1968/69. During the -155-

first winter eggs were killed at regular intervals up to the 140th day of development. All the eggs laid by a single female were fixed on each occasion. The difficulties with mating, previously mentioned on p. 15 9 resulted in only three of the females laying fertile eggs. Eggs were therefore incubated successfully fot 7, 56, and 112 days. The embryonic stages reached are shown in Table 22.

-156-

Table22 — Embryonic Stages reached during Development in an outdoor insectary

Stage XV 26 XIV 22 xIII 18 xi' 65 x1 15 25 x 71 11 Ix 46 3 VIII 21 VII 26 VI 10 1

1TH 2 2) IV3 III 137 1 21 II 13 I 11 1 3 7 56 112 Age in Days

Stages IV and V are recorded together because no distinction was made between them at the time of the experiment. -157—

The results show that morphogenesis does proceed during the winter, but there is not sufficient evidence to state whether this development is continuous or not. One striking feature is the considerable variation after two and four months development.

Eggs were collected from broom plants at

Silwood Park fortnightly from January 16th to April 23rd

1968; each sample of 20 eggs was killed and fixed on the day of collection, and the embryonic stages were recorded. These collections were made partly to test the usefulness of the insectary in simulating field conditions, and also to check if the development in the insectary was affected by the antifungal agent in the cotton wool pads. The frequency distribution of stages in each sample is shown in Fig. 37. On the whole there appears to be little difference between the insectary and field data. The median stage after 56 days incubation in the insectary was VII, and these eggs were killed between December 12th and 23rd. The median stage for the first field sample on January 16th was XI. The 112 day median stage was X, and these eggs were killed between February 5th and 19th.

Field samples for February 1Iith and 27th had medians Fig 37Embryonic StagesofFieldSamples -1968(C.legatella)

Percentage of Sample 40 40 20 20 60 60 80 20 60 40 40 80 20 60 80 80 0 0 0 0 Embryonic Stage 16:1:68 31:1:68 272:68 14:2:68 60 80 20 40 40 80 60 20 20 20 60 40 40 80 60 80 —158- 0

0 0 10 15H 12:3:68 N.20 23:3:68 N=20 23:4:68 9:4:68 N=20 N=20 H. Hatching N= NumberofEggs in Sample -159-

of XIV and XII respectively. The small size of the

field samples, and the fact that the eggs in the samples

could have been laid at any time during the adult season,

provided rather erratic results. The insectary eggs appear to have been retarded slightly, particularly in

the 112 day group in which there was a definite "slow"

fraction, and this may have been due to the effects of

the antifungal agent.

In the winter of 1968/69 eggs were again

incubated in the insectary and killed at regular intervals.

At least 100 eggs were killed on Qach occasion. 11

females contributed eggs to the experiment; the eggs of

each female, which were laid during the first three days

of oviposition, were distributed randomly into ten groups.

All the eggs in the experiment were laid between

October 13th and 18th, 1960. Table 23 shows the age—

stage data produced. -.160—

Table 23 — Details of Embryonic Development in an outdoor Inseetarire. legate1141-

Stage

XX 2 1 3

XIX 5 4 13 XVIII 1 4 XVII 1 34 48 39 XVI 2 14 18 7 XV 2 9 17 6 6 XIV 1 1 14 XIII 1 7 4 3 XII 1 5 14 30 10 11 6 XI 2 24 35 27 8 2 13 X 3 21 31 30 15 9 4 17 IX 11 29 5 8 3 1 1

VIII 1 11 4 6 7 2 2 1 VII 5 29 13 9 2 4 4 1

VI 4 25 22 16 9 8 6 3 1 V 7 35 24 14 24 12 6 1 1

IV 78 51 17 18 5 III 24 II

1 8 2 4 9 6 3 10 4 9 6 14 28 42 56 70 84 98 119 126 140 Age in Days -161—

The results confirm that the eggs of this species develop slowly during the winter months, but at extremely variable rates. The scale on which develop— ment has been measured consists of a set of arbitrary stages and is consequently an ordinal or ranking scale.

The appropriate statistics for this level of measurement are the median and the percentile (Siegel, 1956). The former has been used to describe the central tendency of each of the distributions, and the quartile coefficient of variation to describe the dispersion (since this is derived from the semi—interquartiIe range). Table 24 presents the median and quartile coefficient of variation for each of the distributions in Table 23. -162-

Table 24 - Summary of the Embryonic Development in an Outdoor Insectary_iC. legatelliTT

Incubation Median Approximate Quartile period in otape Median Coeff. of Days Stage Variation GO

1/4 3.9 IV 7.6 28 4.7 V 1 5.5 42 6.3 VI 19.0 56 7.4 VII 27.3 70 9.5 X 26.8 8L. 10.3 X 8.3

98 11.2 XI 10.5 119 15.5 XVI 19.1 126 16.5 XVII 5.4 140 16.5 XVII 20.1

The variation in developmental rates is extremely high, especially since the quartile coefficient of variation represents approximately two thirds of the coefficient of variation based on the standard deviation

(Spiegel, 1961). All the stage distributions from the

70th day onwards are negatively skewed (cf. C. rufata). -163-

In Fig.38 the median stage has been plotted

against the proportion of the total developmental time

in the insectary. The latter has been taken as the mean

developmental time for the eggs laid in the insectary at

the same time as the eggs in this experiment (i.e. eggs

laid between October 13th and 19th; see Table 21). Thus

the total developmental time was 165 days. Approximately

30A of the developmental time is taken up by the formation

of the germ band and its early development prior to

segmentation, which is completed after 50% of the time.

Blastokinesis is completed at the 701, level. This is a description of development under insectary conditions, and not at a constant temperature as with C. rufata. In any comparison between the two species this must be taken into account. However, the principal difference between them is the prolongation of the early stages of develop— ment in C. legatella; in relative terms the development of the embryo up to the completion of segmentation is 2.5 times slower in C. legatella than in C. rufata. Dorsal closure and blastokinesis occupy similar proportions of the developmental time in both species, and the remainder of development is somewhat condensed in C. legatella as a result of the extension of the earlier stages. The causes Fig. 38Relationshipbetween EmbryogenesisandDevelopmental Time- Median Embryonic Stage XVII Insectary 1969 (C.legatella) XIII XIV XVI VIII Xll VII X V IX IV 111 VI Xl ll V X 0 102030 • • • . 405060708090100 • Developmental Time(%) • • • • • • -165—

and implications of these differences will be set out and discussed later.

Summary of (a), (b) and (21

(i) C. legatella passes the winter in the egg stage. The eggs are laid in October and early November, and hatch in late Larch, April and early May.

(ii) The duration of the egg stage is very variable, but with a mean of approximately 180 days.

(iii) When eggs of thi species were kept in an outdoor insectary the duration of development was far more variable and, on the whole, shorter than under natural conditions.

(iv) Embryogenesis proceeds slowly during the winter, but at extremely variable rates in individual eggs.

(v) In contrast to C. rufata the early part of embryogenesis is prolonged, so that development of the embryo up to the completion of segmentation occupies approximately 50% of the total developmental time of the embryo.

(d) Embryonic Development under Controlled Conditions

During the winter of 1966/67 four experiments -166—

were conducted to assess the effect of different temperature treatments on embryonic development. In the first experiment over 400 eggs, laid by six females at

20°0. and in a 1 6/24 hour light regime, were incubated under these conditions. The results are shown in

Table 25 .

Table 25 — Duration of Development at 20°C. C. legatella)

ho. of 1 s Median Ovip. Hatching Period: Female Laid Hatched yi) Hatch to Ovip. Period Median Hatch (Ratio) (Days)

A 39 19 48.7 61 20:1 B 117 39 33.3 60 8.5:1 0 94 41 43.6 64 6.4:1 D 81 13 16.1 57 7.8:1 E 97 61 62.9 61 10.3:1

TOTAL 428 173 40.4 61 9.8:1R

'deighted means -167—

The effect of a constant high temperature was to reduce the developmental period markedly (cf. insectary or field conditions), but the proportion of eggs hatching was also reduced by approximately 50%. A similar number of eggs, laid by six females in a greenhouse with open sides, were kept outdoors until December 21st, 1966, and then transferred to 20°C. and a 16/24 hour light regime. The results are shown in Table 26.

Table 26 — Duration of Development at 20°C. after ,Period of cold outdoors (T. legatella)

No. of Eggs Median Ovip. No. Hatching to Days Period: Female Laid Hatched iv, Hatch Median Hatch at Ovip.Period Da, s 20°C. Ratio 116 41 35.4 69 10 1.7:1 91 79 86.8 70 19 1.8:1 116 40 34.5 66 16 2.0:1 19 7 36.8 62 18 3.2:1 40 14 35.0 50 19 1.2:1 80 4 5.0 52 20 0.8:1

TOTAL 462 185 40.0 67H 16 1.8:1

3€ weighted means -168-

The proportion of eggs hatching was identical

to that in the previous experiment, but the variability

in rate of development was considerably reduced; the latter is indicated by the ratio of the hatching period to the oviposition period (Tables 25 and 26).

The median stage reached after 56 days develop-

ment in the insectary in 1968/69 was VII (Table 24).

In the present experiment the eggs were outdoors for

approximately 50 days; it is probable, therefore, that most of these eggs were still in an early stage of development when placed at 200C. The median developmental time at 20°C. was only 16 days; thus, a constant temperature

of 2000. reduces the time required for embryogenesis from the beginning of segmentation to hatching from over

100 days under insectary conditions to a mere 16 days.

The reduction in variability by a factor of five (cf.

Constant 20°C.) suggests that low temperatures are more favourable to the earlier part of development than high temperatures.

In the third experiment 400 eggs, which were laid outdoors between October 20th and November 28th, 1966, were placed at -1 °C. on December 5th. After 14, 28, 42 and 56 days 100 eggs were removed and incubated -169—

at 20°C. in a 1 6/24 hour light regime. The results are shown in Table 27.

Table 27 — Duration of Development at 20 C. after treatment at —1 00. legatella)

No. of Period at Median Hatching Period Group Eggs —1°C. 7 Hatch Time at (Days) (Days) 20'C. (Days)

100 14m 73 13 41 100 28 55 14 45 100 42 58 15 34 100 56 47 14 20

K This group was accidentally placed outdoors for 14 days after treatment at —1°C. and before incubation at 20°C.

This experiment confirms that a period of cold treatment is favourable early in development. The hatching period was reduced with increase in length of treatment at —1°C.; but the proportion of eggs hatching, which in group 1 approached that found in the insectary, also decreased with increase in the length of the cold treatment, and it was very similar to that in group 4 in the previous experiment. -170—

The ability of the eggs of C. legatella to withstand very low temperatures was investigated by placing eggs at -8.7°C. and -17.3°C. for different periods. These eggs were laid between October 20th and

November 28th and were kept outdoors until the start of the cold treatment on December 5th. After treatment they were returned to the open greenhouse. The results are indicated in Table28, and clearly show that the eggs of this species can withstand very low temperatures early in development. The proportion of eggs hatching only dropped below 50A in one treatment (14 days at -17.3°C.) . It seems likely that a certain stage early in embryogenesis is able to withstand low temperatures rather better than other stages. The variation in the stages reached after any given time, and the fact that the eggs in this experiment were laid over a period of several weeks, could well account for mortality in some eggs and not others. -171—

Table 28 — The Ability of Eggs to Survive Very Low Temperatures C. legatell,a1

Title.7t Time at Group No. of Eggs -17. C. , Hatch (Days) (Days)

1 50 1 — 64 2 50 7 - 52 3 50 14 - 64 4 50 - 1 88 5 50 - 7 68 6 50 - 14 42

A series of treatments were applied to eggs of C. legatella in 1967/68 to assess the effects of various periods at 15°C. followed by various periods at 2°C. before incubation at 20°C. Net one egg hatched in any treatment and this is attributed to the effect of the antifungal agent incorporated in the substrate on which the eggs were kept (p.78).

Embryogenesis was also studied under these conditions, and some information was obtained concerning the temperature requirements of the eggs at different stages, despite the effect of the antifungal agent. -172-

0 Eggs kept at a constant temperature of 15 C.

for up to 112 days never developed beyond Stage VI, and

the median stage was between III and IV-qm. Development

proceeded to this level within seven days, and then

appears to have been halted with almost all the eggs at Stages III or IV—V. After 28 days a few eggs had developed to Stage VI, but did not proceed any further

in the next 8L. days.

Freshly laid eggs, which were kept for up to

112 days at 2°C., did not develop beyond Stage II, the stage at which the blastoderm begins to condense to form the germ band, When the period at 2°C. was preceded by 7, 14, or 28 days at 15°C. development was halted at the time when the eggs were placed at the lower temperature, and did not proceed any further even after 112 days.

When a period of 28, 56 or 112 days at 2°C. was followed by up to 14 days at 15°C., development rarely proceeded beyond Stages IV — V. However, when the freshly laid eggs were subjected to 7 or 14 days at 15°C.

No distinction was made between Stages IV and V at the time of the experiment. -173-

before treatment at 2°C., development did proceed as far as segmentation and even up to Stage XII in some eggs, when they were returned to 15°C. When the initial period at 15°C. was extended to 28 days, the results were similar to those obtained without any initial period at 15°C.

The effect of the antifungal agent on egg development was much greater in these controlled experiments than it was in the insectary and this may well have been due to the higher metabolic rates at 15°C.

When kept at this temperature the embryos did not develop beyond Stages IV — V, and this suggests an arrest of development at this point in embryogenesis. However, when the eggs were placed at 15°C. for a few days, to allow them to develop to the normal stage of arrest, a period at 2°C. facilitated further development when they were returned to 15°C. This is in agreement with the conclusions drawn from the previous year's work. These results apparently contradict those obtained in 1966/67 on one point, when eggs were incubated to hatching at

20°C. It seems likely that the complete cessation of development at 1500. was caused by the antifungal agent, and that this became effective when the embryos resumed morphological development after a period of natural arrest -174--

at Stages IV - V. The embryos which developed to some

extent after a period at 2°C. may have been in a better

physiological state to resume growth after their period at the low temperature, in which case they were able to develop some way before the inhibitory action of the antifungal agent became effective. It is unlikely that the cessation of development at 2°C. was caused by the antifungal agent, since it appears to have had little effect at the moderately low temperatures in the outdoor insectary; this arrest of development was almost certainly because the temperature was below the developmental threshold.

A detailed study of the changing temperature requirements of embryos of C. leRatella was conducted during the winter of 1968/69. 27 temperature treatments were applied to eggs of this species, and these are summarized in Table 29 -175—

Table29 - Temperature Treatments Applied to Egys of C. legatella

Details of Treatment Treatment H.T. Duration L.T. Duration HAT. Duration Ref. No. (°C.) (Days) (°C.) (Days) ( C.) (Days)

1 - - 20 28 2 - - 15 ii 3 — 60 :I 4 — 20 112 5 — — 15 It 6 — — 6 ti 7 — — 20 H 8 - - 15 H 9 — — 6 H 10 - - 7.5/ 28 4.5 11 — — 9/3 it, 12 - - 7.5/ 112 4.5 13 — — 9/3 ;i 14 — — 7.5/ H 4.5 15 — 9/3 H 1 6 20 3 9/3, 28 — 17 15 5 tf — 18 la 7 ti II — 19 it 12 12 fl _ 20 20 3 it 112 - 21 1 5 5 it it — 22 If 7 If If - n If II 23 12 - 24 20 3 it it 20 H 25 15 5 If if If H 26 If 7 It It If H 27 It 12 n n n H

H.T. = High Temperature L.T. Low Temperature H Incubation Until Hatching The experiment was divided into three sections: in the first eggs were incubated for 28 days, killed and then fixed; in the second eggs were kept for 112 days before fixation, and in the third incubation was continued until eclosion. In each section nine treatments were

applied to approximately 1,000 eggs contributed by nine females. The eggs of individual females were distributed at random to the nine treatments, with the result that each treatment was applied to at least 100 eggs.

Within each section eggs were incubated at three

constant temperatures (20°0.: 15°C.: 6°C.) and two ,o alternating temperature regimes, both with a mean of o C.

(7.5°C. for 24 hours/4.5°C. for 24 hours: 9°C. for 24 hours/3°G. for 24 hours). In the four remaining treat— ments incubation at 900./3°C. was preceded by a few days o at either 20°C. or 15°C. (3 days at 20 C.: 5, 7, or 12 days at 15C.). All eggs incubated to eclosion in the last four treatments were transferred to 20C. after 112

days at 9°C,/3°C. At all temperatures the eggs were subjected to a light regime of 10/224 hours.

The eggs used in the experiment were laid at

15°C. in a 10/24 hour light regime, collected daily, and observations on hatching were made with the same frequency. -177-

Collections were always made at the end of the scotophase

on each day; thus the eggs, which were always laid in the dark, were 0-14 hours old when collected. In those eggs

which were fixed, the embryonic stages were recorded.

The results of this experiment are summarized in Tables

30 -32 and Figs. 39 -44. Since development has been recorded on an ordinal scale of measurement (i.e. arbitrary stages), two non-parametric statistical tests have been employed to analyse the results; these are the Kruskal-Wallis

one-way analysis of variance and the Lann-Whitney U test. The former tests differences between °k" independent samples and is the most efficient nonparametric test for this purpose. The latter tests if two independent samples differ in location (central tendency) and is the most sensitive test for large samples with ordinal measurement (Siegel, 1956).

The results of incubation to eclosion have been analysed by one-way and two-way analyses of variance

on means, together with "t" tests where necessary.

Significant differences between the proportion of eggs hatching in various treatments were tested by the method for comparing two percentages based on two large samples

(Bailey, 1959). Table 30 — Embrunic Stages after 28 Days Incubation at Different Temperatures (C. legatella)

Treatment Median Stage Ve (1,)

1 IV (4.0) 9.2 2 V (4.6) 16.2 3 V (5.1) 17.8 10 VI (5.5) 21.9 11 VI (5.9) 21.4 16 VI (5.9) 13.7 17 Vii (6.6) 15.2 18 VII (6.6) 15.3 19 VII (6.9) 12.7 ,...

Vg = Quartile Coefficient of Variation -179-

Table 31 — Embryonic Stages after 112 Days Incubation at Different Temperatures (C. legatella)

Treatment Median Stage Vg (20 % Hatch

14.mm 68.3 + 0.56 11.33 78.6 5= 87.5 ± 1.1 .5 12.72 62.5 6 XII (11.8) 10.6 — 12 XV (1L..6) 13.5 — 13 XIII (13.4) 17.2 — 20 XIII (12.8) 16.0 — 21 XV (14.8) 14.2 — 22 XII (12.3) 15.5 — 23 XV (15.1) 13.6 —

Vg = Quartile Coefficient of Variation KM Figures given for these treatments refer to mean duration of development in days and coefficient of variation, V. -180-

Table 32 - Duration of Egg Development at Different Temperatures (q. legate11a),

Treatment Mean duration of VR (%) 5i; Hatch development (Days)

7 66.0 + 3.5 12.9 54.6 (69.11 8 87.7 + 2.6 15.0 75.0 9 216.2 + 1.5 4.3 34.3 14 152.7 + 1.8 7.6 40.7 15 162.5 + 2.5 13.3 66.7 (79. 24 122.6 + 0.5 3.3 79.0 (89.01 25 123.7 + 0.4 3.1 93.8 26 125.6 + 0.3 2.2 90.8 27 131.9 ± 0.5 3.3 76.6 (91 .g,

K V = Coefficient of Variation.

RAE Nos. in brackets are corrected percentages. -181-

a. 100

Median b. 60 15°C.

20 os bry

f Em 6°C.

o 40 ber m Nu d. 7.5°C./4.5°C. 40

e. 9°C. /3°C.

Stage

Fig. 39 Embryonic Stage Distributions - 28 Days (C. legatella) -182-

Median a. 60 9°C. /3°C.

b. 20°C.:9°C./3°C.

c. 15°C.(5):9°C./3°C. os 40- bry Em f o ber

d. m 15°C.(7):9°C./3°C.

Nu 40

0 e. 15°C.(12):9°C./3°C.

III IV VI VII VIII IX X

Stage

Fig 40 Embryonic Stage Distributions - 28 Days (C. legatella) -183-

1 1 1 1 1 1 1 1 1 I 1 1 1 1

d. 7.5°C. /4.5°C. 20

f. 9°C. /3°C. 201-

VII IX Xl xIa XV XVII XIX Stage

Fig 41 Embryonic Stage Distributions -112 Days (C. legatella) -184--

a. Median 9°C. / 3°C. 40

1 I I b. 20°0: 9°C. / 3°C. 40

Fa1,11 [VW I I I

15°C.(5): 9°C. /3°C.

4z) 0 . I I I LJJ d. ° 15°C.(7): 9°C. /3°C. 40

e. 15°C.(12):9°C. /3°C. 40-

VII IX XI XIII XV XVII XIX

Stage

Fig. 42 Embryonic Stage Distributions - 112Days (C. legatella) Mean a.10 20°C.

50 60 70 80 90 b. 15°C. 5

70 80 90 100 110 120 c. 15

6°C. o5 n 210 220 230 240 d. 15[ 75°C/4•5°C. 5 1 Jaj 11 130 140 150 rso 170 180 9°C. /3°C. e. 5 1.41. ra 7 • me 130 140 150 160 170 180 190 200 210 220 Developmental Time (Days)

Fig. 43 Hatching Distributions at Controlled Temperatures (C. legatella). -186-

a. 25 20°C.:9°C. /3°C.:20°C.

b. Mean 15°C.(5):9°C./3°C.:20°C.

15°C.(7):9°C./3°C.:20°C.

d. 15°C.(12):9°C./3°C.:20°C.

2 4 6 8 10 12 14 16 18 20 22 24 Developmental Time at 20°C. (Days) C. Legate Fig. 44 Hatching Distributions - Controlled Temperatures -187—

(1) The Effects of Three Constant Temperatures on E_gg Development The three constant temperatures 20°C., 1 5000 and 6°C. have significantly different effects on the rate of embryonic development as shown by Kruskal-Wallis one way analyses of variance (p < .001 after 28 and 112 days, Appendix 5 ) and a one-way analysis of variance on the mean developmental times (p < .001, Appendix 6 ). After 28 days incubation eggs at 15°C. were significantly more advanced than those at 20°C.

(p < .001 0 Appendix 7 ) and those at 6°C. were also more advanced than those at 15°C. (p < .05, Appendix 7 ). But the medians for the three treatments were all within Stages IV - V, between which there is very little morphological difference (p. 87 ). An examination of the distributions however (Fig. 39 a,b, c) shows that those at 15°0. and 600. are very different from that at 20°C. In the latter the mode (Stage IV) has a very large frequency and there is very little variation, the quartile coefficient, Vq, being 9.2%. At 15°C. and 6°C. the frequencies of Stages V and VI are greater with a consequent Vq of 16-18. Thus the proportion of eggs developing beyond Stage IV in one month decreases with an increase in temperature over the -188—

r 0 range 0 — 20°C. (Table 33); a similar trend was found with regard to Stage III, but the proportion at this stage was much smaller in all three treatments.

Table 33 - The Proportion of Eggs Developing beyond Sta e IV after 28 days incubation C. legate1165

Treatment Proportion beyond Stage IV ( ) Ref: Temp. (°C

1 20 12.5 2 15 52.7 3 6 63.3 10 7.5/4.5 66.9 11 9/3 72.3 16 20:9/3 90.5 17 15(5):9/3 90.7 18 1 5(7):9/3 92.5 19 15(12) :9/3 95.3

Visual observations have shown that freshly laid eggs develop to Stage III in approximately two days at 20°C. and in three to four days at 15°C, Development -189—

then slows down, the embryo condensing and sinking into

the yolk (Stage IV) whilst the orange pigment of the

serosa is laid down. After 28 days at 20°C. only 12.576

of the eggs had developed beyond Stage IV.

The evidence clearly indicates an arrest of

embryonic development at the time when the germ band

sinks into the yolk (Stage IV). This arrest occurs even

at temperatures which are normally favourable to

morphological development, and is therefore a physiological

state rather than a quiescence brought about by conditions

unfavourable for morphogenesis. Such an arrest of

development is termed a diapause. In the case of this

species the duration of diapause is short, since after

28 days some eggs had recommenced morphological develop—

ment at all three temperatures. However, diapause develop—

ment proceeds more quickly at low positive temperatures

than at temperatures in the region of 20°C. This is

indicated by the higher proportion of eggs developing

beyond Stage IV after 28 days at the lower temperatures,

and also by the significantly greater median stage at these

temperatures.

After 112 days incubation very different results o were obtained. At both 20°C. and 15 C. eclosion had -190—

taken place, whilst at 6°C. the eggs had reached a median stage of XII (Table 31 and Pig. 41 a, b,c). Thus, the higher temperatures were more favourable to development following the 28 day stage. Table 34 shows the proportions of eggs which had passed Stages V - XV after 112 days at the three temperatures. Development proceeded more rapidly at higher temperatures in all these stages and there is no indication of an arrest of morphological development as found in Stage IV.

Table 34 - Proportion of Eggs (M Developing beyond Each Stake after 112 days incubation (C. Iegatella)

STAGE Temperature (°C.) V - VII VIII IX X XI XII XIII XIV XV 20 100 100 100 100 100 100 100 100 100 15 100 100 100 100 98 95 94 93 92 6 87 84 83 66 61 2L 20 10 0

The mean duration of development at 2000. was 66.0 + 3.5 days and this is significantly shorter (p < .001) than the developmental time of 87.7 + 2.6 days at 15°C. 216.2 + 5.6 days was the duration of development recorded at 6°C., but this result

is probably somewhat artificial. A fault in the apparatus 0 resulted in an incubator temperature of 7-7.4 C. for

several days (indicated in Fig.43 by arrows); most of

the hatching in this treatment occurred during this period

and the next few days. ho eggs hatched prior to the

period of elevated temperature and it is questionable

whether any would have hatched if the temperature had remained at a constant 6°C. Thus, although the mean

developmental time recorded at 6°C. is significantly

longer than that at either of the other temperatures

(p < .001), the effect of a constant temperature

of 6°C. on the duration of development and eclosion may

well be even more unfavourable than has been demonstrated.

The proportion of eggs hatching at 6°C. was

only 34% and this should probably be much nearer to zero

for the same reasons as given above. The proportions

hatching at 20°C. and 15°C. are shown in Tables 31 and 32,

since the eggs intended for 112 days incubation also

hatched. At first the results in the two sections appear

to be contradictory. However, a close examination of the

original data revealed that the low figure in treatment 7

has been caused by the failure of one group of eggs. -192—

When the results of treatments 4 and 7m, and treatments

5 and 8 were pooled, the proportion hatching at both

temperatures proved to be 66%. Alternatively when the

data from treatments 7 and 8 were pooled, omitting the abnormal eggs in treatment 7, the proportion hatching

(pooled data) was 72.4A. The failure of individual groups of eggs to hatch has affected some of the results shown in Table 32; this will be mentioned where appropriate. The mean duration of development, however, o has not been affected at either 20 C. or 15°C. as can be seen by the close agreement of the results in Tables 31 and 32 )

The coefficient of variation was 11-15% at the two higher temperatures, and the low value of 4.3;6 obtained at 6°C. again indicates that eclosion occurred in response to the short period spent at 7-7.4°C.

(ii) The Effect of a Low Temperatur8 Regime on --Uubsecuent Development at 20 C.

The effect of 112 days at 9°C./3°C. between two periods at a high temperature is shown in Table 321 treatments 2L. — 27. The freshly laid eggs in those

20 C. 3EM 15°C. -193-

treatments were incubated'at a high temperature

(20°C. or 15°C.) for a few days and then at 9°C./5°C. for 112 days, after which they were placed at 20°C.

until eclosion. The coefficient of variation in these

treatments was 2-3A (cf. 11-13A at constant 2000. and o 15 C.). Thus a period of low positive temperatures

dramatically reduces the dispersion of the emergence

curve in this species (Fig.L , a-d cf. 1ig.43 a-b).

The proportion of eggs hatching is higher in these

treatments than at 20 C. or 150C. The percentages

shown for treatments 2L1 and 27 are lower than those for

treatments 25 and 26. Once again these low values have

been caused in each case by a single group of eggs;

corrected values for the proportions hatching in

treatments 24 and 27, which have been calculated

omitting the eggs concerned, are shown in brackets, and

are in close agreement with treatments 25 and 26. The

proportion of eggs hatching in these four treatments

(pooled data, corrected) is 884. (iii) The Effect of High initial Temperatures - on Embripnic Development 0 The effect of high temperatures (20 C. and

15°C.) on freshly laid eggs was investigated and a

comparison made with the results under a continuous -194—

temperature regime of 9°C./3°C. After both 28 days and

112 days incubation at 9°C./3°C. Kruskal-Wallis one-way

analyses of variance indicate a significant treatment

effect (p < .001 at 28 days; p < .01 at 112 days, Appendix 5 ).

After 28 and 112 days at 9°C./3°C. there

was no significant difference, with respect to central

tendency, between the eggs incubated continuously at

that temperature and those which were previously kept at 20cC. for three days. This is illustrated in

Tables 30 and 31 by the close agreement between the medians of treatments 11 and 16, and of treatments 13 and 20. However, after 28 days treatments 17, 18 and

19, which were kept at 15°C. for 5,7, and 12 days respectively before transference to 900./3°C., were all significantly more advanced (p < .001 in each case, Appendix 7 ) than either treatment 11 N or treatment 103/ (Fig. 40, c-e cf. a-b). There are no significant differences between treatments 17-19.

It seems likely that the above differences are functions of the extra time spent in incubation rather than specific temperature effects; for instance treatment

19 in which eggs underwent 12 days at 15°C. prior to

y°C•/3°C. 20°C. : 900./3°C. -195—

0 the 28 days at 9C./3 C., had 4% more incubation time

than treatment 11. This is supported by the results

after 112 days, but the differences between treatments

have become reduced. Treatments 21 and 22 in which

eggs underwent 5 and 7 days at 15°C. prior to the 112 o days at 9°C,/3 C. were not significantly different

from either treatment 13N or treatment 20x31. Eggs

which underwent treatment 23 (15°C. for 12 days)

were still significantly more advanced than those under treatment 13 (p < .001, Appendix 7) and treatment 20 (p < .05, Appendix 7 ). Certain significant differences also exist between the three treatments in which eggs were kept at 15C. initially.

All these differences after 112 days incubation must be viewed with caution. In each of the five treatments in question, the median develop— mental stage is one of the stages of blastokinesis.

This is a comparatively rapid process as has been shown by the incubation of eggs in an outdoor insectary

20 C. mm 0 20 C. : 9°C./3°C. -196-

(p. 162 ). Its rapidity is also shown in Pig. 42

a—e; the frequencies of Stages XIII and XIV

(blastokinesis) are very low in the eggs subjected

to all five treatments, compared with the frequencies

of the higher and lower stages. Thus, although a

median stage of XV may be significantly different

from one of XII statistically, the biological

significance is by no means certain.

The value of Vq is very similar in all five

treatments after 112 days, lying between 12.1 and 17i).

After 28 days, however, the eggs in treatment 11

(9°0./3°C. continuously) had a Vq of 21% in comparison

to 13-15 in the other treatments. Examination of

the frequency distributions (Fig. 40 , a—e) shows that

eggs in treatment 11 differ from the others in that

the frequency of Stage IV is much higher. Table 33

shows that the proportion of eggs which passed Stage

IV under treatment 11 was 72p compared with 9aA

under the other four treatments. It may well be that

the higher temperatures enabled freshly laid eggs to

develop to the point when they enter diapause more rapidly and uniformly; this would have the ultimate

effect of an earlier termination of diapause as is shown -197-

in Fig. 40, b—e, in which the frequencies of Stage IV are all relatively low.

A two way analysis of variance showed that 'here was no significant treatment effect ( p > .2, Appendix 8 ) on the mean duration of development in treatments 2L1. — 27 in which the eggs were subjected to a high temperature initially and again after 112 days at 900./30C. However, the analysis did reveal a significant parental effect, which will be discussed later. As previously stated (p.193 ), the corrected proportion of eggs hatching was very similar in each of these four treatments, as was the coefficient of variation, V. It appears that the rate of development of freshly laid eggs increases with rise in ambient temperature until they enter diapause. However, the effect of this on the total development of the eggs has been shown to be only slight and probably insignificant. Thus it may be concluded that high initial temperatures are not essential, and that low positive temperatures during the first few days of development do not have a detrimental effect. Nevertheless, the evidence suggests that the embryos -198— do not enter diapause fully until they reach Stage IV. (iv) The Effects of 2000. and 9°0./3°C. on Embryonic Development after Blastokinesis

The mean developmental time of eggs kept at 900./30C. continuously was 162.5 ± 2.9 days (Table 32, treatment 15). The weighted mean developmental time for treatments 24 — 27, in all of which the eggs were transferred to 20°C. after 112 days at 9°C./3°C., was 125.9 days. After 112 days at 9°C./3°C. the median stages reached in treatments 13 and 20 — 23 were all stages during the process of blastokinesis. Thus the effect of the higher temperature is to accelerate embryonic development after blastokinesis. This part of embryogenesis, which took approximately 40 days at 9°C./3°C., took only 7.2 days at 2000. (weighted mean of means for time spent at 20°C. in treatments 24 — 27). The proportion of eggs hatching in treatment 15 (9 0./300.) was 67i,. Once again this figure was reduced by the non—viability of one group of eggs; the corrected value is 79,,z, but this is still considerably lower than the proportion hatching (8k:;) in the treat— o ments in which the eggs were kept at 20 C. during the later part of embryogenesis. -199—

The value of V in treatment 15 (9°0./3°C.)

was 1.%, which is similar to that obtained at a constant

temperature of 2000. (13i,, treatment 7). However, in 0 , all the treatments, which included 112 days at 9 C./3-n C. followed by 20°C., V was only 2-3. Thus, the period at 900./300. reduced the dispersion of the emergence

curve at 2000. It is equally true that a temperature of 2000. during the later part of embryogenesis and during eclosion also reduces the dispersion of the emergence curve.

(v) The Effects of Different Temperature Regimes with a common mean of 6uc.

The development of the eggs of this species under three temperature regimes, each with a mean of 6 00., was investigated, and it was found that embryogenesis proceeds at these low temperatures and eclosion occurs.

There was a highly significant treatment effect at all stages of development (p < .001, Appendices 5 and 6). After both 28 and 112 days the eggs incubated in alternating temperatures (treatments 10, 12, 11, 13) were significantly more advanced than those incubated at a constant temperature of 6°C. (treatments 3,6). After 28 days treatments 3 and 10 were significantly different at the level and treatments 3 and 11 at the -200-

001% level. Neither treatments 10 and 11 nor 12 and 13 differed significantly (Table 30, Appendix 7)0

The stage frequency distributions after 112 days under the two alternating regimes are very similar

(Fig. 41 d e), and both differ from the distribution after 112 days at 6°C. (Fig. 41 c). In the latter, the frequency of Stage XII is relatively high but only 24:A of the embryos commenced reversal. In treatment 12

(7.5°0./4.5°C.) 59A passed the beginning of reversal and in treatment 13 (90C./3°C.) the proportion was 57j. After all three treatments the frequencies of Stages XIII and XIV were low, which again indicates the rapidity with which the process of reversal is accomplished. The mean durations of development under the three treatments differed from each other to a highly significant extent (p < .001). The two alternating regimes produced the shorter developmental times, 152.7 ± 24.7 days at 7.5°C./4.5°C. and 162.5 ± 2.9 days at 990./3°C. As pointed out previously (p. 191 ) the developmental time of 216.2 ± 5.6 days for a constant temperature of 6°C. is probably misleading, and it is doubtful whether any eggs should have hatched. However, if hatching had taken place at a constant temperature of 6°C. it may well have been after -201—

a longer period of incubation.

Table35 summarises the results of incubating the eggs of C. legatella under the various temperature conditions previously described.

Thus it has been established that embryo— genesis can take place at temperatures as low as 6°C.

It is not clear whether the alternating regimes accelerated diapause development, or if the acceleration occurred only in post—diapause development; both possibilities could produce a higher median stage after

28 days than a constant temperature of 6°C. An increased rate of post—diapause development did occur under alternating temperature conditions, although there was no significant difference between the two alternating regimes until eclosion. The differences between the developmental rates of eggs under alternating and constant temperature conditions suggest that the post—diapause embryo responds to the maximum temperature, even though the time spent at that temperature is short. However, the eggs require higher temperatures during the later part of develop— ment (after blastokinesis) and those subjected to 3°C. on alternate days were slowed down in comparison with -202--

Table 35 Summary of Embryonic Development under Different Temperature Conditions (C. legatellal

Temp. Days of Incubation (°C.) 0 28 56 84 112 140 168 196 224

20 IV XX

15 IV XX

6 V XII (XX)

7.5/4.5 V XIV XX

9/3 VI XIII XX

20:9/3R VI XIII XX ( 3)

15:9/3R VI XV XX ( 5)

i5:9/3R VI XII XX (7)

15:9/ 1 VII XV XX (12)

Transferred to 20°C. after 112 days at 9°C./3°C. -203—

those subjected to 4.5°C., even though the former experienced a higher maximum temperature. It is difficult to draw conclusions from the reported proportions of eggs hatching at the two alternating regimes because of the inconsistency of the results in individual groups of eggs, particularly at 7.5°C./4.5°C.

(e) Further Aspects of Embryonic Development under Natural and Insectary Conditions (i) The Effect of Temperature early in Development on the Dispersion of the Emergence Curve The results of the experiment in which eggs were kept in the field and in an insectary for the winter of 1968/69 are summarized in Table 20(p. 151 ), Table 21 (p. 154 ), and also in Figs. 45,46 and 47. Two groups of eggs were kept in the insectary, and another two groups in the field. One group in each case was laid in mid—October, and the other two groups were laid at the end of that month. The former will be referred to as the "earlyu eggs, and the latter as the "late" eggs. The most noticeable feature of the emergence curves, as shown in Fig.45 in an accumulating form, and in Figs.46 and 47as histograms, is the widely differing hatching periods. Both the insectary groups had longer hatching periods than the field groups, and the "early° 100 "tee 0000000000C°

0° • D DI 0 • D 0°C) 0.00CCP

0 -Early" 1 00020 0 80 Insectary oo • "Late" 1 0 •• 0° " 0 00 • h 0 Field •

tc 000

Ha 0o • 60 00 e

tag 0 •

en ❑ 0 rc 00 • 3

Pe 0

40 0

e 0 • iv

t 0 pc la • 0

mu • 0 cu 20 o • Ac 000 ,00° • 0.00°-

0000 • 0 cp000000000d3°° 000000"' 0

04 7 14 21 410. 7 14 21 2840. 7 14 21 2840. 7 14 21

Feb. Mar. Apr. May

Fig. 45 Hatching under Insectary and Field Conditions - 1969 (C. legatella) 8

hing 4 tc

Ha 2 r

be 0 Num

8 Female 3 6

0 im

120 130 140 150 160 170 180 190 200 210 220

Developmental Time (Days)

Fig. 46 Hatching Distribution of "Early" Eggs - Insectary 1969 (Qjeaatella) Number Hatching Fig. 47 HatchingDistribution of"Late"Eggs -Insectary 1969 10 2 4 4 6 0 6 8 8 0 2 110 I

120 I

130 I

140 I Developmental Time(Days) 150

160 ( C.

170

180

n 190 Female 6

200 -207—

insectary eggs had a much longer hatching period than the "late" insectary eggs, The two field groups hatched over a relatively short period and are plotted together in Fig. 45.

A two way analysis of variance on the standard deviations of the emergence curves of the insectary groups shows a significant difference between the "early" and

"late" eggs. (p < .05 , Appendix 9).

The block effect, representing the effect of different females, is insignificant ( p > .2, Appendix 9).

Thus the eggs laid at the end of October had a significantly shorter hatching period than those laid in mid-October. No analysis was performed on the field data becauae the numbers involved were rather small

(Table 20).

Since the optimal range of temperature during the early part of development (say, the first month) is in the region of G-1000., it was thought that the variability in the developmental rates of eggs kept in the insectary may be the result of higher temperatures during the first month of development than would normally be experienced in the field. Fig. 48 indicates the relationship between the standard 25

11 ."Early") Insectary 12 ."Late" ) 20 • F1 ="Early" ) Field F2 ."Late"

n 15 S.D.. Standard Deviation io t ibu tr is

D 10

ing • 12 h tc 0 Ha co • F1 D.

S. 5 • F2

I I I I I I 0 1 I 6 8 10 12 14 16 18 20

Hours below 10°C. (Mean Daily)

Fig. 48 Effect of Temperature during Diapause on the Standard Deviation of the Hatching

Distribution (C. leaatella) -209—

deviation of the emergence curves and the mean daily o number of hours below 10 C. during the first 28 days of

development. The insectary mean temperatures were

calculated from thermograph readings and the field mean

temperatures were calculated from the readings

obtained from thermocouples placed on broom bushes

(p. 81 ). It is clear that the eggs laid in the

insectary in mid-October experienced much higher tempera-

tures than those laid in the field at the same time, and

likewise for the "late" eggs laid at the end of October.

The "early" field eggs even experienced lower tempera-

tures than the "late" insectary eggs. The variability

in developmental rates of individual eggs increased

markedly at the higher average temperatures. This reaffirms the idea that low positive temperatures are

optimal for diapause development in the egg of

C. legatella; higher temperatures at this time result

in an erratic termination of diapause and consequently

great variability in the duration of embryonic develop-

ment. The effect of temperature early in development

on the dispersion of the emergence curve cannot be

compgred in different years, because the temperatures at the time of hatching also influence the length of the hatching period, and these differ from year to year. -210—

(ii) The Effect of Temperature on Eclosion

The final part of embryonic development and the process of eclosion proceed more rapidly and successfully at high temperatures (p. 190 ). Hatching in the insectary and under natural conditions takes place in the spring at a time when the daily mean temperature is rising. Figs.L9 and50 show the hatching of eggs kept in the insectary in 1967/68 and 1968/69 at a time when there was a rise in the daily mean tempera— ture. The latter has been calculated from the maximum and minimum for each day, as recorded on a thermograph.

The pattern of hatching was different in the two years, but in each case a high proportion of the sample hatched during a period of rising temperature. The bimodality of the emergence histograms in 1969 for the eggs of females 5 and 6 (Fig. 47) was almost certainly due to a considerable drop in temperature during the third week in April (Fig. 50). Fig.51 shows the hatching pattern in the spring of 1 969 under field conditions. This field sample hatched much later than the insectary sample in the same year (Fig. 50 ) but this again occurred when there was a considerable rise in temperature. Fig 50 ily ( Fig 49HatchinginRelationtoTemperature-Insectary1968 ily Mean Da Temp. 'C. Mean Da Temp. (T.) 15 13 11 17 19 Hatching in Relation toTemperature -Insectary1969(C. 21 21 28714 March Mach 1 I

28 i

25 5075 7 I

14 April I

April

-211-

21 I

28 I

v II 1

100 %Hatch May May 100 (C. legatella) I

- legatella) % Hatch

-212—

ture a er

Temp % Hatch ily Da n a Me

I I I I I 22 26 30 3 7 11 15 19 23 27 1 5

March April May

Fig 51 Hatching in Relation to Temperature - Field Conditions 1969

(C. legatella ) -213—

The nature of the relationship between hatching and temperature was investigated further. A partial correlation was used to measure the association between the daily number of eggs hatching in the insectary in

1969 ("late" group) and various aspects of the tempera— ture. The method employed was that of Davidson and

Andrewartha (1948). Polynomial curves were fitted to express the relationship between the logarithm of the daily number of eggs hatching, and the daily maximum and minimum temperature, and time. These curves were plotted for the period from the beginning of hatching to the peak of the emergence curve, and indicate the natural trend with time of each of the variables

(Figs.52 and53 ). The trend in log. daily emergence is adequately described by a straight line, but the trends in both maximum and minimum daily temperatures are best described by second degree polynomial curves.

The deviations from the curves were then used as the variables in a partial correlation, thus permitting the association between egg hatching and temperature to be examined free from the complication of the time factor. Six variables relating to temperature were correlated with the deviations from the egg hatching curve (y), namely the deviations from the maximum and

-214--

2.0- Y = 0644 +0.037 ei 1.6 - ) 1.2 - • • Peak of Emergence (Log. 0.8 - • • hing • tc 0.4 -• • • • •

Ha 0.0 ber m

Nu -0.8 -

0 2 4 6 8 10 12 14 16 18 20 22 24

Time (Days)

FIG. 52 Relationship between Number of Eggs Hatching and Time -

Insectary 1969 (C. legatella)

18 • Y =11.350+0-13901+0.07902 16

14 Li 12

10 8

4 • Max. Temp. 08.00-08-006MT 2 o Min. Temp. 08-00-08006MT 0 I I I I I I I I I I I I 2 4 6 8 10 12 14 16 18 20 22 24 19:3:69 10:4:69 Time (Days)

FIG.53 Relationship between Temperature and Time during Hatching Period -

Insectary 1969 (Gjegatellg) -215—

minimum temperature curves for the day on which hatching

occurred, and also for both the two days preceding hatching. The values of the variables used in the partial correlation, together with the correlation coefficients, are given in Appendix 10. There is only one significant correlation coefficient between y and a temperature variable in these data, and that positively correlates egg hatch— ing with the minimum temperature two days before the hatching date. (Correlation coefficient for x6 and y

= 0.62 : p < .01, Appendix 10 ).

These data confirm, therefore, that the final stages of embryogenesis and eclosion are more successfully completed at higher temperatures, and they suggest that one of the governing factors is the minimum temperature two days or so before eclosion. Environmental temperature appears to have little effect after this time.

(f) Parental Effects on Egg Development (i) Parental Effect on Duration of Development The form of the data obtained on the duration of egg development at 20°C. following 112 days at

9°C./3°C. facilitated a two way analysis of variance -216—

on the mean durations of development, as previously mentioned on p. 197. Whilst no significant treatment

effect was evident, the analysis did reveal a significant parental effect on the duration of the egg stage (p <

.05, Appendix 8 ). A parental effect on the duration of the egg stage is also indicated in the data obtained by incubating eggs in an outdoor insectary during the

winter of 1968/69. The mean duration of development

for the eggs of each female is shown in Table 36. A comparison of the mean developmental times of the eggs in the "early" group (females 1-3) reveals that they all differ significantly (p < .001). A similar comparison in the "late" group shows that the mean developmental time of the eggs of female 4 is significantly less < .001) than those for the eggs of the other two females, which are not significantly different from each other (.22 > p > .21). The values of the normal deviate, d, in these tests are shown in Appendix 11. -217—

Table 36 — Parental Differences in Duration of Egg Stap in Outdoor Insectary LC. legate lie)

Mean duration of eggs + S.E. (Days)

1 165.45 + 1.46 2 146.56 + 2.09 3 174.56 ± 1.58

4 157.73 + 0.56 5 165.78 + 0.89 6 164.34 ± 0.73

S.E. . Standard Error The above results indicate that the duration of the egg stage is influenced by certain parental

factors; the possible nature of some of these factors and the way in which they affect egg development are discussed below.

Three female moths, which emerged on the same day and mated the following night, were kept in an outdoor insectary. The eggs were collected each day and samples were taken to represent the eggs laid by each female over a period of nine days. Each freshly -218—

laid egg was weighed on a Cahn Gram Electrobalance to the nearest microgram, and then incubated to hatching in the insectary: (ii) Parental Effect on Egg Weight The mean weights of the samples taken from the eggs of each female are shown in Table 37.

Table 37 - Lean Egg Weights of Three Females (C. legatellai

Age of Female . L-ean weip- ht of eggs in micrograms (Days) Female 1 Female 2 Female 3 1 119 (30) 2 98 (30)N 109 (30) 117 ( 8) 3 101 (30) 114 (22) 4 - 5 93 (10) - 102 (29) 6 93 ( 5) 89 (15) 7 96 ( 3) 90 ( 7) 102 (12) 8 91 ( 9) 93 (10) 112 ( 6) 9 83 ( 8) 85 ( 8)

Grand Mean 94 + 6 (65) 99 + 9 (100 111 + 9 (107)

Number of eggs in each sample -219—

A comparison of the mean weights of all the eggs taken from each female shows that in the three groups of eggs examined there was no parental effect on mean egg weight (p = .12 — .66, Appendix 12 ). However, Table37 clearly indicates a decrease in mean egg weight during the oviposition period of each female. Regressions of egg weight against age of female in days, for the eggs of each female, have significant negative coefficients (p < .001 0 Appendix 12). Fig. 54. shows the regressions, the coefficient for female 1 being significantly less than those of females 2 and 3 (p < .001 and p < .01 respectively, Appendix 12 ). (iii) The Effect of Paternal Age on Egg Viability The proportion of the eggs laid in the insectary which hatched did not decrease in the eggs laid late in the oviposition period. Table 38 shows the proportion hatching in the eggs laid by each female during the first three days of oviposition, and in those laid from the fifth to the ninth day. The proportion hatching did not change significantly in the eggs of females 2 and 3 (p > .6 and p > .1 respectively ). However, the proportion

-220-

Female 1 110- • Y= 100.9 -1.5X

•

100 • • a •••

• • 90 • . • • •

80 a I I I I I I I I I

Female 2 120r Y = 112 8 - 3.2X

• 110 U)

uCh 10 0 0) 0) LIJ 0 le...o -▪ 90 E • .0) 80

Female 3 130 Y= 120.9 - 2.8X

120 -

110 . z . . 11 100 : : Z 90 I I I I I I I I I I 1 2 3 4 5 6 7 8 9 10

Age of Female (Days)

Fig. 54 Relationship between Egg Weight and Age of Female - Insectary 1968 (C, legatella) -221—

hatching did decrease markedly in the eggs of female I (p < .001); since this female laid a large number of infertile eggs, it is likely that either mating was not entirely successful or there was some reproductive abnormality. Table 38 — Parentaliffict gaio Viability

Days 1-3 Days 5-9 Female No. Eggs it Hatch Nb. Eggs c Hatch Incubated Incubated

1 30 63 35 9 2 60 90 40 88 3 60 92 47 98

In the experiment in which the eggs of six females were incubated in the insectary, there was also no evidence of a significant decline in egg viability during the oviposition period. Table 39(a) shows the proportion hatching in the eggs laid on the first two days of oviposition, and in those laid between the third and seventh days; these proportions are not significantly different (p > .4 ). -222—

Table 39 Egg Viability in an Outdoor Insectary (C. legatella)

Time of oviposition No. of Eggs Laid % Hatch (Days)

(a) 1 — 2 717 86 3 - 7 270 84

(b) I 193 87 5 - 7 107 82

In Table 39(b)the results refer to the eggs laid on the first day of oviposition and on days five to seven, thus leaving a gap of three days between the two groups. Again the proportions hatching are not significantly different (p > .2 ) These results are similar to those for o C. rufata at 20 C. and 15°C. (p. 147), and indicate that egg viability is not a function of egg weight, since the viability of the eggs remains constant as oviposition proceeds whilst egg weight decreases. These -223—

observations have been restricted to the first week of oviposition, and provide no evidence concerning the viability of eggs laid towards the end of the oviposition period, since this may continue for two weeks or more. However, very few eggs are laid during the latter part of the oviposition period, so that the eggs laid at the end of the first week can be regarded, in quantita— tive terms, as some of the last eggs to be laid* (iv) The Influence of Egg Weight and Time of Oviposition on the Duration of Development The small proportion of the eggs laid by female i which hatched precluded the use of data from that source. The data relating to the eggs from females 2 and 3 were used in a partial regression (for the eggs of each female) of developmental time (y) against time of oviposition (x1) and egg weight (x2); the corresponding partial regression coefficients b, and b2 are shown in Table40. -224-

Table 40 Coefficients for Partial Regression of Developmental Time against Time of Oviposition (b1) and Egg Weights (b2)

Fe— b1 +S.E . . 3 b 2 + S.E.ff p male - • p — , 1 -1.3143 ± 0.7886 >.5 -0.5947 ± 0.2141 < .01 2 —1.8069 + 0.7573 <.05 -0.1263 + 0.1876 > .05

S.E. . Standard Error The two partial regressions show conflicting characteristics. In the regression for the eggs of female 1 developmental time is shown to have decreased significantly with increase in egg weight, but the relationship with time of oviposition is insignificant. In the other regression the coefficient for developmental time against egg weight is insignificant, whereas develop— mental time did decrease significantly in those eggs laid later in the oviposition period. Since egg weight decreases significantly during oviposition, a negative value for both b and b is a contradiction. 1 2 These results are inconclusive and the experiment needs to be continued with greater replication before any firm conclusions can be made -225—

as to the way in which the duration of the egg stage is affected by the parents.

(g) Summary

(i) The eggs of C. legatella take approximately five to six months to develop and hatch under natural conditions. (ii) The mean duration of development is reduced to 87.7 ± 2.6 days at 15°C. and 66.0 ± 3.5 days at 20°C., but variability in the rate of development is increased considerably at these higher constant temperatures. (cf. C. rufata: Pig. 55 and p.140 ).

(iii) Embryogenesis is interrupted under natural conditions for a period of approximately four weeks early in development, but proceeds continuously at all other times. The interruption in embryonic development is brought about by a state of diapause. Diapause development can take place at all temperatures between at least —1 °C. and 20°C., but the rate of this development increases with fall in temperature at least between 20°C. and 600.

(iv) Post—diapause embryogenesis can proceed between at least 600. and 2000., and the rate of development increases with rise in temperature over 100

80 0 C. rufata • C. legatella

60 h tc Ha

e 40 tag Percen

0 10 20 30 40 50 60 70 80 90 100 Developmental Time (Days)

Fig. 55 Duration of Embryonic Development at 20°C. -227—

this range. 6°C. is near the threshold for develop—

ment from blastokinesis to eclosion.

(v) Egg viability is related to the

changing temperature requirements during embryogenesis.

Thus, eggs which are incubated at a low positive

temperature during early development, and at a high

temperature (say, 15°C. or 2000.) later in develop— ment, are more viable than those incubated at either a constant high or constant low temperature.

(vi) Considerable variation in develop— mental rates is evident throughout embryonic develop— ment, and this consists mainly of a retardation of development in a proportion of the eggs,

(vii) The variation in developmental rates and the dispersion of the emergence curve are influenced by the environmental temperature during the first month of development, when the embryos are diapausing. High temperatures at this time result in an extended hatching period, whereas low positive temperatures result in a more uniform development and comparatively short hatching period.

(viii) The rate of development of the eggs is influenced by the parents. Egg weight decreases during oviposition, and egg weight or time -228—

of oviposition may have some effect on developmental rates, although this is not yet certain. Egg viability, however, does not differ in eggs of differing weight or in eggs laid at different times during the oviposition period. -229-

(vi) Discussion The various aspects of the egg development of C. rufata and C. legatella can be discussed conveniently under separate headings. The External Morphogenesis of the 'Embryo The continuous process of embryogenesis has been divided up into a number of arbitrary morphological stages to provide a means of describing the progress of development under different conditions in a quantitative way. The weight of the embryo could have been a good measure of development, but the practical difficulties of dissecting and weighing thousands of embryos precluded the use of this criterion. The procedure of describing development by stages corresponding to each day of development (Slifer, 1932; Salt, 1949; Salzen, 1960; Riegert, 1961) has not been adopted here because a uniform system was required, so that comparisons could be made between the two species. The system of arbitrary stages, based on morphological characteristics easily seen in whole mounts of dechorionated eggs, facilitated a comparative study of egg development in the two species (one of which diapauses), and of the eggs of each species under different temperature conditions. The morphogenesis of these two species has been -.230-

shown to be basically similar, but certain important differences were observed. The considerable differences during the early part of embryogenesis appear to be connected with diapause at an early stage in C. legatella. The contraction of the germ band, which is very similar to that of S. triquitella (Lautenschlager, 1932), its movement into the yolk, and the formation of a deep orange pigment in the serosa, all of which occur by Stage IV in C. legatella, are in sharp contrast to the development of the embryo of C. rufata on the surface of the yolk until the completion of segneatation. It is at this time that a pale yellow serosal pigment is laid down. The general compactness of the embryo of C. legatella is another feature which distinguishes it from that of C. rufata. A similar phenomenon has been reported by Hogan (1960) in the eggs of A. commodus; he found that embryos of this species in diapause were smaller and more compact than non-diapause embryos in the same stage of morphogenesis, The difference between embryos at the same stage in the two species studied is evident during much of egg development, and not just at the time when one species is diapausing. Anderson and Wood (1968) have shown that many of the differences between embryos of different species of -231-

Lepidoptera can be explained simply by the varied shape of the egg. They refer to three basic types of egg: the upright ovoid egg in which the embryo curves in a single vertical plane as it elongates, the upright spherical egg in which coiling is more complicated, and finally the flattened egg in which the coiling becomes very complex. They make no reference to ovoid eggs which lie on one side, and which are flattened slightly; this type is common among the Geometridae (Peterson, 1962 b). However, in both species of Chesias the general orientation of the embryo relative to the long axis of the egg is remarkably similar to the orientation of the embryo in the upright ovoid type described by Anderson and Wood (1968). Consequently the main features of development in these two species are also found in P. rapae (Eastham, 1927, 1930). Even more striking is the resemblance between the development of these species and that of the winter moth as described by Gaumont (1950); this is to be expected, since both belong to the same family and their eggs are very similar in shape. Finally, a striking transformation was noticed in the embryos of both species between Stages XVII and XVIII. This takes the form of a quite sudden appearance of well defined claws and setae on the thoracic legs and of setae -232-

all over the body. A possible explanation for this could be embryonic moulting. Okada (1958) states that embryonic moulting is common among the Hemimetabola, but much rarer in the Holometabeaa. Nevertheless he reports finding it in B. mori, and states that the setae do not make their appearance until after the moult. A detailed examination of lepidopteran embryos at this point in morphogenesis could well provide some interesting information on this phenomenon. The Duration of Egg Development The basic difference between the development of the egg of C. rufata and that of C. legatella is that under natural conditions the former is completed within two weeks whereas the latter takes approximately five to six months. Since the egg of C. legatella overwinters, its long period of development is not surprising; however, even at fairly high temperatures this egg takes a very long time to develop in comparison with that of C. rufata (Fig. 55). Bigelow (1960) showed similar differences between the eggs of two species of Acheta Linnaeus, one of which diapauses whilst the other does not. The presence of a short diapause in the egg of C. legatella, and the accompanying differences in temperature requirements, account for the vast difference in the duration of development under any given temperature -233-

conditions in comparison with C. rufata. Both species, however, show a tendency towards a positive skew in the emergence curve under favourable conditions. Howe (1966) records that the distribution of developmental periods is positively skewed in all species of stored products beetles for all conditions.

Timetable and Variation in Develojmental Rates A comparative study of the embryonic development of these two species was made possible by the description of embryogenesis in terms of arbitrary stages essentially common to both species, and chosen for their morphological differences and not on a time basis. It was therefore possible to study the vast differences in developmental rates between the two species, and within each species at different temperatures. The great morphological variation found in embryos of the same age, particularly in tho.5e of C. legatella, has been recorded in other insects. Van Horn (1966 a) found variation in the embryos of A. elliotti, and as a result described embryogenesis in terms of arbitrary stages. Slifer (1932) recorded variation in embryos of M. differentialis in terms of days behind or ahead of the mean stage reached by embryos of the same age. Popov (1959) reports underdeveloped eggs of L. migratoria migratorioides present in pods containing normal eggs, these having been -234-

collected from the field. Salt (1949) established developmental timetables for three species of Melanoplus StRl using the optimum stage reached after each period of time, but noted considerable departure from the optimum. Amaro (1963) records much variation in the eggs of A. siro and even concludes that there is no constant rate of development, since slow eggs at one point in development become fast eggs, and vice versa. King (1959) illustrates that eggs laid by genetically different populations of D. melanogaster have different developmental patterns since the relative rates of development appear to shift constantly as development proceeds. The work of these two authors indicates that more attention should be paid to this aspect of insect development. In the present work lack of developmental variation was used as a criterion, together with the proportion hatching, total developmental time and embryonic stages reached after given periods of time, in assessing the effects of various temperature treatments on embryonic development. In the non-diapausing eggs of C. rufata embryogenesis adheres to a rigid timetable with little variation. The slight variation that does exist is not influenced by temperature, but is affected by parental influences. However, fluctuating temperature conditions do increase -235-

variation; Van Horn (1966 b) records that the development of embryos of A. elliotti in a greenhouse was faster and the variation was greater than in a constant temperature room, and concludes that the wider temperature and photo- period ranges in the greenhouse are likely to be responsible for the differences. In order to compare variation at different times during development, the arbitrary stages had to be grouped into units representing equal periods of time. It then became evident that the variation was similar to that found in eggs of Melanoplus bilituratus Walker ( = sanguinipes Fabricius) by Riegert (1961). He reports that there was very little variation in the eggs of the non- diapausing form at 30°C., most of the variation being the result of a few eggs developing more slowly than the rest. Slifer (1932) reports that, compared with the mean stage, the development of eggs of M. differentialis can be retarded as much as ten days, but not advanced more than two days. In C. rufata the degree of variation increases markedly after the embryos have passed through blastokinesis, and the slow eggs during development are probably synonymous with those that are late in hatching or which fail to hatch, unlike the eggs of A. Biro (Amaro, 1963). In the diapausing eggs of C. legatella development does not proceed throughout at a constant rate at any one -236-

temperature. The prolonged development occurring under natural conditions can be reduced to a mean of 66 days at 20°C., but with a coefficient of variation of 11-13%. In contrast to the eggs of C. rufata variation in developmental rates is greatly influenced by temperature. Low positive temperatures early in development, followed by high temperatures (20°C.) for the remainder of development, reduce variation by a factor of 4-5 from that at a constant high temperature (20°C,:11-13%) or constant low temperature (9°C./3°C.:13%). This point is further emphasised by keep- ing eggs in the field and insectary. Those eggs which were subjected to the lowest temperatures during the first month of development had the shortest hatching period. Thus, this species reacts to unfavourable (high) temperatures during embryonic diapause by increased variation in the rate of subsequent embryonic development. Likewise continuously unfavourable (low) temperatures during the post-diapause period also result in greater variation. Gaumont (1950) concludes that in the eggs of 0. brumata low temperatures have different effects on the developmental rate at different times during embryogenesis, and that the length of the hatching period varies according to the time when the cold is applied. The changing temperature requirements of the embryos -237-

during development made the grouping of stages to represent equal periods of time at any one temperature rather meaning- less. However, even without this transformation, there is obviously a negative skew in any stage frequency distribution from the 70th day onwards in the outdoor insectary. The 70th day of development corresponds approximately to a median stage of X (the completion of segmentation). Much variation was recorded between the progeny of different females and even within the progeny of a single female. Similar observations have been made on the overwintering eggs of 0. brumata (Gaumont, 1950; Briggs, 1957; Wylie, 1960 a). Variation in the overwintering eggs of insects is not Rn uncommon phenomenon, for instance Moore (1948) reports a wide range of embryonic stages, from the germ band to the definitive stage, in overwintering eggs of Acrididae. He also observed population differences from one year to the next, and between populations in different locations. Van Horn (1966 b) remarks that the degree of variation in embryonic development in different wild overwintering populations of eggs of A. elliotti was more than expected, especially in the spring.

Temperature Requirements The temperature requirements of the embryos of the two species are very different. Whereas the eggs of C. rufata -238--

develop satisfactottlyat constant high temperatures, those of C. legatella respond to temperature differently as development proceeds, and no one constant temperature is entirely satisfactory for all stages of egg development. The curve obtained by plotting the developmental periods of the eggs of C. rufata against temperature (Fig. 33) appears to be part of a J-shaped curve; this type of curve is typical of most insects when the developmental periods for a stage or for the whole life cycle at a series of constant temperatures are plotted against those temperatures (Howe, 1967). Also typical is the shallow sigmoidal curve obtained by plotting developmental rates against temperature (Fig. 33). Howe (1967) states that this curve is almost straight over a range of some 10°C. just below the optimum for most species, and certainly this is applicable to C. rufata between 15°C. and 20°C. Both the shape of the developmental curve and the positive correlation between the proportion of eggs hatching and temperature (between 15°C. and 20°C.) indicate that the optimum constant temperature for the egg stage in C. rufata is in excess of 20°C. The suitability of constant temperatures for the embryonic development of C. rufata does not rule out the possibility that different embryonic stages may have slightly different temperature requirements. This -239-

is a topic which could usefully be the subject of future work on insect embryonic development. The changing temperature requirements of the embryos of C. legatella during their development are probably an adaptation to the environmental conditions which they normally experience, the latter being in sharp contrast to those experienced by the embryos of C. rufata. Way (1959) showed this type of adaptation in the eggs of L. coarctata. The eggs of C. legatella are laid between the end of September and the beginning of November, a tine when the daily mean temperature is falling. Thermocouples placed on broom bushes in the form of artificial 'eggs' have indicated that throughout the winter the eggs are subjected to extremes of temperature, often low minus temperatures at night and high positive temperatures during daytime (especially in exposed sunny positions). The eggs complete their development and hatch in the spring when the daily mean temperature is rising. The eggs of C. legatella are adapted to these temperature conditions in several ways. Firstly, there is a short period of pre-diapause development which takes approximately three days at 20°C. and which can take place even at a constant temperature of 600.1 although under the latter conditions it takes much longer. There is no evidence to -240-

suggest that the intensity of the diapause is affected by the temperature during pre-diapause development within the range 6-20°C. High initial temperatures induce a more intense diapause in A. cruciata (AndravIrthal 1943), G. commodus (Browning, 1952 b) and 0. brumata (Kozhanchikov, 1950), but not in L. coarctata (Way, 1959). However, the latter author indicates that such temperatures seem to shorten the subsequent diapause, a feature not apparent in C. legatella. Continuous low positive temperatures in the region of 6°C. during pre-diapause development do not appear to be harmful, but a continuous temperature of 2°C. prevents development to the diapause stage. These temperature requirements correspond closely to the conditions which often prevail soon after the eggs are laid; temperatures may fluctuate from above 20°C. to well below freezing, but the relative time spent at the extremes is very small and the daily mean temperatures of artificial 'eggs' at this time in 1969 were in the region of 4-15°C. When the embryo attains Stage IV it ceases to develop morphogenetically and enters an obligate state of diapause. This is the stage at which some other lepidopteran embryos are known to diapause, notable examples being N. thyellina, D. undans excellens, A. )cylostearia (Umeya, 1950) and 0. brumata (Kozhanchikov, 1950). The duration of diapause is -241-

dependent on the ambient temperature; low positive temperatures, such as those to which the eggs are often subjected during October - November, facilitate a uniform termination within 28 days, whereas higher temperatures lengthen the diapause period and the subsequent resumption of morphogenetic development is a very variable process within any group of eggs, even those laid by one female. Eggs incubated at -1°C. during diapause complete diapause development rapidly so that a uniform hatch occurs when they are subsequently placed at 20°C. However, if the period at -1°C. is extended beyond approximately two weeks many eggs fail to hatch; it may be that when this temperature is applied continuously it has a detrimental effect on the eggs once diapause has been terminated, or that this temperature is simply below the favourable range for the whole of diapause development. Andrewartha (1952) and Andrewartha and Birch (1954) point out that the range of temperatures which favour diapause development varies with different species and is related to the temperatures normally occurring under natural conditions. Thus, as in many insects from temperate latitudes (lees, 1955), low positive temperatures are optimal for diapause in C. legatella. In M. disstria the optimum for diapause is 2°C. (Hodson and Weinman, 1945), in L. coarctata it is 3°C. (Way, 1959), and Lees (1955) refers to many -242-

examples of insects from temperate climates responding most readily during diapause to temperatures within the range 0-12°C., included in which are many European Lepidoptera. Diapause, then, serves as a safety mechanism prevent- ing eggs of C. legatella from developing and hatching during the late autumn. It halts the development of the embryos until the environmental temperatures fall to the normal winter levels. Thus, eggs laid early in the adult season are prevented from developing and hatching before winter if the climate is particularly mild during October. Unlike many species of insect which overwinter in temperate regions the diapause in C. legatella is not primarily a mechanism for surviving extremes of cold, since these do not normally occur in this country during late autumn; however, it has been shown that these eggs can survive short spells at temperatures down to -17°C when they are diapausing. The temperature relations described pertain to eggs of C. legatella laid by moths at Silwood Park. It is likely that eggs of the species in other parts of its geographical range have different requirements. This geographical variation has been illustrated well in 0. brumata. It has been shown that in the eastern extremity of the European range of this species quite long periods at temperatures in the region of 0°C. are required to terminate embryonic -243-

diapause successfully (Kozhanchikov, 1950). However, in Western Europe complete embryonic development can take place at quite moderate temperatures, and several authors have concluded that no true diapause exists (Grison and de Sacy, 1948; Gaumont, 1950; Briggs, 1957; Wylie, 1960 a,b) although Wiesmann (1937) suggests an obligatory arrest at the end of anatrepsis. The diapause in 0. brumata, as recorded by Kozhanchikov (1950), occurs at a morphological stage equivalent to that in C. legatella. He states that egg development in the autumn always stops at a definite stage, when the embryonic rudiment becomes enclosed in the egg membranes and the serosa beceomes coloured orange-yellow. Thus, in both species the change in colour from green to orange-yellow is completed at the same point in the morphogenetic and physiological development of the embryo, that point being the commencement of diapause. The existence of more serosal pigment in the diapausing egg of C. legatella than in the non-diapausing egg of C. rufata is paralleled in B. mori. In the latter species two types of egg are laid, heavily pigmented ones which diapause and pale coloured ona5which do not (Kogure, 1933). Lees (1953) records that the overwintering eggs of Metatetranychus (= Panonychus) ulmi (Koch) are heavily -244-

pigmented, whereas the summer form is only lightly pigmented. Yamashita and Hasegawa (1964 a,b) claim to have shown, during their work on the mode of action of the so-called diapause hormone in B. mori, that injection of the hormone into a female pupa facilitates the penetration of (among other compounds) 3-hydroxy-kynurenine into the developing eggs. This substance is the precursor of the serosal pigment (one of the ommochrome group), which is so abundant in diapausing eggs and much less abundant in non-diapausing eggs. Although no tests have been carried out on the serosal pigment .in either of the Chesias species, it may well be an ommochrome, and a mechanism similar to that in B. mori may be operating in these two species, the only difference being that there does not appear to be the facility for a change from one type of egg to the other within each species. Further investigation in this field could be rewarding. The third adaptation of the eggs of C. legatella to the field temperatures during the winter is manifest during post-diapause development. This is their ability to develop slowly at temperatures as low as 6°C. and become quiescent at lower temperatures. During the. quiescent phases the eggs are resistant to temperatures well below zero. Thus, from the end of diapause (i.e. late November - early December) periods of slow morphological development alternate with -245-

periods of dormancy. The rate of development during this time has a normal temperature relationship, but under field conditions it is kept low by the low ambient temperatures. This type of development throughout the winter months is evident in eggs of 0. brumata in Western Europe (Gaumont, 1950) and D. undans excellens (Umeya, 1946). Development beyond reversal (Stage XV onwards) requires higher temperatures and does not normally take place until the spring rise in temperatures. Thus, there is an accumu- lation during late February and March of embryos which have just completed reversal. This requirement of higher temperatures during the latter part of embryogenesis serves to synchronise hatching by providing a means for retarded eggs to catch up. Post-blastokinetic development will take place at temperatures as low as 6°C., but extremely slowly, and results in little or no hatching. Thus, under natural conditions hatching is delayed until temperatures favourable for the larvae prevail, and the broom buds are bursting to provide a plentiful supply of suitable food. There is slight evidence that the activity of the young larva prior to hatching is affected by the minimum temperature two days before hatching, and that once this activity has commenced temperature has comparatively little effect. -246-

Parental Effects on Egg Development The existence of some parental control over the embryonic development of the progeny has been established in both species. Intrinsic differences have been observed between the progeny of different females and between individual progeny of one female. Similar variations have been recorded in 0. brumata (Briggs, 1957; Wylie, 1960 a). In C. rufata the rate and variability of embryonic development are affected in this way. One parental factor which has an effect is age. The rate of embryonic development decreases with increase in the age of the maternal parent when mated, but this does not become apparent until After blastokinesis. Van Horn (1966 b) found quite the opposite age effect in A. elliotti; she found that the age of the maternal parent has a highly significant effect on the embryonic development of the progeny, but that the eggs laid by old females develop at a faster rate than those laid by young females. She points out that this is probably a survival advantage to a species in a northern environment, and this is undoubtedly the case in A. elliotti since the embryos diapause at the end of anatrepsis. There is evidence that the variation in developmental rates is greater in eggs laid by older females. The eggs laid in an outdoor insectary and incubated for six days developed far more uniformly than those at a constant 20°C. -247-

The females in the insectary were all young whereas some of those at 20°C. were much older when they laid their eggs. These older females appear to have laid more variable eggs, but a rather more careful study of this aspect is required before firm conclusions can be made. There is a positive correlation between the number of infertile eggs produced and the age of the maternal parent at the time of mating. However the proportion of eggs hatching does not vary significantly during the oviposition period of a female. The paternal age effect has not been investigated, as yet, and this is still an open question. In his work on the eggs of Malacosoma pluvialis (Dyar) Wellington (1965) showed that different types of progeny are found in different parts of each egg mass. Eggs produc- ing the most agile larvae are laid first and the least viable are laid last. He concludes that unequal partition- ing of maternal food reserves during egg production is responsible for this. However the effects of the unequal distribution of nutrients do not become apparent until after the eggs have hatched. This suggests that observations of this kind on C. rufata should be continued beyond the egg stage, since the proportion of eggs hatching is not necessarily a good indication of the viability of those offspring. Wellington (1965), for instance, found that the few sterile -248-

eggs laid in an egg mass were not particularly associated with either the first or the second halves of the mass; they were almost always in the first or last half dozen eggs laid. In a similar context Richards (1959) decided that hatching itself is of no significance in determining the effects of near threshold and sub-threshold temperatures on the egg development of 0. fasciatus. This is because of delayed lethal effects which come to light as debilities in the larvae. Laboratory and field data have indicated that there is a parental effect on the duration of embryonic development and on egg weight in C. legatella. Egg weight, which has been shown to vary significantly from one female to another, is in all probability largely under genetic control. Taber and Roberts (1963) came to the same conclusion concerning the eggs of Apis mellifera Linnaeus; the weights of eggs laid by different females were shown to vary significantly, and it was suggested that, in addition to genetic control, environmental factors contributed to the total variability. One form of variation in the eggs of C. legatella was the gradual decline in weight from the first to the last eggs laid. The rate of this decline varies significantly from female to female. Campbell (1962) found a similar -249-

decline in the spruce budwol Choristoneura Lederer. His explanation is that as more ovarian follicles are released in each ovariole, the inter-oocyte competition for egg substrate increases, and consequently there is a reduction in the quantity of substrate which consecutive oocytes can as- similate during their period of growth. He also states that a comparison of the eggs laid by two females, which initially produced the same sized eggs but which had different fecundities, will show a lower mean egg weight and greater variation for the female with the highest fecundity. Although there is no accurate data on C. legatella it is evident from a comparison of the regressions of egg weight against time for the oviposition periods of three females, that the female which had a significantly lower regression coefficient than the other two laid the least number of eggs. Turnbull (1962) found that variation in egg weight in Linyphia triangularis (Clerck) was the result of differences in the quantity of yolk incorporated in the eggs. He pre- sumes that this would have a strong bearing on the success of the emergence of the spiderlings. This evidence joins with that of Campbell (1962), Wellington (1965) and the results presented here in establishing an unequal partition- ing of food reserves during oogenesis in some insects. Campbell (1962) states that larvae of Choristoneura which -250-

hatch from small eggs die quicker than those from larger eggs when overwintering at high temperatures. However, results for both C. legatella and C. rufata do suggest that there may be no significant difference between the viability of eggs laid at different times during the oviposition period, and therefore under normal conditions egg weight may not be a significant controlling factor over viability. Van Horn (1966 b) suggests the possibility that neuroendocrine functions are indirectly involved in the maternal effects on egg development in A. elliotti through the vitellogenetic processes during the reproductive period. But the same author also states that during the course of the experiments food was always available and therefore this is unlikely to have been a factor in the differences observed. Norris (1952) reports that eggs of S. gregaria are often heavier later in the season. Van Horn (1966 b) thinks it unlikely that the quantity of yolk affects developmental rate and variation in A. elliotti, but expresses the opinion that correlative data is needed to clear this up. An attempt to relate duration of embryonic development in C. legatella to either egg weight or time of oviposition proved inconclusive. This requires further and more careful work. -251-

If the quantity of available food reserves is not affecting egg viability to a significant extent (at least within the limits observed) then the important factor may be the quality of the reserves or variations in the titre of growth stimulating (or inhibiting) substances within the egg. Van Horn (1966 b) firmly supports the latter and recent work, such as that by Gilbert and Schneiderman (1961), Fewkes (1963), 81.&fla and Williams (1966), Riddiford and Williams (1967), Masner, S16ma and Landa (1968), Novak (1969), Wellington (1969) and Riddiford (1970), indicates the importance of hormonal substances incorporated in the maturing eggs to the development of the progeny.

The Duration of Different Stages of Development The arbitrary definition of the different embryonic stages precludes a comparison of the rate of development at different times during embryogenesis. However, it is possible to compare the relative durations of each stage in different species and at different temperatures, provided that the duration of each stage is expressed as a proportion of the total developmental time and there is uniformity in the arbitrary stages used to descrfbe embryonic development in different species. Embryonic development in Orthoptera has often been described in terms of arbitrary stages based on external .252—

morphological characters. Uvarov (1966) expresses the need for a unified system of describing embryonic stages in Orthoptera and Chapman and Whitham (1968) respond to this. These authors set out a system which they relate to the various systems used by other authors. They point out that comparisons between species can be complicated by slight morphological differences, and their system therefore consists of a series of catagories describing the major developments common to all grasshopper embryos, some of which are sub- divided into stages which are not necessarily comparable in different species. Chapman and Whitham (1968) show that there is a remarkable uniformity in the development of non-diapausing grasshopper embryos. They also indicate that species which diapause late in development experience a slowing down of development prior to diapause, especially in the stages immediately preceding diapause. There is rather less information of this nature available on lepidopteran embryonic development. Gaumont (1950) describes the development of the winter moth embryo in terms of a rather limited number of arbitrary stages, and he does compare the developmental rates of individual stages at two temperatures. Sehl (1931) records some embryonic stages and their time of occurrence in E. kuehniella, and Anderson -253-

and Wood (1968) give a very full account of the embryonic development of E. postsrittana. Fig. 56 shows data for C. rufata (reproduced from Fig. 28) together with data on the embryonic development of E. kuehniella (Sehl, 1931), 0. brumata (Gaumont, 1950), H. z.ea (Presser and Rutchsky, 1957) and E. postvittana (Anderson and Wood, 1968). The embryonic stages described by these six authors have been converted into their approximate equivalents in the scheme used for C. rufata. The points for C. rufata relate to median stage, those for the other species are presumably modes. The figure clearly shows that in these five species, at least, there is considerabI uniform- ^ ity. Since these species represent four superfamilies (Pyralidoidea, Geometroidea, Noctuoidea, and Tortricoidea), the similarity in the timing of major embryonic developments is particularly striking. Anderson and Wood (1968) demon- strate conclusively that although details of the development of lepidopteran embryos may be different in the various shapes of egg occurring, the major developments are common to all. Evidence is now provided that the relative timing of the major events in embryogenesis is also a uniform feature in non-diapausing lepidopteran eggs. Further and more accurate information is required, and this needs to be gathered using criteria which enable species to be compared.

XX 1 0 • XVIII • • XVI •

XIV D ■ XII • 0 • 0 V

e • 0

tag X S

ic • C. rufata 20°C (Geom.)

n VIII o o 0. brumata 16°C. (Geom.) • w bry a E. kuehniella ? (Pyral.) Em VI ✓ • mo • H. zea 25°C. (Noct.) IV V 0 El E. postvittana 28°C. (Tortric.) so // a 0 I 1 1 1 1 1 1 1 1 1

0 10 20 30 40 50 60 70 80 90 100

Developmental Time (%)

Fig. 56 Relationship between Embryogenesis and Developmental Time (Lepidoptera) -255-

It has been difficult to make a constructive comparison between the rate of development of the individual stages in the non-diapausing eggs of C. rufata (Fig. 28) and the diapausing eggs of C. legatella (Fig. 38) since the latter were subjected to the normal fluctuating conditions of winter. Diapause at Stage IV has the effect of extending the earlier stages of development, and at high temperatures (e.g. 20°C.) eggs can still be at Stage IV after 43% of their total developmental time. Figs. 28 and 38 do indicate that the relative duration of Stage I-III inclusive is similar in both species; there is no evidence that the diapause at Stage IV in C. legatella affects the relative rate of embryogenesis up to that stage. -256-

SUMMARY

(i) Although the larvae of both C. legatella and C. rufata feed on Scotch Broom, S. scoparius, the life cycles of these two species are very different. (ii) Adults of C. legatella emerge in autumn, and lay eggs which overwinter and hatch the following spring. The larvae feed on the spring flush of growth and pupate in mid summer. There is a diapause in both egg and larval stages. (iii) Adults of C. rufata emerge in mid summer, and lay eggs which develop without interruption to produce larvae which feed on the late summer flush of growth of the broom. This species overwinters as a diapausing pupa, sometimes for two years. (iv) The adults of both species are nocturnal and are particularly active during the first few hours of darkness. However, females of C. legatella oviposit later in the night than those of C. rufata. The mating behaviour appears to be very similar in both species. (v) The larvae of both species are attacked by several parasites common to both (mainly Hymenoptera; Braconidae). (vi) A comparison of the embryonic development of both species has revealed that the morphogenesis of the embryo is essentially similar, but that the duration of development and temperature relations are quite different. -257-

(vii) The embryonic development of C. rufata is a rigid process, which is completed in approximately nine days at 20°C. with little variation. (viii) The embryonic development of C. legatella is often very variable, takes approximately five to six months under normal winter conditions, and is interrupted by a short diapause as soon as the germ band is formed. (ix) The temperature relations of the developing embryo of C. legatella have been shown to change; low positive temperatures are favourable for diapause development and higher temperatures are normally required for the successful completion of post-diapause development. (x) The variation in embryonic developmental rates in both species consists mainly of a retardation of some embryos. (xi) Parental effects on the embryonic development of the progeny have been investigated in both species. (xii) The relative duration of the different embryonic stages has been compared in the two species, and a comparison between the embryonic development of C. rufata and that of other non-diapausing lepidopteran eggs has revealed remarkable uniformity within the Order. -258.-

ACKNOWLEDGEMENTS

This work was carried out at Imperial College Field Station, Silwood Park and I would like to thank Professor O.W. Richards and Professor T.R.E. Southwood for facilities at the field station. In particular I wish to express my thanks to my supervisor, Dr. N. Waloff, for her interest and encourage- ment throughout the course of the work, and during the preparation of this thesis. I would also like to express my sincere thanks to the following: Mr. R.G. Davies for statistical advice and the use of computer programmes for some analyses. Professor M.J. Way for his constructive criticisms and interest throughout the project. The late Mr. J.W. Siddorn for his technical advice and assistance in constructing apparatus. Mr. G.E.J. Nixon for the identification of parasites. Mr. Goodson for invaluable information concerning collecting sites. Mr. D.S. Fletcher for information concerning the taxonomy and distributions of the species under study. Mrs. P. Summerhayes and Mrs. C.A. Steel for typing the manuscript. Dr. V.K. Brown for assistance with the collection of -259-

specimens, the duplication of the thesis and the preparation of the plates. Finally I wish to thank the Science Research Council for the award of a S.R.C. Studentship. -260-

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STATISTICAL APPENDICES.

1 - 4 C. rufata

5 - 12 C. legatella -273—

Appendix 1 — Embryonic Stages reached after 6 Days at 20°C. by Progeny of Different Females (0. rufata).

Stage XX XIX XVIII XVII XVI 286 195 62 14 XV 13 2 9 31 XIV 3 9 XIII 3 10 XII 1 4 22 XI 1 X IX VIII VII VI V IV III II I 4 15 46 4 6 11 Age of Female (Days) when mated -274-

Appendix 2 - Regression of Pro ortion of EITs Hatchin against Temperature rufata .

S x 217.46 S y = 733.49 S x2 3987.51 S y2 51085.48 S xy = 13690.35 12

yx 8.52 S.E. b = 2.47 (t = 3./1 for 10 df : p <.01) 0.74

x = Temperature (°C.) Y = % Hatch S = Sum N = No. of Observations byx = Regression Coefficient S.E. b . Standard Error of Regression Coefficient df Degrees of Freedom r = Correlation Coefficient -275—

Appendix 3 - One-way Analyses of Variance - Mean EgE Develop- mental Times : Different Females at 20'C. and 15°C. (C. rufata).

Partition of Variation

2000. 15°C. Source S.S. Source S.S.

Female A 19196 Female A 19367 ii B 7594 I, B 28996

TT C 25276 It C 6912 ll D 13961 Between Females 3685 Betwien Females 22580

Total 69712 Total 77855

Analysis of Variance - 20°C.

Source df S.S. M.S. F

Females 3 3685 1228 16.58*** Error 891 66027 74

Total 894 69712

S.S. = Sums of Squares M.S. = Mean Square df = Degrees of Freedom contd./ -276—

Appendix 3 contd.

Analysis of Variance - 15°C.

Source df S.S. M.S. F

Females 2 22580 1129 4.07* Error 199 55275 278

Total 201 77855

S.S. = Sums of Squares. M.S. = Mean Square. df = Degrees of Freedom. -277-

Appendix 4 — Regression of Proportion of Infertile Eggs against Age of Female when Mated (C. rufata).

S x = 103.00 S y .. 182.43 S x2 = 783.00 S 9" = 3850.22 S xy = 1480.39 S . 19

yx 2.1877 S.E. b = 0.2531 (t = 4.23 for 17 df : p< .001) r = 0.7157

x = Age of Female when Mated (Days) y = % Infertile Eggs S Sum N = No. of Observations = Regression Coefficient bYx S.E. b . Standard Error of Regression Coefficient df Degrees of Freedom r = Correlation Coefficient -278-

Appendix 5 - Kruskal - Wallis One-way Analyses of Variance - 28 and 112 days incubation (C. legatella)

Test No. Treatments df Test Criterion

1 1, 2, 3. 2 88.791*** (28 Days) 2 4, 5, 6. 2 239.204*** (112 Days) 3 11, 16, 17, 18, 19. 4 58.278*** (28 Days) 4 13, 20, 21, 22, 23. 4 15.276** (112 Days) 5 3, 10, 11 2 16.003*** (28 Days) 6 6, 12, 13. 2 38.192*** (112 Days)

df = degrees of freedom

For details of treatments consult Table 29. -279-

Appendix 6 - One-way Analysis of Variance on Mean Develop- -mental Times - Controlled Conditions (U. lepatella).

Partition of Variation

Source S.S.

Treatment 7 4233 rt 8 13829 rt 9 3116 II 14 5814 II 15 32940 Between Treatments 758424

Total 818356

Analysis of Variance

Source df S.S. M.S. F

Treatments 4 758424 189606 911*** Error 288 59932 208

Total 292 818356

df = degrees of freedom S.S. = Sums of Squares M.S. . Mean Square For details of treatments consult Table 29. -.280-

Appendix 7 - Mann - Whitney U Tests (C. legatella).

Test No. Treatments Normal Deviate

1 1 : 2 7.35*** 2 2 : 3 2.52* 3 4 : 5 1.53 4 5 : 6 11.74*** 5 3 : 10 2.40* 6 10 : 11 1.58 7 3 : 11 3.98*** 8 6 : 12 5.94*** 9 12 : 13 1.11 10 6 : 13 4.65*** 11 13 : 20 0.57 12 13 : 21 1.90 13 13 : 22 0.41 14 13 : 23 3.10** 15 20 : 21 1.24 16 20 : 22 0.83 17 20 : 23 2.43* 18 21 : 22 2.11* 19 21 : 23 1.27 20 22 : 23 3.25** 21 11 : 16 0.93 22 11 : 17 4.46*** 23 11 : 18 4.25*** 24 11 : 19 6.15*** 25 16 : 17 3.93*** 26 16 : 18 3.63*** 27 16 : 19 5.98*** 28 17 : 18 0.24 29 17 : 19 1.55 30 18 : 19 1.83

For details of treatments consult Table 29. -281-

Appendix 8 - Two-way Analysis of Variance on Mean Develop- mental Times - Treatments 24-27 (C. legatella)

Mean Developmental Times

Block (Female) Treatment I II III Total

24 7.62 20.25 5.59 33.46 25 6.86 13.57 4.33 24.77 26 7.10 4.67 5.28 17.05 27 7.60 16.00 5.82 29.41 Total 29.17 54.49 21.02 104.69

Summation Terms:- Total Term T = 1197.01 Block Term Tl = 1065.53 Treatment Term T2 = 962.90 Correction Term To = 913.28

Analysis of Variance

Source df S.S. M.S. F

Blocks 2 152.25' 76.13 5.58* Treatments 3 49.62 16.54 1.21 Error 6 81.86 13.64 Total 11 283.74

df = Degrees of Freedom S.S. = Sums of Squares M.S. = Mean Square For details of treatments consult Table 29. -282-

Appendix 9 - Two-way Analysis of Variance on Variances of Developmental Period - Insectary 1968/69 (C. legatella).

Variances

Block (Female) Treatment I II III Total

"Early" 320.58 308.48 338.34 967.40 "Late" 48.71 132.36 85.58 266.65 Total 369.29 440.84 423.92 1234.05

Summation Terms:- Total Term T = 339621 Block Term Ti = 255210 Treatment Term T2 = 335655 Correction Term To = 253812

Analysis of Variance

Source df S.S. M.S. F

Blocks 2 1398.06 699.03 0.54 Treatments 1 81842.97 81842.97 63.74* Error 2 2567.96 1283.98 Total 5 85808.99

df = Degrees of Freedom S.S. = Sums of Squares M.S. = Mean Square /ep4uoo

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Appendix 10 contd.

Partial Correlation Coefficients for Daily Egg Hatch and Different Aspects of Temperature (C. legatella).

x1 X2 x3 x4 X5 x6 Y

x:1 +1.00 +0.62 +0.21 +0.66 +0.47 -0.02 -0.08

+1.00 +0.65 +0.52 +0.70 +0.51 +0.33 x2

x3 +1.00 +0.27 +0.49 +0.69 +0.34

+1.00 +0.56 -0.12 -0.24 x4

+1.00 +0.55 +0.31 5

+1.00 +0.62 x6

+1.00 Y

Significant Correlation Coefficient = 0.42 (p = .05). F = 1.87 for 6/13 df. (p -285-

Appendix 11 - Comparison of Mean Developmental Times for Eggs of ITerent Females - Insectary 1968/69 (C. legatella).

Female E (Days) Variance N Variance t 1 165.45 320.58 150 2.14 2 146.56 308.48 71 4.34 3 174.56 338.34 135 2.51 4 157.73 48.71 158 0.31 5 165.78 132.36 166 0.80 6 164.34 85.58 162 0.53

Comparison of Means

Females Normal Deviate 1 - 2 7.42*** 2 - 3 10.70*** 1 - 3 4.23*** 4 - 5 7.65*** 5 - 6 1.25 4 - 6 7.23***

E = Mean Developmental Time. N = No. of eggs. -286-

Appendix 12 - Caparison of Mean Eggs Weights and Egg Weight/ Age Regression Coefficients for Different Females - Insectary 1968/69 (C. legatella).

Female R(mg.) Variance N S.E.X

1 0.0938 0.0025 65 0.0062 2 0.0988 0.0085 100 0.0092 3 0.1109 0.0090 107 0.0092

Comparison of Means

Females Normal Deviate

1 - 2 0.45 1 - 3 1.54 2 - 3 0.93

Comparison of Regression Coefficients

Females Normal Deviate

1 - 2 6.04*** 1 - 3 3.52*** 2 - 3 1.18 X . mean weight of eggs. S.E. = standard error.