Bas Metabolism of Caroteaoida la Pierls brassioae Is* (The large White Butterfly) ia relation to its foodplant Prassica oleracea var -ca.'oitata L* (The Cabbage)*^

A thesis siabmitted for the degree of Doctor of Philosophy ia the TMlversity of London '1 ; ' . , - 11- y ii t

by ' » Q G U j R . ' __#LJ John Stewart Edaonda Feltwell \

Eoyal Holloway College, ^ Cniversity of London

1973 ProQuest Number: 10096797

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9 underside

K H. c. LfSRAHT A J i l U (u^crij of üÀ)XurU'S Uiseds luirtfuli in. mt jcurcUtv^, auoL licu^e, ail hunvu., a u d Ukelij m.e

JL^tH è V* XX X I tV'

Sel^ame fAa>ocU 30

J - m i giifor U/fai6 ( l J 8 i ) T(ié iS/aiwrtxi Mishir^ ^ Self

~T. Beusle^j L.

“ Lge no I

Symbola used In the text 1

Abstract 2

Introduction 3 29

Materials and Methods 30 60

Carotenold analysis 30 m 37 FOodplant 38 Livestock 39 40

Carotenold studies In P.brasslcae 41 m 43

Vitamin A 44 m 46 Labelling experiments 47 33 Carotenold studies in other species of lepidoptera 55 60

Results 6 l m 131 B* oleracea 64 4# 73 P.braseicao 74 85 Feeding experiments 85 «ft 99 Vitamin A 100 ft» 107 Labelling experiments 108 ft» 121 Carotenoids in other species 122 ft» 131

Discussion 132 4ft 146

References 147 167

Appendix 168 175

Aclmowledgements 177 Symbols used in the text

ABA Abscisic acid

BDH British Drug Houses

T/C Total carotenoids

TLC Thin Layer chromatography

TFA Trifluoroacetic acid

I*U* International Units of vitamin A

0 Ova

L Larval instars, i.e. ^

PP Pharate pupa

P Pupa

A Adult/Imago

KVA Mevalonic acid

ESS External Standards Ratio cpm Counts per minute dpm Pieintegratieris per mj.nute

MGS Trade nans© of tissue solubiliser

FFO 2,5-Di plieny Icxazole

CPM 100 Scintillation Counter (Beckman)

SP 800 Spectrophotometer ( Unicam ) ABSTRACT

The difficulty of obtaining accurate and coo^arable aeasurements of the carotenold content of plant and insect specimens is discussed*

The carotenoids present in cabbage and in all stages in the life cycle of P* brassloaa were investigated; the carotenoids in the insect reflected those in the plant; male and female adults of Ptbrassieae seemed to possess similar amounts of carotenoids*

In the plant there was a considerable vairiation in the carotenoids, both in quantity and in specificity* Age of the plant and season of growth were two factors, probably of several others, which were shown to influence the carotenold content of the plant*

The variation in the plant made it difficult to assess the significance of any variation in the carotenoids present in the Insect*

Feeding experiments showed that one larva during its life assimilated 77»79fS of P-carotene, which was 57*41^ of the total ^-carotene ingested* Of the total assimillted at least 66*10ng, that is 84*97^# was , Analysis of the frass indicated that greatest assimilation of ^-carotene occurred at the end of the larval stage* labelling experiments seemed to show that carotenoids were sequestered from the haemolymph by the f^t body four hours after ingestion*

The carotenold content of several other species of lepidoptera was determined* Hie results were often based on very different samples and therefore could not give reliable information about differences in amounts of carotenold in the different species* However, they showed that carotenoids were present in all species studied* Lutein and ^-carotene, two of the major plant carotenoids were found in all species in which individual carotenoids were determined and in all but examples either lutein or ^«carotene was the principal carotenold in the insect* In species where males end females were available there was no apparent difference in the amount of carotenoids in the two sexes* Total carotenoids peroinsect was shown to be positively correlated with the dry weight of the insect*

Possible functions of carotenoids in insects are discussed, in particular as a source of vitamin A, in colouration and involvement in olfaction and toxicity*

Key wordsPleris, Carotene, Brassica. Toxicity* INTRODUCTION

Carotenoids are tetraterpenoida of aliphatic or alicyclic structure made up of isoprena units (usually eight), p-carotene consists of a central chain of alternating double bonds with two

P-ionone rings, one at each end (fig# 1)* Carotenoids are divided into two groups, the carotenes which are hydrocarbons and the xanthophylls which are oxygen containing derivatives of carotenes#

The cyclic structure ab each end of the chain of p-carotene is the p-ionone ringy an essential component for vitamin  potency# a-Kïarotene has one p-^ionone ring and one a-ionono ring and is about half as vitamin A potent q S p-^arotene.

It has been shown by Williams et al», (1967a,b) that carotenoids are synthesised in the plant along t#o distinct rou^, the a« and p-*ionone pathways (îig# 2), The stages in the biosynthesis have been reviewed by Goodwin (1971&)* Both pathways have common precursors t but diverge possibly at the neurosporene stage#

As electron donors the methyl groups can undergo substitution

(Sykes, 1970) enabling them to bind with many different molecules including proteins, fats and bile pigments#

Nomenclature of all carotenoids mentioned is in accordance with the 13 Tentative Rules laid down by the lUPAC Comcdssinn on the

Nomenclature of Organic Chemists and lUPAC lUB Commission on

Biochemical Nomenclature (Isler, 1971)* Tlie trivial names of carotenoids are used throughout# 3,6*5^6^ Diepoxy-p-carotene is referred to in the text as 5,5-Diepoxy-p«'Carotene# F ig . 1

B-CAROTENE

H^C

H3C CHo

3 '-ng

LUTEIN

% F«3

CHo C H 3 *^3^ ^^^3 HO' CM-

B-iONONE OC-IONONE

CM CH CH CH

CM Figure 2.

BTOSYT^TIIFTIC PATH'JAY FOR CAPOTENOIDS based on Valadon and Mummery, 1969#

Phytoene ^ Fhytofluene----- ^So-carotene---- —^ Neurosporene

(p~ionone pathway) (a-ionone pathway)

p-seacaroteneac< 00 •zeacarotene 1 1 y-caroteneroi ------> Eubixanthin ^-carotene i 1 B-carotene- Monoepoxy ^ -carotene 4-^jX:-carotene 1 Cryptoxanthin Flavochrome

eaxanthin ""'^'^fSSnoeiZxy p-'Carotene*"-""'^ Lutein epoxide Dark] | lAght '' Dle^xy p-carotene V Anther axanthln Mutatuchrome Chrysanthemaxanthin + DarkJ | Light Aurochrome Flavoxanthin Violaxanthin

Auroxanthin Neoxanthin

Possible interconversions ( but not generally accepted)

1# Hager and Perz (1970) 5# Blass et al.,(1939) Yamamoto et al,,(1962) Keister and Maslova (I968 ) 6 # Cholnoky et al.,(1936)

2# Costes (1965V 1968) 7# Liannen and Sorenson (1936)

5# Sapozhnikov and Bazhanova (1998) 8 . Claes (1939)

4. Krinsky (I966) Krinsky (1971) reviewed the functions of carotenoids in nature and dealt larlncipally with plant functions. Ke considered

there were three main functions, photofunctions concerning

accessory pigments and energy transport in photosynthesis, non**

photofunctions involving reproduction, protein and membrane

stabilisation and metabolic functions such as the i^mthesis of

vitamin A. Carotenoids have been noted in the pollen of entomo-

philous flowers (Brooks and Shaw, 1968)jH^ n&t in the pollen of

aneraophilous flowers. Carotenoids play an important part in the

colouring matter of flowers and in this respect they serve in

helping to attract pollinators. Carotenoids may also serve as a

possible food source for honey bees (Schuette and Bott, 1923).

Herou^t (1970) suggested that some secondary plant substances may

act as attractants for pollinators and in other cases protect

plants from animals.

Carotenoids have been suggested to be precursorscôf abscisic

acid (ABA) (Taylor, I968 )* Taylor and Burden (1970) have shown how violaxanthin could be photoxidised to a neutral inhibitor which has

a biological activity close to ABA. They suggested that xanthophylls

may be transformed to xanthoxin, a plant hormone inhibitor and then

to ABA. The effect on the animal after ingestion of ABA is unknown

(Osborne, 1973)♦ Goodwin and Mercer (I963) suggested that ABA was a

derivative of a sesquiterpenoid synthesised from mevalonic acid.

ABA is a very important compound in the plant and Wareing and

Eyback (1970) described how it is involved as a growth regulating

hormone which induces senescence, abscision, dormancy in buds

and seeds and may act as a growth inhibiting hormone, probably as

an inhibitor of nucleic acid and protein synthesis.

Comparative studies in pasture plants of domestic animals has

shown that the highest concentration of carotenoids is present in spring than at any other time of the year (Atkeson et al. 1937)*

Oxidation of carotenoids in various deciduous trees has been investigated by Goodifin (1958)# Swain (I966) has suggested t)iat there is a tendency for epoxides to be present at the end of the season.

The cabbage ’Heart* is thought to have evolved since primitive Han first cultivated cabbage plants over 2,000 years ago

(Eailey, 1927)* The migration patterns of P. brassicae have been suggested by Poor (1559) to have been brought about by the spread o| Fan and his crops across Europe from the Palaeartic region.

De (1936) investigated the ’carotene* content of five different parts of the cabbage, finding most in the outer leaves.

Carotene Content in vs/s

Outermost green leaves 39*2?

Outer green leaves 31*60

Inner green leaves 23.80

Inner white leaves 0.90

Innermost white portions 0.32

Carotenold studies in Braasica species have been carried out by Bondi and Meyer (1946) who investigated the carotenoids in the outer and inner leaves of B. oleracea var# canitatatL.j in an Asian variety^

Fak-choi, B. chinensis L. by Karris et al.,(1949) and in Field

Mustard, B. campes tris L. by Konieki et al., (1963) who identified neoxanthin, lutein, violaxanthin and two new xanthophyll^alled xanthophyll 443 and 439* The carotene content of B» oleracea var. capitata and other Brasslca species from Central American countries has been thoroughly investigated by Harris and his colleagues, a review of this literature was given by Goodwin (1952a). The carotene content was sliown to vary from 4-14o pg/g. 8

It has been known for a long time that pigments derived from the plant provide colour to lepidopterous larvae (Poulton, IS93)*

Colour in insects may be formed in three ways. First, structural colour which is an optical effect brought about by light sliining on corrugated scales such as are found on the wings of the blue

Horpho butterflies of South America. Second, the accumulation of uric acid excretory products and third, secondary plant pigments.

Many insects use some or all of these methods. Reviews on colour in animals have been made by Gott and Huxley (1940), Fox (1953) and Fox and Vevers (I96d).

As pigraentors carotenoids are very often bound to other rsole- cules, wliich in the case of carotenoproteins make the colour more stable (Cheesrnaa et al.,(1967) or when complexed with bile pigments a different colour is produced, little work has been done on the effectiveness of specific carotenoids to produce colouration in animals but Quackenbush et al.,(l963) stated that lutein, zeaxanthin and cryptoxanthin produced a golden hue in the shanks of chickens while p-corotene did not. In other experiments with chickens

KumLcky et al«,(l969) showed that lutein was a good pigmentor, violaxanthin was ineffective and neoxanthin was G;? as effective as lutein. Other work on pigmentatlon:in fowls has been done by

Livingstone et al.,(1969)»

Towards the end of the ninteenth century a lot of work was done on the colouring matter of insect tissues and the colour polymorphisms found in various insects. The first account in relation to P. broscicr?e was by Wood (I867 ) who suggested that the newly exposed skin of the pupa was ‘photographically sensitive* to light for a few hours after ecdysis, and the final colour was then determined. Ik)ulton (1887 a) was the first to investigate the pigments responsible for the colour in P. brassicae. He believed that xanthophylls were present ia the ova and the subcutaneouG tissuGs of P» brasoicae and t M t chlorophyll ‘or some modification of it* passed through the digestive tract and gave colour to the haemolymph. The green colour of phytophagous larvae was thought to be due to blue bile pigments and yellow carotenoids (Junge, I9M ; Goodwin, 1951)* producing a Tyndall effect. However this is not believed to be the case by Villaume (I968 ) who advocates that it is the bile pigments which produce the green colour. Csanai (1966) found p-chlorophyll, bile pigment and xanthophyll in the cuticle of the butterflies,

Hestina japonica Felder and Felder and Sasakia charonda Hewitson

(F. Nyraphalidae). Gc^rould (1921) noted the presence of lutein in the green pupal colour form of Pieris ranae crucivora Boisduval

(an outsize form of the Small White, Pleris rapae L. occurring mainly in Jaj>an (Seitz, 1929) und regarded as a true P. rapae, not a closely-related species [de Worm s , 1972]). The bile pigment biliverdln has been identified in P. brassicae by Welland and

Tartter (1940), Lemberg and Legge (1949) and Pwdiger (I968 , I969)*

Biliverdln may impart a green colour in human bile while its form reduced/bilirubin may give a yellow colour (Hawk, 1954)#

*Xanthophyll* was used as a widely descriptive term in early works and included not only xanthophylls but also the carotenes.

Lutein was often referred to as xanthophyll. Foulton (1887a) investigated the development of colour in the pupae of F« brassicae (1867 ) disproved Wood*o/thecry and suggested the wandering stage was the important one in determining the final colour. Foulton (loc. cit.) differentiated between species in which colour production wag dependent àh, light and those where light was not necessary. He

(1887 b) showed tiiut reflected light, particularly black gave greatest pigmentation even when the larvae were blinded. io

Hopkins (1895 ) Investigated the pigmenta of the Pieridae, but made no mention of the carotenoids* Both Steche (1912) and

Geyer (1913) noted sexual differences in larval haemolymph of

P.brassicae* Steche stated that the female contained slightly modified chlorophyll only, whereas the male haemolymph had _ xanthophylls only, the chlorophylls having been already degraded*

î^ ken (1916) noted the pigment distribution in the pupal cuticle of P.bra-sicae , the black pigment in the chi tin, the white pigment in the hypodermis and the green pigment in the deeper tissues*

Brecher (1938) investigated the effect of coloured lights on pigment, particularly melanin, synthesis in P.bressicae larvae and demonstrated that in the late I5 larvae light acts through the eye and central nervous system; yellow light giving weak black pigmentation and blue light giving strong black pigmentation*

Brown pigmentation la P.brasslcae has been shown by ligaturing experiments to be under the control of a pigment promoting hormone secreted from the anterior end of the larva (Oltmer, 1968 ).

Paillot and Noel (1926, 1928) stated that melanin and carotenoids are stored in the hypodermal cells of the cuticle of P.braisicae* Meyer (1930) identified carotene in the haemolymph of P»brassicae and stated that the sexual difference in haemolymph colour was due to the presence or absence of an aldehyde or other compound* Steche (1912) could not determine whether carotenes were synthesised in the larva or were of plant origin, but he showed chemical that the carotene of the larva had similar/properties to carotene from Carrots* I^derer (1933) also noted the presence of carotenoids ia P*brassicae* Kanunta (l94l) identified p-carotane and taraxanthin 11

(lutein epoxide; Egger,' I568 ) in the haemolymph# He also showed

(1S’42) that the hymenopterous parasite Microgaster conrlomerattis contained the same carotenoids as its host, the larva of P. brassicae #

When Van Der Geest (1963) looked at the proteins of

P. bressicae he identified ^-carotene and lutein as the prosthetic groups of the yellow chromoprotein sequestered by the fat body from the haemolymph. Chippendale and Kilby (I969) showed that the two ma’jor proteins from the haemolymph of P. brassicae were

Sequestered by the fat body and that one of these was a chromo­ protein, probably involving a carotenoid (Chippendale, 19?1)*

Feltwell and Valadon (1972) identified thirteen carotenoids in

F» brassicae.

Investigations into the utilisation of food and energetics of

P. brassicae have been carried out by Chlodny (196?), I Vans

(I95S, 1939, 1940), Johansson (1951)* Boeder (1953), Aichtar (I966) and David and Gardiner (1962); into the effect of population density and gregariousness by Symczah (1950), Long (1953, 1955),

Zaher (1957); into growth and developnent by Jones (196O), Burgerjon

(1962), Dusaussoy end Belplanque (1964), into oogenesis by ?:lein

(1932), Kaiser (1949), Karlingsky (I962), Benz (1970), and into mating behaviour, ovipositing and orientation by Hertz (192?).

Kobayashi (1957) Investigated the egg laying habits of P. rapae.

Excellent reviews on insect nutrition have been made by House (I96I),

Waldbauer (I968 ) and Dadd (1973).

Carotenoid studies in other members of the Fieridae have been made by Gerould (1921) on the Roadside Sulphur/Clouded Sulphur

Colias philo dice Go dart; Hackman (1952) on P. rapae and Ohtalii end CImisM (196?) on P. r a m e c rue Ivor a. Gerould found that the green form of Colias did not absorb dietary carotenoids but yet it laid yellow ova. Hackman identified p-carotene and lutein in the haemolymph of P. rapae and Ohtaki and 12

Chûishi (1967 ) identified lutein in Jh© green pupa of P. rapae crucivora. The Saturn!da© (Silkmoths) have been studied intensively because of their previous economic importance, and considerable information about carotenoid metabolism can be derived from the literature. It has been shown that dietary carotenoids are absorbed by the gut and appear in the haemolymph (Manunta, 1935»

Cmelik, 1969); in the silk glands and cocoons (Cku, I93O; Uda,

1919; Kanunta, 194?» Jucci, 1949; Karizuka, 1953), ®nd carotenoid differences Îîave been studied in the sexes (Yoshida, 1955) # Food energetics and nutritional studies ia Bombyx mori L. have been carried out by Iliratsuka (1920), Legay (1958a,b) and Ito (1962).

Carotenoids may be modified when they enter the animal body

(Thommen, 1971)* Goodwin (1952b) showed how g-carotene was converted to astaxanthin in the ova of the locust and Leuenberger and Thommen

(1970 ) have shown how the adults of the Colorado Beetle,

Teptlnot^rra decerrlineeta Say accumulated the keto-carotenoids, echinenone and canthaxantliin, which are converted from carotenes and possibly from xanthophylls. Carotenoid identifications in the class

Insecta are very numerous and are listed in reviews by Goodwin

(1952a,b; 1971).

Davies (1964) mentioned that in the Fieridae the major pigment class was the pteridines. île considered the carotenoid content to be too small to be worth looking at.

In the adults of P. brassicae the pigments of the predominantly white wings consist of pteridines (Kakino et al., 1952). Hnrmsen

(1966 a, b, 1969) found tliat the pupa of P. brassicae sjmthesiæd some of these pigments and that the pteridines were concerned as eye pigments. Synthesis of pteridines has been demonstrated in the 13 wings and ovaries of P. brassicae (Lafont et al, 1971; lafont,

1972; Lafont and Papillon, 1972 and Kabos, 1967)# Electron microscope investigations of the pigment contained within the scales has carried out by Tagi (1954), Sellier, M, (1971) and Sellier, E.

(1971 )# Sellier, M. (1971) suggested that the yellow pigment contained within the scale might be xanthopterin# Mayer (I896 ) studied the development of lepidopteran wing scales and observed in P# rapae that when the protoplasm recorded from the scales, yellow liaemolymph flowed in. He noted that in some moths

^Xanthophylls” were present in the Imemolymph.

Wing patterns have _>been recognised as a means of sexual recognition in P. rapae crucivora by Obara and Hî-âaka (1968 ) and

Cbara and Hidaka (1964). Cbara (1970) stated that the ultra-violet light reflected off the wings at 380-400 nm is the specific region to which the males resi^ond. The female reflect 30-40^ u V while the males only reflect about 5#. Thompson et al.,(1972) have sugges­ ted that the flavonols are responsible for the demarcation of ultra violet petal patterns visible end relevant to insects. The principal maxima of flavonols are 350-390 nm and 250-270 nm.

There are, however, some carotenoids which absorb within this wavelength and they also may be implicated. Fishvâck (I962) who investigated thé major oxidation products of p-csrotene found that all of the products absorbed only in the ultra-violet. Wing patterns in the Fieridae have been investigated by Nekrutenko

(1964) and Kazokhin-Porshniakov (1957)#

The legs, antennae and maxillae have been shown to be chemoreceptor sites in members of the Fieridae. The legs of

F. rapae are receptive to sugars (Anderson, 1932, Fox, I966); the antennae, maxillae and oral areas of P. brassicae larvae have 14

been shown to be sensitive to benzaldehyde (Dethier, 1941). Ke

found that there was no response to this chemical after maxillectomy.

Waldbawer and Fraenkel (1961) found that larvae of the Tobacco

Eomworm (Kan due a sexta Johan.) (F. Sphingidae) fed on plants

normally rejected after maxillectomy. Schoonhoven (1969a,b)

demonstrated by ingenious experiments how the sensilla on the

maxillae of P. brassicae larvae are sensitive not only to mustard oil glycosides (KOG) and amino acids (1970) but also to anthocyanins,

a class of secondary plant substances. The significance of

Schoonhoven*8 work is that aa anthocyanins can be detected, other

substances such as carotenoids may well be detected by the same

manner* The MOG in cabbage have been recognised since 1910 by

Verschaffelt as feeding stimulants for P. brassicae. HOG occur in

cabbage plants (Schultz and Gmelin, 1954) but particularly in the roots, stem and heads (Josefsson, 196?)# David and Gardiner (1966 a,b) and Wei-Chun (I969) experimented with lîOG, including einigrin and found that these could be used as feeding stimulants in semi­

synthetic diets for P. brassicae.

Carotenoids are one of scveral^tlasses of plant pigments

Including clilorophylls, anthocyanins and flavones which are taken

in by most photophagous larvae in their food. Plant substances are

divided into two categories with respect to the animal, namely

primary and secondary. The former include the essential proteins,

fats and carbohydrates. Carotenoids and other jpLant pigments

are in the second category. Fraenkel (1959) stated that "primary”

substances are found in all living material, and that the role

of secondary plant substances in the metabolism of plants "lias not been satisfactorily explained”. 15

It has been regarded by many workers, including Beck (1956), that plants, during their co-development with insects, have become unpalatable to the insect by the presence of deterrent substances, or inedible by such physical means as hardened cuticles or fast flowing sap (Southwood, 1972). The absence of certain important amino-acids from the proteins of some plants (Boyd, 1970) has been suggested as a means by which plants have evolved unacceptability to larvae. In a study on the sueceptibility and resistance of 40 varieties of cabbage to P. rapae Benepal and Eall (1967 ) noted that susceptible varieties had greater amounts of total free amino acid and total nitrogen. Allen and Selman (1957) noted that the absence of elth^ nitrogen, potassium, phosphorus or iron from the diet of P. brassicae caused a severe check on growth.

Fraenkel (1959) discussed the raison d ’etre of secondary plant substances and suggested that insect food specificity was based on the presence or absence of secondary plant substances.

Schoohoven (1969b) stated that monophagous and polyphagous larvae detected their foodplants on the basis of a subtle combination of common plant substances, fraenkel (I969) opposed Schoonhoven*s idea because of the enormous quantitative clianges that can take place with secondary plant substances in plants grown under different conditions. Of the 30-40 secondary plant substances available for insects, many combinations and proportions are possible. An excellent paper which demonstrates the Co-development of animals with plants is that by Watanabe (1958 ) who showed that young larvae of B. morj are first attracted to p-y-hexonol in their foodplant and then when they are older they are attracted to a-p- hexenol. Another example was given by Walls (1942). He has sliown 16 that females of P. brassicae are first attracted to blue, purple and yellow colours and as a result find flowers from which to feed» When they are mature they are attracted to green, green-blue colours and so find leaves on which to oviposit.

Goodwin (1952a) believe^ that carotenoids may exist in the insect body either in a free or modified state* He believes that carotenoids in some insects are a reflection of what pigments are in the food-plant because many carotenoids appear to be present in eruch a wide range of insects (Goodwin, 1972). No one particular carotenoid is found in the insect body suggesting that no one pigment is specifically concentrated. Genetic control over carotenoid absorption from the gut was believed by Legay (1958a) who suggested that three genes were responsible in B. mori. In the blue variety of C. philodice Gerould (1921) thought that there was a recessive factor responsible for either the prevention of absorption of carotenoids or their destruction once assimilated.

Xanthophylls he suggested were broken down or decolourised by a specific enzyme.

Watt (1969) considered that the seasonal light and &irk forms of the Alfalfa/Grange Sulphur butterfly Collas eurytheme Boisduval had been evolved to maximise solar heating in cold seasons and minimise overheating in warm seasons. The differences in colour were attributed to melanin. Hansen and Kerivee (1971) found that the critical temperature below which pupae of P. brassicae were killed was -22i3^C in Russia. However, the local climate ia c Estonia (in /r^ic circle) was more severe than this and they suggested that factors other than cold hardiness must explain the winter resistance of P. brassicae. Danilevî^ii (19&5) has also noted that P. brassicae occurs every year north of Leningrad where winter temperatures always drop to -36^0. He stated that it 17

is not known whether P. brassicae is issintained by migrations from

the south or whether the pupae diapause over winter. Other

workers have investigated the cold-liardiness of P# brgssicae pupae

in the aj^ic circle and have suggested that the critical temperature

below which the pupa dies is between -22*3^ and -33^C (Kozhanchikov,

1936; Somme, 1567 ).

Some kind of p^otosensitizcr in insects cuticles responsible

for the effects of day length activities have been looked for by

somo workers and carotenoids have been suspected (Lees, 1972).

Barbier et al.. (1979) suggested that bile pigments may be involved

in photoreception in P. brassicae larvae, but Fuseau-Braesch (1972)

suggested that the other pigments contained by the larvae should be

investigated. Claret (I966), however stated that in diapausing

P. brassicae pupa© light penetrates the integument and has effect

dii'ectly upon the brain, causing release of a hormone*

Vitamin A has been reported by Koore (1957) and Goo divin (1963a)

not to be present in plants, however, it has been reported in

plants by Todhunter (l939)* in the mulberry as provitamin A by

Yoshida (1945) and in grasses by Winterstein et al. (I96O) andWinterstein and /Eegedus (I96O).

Vitamin studies on the members of the genus Brassica have been

centred on the nutritive value of these plants as food sources.

Davis and Stillman (1926) tested the juice of winter and summer

cabbage on children and found thatonly summer juice supported growth.

Collison et al.(1929) tested the extracts from inner and cuter leaves

of cabbage on growth of rats and found t:mt the vitamin A factor was

principally in the outezjLq^ves.

While vitamin A is not considered essential for the survival of

invertebrates, as it is for vertebrates, it is thought that the

function of vitamin A 18

in invertebrates is essentially similar to its function in

vertebrates. In vertebrates vitamin A is principally involved in

the visual cycle and body growth (Moore, 1957; Pitt, 1971)*

Without vitamin A vertebrates lose their night vision, develop

dermal lesions and then may die due to a lack of growth. Vitamin

A has also been implicated ia vertebrate olfaction (McCartney,

1968 ; lucîaier, 1972), in taste (Dingle and Lucy, I965), in

enzyme synthesis (Sundaresaa and Wolf, 1963)1 in detoxification

(Haley et si, 1943; Forrando, 195G) 2nd in gametogenesis (Sheer,

1940). Clare (1952) stated that domestic animals which lack pigfften-

tation rnay be susceptible to death from exposiire to sunlight

(xeroderra. pig'>iantosusî) and from an inability to detect plant toxins.

However, Moulton (1971) is sceptical about repcrts in the literature

that albino animals cannot detect toxic plants by small. Ee

suggests that aibino animals are merely more susceptible to certain

plants tîu?.n pigmented members of the same species. Tills is termed

phctod^mamic sensitisation, a phenomenon also noted by Bluir (1964).

It is interesting to note that administration of carotenoids or

vitamin A can alleviate humans from tdcin photosensitivity (Mathews-

Eoth et al, 1979), an inability to smell (anosmia) (McCartney, 1963;

Duncan et al, 1962) and from several forms of deafness (puedi, 1954)

including Kenieres disease and endogenous ear deafness. Kuedi,

(1954) also noted that vitamin A inhibited the toxic effect of certain

antibiotics in the inner ear of the guinea pig. Moulton (1970$

1971 ) believed that pigments in the ear wax do not have any apparent

importance.

Certain carotenoids are recognised vitamin A precursors, in

particular a-carotene, {3-carotene, p-zeacarotene, cryptoxant|dn end

mutatochrome (Goodwin, 1963a). Their percentage potencies for 19

conversion into vitamin A are 100^4, 40^$ 57/ and 50

respectively. Theoretically two molecules of vitamin A are

synthesised from one molecule of p-carotene and the enzyme

responsible is p-C£rotene 15t1 5 *o3{ygenase (Goodman and Olson,

1969)#

It has been argued for a long time that vitamin A is of

limited importance in insects. Wald (1943) thought tlmt it had

only an eye function; Gilmour (1961) said that carotenoids were

needed in insect nutrition but the interpretation of the role of

vitamin A required revision; Goodwin (1963b) considered that

animals were entirely dependent on plants for their supply of

vitamin A because they cannot synthesisecarotenoids do novo.

Goldsmith (1964) stated that the question of whether insects

require vitamin A or carotenoids for growth is not easily answered

while Wigglesworth (1972) stated that it was not known what if any

part carotenoids play in insect phyid.ology*

Figure 3

TZLMDJOTXDGY CF VITAMIN A

f "VITAMIN A” RFTINGL

yiTmiti A ALCOHOL ,PalinitriC acid

Cxi dation

VITAMIN A FALi{ITR,1TS

/RETINAL I R ETIH EI3 KETE.OIG ACID (growth) \ VITAMIN A ALDNIIYDi ( RETIHAIDEIIYDE y Opsins

VISUAL riGNilTS 20

Although vitamin A was not detected in insects in the past, presumably due to difficulties in teclmiquea, it has now been found to occur quite widely and has been shown to play an active pert in vision. Retinal (s vitamin A aldehyde) has been found in a number of insect orders, for example: Crthoptera, Coleoptera, Cdonata,

Lepidoptera (Briggs, 1S’61), hymenoptcra (Goldsmith, 1958; Goldsmith and Warner, 1964) and Diptera (Wolken et al, I96O). Until now no identification of vitamin A in P» brassicae lias been made as far as is known.

P. brassicae larvae when reared on the colourless heart leaves of cabbage have been found to di.e from granulosis virus or from bacterial causes (Griscn and Lilvestre Do Uacy, 1957» David and

Gardiner, 1965)* Synthetic diets have been used by several workers to determine the effect of omitting {5-Ccr‘otene or cabbage leaf powder.

David and Gardiner (1965b) reared F. brassicae on a send-synthetic df.et without cabbage pov/der and found tiiat the length of time for development was increased by two days and larvae were lighter coloured and under weight, /ikhtor (1966) reared instar P. brassicae larvae on a semi-synthetic diet and found that the addition of p-c-\rotene actually retarded growth; larvae fed on carotene free diets were normal. Van der Geest (1963) also reared ?. brassicae on a semi-synthetic diet but he omitted cabbage leaf powder. He found t’nat the haemolymph changed from yellow to blue and that the fat body was blue-green instead of yellow. The larvae were also irruch paler than those fed on diets with cabbage ix>wdor. In this work blue adults were reared as the survivors from a virus outbreak,

Rothschild (1972 , unpublished results) has also obtained blue pupae of F* brassicae. 21

Figure 4

(kindly presented by The Hon. ftlriam Rothschild)

Blue coloured pupa of P.brassicae.

Wardojo (1969) reared P. brassicae on a synthetic diet which lacked cabbage ix>wder but was supplemented with vitamin A palmitrate. He found that ^^rowth was suboptimal and the females were not all fertile.

Dadd (1957* 1960, I96I) reared the Desert Locust,

Scliistocerca gregaria Forsk. on a semi-synthetic diet and found that

^-carotene was required for two independent effects, namely growth and pigmentation. Bowers and McKay (1940) reared the German

Cockroach, Blatella germanica L# on a vitamin A free diet and found no effect of its absence. When Goldsmith et al.,(1964) gave the 22 House-Fly, Fusca domestic»-L» a cîîrotencid free diet they found

that the visual receptors had a lessened sensitivity, Carlson et al.

(1967 ), also found that a lack of vitamin A in M* sexta caused severe

visual impairment but no apparent effect on growth* p-carotene was

found by Morere (1971) to play an essential role In the metabolism

of the Indian Meal Moth, Flodia interpunctella Hubn., (F* ^ralidae),

Olfaction in insects has been recognised for a long time

(Muller, 1877 )* odours being used notably either as sexual attrac-

tants or as defensive secretions* A strong smelling defensive

vapour from the cervical glands has been shown to be emitted by

adults of the Garden Tiger, Arctia ca.ja L* when they are disturbed

(Rothschild and Easlcell, I966)* However the vapour was not

identified by gas chromatography (Rothschild and Feeny, I96O)*

Rothschild (I960, 1964a) has reported many orders of insects in

which similar smelling odours have been produced independently ;

a form of Mullerian mimicry* Wright (1964) investigated the

chemical structure of some sexual attractants and found that they

were complex molecules with a high biological activity.

Hertz (1927 ) stated that cabbage butterflies recognise

opposite sexes at a distance of two metres* When they flutter

around each other in circles they do so at a distance of 10-40 cm

but rarely make a direct touch. This suggested either a visual

or olfadory mechanism* U s e (I928 ) considered that olfaction

was not involved in sexual recognition in P. brassicae. and

Obara (1970) demonstrated that male P. rapae crucivora sought out

females in the wild purely on a visual stimulus* Muller’s

suggestion (1877) that larvae may possess odours is backed up by

Wojtusiak’s (1930) work on P.brassicae larvae in which he found

that olfaction played a role in their gregariousness* He suggested

that larvae produce a specific smell which attracts others and is

detected up to 5 cm distant. The haemolymph of insects which

produce defensive sprays or secretions is very often yellow while 23 that of species which do not produce sprays or secretions is often green (Rothschild, 1972a)* In some insect species the yellow haemolymph has been found to contain carotenoids while in the haemolymph of an orange coloured aphid other compounds have been identified (Brown et el, 1969)* It has been tentatively suggested that carotenoids may act on the vertebrate nasal mucosa and enhance the repellant nature of secretions(Rothscliild, 1971a),

The nasal cueosa of many vertebrates has been found to contain carotenoids but Moulton (I962) has suggested tliat carotenoids may be restricted to certain species only* Carotenoids do not constitute all the yellow pigment in the nasal mucosa of cows

(Kurihara, I967 )* Bang (1971) investigated the nasal clefts of many bird species and suggested that a considerable number of species are capable of olfaction. Rosenberg et al, (I96S) put forward a theory of "olfactory transduction" that depends on the carotenoid molecule being easily absorbed upon the nasal membrane of vertebrates. Leblanc and Orger (1972) have noted tliat when p-carotene absorbs upon a water-air interface it can form a single layer*

The involvement of carotenoids in olfactory processes in insects has nbt been clearly demonstrated. Melnwald et al., (I968 ) and Meinwald and Hendry (I969) found a degraded carotenoid in the defensive secretions of the poisonous grasshopper, Pcmelea microptera

Beauvais, and characterised it as a sesquiterpenoid, possibly degraded from neoxanthin. ^esquiterpenoids have also been identified by Eisner et al., (1971) iu secretions from the osraoteria of larvae of the Black Page/Gold Rim, Battus polydarmis Rothschild and Jordan, (F. Papilionidae). These compounds were not present in the foodplant and were presumed to have been synthesised by the larvae. This work suggests that carotenoids may be involved in certain insects defensive secretions* 24

Aposematic insects are those which have warning colouration and the definition has now been extended to cover all display associated with bright colours (Rothschild, 1973), although this is from the point of view cf human vision* Insects can see

further into the ultra-violet tlian Man (Thomx^tiori et al^ 1972).

Cctt cTid Huxley (1940) stated that aposematic insects may have poisons, defensive secret ions, a naus:^ou3 taste, a protective cuticle and a great tenacity for life* Kot all insects which are toxic are aposematic for example ants* However, aposenatic insects of rrany lepidopteran families contain distasteful substances or secrete toxins (Rothschild et al, 197G).

As a family the Fieridae are regarded as intermediate from the point of view cf palatability, between the unpalatable groups

Danainas, lapllioninae, Ithomiinae, Acraeinae and Meliccniinae and

the palatable groups lapilionini, Satyrinas, Nymphalinae and

Lycaenidae (Brower and Brower, 1964), HothschJLld (1973) suggests that the fieridae have inoro affinities to the unpalatable groups.

Cryptic insects, such as the Hawks (B* ophingidae) usually have rather dull colours which either match the colour of the background or, by means of disihiptive colouration, brealc up the visual picture and serve to mdce the insect inconspicuous* I-eterson (1S>60) stated that tlis resting site, the resting position and the cryptic 4ouc^no

Fiaving co-evolved together, instar larvae of P. brassicae have been shown to be aposematic while oi: cabbage leaves but cryptic

When on the ground (Baker, 197B), Rothschild et al,,(1972) suggeslfid t'nnt primarily it is important for an insect not to be seen by its predator but if scon to keep tho i:rodator at a safe distance, Baker (1'>7C) gives evidence which suggests that instar larvae of P. brassicae may be unpalatable to the Blue Tit 25

(partis caeruleus PrazJ and to the Great Tit ( Partis m-t.jor Praz»).

He shows that larvae of P. brassicae experience different predators

depending on whether they are on cabbage or on the ground, and how

old they are* He also points out that P, brassicae larvae are gregarious and aposematic while P* rapae are solitary and cryptic*

Weisraann (1832) and Pocock (I9II) both demonstrated in

feeding experiments with birds that the larva© of P* brassicae were unpalatable but the adults were relatively acceptable. Pocock tested the palatability of P. brassicae. the Green-veined White,

Fieri s n-apl L., P. ranae and the Grange Tip, Anthocharis car darning s

L. larvae, pupae and adults to many species of bird and mammal*

He found that P. brassicae, the only one which he regarded as aposematic, was the least palatable of the four, and that P. napi and A* cardamlnes which are both more cryptic than P. brassicae and

P. ranae appeared to be the most palatable* also found that when larvae of P* brassicae were reared on different foodplants

(Nasturtium (Tropaeolum) and Cabbage) the behaviour of the predators was not altered* Cabbage fed laryae were talien and eaten by 11 species of bird and taken and rejected by 10 other bird species.

Pupa were eaten by only one out of ten species of bird. Adults were eaten by 14 and rejected by 4 speciescf bird. Roer (1957) showed that up to 10^ of marked P. brassicae adults were eaten by wild birds* The Tree-sparrow (Passer montanus L.) ate more adults than the Whitethroat (Sylvia communis Latham.) which was better at catching adults on the wing than the Tree-sparrow. I,ane (1957) noted a difference between the palatability of stages of P. brassicae and P. rapae. He found that the Shama (Kittacincla malabarica Gm.') rejected the larva, pupa and adult stages of P. brassicae but 26 ate the cryptically coloured larvae of P. rapae, Frazer and

Rothschild {1962) noted the effect of feeding several different species of lepidoptera to different insectivorous mammals, birds and reptiles. A palatability rating was given to each species of insect so that the most acceptable was 0 , and the most unacceptable

7. Adults of P. brasslcme, P« ranae and P. napi were rated at 4, while adults of the most unpalatable species the Cimiabcor (ThTia jacobaeae L.) had a rating of 7* The Long Eared Bat (Plecotos err- itua L.) liad a noted dislike for adupts of P. ranae. The most palatable species, the Buff Ermine (pnllosoma luteura Hufnagel) had a rating of 3. Rothschild (I96I) looked for volatile substances in P. napi but did not find any evidence fcr their presence* Rothschild (1964b) stated how the Crow cor one L*), which found ?« brassicae larvae distasteful, remembered its aposematic colour pattern for nine months after being fed this insect. Karsh and Rothschild (1973) found that adults of P» were mere toxic to mice trian P. ropae.and pupae of F, brassicae were more distasteful tlmn adults to the Magpie (Gorvus pica L.). They also found that when, larvae of P. brassicae wore fed on a diet without cabbage or sir 1 grin the pupae were about half as toxic to mice as normally reared pupae. Pupae reared on a normal diet were more toxic than adults,killing mice after a few hours. Kale adults were not so toxic as females.

Benz (1962) investigated a toxic factor in the gut of P. brassicae larvae but was unable to make any identifications. The toxic factor was sufficient to paralyse six out of seven Ihckey moths (Kolacosoma neustria L.) and Benz(loc> cit)believed that it was caused by the bacterium, Bacillus thurirpiersis Berliner. Benz

(1966) showed that injections of this bacterium increased the permeability of the gut 27

wall of P. brassicae to pigments of the red kale.

In vertebrates vitamin A may act as a detoxifying agent

(Moore, 1957)# Experiments with rats which were fed the poison sodium benzoate showed that rats which were on a diet with extra vitamin A added survived and those on a normal diet died (Meunier et al, 1949)* Haley et al.,(1943) injected rats, fed on diets with and without vitamin A, with monobromobenzeue and found that only those with a vitamin A supply survived* They also showed that when Bionobroraobenzen© was injected less vitamin A than usual was stored in the liver* They concluded that vitamin A was not directly associated with detoxification but that the monobromobenzene prevented its absorption*

A detoxifying effect of vitamin A in insects could be important*

Many insects sequester poisonous plant substances which are used by the insect in some instances for defensive mechanisms against predators* The insects can apparently neutralise the effect of the poisons} a particularly impressive example being the Five-si)Ot

Burnet.Zygaena feygaena)trifolll Esper (lane and Rothschild, 1959) which can release hydrocyanic acid when crushed.

During the last 100 years much work has been carried out on toxic substances in mimetic and cryptic insects, resulting in a surfeit of literature relating to eight insect orders.

In the lepidoptera ten different toxins have been identified in eleven families, three of which belong to the Shopalocera*

Six of the toxins were found only in the families listed below*

Aristolochic acid-1 in Papilionidae Von Euw et al.,(1968)

Pyrrolizidine alkaloids in Arctiidae Rothschild and Aplin (1571)

Amajyllidaceae alkaloids in Moctuidae Aplin and Rot)i3ChiId (in prep) Mustard Oil Glycosides in Fieridae Aplin, d’Arcy Ward, March and Rothschild (in prep.) 28

0,0-dlmethylacrylychollae in Arctiidae Bisset et al., (I96O)

EyseSn^^"^ Zygaonidae Boccl (I916)

lane and Rothschild (1959)

Frazer end Rothschild (1962)

Jones et al., (I962)

Rothschild and Aplin (1971)

Of all toxins histamine was found most widely and was present in five families (/’arctiidae, Sphingidae, Bygaenidae, ly^mantridae,

Saturnidae) while acetylcholine and cardenolides were each found in four families (Acetylcholine in Arctiidae, Sphingidae,

Lyraantridae, Papilionidae; Cardenolides in Arctiidae, Danaidae,

Papilionidae and Ctenochidae). /ui unidentified toxic protein was also found in the Notodontidac, Sphingidae and Bygaenidae. A comprehensive survey of the literature relating to toxins in the lepidoptera and other insect orders has been made by Rothschild

(1972a). Many different combinations of these toxins may occur in different families cf insects (Rothschild, 1572b). Some of the toxic constituents of plants have been reviewed by Leiner (1569),

Parke (I96S) suggested that foreign compounds may be bound to proteins of the haemolymph and tissues thus making them incapable of crossing membranes. Parke (I568 ) also considered that the bonding site of the foreign compound and protein could be on the

K-terminal end of aspartic acid. Zaga^sky and Herring (1972), working on the carotenoproteins of the copepod, ladldocera acutifrons found aspartic acid was the binding site for carotenoids. Kqy et al.#

(1969) found only small sized protein particles with different shapes, ranging fi*orn IOO-I3O a in diameter, in the haemolymph and defensive secretions of A. caja and other insect species. Thommen

(1971 ) believed that carotenoids function as conjugates rather than as free molecules. 29

The purpose of this research was to investigate the role of carotenoids in insects. It was thought that an investigation into the carotenoids of a single plant herbivore relationship might provide further information on the importance of carotenoids. A typical insect which could be worked with throughout the year was chosen and an investigation into its carotenoid metabolism was conducted#

All stages in the life-cycle of P. brassicae were analysed for carotenoids and a qualitative and quantitative examination of the fate of carotenoids in the insect was nmde. It was thought that tracing labelled ^-carotene in the insect might indicate whether any changes in the carotenoid structure had taken place. Originally it was planned to synthesise labelled p-carotene from the fungus

Phycomyces blakesleeanus Burgess (Lilly et el». 1958)# However, it took a long time to select a strain of the fungus which produced a satisfactory amount of ^-carotene so when a free supply of initiated p-carotene was offered by Koffmann-La Roche & Co. Ltd., the work with P. blakesleeanus was discontinued and the radioactive work whs restricted to getting either labelled p-carotene or labelled

KVA into the plant and following their fate when ingested by the insect.

There was considerable variation in carotenoid concentration in the foodplant, B. oleracea. therefore a study of the carotenoid changes in the plant throughout the year was carried out.

For comparison with P. brassicae the carotenoid content of several other species of lepidoptera was investigated. It was thought that such a survey might throw some light on the importance of carotenoids in insects. 30

MATERIALS AMD METHODS

Carotenoid Analysis

The standard method for isolating carotenoids involves

extraction, saponification, partition and purification.

Purification may involve several techniques, either used separately or in conjunction with each other (fig. 3). In general the methods used for the isolation of carotenoids were those reviewed by

Goodwin (1955)* and those carried out by Jungalwala and Cama (1962).

Extraction

Total carotenoids

Large samples were macerated in a Waring blendor and snail ones were crushed in a test-tube or mortar. The samples were extracted with 95^ methanol and acetone until no more colour came out. Dry weight of samples was estimated after drying in an oven at 60®C for

48 hours*

Free carotenoids were removed by exiiaustively extracting the

samples in n-hexane. The material was then extracted in acetone to remove the bound carotenoids; acetone releases carotenoids from

proteins (Goldsmith, 1958; Goldsmith and Warner, 1964).

Saponification

With larval frass and plant material saponification was necessary to remove the large amounts of chlorophyll and lipids.

The collected filtrates were transferred to a separating funnel and about one third (v/v) (Koffmann-La Eoche, I960), of 70# KOI! in methanol was added. This was shaken and allowed to stand for half an hour. Saponificotioa was repeated until all the green colour had been removed. 31

Fig 5

FLOW DIAGRAM

COLLECTION OF MATERIAL I ------EXTRACTION Methanol Acetone

FILTRATION

SAPONIFICATION KOH

ADDITION OF TWICE THE VOLUME ______OF DIETHYL ETHER______WASH THREE TI ΠS IN DISTILLED WATER

EPIPHASIC HYFOFHASIC Carotenoids Chlorophylls, fats and proteins

DRY E'l AI'iHYDROUS SODIUM SULPHATE X EVAPORATE TO DRYNESS X PARTITION IN EQUAL VOLUÎ<ÎES OF 90% METHANOL AND HEXANE

EPIPHASIC HYPOFH/\SIC Carotenes *• Xanthophylls méHdCiiJinrxu- yaufic^Uullf. “ I"- . 1... . ----—* EVAPORATE TO DRYNESS EVAPORATE TO DRYNESS

COLUMN ON ALUMINIUM OXIDE COLUini ON ALUMINIUM OXIDE AND CSLITS AND CELITB

ALIQUOTS SP 800 ALIQUOTS spec trophotometer RECHROMATOGRAPHY RECHROI-LATOGRAPHY

PAPER CHROMATOGRAPHY OR PAPER CHROMATOGRAPHY OH TLC WITH KNOWiS TLC WITH KNOWNS

IDENTIFICATION WITH KNO'^S 32

PrylrR and Concentration

Approximately two thirds (v/v) of anhydrous"diethyl ether was added to the saponified extract# This was partitioned and freed of alkali by washing in three changes of distilled water#

The epiphasio layer containing the carotenoids was dried by passing it through a funnel containing anhydrous sodium sulphate#

The filtrate was reduced to % few ml in a rotary evaporator and a reading of its absorbance in n-hexane was taken in a SP 800 spectro­ photometer# The amount of filtrate was noted for the calculation of the total carotenoids of the crude extract# The filtrate was then completely reduced to diyness#

Partition

The evaporated filtrate was taken up in equal quantities# usually 20 ml of each of n-hexane and 90% methanol and transferred to a separating funnel. This was allowed to stand until the two immiscible layers had equilibrated# when the hypophaeic layer was drawn off# ether was added to it# so that all the colour was transferred to the ethereal layer# The epiphasic layer was drawn off separately and both were then evaporated to dryness under reduced pressure at about 35°C# The epiphasic layer contained carotene hydrocarbons, monohydroxy-xanthophylls and mono- and di­ epoxides of carotene hydrocarbons# while the hypophasic layer the di-and poly-hydroxy-xanthophylls and their epoxy derivatives.

Purification

Column Chromâto"Taphy

Glass chromatography columns# ^ x 1.8 cm., were used for all separations. Kagnesium oxide and cclite# (1:2 v/v) was tightly 33

packed into the columns to a depth of 12 cm on top of a piece of non-absorbant cotton wool. Hagnesiura oxide and celite (2*1 v/v) was used for rechromatography.

Carotene and xanthophyll extracts were taken up in ml of n-hexsjis and transferred to the colurms# where theyv,ere eluted in n-hej:ane and then in increasing concentrations of diethyl ether in n-hexane# 1%# 10^a up to 100%. Elution of more polar carotenoids was achieved by using increasing concentrations of acetone in diethyl ether, followed by acetone^ in methanol (Goodwin, 1955)*

Attempts to separate the colourless carotenoids phytoene and pîiytofluene were made by eluting in spectroscopic hexane and collecting the eluate immediately below the first coloured band* The looked for characteristic fluorescence of phytofluene was/inder the ultra- violetllight.

Paper Chromatography

The technique of circular paper chromatography developed by

Jensen and Jensen (1959) and used by Valadon and Mummery (1972) for separating carotenoids was used to confirm the purity of samples.

The papers used were Whatman’s Chromedia AH 8l and SG 81 loaded with aluminium hydroxide equivalent to 7«5% Al^O^ axid with 22% SiO^ respectively. Solvents used were 1%, 5% and 2Cy% acetone in n-hexane•

Thin Layer Chromatography (TLC)

Three types of plates were prepared using silica gel G (Targan et al. 1969? Kahan, 1967 ), kieselguhr (Snyder, 1964) and polyamide

(Egger and Voigt, I965). 34

The method of preparation of plates was that suggested in the booklet by Shandon for use vd.th their equipment. Gleaned glass plates, 20 x 20 and 5 % ^ mm, were put into a ’Unoplan* leveller and flattened off by using compressed air# A mixture of required absorbant was blended to a smooth paste in a Waring blendor, transferred to an adjustable spreader, and the slurry then drawn across the plates in one quick and continuous movement# The plates were then placed in an oven at IIO^C for thirty minutes to dry#

They were stored in an airtight Perspex container until use#

Samples for TLC were concentrated in n-hexane or diethyl ether and spotted onto the plates from capillary tubes# Care was taken to ensure tlv.it the spots had a small diameter# The plate v/as placed in a cîiromatography tanl'i and run in a suitable solvent#

Ti-ree solvents were used; benzene and diethyl ether, 90:10 (v/v)

Targan et al. 19^9)î petroleum ether, diethyl ether and acetic acid 90:10:1 (v/v) (Kahan, 196?) and hexane and diethyl ether 1:1

(v/v) (Goodman and Clson, 1967)* For polyamide plates methanol in methylketone (1:1) in hexane 1:10 (v/v) (Egger and Voigt, 1965) was used.

When the solvent had run about three quarters of the length of the plate the plate was removed, air dried and Bf values talten.

Removal, Sanorificatlon and TLC of Haemolymph Samples

Speed of operation was essential because oxidative processes and darkening of the heaemolymph takes place almost immediately on contact with air (Goodwin, 1955)* Efficiency of the removal process was therefore particularly important where the supply of haemolymph was small, e.g. srviall larvae and adults. Larvae were pinned upside down on a cork, with one pin passing through the head and another through the last segment so that the whole body was 35

stretched out. Care was taken not to rupture the gut wall. The tip of one of the prolegs was cut off and the end of a capillary tube was quickly put over the wound. Haemolymph was allowed to rush up the tube until no more was seen to come out after gentle pressure was applied to the body. The tip of another proleg was cut off to see if any further haemolymph could be collected. Any specimens in which the gut wall was punctured were discarded.

H^'Smolymph was removed from pharate pupae and pupae by puncturing the thorax with a capillary tube* It was found best to remove a forewing of a freshly emerged adult to obtain the haemolymph.

After removal of the haemolymph the liquid was put into 1” x

0.29” diameter tubes and mixed with one drop of 90% methanol. Half a pellet of potassium hydroxide and two drops of diethyl ether from a Pasteur pipette were then added. The tubes were shaken vigorously to allow saponification to take place more rapidly, sealed with

Para film and allowed to stand, in the refrigerator for ten minutes.

The epiphasic carotenoids could then be drawn off with a capillary

tube and used directly for TLC.

Polyamide plates were used (Bgger and Voigt, 1965), each sample

being applied as a small spot half an inch from one end of the

plate. The plate was developed in 1% acetone in n-hexane. To

prevent any oxidative effects of light on the carotenoids the tank

was covered with a black cloth during development. After the

solvent had run about two thirds the distance of the plate, the

plate was removed and the position of the solvent front marked.

The position of coloured carotenoids was quickly maiiced before any

photoxidation occurred. Ef values were calculated for all the spots. 36

Identification

Identification of individual carotenoids was determined from many criteria; the relative position of the band during chromato­ graphy, the colour of the band, its Ef value (TLC only), its spectrum in the SP 800 spectrophotometer and its reaction with acid.

When column chromatograpliy was used aliquots of eluate were taken and read in the SP 800 against a blank containing a pure sample of the solvent used. The maxima ;, and absorbance of the spectra were recorded. Impure samples could not be identified from their absorbance. These samples were rechroraatogramed.

HCl Test

Carotenoids having epoxy groups were characterized by a modified concentrated IlCl-ether testj^ 9 ,6-Monoepoxy-p-carotene, 9+6*

9#,6*-diepoxy-g-carotene, 5 ,6-monoepoxy-lutein, violaxanthin

(5 ,6 *9 *,6 *-diepoxyseaxanthin), and neoxanthin, were converted into the corresponding 9 ,6 or 9 ,6 :5 *8 * furanoid oxides, namely mutatochroroe, aurochrome, flavoxanthin, auroxanthin and foliachrome respectively. (See Appendix Figs. land 2 for spectra of common

Carotenoids and spectral shifts with acid).

When identified each carotenoid could be designated either a carotene or xanthophyll as follows*-

Carotene Xanthophyll a-carotene Cryptoxanthin P-carotene 9 ,6-monoepoxy-lutein jf,-carotene Lutein P-zeacarotene Neoxanthin Violaxanthin Chrysanthemaxanthin and Flavoxanthin Auroxanthia 9,6-monoepoxy-p-carotene 5 ,6 t9*6 *-diepoxy-p-carotene Hutatochrome Flavocbrome Aurochrome 37

The isomers chrysanthemaxanthin and flavoxanthin were grouped

together in this work because they were difficult to separate.

Concentration of Carotenoids

Concentration was calculated using Goodwin’s (1955) method

which states that 1 gram of tissue contains y x z x 10^ pg of 100 X w X d X b

pigment, where y « absorbance, z » volume of solvent (ml), w » weight of material (g), d = size of the cell (1 cm) and 1% b » E value for the pure pigment. 1% Each carotenoid in a particular solvent has a specific E value, e.g. for ^-carotene in n-hexane the value is 2580. The 1% E value of other common carotenoids in n-hexane does not vary

greatly from 2500 so this value was used for all of them (Goodwin,

1955)* The equation can then be reduced to y x z x 4 pg/g dry w

material. Using the formula the concentration of each carotenoid

in a sample was calculated. Normally the total carotenoids were

determined from the sum of all the individual carotenoids in the

sample, but where only small amounts of material were available

the total carotenoids were calculated from the absorbance of the

crude extract at a specific wavelength. 38

Foodplant

Brassica oleracea var* capitata

General conditions of prowth

Seeds of Primo type cabbage (Messrs. Thompson, and Morgan) were grown in a greenhouse at an average temperature of 25°C - 5®C and at a relative humidity of 75 - 15$ on John Innes Mo. 1 soil.

The plants were allowed natural daylight. The seedlings were potted out after three weeks and were repotted thereafter whenever necessary.

Carotenoid changes over twelve months

Seeds were sovm on November 1st, 1971 under conditions already described. Samples were removed for analysis weekly for the first four weeks, for the next four weeks and then monthly for a total twelve months. During the first month whole seedlings without roots were extracted. At a later age only outer leaves were extracted. Carotenoids were identified in the usual manner. The mean concentration of each carotenoid from all the extractions was calculated.

Carotenoid changes in Spring and Summer Cabbage

Spring cabbage was sown on Kay 12, 1972 and summer cabbage on

August 15th, 1972 . Samples were collected at weekly intervals for a month end comprised whole seedlings without roots.

Wet weight, dry weight ratio of Cabbage

Different weights of B. olerarea leaves with midribs removed were used, placed in an oven and dried until constant at 60^C. A graph of the wet weight vs. the constant dry weight was drawn from wt. wliich the wet - dry/equivalents could be obtained (Appendix Fig. 5 )* 39 livestock

Pierls brassicae L .

Source of Stock

Initial stocks of P, brassicae were supplied by The Butterfly

Farm, Kent, but later these were replaced by a generous supply from Dr. C. Rivers, Department of Forestry at Oxford. Many live­ stock samples were provided by the I.C.I. laboratories at oealott’s

Kill, Berkshire; in particular frass, larvae,pupae and adults were given throughout the years of study. Dead adults were obtained from the Shell laboratories, Sittingbourne, Kent, Dr. C. Rivers and the I.C.I, laboratories. Some adults of P. brassicae were collected on the wing during the summer months of 1971 and 1972 .

Culture

A stock culture of P. brassicae was maintained from September

1970 until September 1972, except for periods when attacks of polyhedral virus, (Smith and Brown, 1965), necessitated complete disinfection of equipment and laboratory with formaldehyde. Samples of all stages of P. brassicae were therefore available for experiments at most times of the year.

The insects were kept in a cage in the laboratory (Fig. 6).

Tliey were exposed to sixteen hours of artificial daylight per day

(David and Gardiner, 1952) from two Mazda 20 W floreccent tubes at a temperature of 20^ - 2^ and a relative humidity of 60. During the summer months of 1971 nnd 1972 the cage was placed outside in direct sunlight, where it was found that copulation was induced more readily. The insects were given mature greenhouse grown potted plants on which to oviposit. The ova were ailowed to hatch on this plant and the larvae to consume the leaves. Two or three beakers 40

F is * 6

P.br&Gsicae adults in breeding ca^e 41 containing cotton wool soaked in sucrose and honey were introduced to the cage so that the adults could feed#

Carotenoid Studies in ?♦ brassicae

The carotenoid content of all stages of P# brassicae were investigated# Feplicates of each sample were carried out whenever sufficient material was available# Minimum numbers of ova, L^, 1^,

1^* and Instar larvae on which to carry out extractions were approximately 6 ,500, 1,000-2 ,000, 500, 250, 50 and 50 respectively#

Samples were replicated two or tliree times depending on availability of material. Pharate pupae and pupae were used in groups of 25#

Adults were analysed on nine separate occasions, the numbers ranging from 8 to 2 ,500*

Frass was collected from insects reared in the continuous culturing system at I.C.I# Any sample of frass could therefore be related to the age of the larva#

Carotenoids in the heads and bodies srd the wings, and also in each sex cf P. brassicae were investigated# Samples were supplied from the Shell laboratories, Kent#

Mean body weights of all stages in the metanorrhosis of P. brassicae were recorded.

Bioener^etic studies

Four feeding experiments were carried out to calculate the total

(3-carotene ingested by P. brassicae larvae during their lives. In all experiments the total 0-carctene ingested was calculated from the product of the p-carotene concentration in the plant and the dry weight of food consumed.

In the first experiment one hundred larvae wore used and fed on both greenhouse grown and greengrocer's cabbage# The total 42 aooimt of fresh food eaten was calculated from the difference between the fresh weights of food offered and the fresh weight of food uneaten. Control experiments in which Identical pieces of cabbage were left in dishes without larvae were done so that the percentage loss due to evaporation could be subtrated. The total dry weight of the food consuced was then read off from the dry weight - wet weight graph previously constructed* The p-carotene value for cabbage was taken from the cean annual figure calculated from the results of analysing the plant pigments for twelve months.

In each of the other three feeding experiments samples of

L*. and II.-lervae were fed on leaves from the same plant from which both the dry weight - wet weight ratio and the p-carctene value had been calculated. Larvae were fed on similar outer leaves to those from which these measurements were cm de. Ten larvae of each instar were collected from I.C.I. when they were in the non-feeding stationary stage just prior to ecdysis. They were carefully put inside the petri dishes in which wore weighed amounts of fresh cabbage. Every 24 hours the remaining cabbage was collected and oven dried at 60^C to a constant weight. The frass was also collected and weighed pieces of fresh cabbage were introduced into the dishes. When the larvae began to change skins and stop feeding the food was removed and oven dried.

All food was taken from Primo variety cabbages (grôv/n at I.C.I.) which had developed without a 'heart*. The total frass from each instar was extracted for carotenoids in the usual manner.

Experiments with the and instar larvae proved difficult because of their minute si%e. For the first 6 'hours the instar larvae only eats the cuticle of the leaf in which there are no carotenoids. Thereafter it eats through the lamina. However, the 43

faecal pellets are so small that obtaining enough for an extraction would have required having several thousand larvae*

The assimilated amount of p-carotene was determined from an

Instantaneous Budget (Klekowdil and Duncan, in press), where assimilation (A) is equal to the oonsumrtion/ingestion (C) (which

is called I in this work) less the faeces (F) and the urine (U)*

In terrestial arthropods such as P* brassicae where it is difficult

to determine the urine output the two figures are considered

together. Thus A = I - FÜ.

From the wet weight - dry weight ratio of the cabbage the dry weight of the food offered was calculated* The dry weight of the

food uneaten was subtracted from this figure to give the total dry weight of food ingested. The difference between the amount of p- carotene ingested (vg) and the ©.mount of p-carotene lost in the

frass (%g) gave the amount of p-carotene assimilated in pg. 44

Vitamin A

Quantitative Determination

The Carr-Price Method, described in detail by Hoffmann La

Eoche 8c Co. Ltd. (I96O) was used initially but was supercedded by

the Trifluoracetic acid method (TFA).

TFA

This method, given by Dr. Marsdeu of the Birmingham and

Midland Eye Hospital, was originally designed to tost for the presence of vitamin A in one millilitre samples of blood serum.

It was modified for this work and found to be invaluable because of its speed of operation. From collecting material to measuring

the absorbance (ol8 }nm.) the time taken was about 15 minutes. As vitamin A decomposed rapidly at room temperature speed of operation was a great asset.

Heads of larvae and adults were removed from, live animals immediately before extraction. Ova were collected fresh from cabbage leaves. The samples were put into a test-tube which was then immersed in a beaker of ice and water. To the test-tube was added 1 ml of methanol and 3 ml of diethyl ether and the whole was stirred with a glass rod and shaken vigorously for 2-3 minutes.

Care was taken to ensure that the material had been completely macerated. The solution was allowed to separate out into epiphasic and hypophasic layers. A small amount of the epiphasic layer was taken off by Pasteur pipette and its absorbance read at 450 nm on.the

SP 800 , using silica cdcrocells (path length 10 mm). The petroleum ether was then gently evaporated off over a water bath. It was

taken up in 0 .2 ml of chloroform, transferred to a microcell and placed in the sample holder of a SP 600 spectrophotometer. To the microcell was added 1 ml of a mixture 45 of trifluoroacetic acid and chloroform (1;2 v/v). The absorbance and colour of the résultant solution at 613 nm after 13 seconds was then noted. The blank was made up from TFA and chloroform

(1:2 v/v).

Calibration Curve

Calibration curves wore prepared for both the standards, vitamin A alcohol and acetate, from readings taken at 13 second time intervals. Increasing amounts of the standard samples, from

0.80 international units (I.U.) were evaporated to dryness in vacuo and taken up in 0.3 ml of chloroform. 2 ml of TFA were then added and the extinction of the blue colour was measured at 6l8 nm.

The blank consisted of 0.3 ml chloroform and 2 ml of TFA. Figures of absorbance and I.U. were then plotted (Appendix Fig* 4). 46

Qualitative Determinations

ColuTTtn Chromatography

Two methods were used for P. brassicae heads and bodies extracts, both using columns packed with Aluminium oxide (Goldsmith,

1958 ; Methods of Vitamin Assay, 1966). Extracts from 2,500 and 500 heads and bodies were also chroroatograroed on columns using the methods of Vitamin Assay (I966)*

TLC

Extracts of 100 and 500 heads were run on silica gel plates

(Section on TLC), Preliminary trials showed that the solvent petroleum ether, diethyl ether, acetic acid 90%10;1 (Kahan, I967 ) gave the best separation of p-carotene with vitamin A standards (Appendix

Fig. 5) Tills solvent was therefore used for all vitamin A separations. After chromatography the plates were sprayed with either antimony trichloride or TEA and the colours of the spots noted. Ff values were determined in the usual manner,

Mèlhtë;»re.tion''Of Maxi.ffia at 6l3 nm for Vitamin A Alcohol and Acetate

The reaction of TFA with vitamin A active compounds gives a transitory blue colour which absorbs at 613 nm. The rate at which the absorbance falls off is so fast that standardisation of the method wag necessary# The absorbance after 10, I5 end 50 seconds and the formation of a subsiduary peak at 561 nm were investigated

(Appendix Fig. 6 ). I5 secs was taken as the standard time after from addition of TFA/which calculations were made. 47

Labelling Experiments

Radiochemical3 and Dispensing

[3H] -15-15*3-carotene, (95?j pure, sp. activity I60 Ci/mg) was donated by Dr, 0, Isler of Hoffmann-La Roche & Co. Ltd., Basle,

Switzerland. (5E,^R-^H3+33,^3,-^H^) Lactone Mevalonic acid was purchased from the Radiochemical Centre, Amersham, Bucks, England.

The primary scintillation solute 2,5-Diphenyloxazole, (PIO) was added to redistilled toluene to make a concentration of 5 gm per litre (PPO/Toluene^* MCS Tissue Solubiliser by Amersham/Searle was

supplied from the Radiochemical Centre, Amersham, Bucks.

Dispensing ■5 % Sterilised 1 cm x 0.01 cm disposable pipettes by VCLAC Ltd., were used with a •Griffin Pipette Filler* bulb. Capillary pipettes,

0.01 Cffl^ by T B C m i O Ltd., were operated by a GRIFFIN and GEORGE Ltd., % micropipette filler. 0.2 cm pipettes by E.KIL Ltd., were also used.

Purity

It was necessary to check the radionuclide sources, both old and new, for the presence of impurities. Von Korft (1972) stated that there can be variation in the amount of radionuclide present in commercial supplies. Natural decay and adsorption of the radio­ nuclide on the walls of the container may also account for the loss of specific activity.

Samples were run on khatman*s No. 1 filter chromatographic paper in 5^^ and 10>^ acetone in n-hexane to see whether any separation of impurities had occurred. Paper chromatograms were cut intt strips and counted on a Beckman Scintillation counter (CPM 100). 48

A graph was then prepared plotting counts with distance the strip was from the starting line* If any impurity had arisen these were shown as separate peaks other than the large one of the sample#

3-carotene was eluted from the appropriate strip by immersion in n-hexane and its absorption spectrum checked on the SP 800#

TLC plates using kieselguhr were also.usedtto determine purity

(Snyder, 15&4)* A Tracerlab 4 if scanner was used to detect the position of the radionuclide and any other impurities* The kieselguhr was then i»:raped off In widths of 1 cm* and these were counted in the CPM 100 (Houx, 1969)# The counts per minute (cpm) were then plotted against the distance and the degree of purity was then observed#

Decontamination

All vials and caps were vigorously cleaned and washed in

Decoa-90 as reconanended by in their accompanying leaflet*

In some cases the vials were cleaned in chromic acid before washing in Decon-90* The vials were washed in running water and then allowed to soak in a 5-1^^ solution of Decon-90 for about one hour* The vials were then cleaned, rinsed at least three times in running tap water and then passed through distilled water*

They were then dried in an oven*

In order to determine the effectiveness of washing^all the cleaned vials were filled with $ ml of PPO/toluene and counted in the CPM 100* Any vials with counts higher than 50 were cleaned again and recounted* Caps were replaced when they had been used three times because of their high rate of contamination* In the laboratory W)iatman*s BENCIIKOTE, Polyethylene backed Absorbent paper was used throughout* Disposable Pasteur pipettes were also used* 49

Counting and Background

All samples were counted in a solution of 5 ml of PPO/toluene

(Davidson and Feigelson, 1957), on a BECKMAN CPM 100 Liquid

Scintillation system set at 3% error for a time of 200 minutes*

Each sample was given an External Standards Ratio (ESP) reading*

Each readout from the CPM 100 contained the counts per minute

(cpm), the time it took to count the sample in minutes, the percentage error and the ESN value. Silica glass vials and urea caps by DECKMEN Ltd*, both of which give low counts, were used for all experiments.

The Cllî 100 gave background readings between 20-50 cpm on clean glass vials* A considerable amount of this count wad due to the naturally occurring £; K] present in the glass (Calf, 1959)*

Most experiments were carried out using 0.001-0*6 pCi of radio­ nuclide* In most cases incorporation into samples was sufficiently high for the background to be disregarded (Kerb&rg, I96O).

Quenching

Both the colour of caroteaoids and the solvents in which they are dispersed may decrease the efficiency with which they may be counted* This results in colour and chemical quenching* Solid biological material and inert substances such as silica gel may absorb some of the photons of light from the scintillant and thereby give a lower cpm reading* The photons of light are absorbed by the photomultipliers at 58 O nm which is in the lower range of the spectrum where carotenoids absorb* Yellow and orange coloured solutions therefore cause considerable quencliing*

It wqs necessary to prepare quench curves for p-carotene and of different solvents in which ^-cai'otene was dissolved* Biological 50 material first had to be solubilised and then the resultant liquid, usually coloured had to be decolourised before counting.

Colour Quenching

Sets of ten vials were prepared to determine the colour quenching effects of 3-carotene* Each vial was given *’cold'* p-carotene in increasing 0.1 ml portions so that a series of vials were used, showing a range from a pale straw colour to a deep orange* This represented a p-carotene concentration from approximately 5*6 vs/s sfau»Uvr*t to 57*1 pg/%* To each vial was added an interna]^of 0.1 ml of

3-carotene (0*26 %C1) and 5 ml of PPO/toluene* Tlie vials were then counted.

Chemical Quenching

The effects of n-hexane, diethyl ether and 5p diethyl ether in n-hexane on counting were investigated* Sets of vials wore prepared using increasing 1 ml portions of the above solvents* The effect of silica gel on counting was also investigated* Vials were given increasing 0*1 era portions of silica gel scraped from 5 % 20 cm 3 plates. To all tlie vials was added C*2 ml of [^H] p-carotene

(0*002 pCi) and 5 nl of PPO/toluene before counting.

Tlie effect of evaporating to dryness a series of vials quenched with cold p-carotene was carried out* The vials were gently heated by warm air from an hair dryer until all the solvent had evaporated*

5 ml of PPO/toluene was added to each vial which was then counted*

Solubilisation

In all cases where solid plant or animal material had to be counted solubilisation was carried out using IsCS Tissue Solubiliser* 51

Baemolyrsj)h also had to be solubilised as it was immiscible with

toluene* Two drops of NCS from a Pasteur pipette were necessary for

a haemolymph sample whereas about tea drops were required for a

whole gut sample from a P, brassicae larval gut* After addition of

NC3 all samples were shaken thoroughly and left until solubilisation

was complete* Large samples such as whole guts were warmed under a

gentle stream of air from an hair dryer so that the action of IiC3

Gould progress r^ore rapidly (KCS accompanying leaflet)*

Bleaching

Many methods have been used for the decolorieation of carotenoids.

For a review see Walter and Purcell (I966) and the Radiochemical

Review 6 * Two of the methods were tried in this work5 decolorisa-

tion by ultra-violet light (Yokoyama et al* I962) and bleaching

with benzoyl peroxide (Walter and Purcell, i960). Bleaching with

benzoyl peroxide was found to be the most satisfactory.

Ultra-Violet Light

Samples were bleached under Ü V . light by laying the vials

horizontally for about one hour# The vials were then kept in the

dark for six hours so that excitation of p-particles would return

to normal*

Benzoyl Peroxide (C^H^CO)^ 0^

A solution of benzoyl peroxide was prepared according to the

technique of Walter and Purcell (I966)* It was made freshly before use as suggested by Amersham/SearI0 in their leaflet on NCS* 1 gm

of benzoyl peroxide was heated to 60*^C in 5 ml of redistilled

toluene, then cooled end filtered before use* About 2 ml of this

solution was required to bleach a yellow solution of 3-carotene 52

(5,6 vs) while Approximately 3 ml was needed to decolorise a deep

orange solution (37,1 vg).

Calculations

By reading the ESS valve of a particular sample on a quench curve the percentage efficiency of counting could, be determined.

The E3S also reflected the degree to which the sample had been

quenched. If it were high then the sample was counted efficiently and if it were low then the sample was counted inefficiently.

All counts were converted to disintegrations per minute (dpm) on the basis that the counting efficiency of tritium is

Radiochemical Review 6 (1971)* 53

Radioactive Experiments

Acceptability of B-carotene to P, brasgieae larvae

instar larvae were used for all experiments. Several ffietliods were investigated to incorporate tritiated p-carotene into the larvae. The following one was used for all experiments i^^. which squares of cabbage were offered to the larvae.

1. Larvae were isolated in ventilated plastic containers without

food for four hours. 1 cm squares of cabbage were cut from the

lamina of leaves. Using a micro syringe 0.05 ml of

p-carotene was dropped onto each square and allowed to

evaporate to dryness. The starved larvae were then placed

individually into the containers and given one piece of the

treated squares. When the squares had been consumed the

larvae were ready for experiments.

Other methods were investigated

2, Using anhypodermic needle (503 % 32 mm) 0.02 ml of [^H]

3-carotene in n-hescine was injected into the body cavity of

the larvae,

3 # An hexane solution of [^H] p-carcteae was evaporated into 1 ml

of linseed oil and then injected as in method 2 ,

4, Using a Pasteur pipette p-carotene was placed onto the

dorsal surface of the larva and allowed to be absorbed,

5, Using a Pasteur pipette one drop of [ H] p-carotene in linseed

oil (as in 3 ) was placed on the dorsal surface of the larva and a 'lo'allowed to be absorbed,

6 , 0,02 ml of [^H] p-carotene in linseed oil (as in 3 &nd 5 ) was

applied to a 1 cm square of fresh cabbage and allowed to be

absorbed. The treated square was then offered to the starved

larvae. 54

7 * 0,02 ml of p-carotene was injected into the stem of a

cabbage seedling which was then fed to the starved larvae,

8 , 0,02 ml of [^H] p-carotene in linseed oil was injected into

the stem of a cabbage seedling which was then fed to the

starved larvae,

9*' Roots of 1 week old cabbage seedlings were immersed in an

emulsion of [^H] p-carotene in water and fed to larvae

from normal diet,

10, Seeds of cabbage were germinated in [^E] MVA water and fed to

L,- larvae from normal diet, y 11, Roots of 1 week old cabbage seedlings were immersed in a

solution of p-carotene in water and fed to larvae from

normal diet.

The reactions of all the larvae to their food was then observed,

Methods 9* 1G and 11 were used when whole seedlings were offered to

the larvae#

3 5 Incorporation of T HI B-carotene and [ H] KVA into P. brasstcee larvae

In two experiments 20 end 50 instar larvae were fed

cabbage seedlings which had been grown in tritiated p-carotene. In

another experiment cabbage seedlings grown in 0,5 pCi of MVA wen& fed

to 20 larvae, When all the food had been consumed the largae were

extracted for carotenoids, Carotenoid bands from the column

chromatography were bleached before being counted.

Five instar larvae were individually fed C,05 V-Ci of

tritiated p-carotene evaporated onto cabbage squares. When this

was consumed the heads and bodies of the larvae were individually

solubilised, bleached and counted. Five larvae were also fed "cold"

food and their heads and bodies prepared for counting as a control. 55

3 Incorporation of T Hi p-carotene into Haemolymph, fat body and gut of P. braasicae larvae

14 larvae were given squares of cabbage containing 0.002

]iCi of tritiated p-carotene. After 2, 4 and 6 hourly intervals five larvae were taken out and their haemolymph, fat bo(iy and gut removed. The body wall was pinned back to remove the fat body from the inside surface. The gut was then removed by cutting each end close to the body wall* Each sample was solubilised with NCS, bleached and counted. Five larvae were given "cold" food and were treated in the same way.

3 Rate at which f HI 6-carotene Is passed out in the Frass

30 larvae were given squares of cabbage each containing

1.126 pCi of tritiated p-carotene. Each larva was kept in an

Individual dish. After regular time intervals all the frass pellets were removed and put into vials for counting. Ten larvae were given "cold" food and were treated in a similar way. A note was kept of the number of larvae defecating and the number which had entirely consumed their food.

3 Incorporation of MVA into B. oleraçea Seedlings

Under similar conditions the roots of B. êleraçea were immersed in 0.9 pCi and 0.31 %iCi emulsions of MVA for one and tîriree days respectively. The stem and green parts of the seedling were then extracted for carotenoids. 56

Carotenoid studies in other species of lepidoptera

The carotenoid content of 4 other members of the British

fieridae (Fig. 7) was investigated. Adults of P, napi and

P» rapae were collected on the wing at Egham# Surrey in the summer

of 1972, Adults of G, rhami and pupae of A, cardamines L# were

obtained from Worldvd.de Butterflies Ltd,, Dorset,

The carotenoid content of 2 0 other species of lepidoptera including Eeterocera and Rhopalocera was also analysed. The pupal and adult stages of many species were extracted, in some cases both

had originated from the same known foodplant.

The follovdng list of species includes all those which were extracted. Nomenclature of the British lepidoptera follows that of

Kloet and Kincks (1972) in which the Law of Priority was used throughout. Sub-generic names appear in brackets after the generic name. Nomenclature of foreign Saturniidae is according to

Crotch _ (1956)5 Australian Fieridae according to Coirmon and

Waterhouse (1972), African butterflies according to Van Son

(1949), Williams (19&9) and Japanese butterflies by Yokoyama

(1955),

C, iacobaeae and C« chrysorrhoea were reared from larvae collected in the field, L, populi. A, exclnmationis. N, pronu.ba,

L, comma, M. trigrammica, S* luteum end S, lubricipedn were collected from a mercury moth trap operated in the grounds of the Botany

Department, ^oyal Holloway College, during the summer of 1972, All remaining species were kindly provided by the Hon, Miriam Rothschild from Worldwide Butterflies &td., Dorset, 57

n« > 7

British H m t i Â Ê m

i

1 Wrwiqa# Ü j u u m u s (larg# Whit#) 2 H#ri# uspj H m m m m <0re«ki»T#lA#4 Whit#)

) jBteSsjaeai u###### (s m o x whit#) 4 imth##h#ri# # w 1##tn## U buasu# (Oraag# tip) 5 qsoiiffltagya rJMwiiai phwrnl M m m * # # (BriMton#) 6 gj^yyui #rp##

LIST OF SPECIES AJTALYSED

SPECIES COmON NAME

RHOPALOCEHA

DAÎÎAIDAE

Danaus plexj-ppus Lirmaeua Milkweed/i-1onarch

PIEEIDAE

PILSINAE

Fieri3 brassicsQ Linnaeus Large White

Fieri B rap^

Pier is rapi Linnaeus Green. Veined White

Anthoeharis cardandnes Orange Tip Linnaeus

DIoNOKPHIINAE

leptidea sinanls sinapis Wood Wliite Linnaeus

Pr T ' T'Tv Î « -ri

Collas c r o c e u s Geoffrey in ClcuciâtYellow Fourcroy

Coniapteryx rkaimi rhami Brimstone Linncveus

Catopsili a ponona Fabriciu3 Lcnon Migrant

C?toprdlia c r o c a l e Fabricius (Green and Yellow colour forms of pomona with black antennae) see Common and Waterhouse, (1972)

E e l o n o i s cslyjssg Drj ry -

F.ylotbris chlo^is Fabricius Dotted Border

HETLRXI'KA

3FKIWGIDAS

SFUiL’GINAE

Laothoe poT’iili Linnaeus Poplar Hawk 59

MA.CEOGLOSSIKAE

H.yles euphorbiae Linnaeus Spurge Hawk

SATURNIIDAS

Satnrnia pa.vonla Idnnaeus Emperor

Actias lu.na • Linnaeus American Moon.

I-oepa kff.tinka Westwood Golden Emperor

Aglia tau Linnaeus Tau Emperor

Antherae pernyi Guerin-Keneville Chinese Oak Silk/

Hyalophora cecropia Robin

ARCTIIDAE

AECTUKAB

Arctia caja Linnaeus Garden Tiger

Tyria jacobaeae Linnaeus Cinnabar

Callimorpha dominula Linnaeus Scarlet Tiger

Spilosoma luteum Hufnagel Buff !irraine

Spilosoma lubricipeda White Ermine Linnaeus

NOCÏUIDAE

NOCTUirbuS

Agro-^ti .9 exclaraationi s Linnaeus Heart and Drrt

^;octua pronuba Linnaeus Lrrge Yellow Undorwing

HADENIIUE

Mythimna (Leucania, Cchsenheimer) Shoulder striped comma Linnaeus Wainscot

AkPEIPIRINAB

Chorsnyca trigr»mnica lîyfnagel Treble Lines

ELN0MIEA3

/brexas grostrilariata Mag%.'ie Linnaeus 60

Operophtera brumata Winter Linnaeus

NOTODOLTIDAE

Phalera bucephala Linnaeus Buff Tip

Cerura vinula Linneaus Puss

ZYGAENIKAE

Bygaena (Zygaena, Fabricius) 6-spot Burnet fillpendulae angllcola Tremewan

LYMANTRIÎDAE

Fuproctis chrysorrhoea Brown Tail Linnaeus

DaflYMra pu dibun da Linnaeus Tussock 61

EESUtTS

Ceroteitoid.s

Many factors may influence the qualitative and quantitative identification of carotenoids both in plants and animals. Some of the points which have to be taken into account when assessing the results are considered below,

1, The amount of material

Because carotenoids are present in insects in very small

amounts it is necessary to obtain sufficient material in order

to iSèntify the individual carotenoids present, When the

size of the insect stage is small as in ova or young larvae,

difficulties arise in collecting enough material. When there

was not sufficient material for chromatography, the crude

extract was used to estimate the total carotenoids. However,

when it was possible to obtain and analyse several thousand

ova end young larvae the major carotenoids could be identified.

For the analysis of the relatively large stages smaller

numbers were required. However, even with them in order to

identify carotenoids present in trace amounts large samples

had to be used, for example see adult sample No, 4 in

Table 8,

2, The age and state of the material

It was found that the age of living specimens was an

important factor to consider when comparing carotenoid results.

For example when considering the carotenoids of female adults

the state of oogenesis ard whether ovipositing had taken place 62

were factors which have to be known* Females of some species

such as 0 # brumata hatch with ova fully developed while

P* brassicae starts oogenesis only after emergence* Dried adult specimens of P* brassicae were found to contain less carotenoid than fresh material (Table 8 ), Because oxidation

of carotenoids occurs readily at room temperature and in light, samples were if possible extracted live and when adult as soon as possible after emergence* If samples had to be

stored overnight they were placed in the freezer compartment

of the refrigerator. During the life of the adult loss of carotenoid may occur due to scale loss and natural oxidation*

3* The foodplant

The carotenoid content of the foodplant was found to be extremely variable, not only during the development of the

foodplant but between plants of similar age and appearance.

This variability must be taken into account when considering the carotenoids in the insect*

4* The teclmique

Although standard methods of carotenoid analysis were carried out certain criticisms can be made. Preliminary experiments showed that successively reducing to dryness a known carotenoid samx^le resulted in a decrease in the absorbance each time* As the absorbance is used to calculate the carotenoid concentration this effect would tend to give a smaller figure for T/C* Reducing to dryness is a necessary step occuring twice in carotenoid analysis, first, of the crude extract and second, the epiphasicand hypophasic extracts* Reducing to dryness is performed two more times if 63 rechromatography has to be carried out* Heat also oxidises carotenoids. SD that the operating temperature of the water bath, used when reducing to dryness, was always kept at room temperature*

Oxidation of carotenoids may occur on the column if it is run for more than four hours* The products of oxidation can be identified on the spectrophotometer* In practise all columns were run for between 1-3 hours* Carotenoids sepirate out on a column more fully if they are left for as long as possible within this time period* Separation is also aided when the concentration of the eluting solvents added is increased gradually* When samples are run too fast or when solvents are changed rapidly the carotenoid bands do not separate out well and tend to become mixed. As carotenoids absorb within a small wavelength range identification is hindered if they are not completely separated* Accurate identification could be made only if such mixtures were rechromatogramed*

Because of the loss of carotenoids during procedures of technique the value obtained for carotenoid concentration of identical samples of either plant or animal material may vary as much as 20^g/g* Thus quantitatively no great importance can be attached to small differences in carotenoid concentrations of samples. Qualitatively the system for carotenoid analysis is reasonably good provided the sample is not too small* 64

S* oleracea

17 carotenoids were detected in B. oleracea plants, although

2 of these were unidentified (Tables 1 and 3 ) * 0-C3rotene,5,6-

monoepoxy-P-carotene, lutein and violaxanthin were always found in

the plant, while neoxanthin v/as absent only once. The fluctuations

of the major carotenoids over 12 months (Table 1 and Fig. 8) showed

that there was a general trend for a decrease after the first few

months. This trend was demonstrated more clearly by adding the

concentrations of all carotenoids together and plotting the T/G

(Fig. 9).

When the results for the 12 months were added together lutein about was shown to be the most abundant carotenoid, representing/^^/ of ■

the total carotenoids (Table 2). p-carotene- was 30/, violaxanthin

14/, neoxanthin 11/, auroxanthiu 3 % and 5*6-moaoepoxy-p-carctene 3/*

However lutein was not always the most abundant carotenoid in the

monthly samples, e.g. in February, June, August and October, p-

carotene was the most abundant. In December, March and September

violastonthin was the most abundant. As an increase in xanthophylls especifi^^ epoK^-~xautii

that the percentage xanthophylls stayed relatively constant at

between 63 - 83 /. From tils data no apparent oxidation had occurred

in the 12 month cabbage plants.

The individual changes of carotenoids were most noticeable

during the first five months (Fig. 8). Carotenoids such as neoxan­

thin and 3*6-raonoepoxy-(j-caroteue allowed an increaoe initially and

then a gentle decrease thereafter. The other carotenoids showed a

steady increase starting at between 3-5 months. There was also a

slight increase in the concentrations of some carotenoids during

the last three months. Most noticeable of the changes was a drop 65

ru co 03 OO K> r- tA lA (N ru Cs vn UA O VD> O O :A lA

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ITM K\ o O CO vo OJ q i 0 \ ru o ir , lA .9 » o \ r û i VO ir \ ■:}■ O VDl r>J K\ x - l

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t I I a

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Tj ü •rl c c O o o C *J H I g o o II k u O ï g ï ü Y vü :3 h 5 ô a r r 11 66

-d" A- UA -d" A- Q CM OV c'O A - VO vo t A O A- MA § >A O -d- Ô O l > R (M Ov vo CM O O MA v o o C3 CJ O O O O t A o -d- r j -d" OO O Q O'V l A MA l A v o

r j i UA CM rj OV AJ 0 3 T - 1 tA VO C v VO Rr r - c o 1 •1 1 • 1 O vI UA ov o l O v O MAI t A t A VO

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CM MAI Q V - MA Ov CM UA LA MAI OV O UA c o -d" a a UA • I 1 CM A - l MA O LA CO UA t A MA O v O MAI MA VO

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V - 1 OV o c o 03 VO OV J - l CO MA A - VO UA 1 • 1 1 • 1 C O 1 -d- - j - CO r - MAI rvl MA VO

t A -d- O l O 03 UA A- -et" n j i A - UA 1 1 • 1 • I • • O tA I O -d- O v g 5 - d - l r - UA

A- UA O CMI A - A - A - l A CM O P CM MA IX n 03 VO Ld- LA IN k • 1 • I • • Ov Pi O A- O 0 3 1 Ov (AI R CM < MA t A I T - t A v o r A- (3v vo tA A- OI OV Ov CO O -d- CO OO B ITv 1 1 • 1 I I -d* r j Ov -d" â CM Rr MAI CM X4) H UAI CM OV G) UA UA OVI co O CO 5 8; O v O > Xi 1 • 1 1 • 1 1 O o -d" 1 CM OV A- -d- LA t ; Ph l A I MA v o

O UA COI A - UA R UA -d-| -d- LA K\ . P . • I • • § ' & Ov MAI MA T- V UAI V-

UA i>- irs CA VO A - l O c o r - MA T- O CV O l & O A - C <\J • • «I C> CV O KA fvj O (V O O A - V - 0 3 1 o vo MA A r - X T - r * t - | Rj r - CO

OO I Ov VO V-1 CV MA R • I • • ' ! ' MA Ovi v o MA MA MAI V- 'Ïa vo

Ov O VO -d-1 LA Ov VO J - CM CM VO -d- O fM A- » Rr lA I O -d- O v CM VO CM T- -d- LA • I • • o rj o MAI CV v o fÙ MA O O CM A - MA -d-1 t A VO

-d* 0 3 OV l A -d- O MA A . (M r - OO

o v 0 vo g ) MA UA -d- 1 MA vo A- J- VOI -d" A - tA OA UA VO tA > 0 - 4 ) . . O G v O VO VO MA z: T- :3 V - CO TJ !• •H O ri g +> O II Î3 e x o 1 (U i '1 □ T) o c a +» o P i g XI T-> -H r i ri o G> 0> o 0) r i O CL, O 3 ri o O o o ri ri I â Q) O O 9 P •rj -p o o o •rl ri 0 h h d E c •U o x> x> E 0) •rl ri O d d C) 1 ri 1 r i A d 1 O h ü Y N VO 0) VO d >ï +> v o ri 4J q d 1 1 1 o u ri r i —1 r i 1 o es CQl e x LA UA O i: u *-4 II I! 67

d Gv OV MA d UA A-O fA I? O W Gv 1 1 1 1 V- OV lA CO ‘O (AJ d vo CO Gv UA tA >9 I +> -d vo -d- d - UA UA Pio IfA T- MA f\j O V-

GV CO O CO CO d vo MA -p fA (AJ UA A- CAJ d CO w (A) vo 1 1 CO O 1 1 1 1 1 UA cc O 1 (d (AJ (AJ Ov UA lA A- d vo O I OJ

Ri O UA UA UA OO lA O A- rj O- UA q A- «M o I OO 1 (AJ P 1 1 1 1 T- CAJ 11 A- CO (AJ d o o tA GV W 1 CO CO CM MA d tc rvj P •H ÎO o 0in

1 ?o •d VO MA UA O Gs UA VO r- o MA § O V- I OO vo (AJ d - •HP MA

(Q

P CO MA •H M a 'R 8 8 A- (U to fA OO DO I I UA O M no GV A - A- OJ •H A- UA d lA O -p I I Gv i S o I ri UA d VO o A- Q (AJ d MA (M o CO O VO o lAJ CO MA vo 1 1 1 1 1 o fA UA 1 1 VO (M X! OV vo CO A- OO O VO AJ d CO d

p 0 0 d p O 0 ri -p p ra o o ri ■g 1 cS -p c x o ri 0 ca rH P X 1 0 0 o a I 'd 0 o +> 0 P i X o +> a •H p P o P 0 a (U I 0 0 ri 0 o P i 0 +> +> ra -p p 0 Ü I p o o t) o o o o P -P1 H H (3 ri a •d 1 •H O (3 3 0 3 1 1 d P i I Q> O O o « O vo vo -p vo -P I 1. 1 t ri - ca â d cx UA UA O UA 3 Ü I. § 68

Fig. 8

CAROTENOID CHANGES IN B.OLERACEA WITH AGE

1200

KEY • LUTEIN o B-CAROTENE A VIOLAXANTHIN A NEOXANTHIN ■ 5.6-MONOEPOXY- B-CAROTENE

Ô 400

10 11 12

AGE IN MONTHS 69 Fig. 9

TOTAL CAROTENOIDS IN B.OLERACEA WITH AGE 7000 o spnng o summer A winter 5000

■D

P 3000

1000 .

1 2 3 4 Age in weeks

TOTAL CAROTENOIDS OF B.OLERACEA OVER TWELVE MONTHS

3000

D 2000

0 ) 1000 O)

a * a

J-----1____ 1____ L 2 3 4 5 6 7 8 9 10 11 12

AGE IN MONTHS 70

In lutein and p-carotene in the 1- 2 month with a corresponding rise in violaxanthin* A subsequent drop in violaxanthin in 2-3 months was accompanied by a rise in lutein#

It was interesting to note that the p-carotene concentration of many extractions of the outer leaves of B» oleracea greatly exceeded the value for outer leaves given by De (19^6) and for the range in ’carotene* content of South American Brassica species given by Goodwin (1952a)#

Winter grown seedlings grew very slowly compared with spring or summer seedlings# In fact 4 v/eek old winter seedlings were equivalent morphologically to 1 week old spring seedlings# Thus in comparing one week old winter and spring seedlings the size and development of the seedlings must be borne in mind# When the seedlings wore small whole trays of seedlings were used for each sample* but as the seedlings grew larger, less specimens were used#

When mature only the outer leaves were used# In all camples sufficient material was gathered to make good extractions#

When carotenoid concentration of spring, summer and %"/lnter grown cabbage.seedlings in the first four weeks of growth, were compared* it was found that in the first week the concentration in the spring groîv'n seedlings was considerably higher than in the other two

(Fig, 0 , 10 and 11)# The winter sown seedlings had the lowest concentration. At the end of two weeks the concentration of carotenoids in spring and summer seedlings fell to the same level#

In the winter sown seedlings the concentration while still rising remained the lowest# In the third and fourth weeks the three groups of seedling had similar concentrations of T/C# The large drop in p-carotene in spring seedlings in the second week was not accompanied by a significant increase in any other carotenoid# In Fig. 10 71

CAROTENOIDS IN WINTER GROWN BOLERACEA

@ lutein o B-carotene

1000 .

CO -g o S

u 500 O) ?

1 2 3 4

Age in weeks

CAROTENOIDS IN SUMMER GROWN BOLERACEA

1000

-g o s CO 500 o

2 3 4 Age in weeks o B -carotene a violaxanthin ® lutein a 5,6-monoepoxy- g-carotene A neoxanthin p aunaxanthin 72 Fig. 11

CAROTENOIDS OF SPRING GROWN B OLERACEA SEEDLINGS

4000 ^ (3-carotene lutein violaxanthin neoxanthin 5.6-monoepoxy- (3-carotene 3500

CO -O o c CD 4 -" O 1000 (Q o

500

-a

0 2 3 4 WEEKS 73 the second v;eek in the Buramer seedlings the concentration of

^-carotene, lutein, auroxanthin and neoxanthin dropped end there was an increase in violaxanthin*

The percentage carotenoids found free and hound to protein in cabbage was investigated in two experiments in which the outer leaves of two plants of identical age and appearance were used

(Table k ) » Although the total concentration of carotenoid in the plants varied by almost a factor of five the percentages which were free and bound were similar. The mean percentage free was 23*09 compared to ?6,91 bound to protein.

Table 4

Free and Bound Carotenoids in £. oleracea

(outer leaves only) Concentration vfr./ff Percentage Free Bound Free Boun d Experiment 1. 202.22 798.35 20.20 79.80

Experiment 2. 1301.62 3703.12 23.93 74.01

Mean 23*09 7^*91 74

P. brassicao

The weight of the insect at yarious stages in its life cycle was detennined (Table 3» Fig* 12). The different stages used were not taken frc(? the same batch of insects so a continuous growth curve cminot be drawn. However, the results indicated a rapid increase in weight from the ova to the stage and a continuing, but less rapid increase until the pharate pupal stage when feeding stops.

14 of the carotenoids identified in 3. oleracea were found in P. brassicao (Table 6). All were present in the adult stage though not in every sample; five of them were found also in other stages, p-carotene, lutein and 3,6-monoepoxy-p-carotene were the most abundant carotenoids. p-carotenelwas detected in all stages except Day 1 larvae, where a large enough sample to detect individual carotenoids was not available, p-carotene and lutein were in high concentrations in the ova, and the T/C in terms of pg/g of the ova exceeded that of all other stages in the life cycle.

During the first two instars p-carotene appeared to be the only caroteuoid present. It was possible that other carotenoids were present in these early instars but in such small amounts that the sample size was not large enough for them to be identified.

Aurochrome and two unidentified carotenoids allefound in

B. oleracea. were not found in any of the stages of P. bracsicae.

Comparing the caroteuoid concentrations of different stages of P. bramricae can be misleading because of the enormous variation shown between samples of the same stage (for instance 10.97,

34*03 and 123#s/g) (Table 7, Fig. 13). Statistical analysis of these results by the Pearson’s fange Method (Snedecor, 1959) was not helpful. The data was therefore expressed as the caroteuoid 75

Table 5,

Mean Dry weights of all stages of P. brassicae IBS

Total number Stage How weighed weighed .SÆ , s s r stage

Ova In 3 gps,$ 40, 30, 30 100 0.003 ± 0.001 In 2 gps., 20, 20 40 0.065 ± 0.010 early Individually 20 1.20 0.224 early M 10 2.23 0.336 early It 10 3.47 0.228 early M 10 11.49 0.790 late H 10 39.88 5.452 PP early M 10 73.50 8.274 PP late tl 10 70.92 8.754 P early ft 10 62.75 4.838 A tf 10 40.60 6.136 A 99 H 10 37.49 3.864 76

Fig. 12

GROWTH CURVE OF PBRASSICAE

100 • •

10

E fZ 10 o) 040 o

001

ova Lj Lg L3 L4 L5 pharate pupa adults pupa Time 77

A l CO o -d- NO -d" AJ 0 3 A- -d" OJ OJ A- OJ 3^ a j A CO O -d* co r - r j kO OO G\ O OO O VO AJ f A T- AJ o o rj ON fco l A CO ON r CO co ON Q CT' NO A A- -d- lA ^A co tA OO O 03 O A- AA- co NO O O -d- O OJ o lA rj OJ ; - -d- rj A Rr

ON -d" A c o A NO A A- K\ 1 NO . 1 CO . . . . lA .. OJKN à a tN I I I CA

lA NO NO o q g . (A I I I I . tN . . c\J ' 'iR tA

o iTv r r \ X \ I I I S-N

CO -d.

S r- 8O OJ il H) .• É gl s f " P co g -d- I co g o (U « . p G o 0) p g o P u O 1 1 d P CQ. ü -8 I 1 •ri o (Cl. 0 G !? 1 o I g •O B 13 o h» Jd E O 'Vj o 0» a P +> *tJ (U CO X p O Pi i l O O o •H G G G o o OG ü 3 -p O O ü o H o a, 3 O d C PP p o G o o G u U u O O o o o o O O 1 O d o ■H ■P H h d c '4 p p e ÔO ■p x> iH O d d o . . d 1 II d o E Pi U ü o o i j NO NO >3 NO 4-) d 3 O d 1 1 1 U 3 n M « ü d co. A A o A y 78

Table ?,

Concentration (uR/g) and content (tir:/irisect) of 0-carotene and total carotenoids in all stages of p. brasslcae. i^ased on dry weights in Table 5)

p-Carotene

Stage *g/g Vg/insect Eepiicates 1 2 3 1 2 3

OVA 201*60 280.00 212,80 0.00061 0.00084 0.00064 102.60 83.20 0,0067 0.0057 52.00 89.28 0.0624 0.107 106.12 88.57 0.237 0.1975 0.1484 0.1485 0.346 4 42.79 42.79 99.85 123.41 10.97 34.05 3.17 0.281 0.875 PP 162.02 35.99 51.95 11.69 2.599 3.751 P 33.64 91.97 44.17 2.111 5.77 2.77 AdkT 57.14# 79 .82 * 2.32 3.24 A99 78 .20# 40.84* 63.94* 2.932 1.531 2.397

Total Carotenoids stage OVA 468.50 511.31 350.00 0.0014 0.0015 0.0012 102.60 83.20 0.0067 0.0057 52.00 89.29 0.0624 0.107 137.32 150.32 0.351 0.335 118.81 99.83 0.263 0.412 0.346 4 73.73 205.95 60.02 34.05 5.291 1.542 0.875 PP 258.81 68.05 72.48 18.69 4.914 5.234 p 51.53 155.81 143.02 3.234 9.777 8.974 tdçS 241.69# 188 .29* 9.813 7.650 A99 245.00# 97 .96* 114.70 ® 9.185 3.673 4.30

• Freshly emerged ® One week old when killed 79

Fig. 13

TOTAL CAROTENOIDS 6-CAROTENE (pg per individual ) 100 CONTENT

10 o o 9 o 0 0 e ® e o 10 & o o 8 «* s o

•01

■oon I»

J______L ova L/| L2 L3 L4 L5 PP pupa adults life history 80 content per individual and plotted against time of life cycle.

All replicates of each stage were plotted. When the p-carotene and T/C concentration of larvae was determined no differentiation between early and late larvae was made. Those chosen were, however, more late than early. The ^-carotene content per individual was therefore calculated from the product of the mean dry weight of early and late larvae and the mean P-carotene concentration of the larvae.

The scatter of the points showed that there was an increase in p-carotene content from the ova to the pharate pupa stage when there was an apparent fall and levelling off. By overlaying the

T/C it can be seen that the same trend is observed* The overlap of the two sets of points shows that p-carotene comprised the major part of the carotenoids present. 81

Marked differences were shown in carotenoid concentration and content in adult samples of P. brassicae (Table 8). (SMzarotene,

5f6-monoepoxy-.p-carotene and lutein were present in all samples while 15 out of the 14 carotenoids were identified in the sample of 2,500 adult specimens# 4 carotenoids were found exclusively in this sample# The large size of this sample made it possible to

Identify carotenoids which were in trace amounts and would not be identified in normal samples# The carotenoid not detected in the large sample was 5,6-*diepoxy-p-carotene; it was found in one sample of 100 females#

Specimens which had died naturally in the laboratory (sample

1-4) and which had been stored in the laboratory at room temperature until sufficient numbers had been collected, were found to have lower concentrations of T/C tloan fresh material# A sample of fresh males had 241 #69 Ps/g while one of females had 245.00 pg/g. On a per individual basis these females had more carotenoid than the males# However, the males in sample 5 had the highest individual carotenoid content of all the adult samples, 7.66 pg# Live females up to one week old, some of which would have oviposited, were found to have 3*$8 and 2.05 pg per Individual. The T/C content per individual ranged from 1.42 vg for a dried sample of mixed adults to 7.66 vg for a sample of one week old living males.

The relative percentages of free and bound carotenoids in males and females was investigated (Table 9). The samples of 100 males ; and females were from dried material collected in the laboratory as previously described. It was assumed tliat the majority of the females had oviposited. Female adults were found to have an average of '$0*73% bound carotenoid, slightly more than males with

22.71 /. In these dried samples males had a greater concentration 82

>>'d H d AJ 2 hO O AJ o 5 W G AJ O AJ 8 O d 1 • l O p E O O co 0 \ P m ü ON A- co AJ

Xi ,—1 A- OA- VO CN -d- r j tN co d .X O vû 0\ lA rA R S' > d •H d VO O O A- rA VO AJ AJ 8:Xi 3 AJ VO fA

"d 1—1 lA o VO lA fA -d* fA O VO o AJ . VO d VO O co ON A> lA VO tA AJ ON > d O •H d O tA O O ON VO ON AJ co fA 8: Xi 3 &

lA VOc'j d S OJ lA ON u G o VO VO o o VO A- -d" rA O VO lA ON co -d" :8 n AJ

w -p •g A- g AJ lA VO VO 0 \ VO g M OO OO CO O AJ VO cd d O 1 • 1 1 • ' • d O -d" O ON lA lA AJ CO CO mc3 IT\ 3 A- fA r* ü Vi w VO CO ON z O CO AJ A- LA O A - AJ r - ON 0 1 O AJ ON CO O Al VO CO ON fA AJ lA ON o M X R • 11 • •H o A- AJ O lA AJ O ON o AI VO "3 PL, g c\T AJ g •S U) VO 'd d d CO fA -d- A VO •H d d AJ O fA ON o X •H O 1 • I 1 • On Al id G OO fA A g O AJ AJ AJ VO +3 o _d- ü lA 'd •d lA (\J -d" co fA fA QO AJ (D rA O VO VO VO A -d" X •H • 11 1 • •H vO O ON co V- N a

-d" VO xS d ON fA ON d d V- O Rr lA P X •H O I 1 I I •si U O oo O rA A- AJ Q lA AJ R? VO â p bo hO 4-> d d O p d O 0) O O d > ■ § g w G p ra d. A d O H .g ü Ph w d g *o •H CQ. ü w •H g t 3 O g «H «H d C i. rH P -p •d O o c k 1 d A g d 0 d O >> S d P g u d o p Pi iN O • | •H O d X •ri O d OG p X 3d G «H bO W O I g g U O Pu 3 P p d o d "H *H +> •p -p ti d d o d O ü T) ü d d g o o ü O d o d d d s 5 3 +> pj E d p •H X •p o d 1 1 d d h o d dd "H> d >» i

Table 9*

Free and bound total carotenoids in P. brassicse adults

Concentration Stage Percentage (vs/e) Free Bound Free Bound

Dried Females 100 (i) 43.54 21.88 66.55 33.45 100 (ii) 56.08 21.81 71.99 28.01 Kean 69.27 30,73 Dried Kales 100 (i) 141.49 33.37 80.00 20.00 100 (ii) 128.41 45.80 74.57 25.43 Mean 77.29 22.71 Dried Heads 500 (i) 125.30 40.80 75.44 24.56 (sexes mixed) 250 (ii) 45.20 51.16 45.78 54.22 Kean 60.61 39.39 Wings and 100 (i) 41.37 41,66 49.82 50.17 Bodies 100 (ii) 555.00 147.00 79.06 20.94 Kean 64.44 35.56

Carotenoids identified in free and bound fractions of 100 wings and bodies (dried)

Concentration Carotenoid percentage ( m / s ) Free Bound Free Bound p-carotene 15.48 1.66 57.90 12.60 5 ,6-mono e po xy-p-c ar o t en e 9.86 5.40 56.90 25.80 lutein 1.36 5.40 5.00 41.00 Kutatochromo - 2.68 - 20.50 Neoxanthin - trace - - 84

üî T/C than females# Carotenoids free and bound in the heads and in the wlnga plus bodies were also investigated in two experiments using similar material. The percentages of free and bound in both parts of the insect were essentially similar.

The constituents of the free end bound fractions, in one sample cf wings plus bodies was analysed. In this sample p- carotene represented the major carotenoid of the free fraction

(57.90/) while lutein was the n?ajor carotenoid bf the bound iYaction

(41.00/)# 5,6-monoepoxy-^-carotene represented 56.90/ in the free fraction, greater than lutein (5,00/)# Kutatochroa© end neoxanthin were both in the bound fraction only# The T/C concentration cf this sample was much lower than ssmy of the others and this apparent difference may be because the sample was a particularly old one#

Cf the 15 carotenoids identified in the wings and bodies cf

Pm brassicaa three were identified in the heads (Table 10)» Koct abundant of the carotenoids in the wings and bodies were p-ceroten© and lutein. A surprising result was that violaxanthin represented more tl'ian 70/ of the T/C In on© of the bead samples. Vhien the exuviae of two and two pupal samples were analysed p-caroteno was the only carotenoid found in three samples. In the other sample lutein was also detected and represented about 4C/. of the

T/C. 85

O 5 s t t I t I I t t I I f I 8 m. o°.

& <30 R 8 # i • i I I I I I I I f ( I • • « 8 O c; •H Tî (ü

« -3 3 8 P 0‘ I 1 ( I t & Æ ■ î & î (O' i ON N R ÎH t I I # I t i 1 t: o (U I rî s «ri «

II OJ -îÿ- OJ Q o tA co (A o lA Q fA 1 !• 5 > 04 C> o 04 c%j VD - co - cb ' O ÎA B * * * * • • • • » • • . O f ON r~ « % o co CM r- ü o o 61 Il oT ê V£> •H : ir, %- T3Xi *r) C I Ê *o 4^- ■§ 4) C tf) «H w 8 Ê 5 «> a I M •H

I •o 3 «H VJ I •H U O o 44 0 4* •KS 4 > O s & •H S g - 0 g 0 q «V 0 X i « 0 •H 0 4 4 ë » S -P €> o © 44 Ci, 44 Q t i 4 a • f j p 4> P» K kl K 0 0 g) 44 xi 44 % Cî O 0 H 0 0 0 g 44 3 g i 0 o •H W <4 44 0 r; X V 0 8 p g 0 w O C U Ü O O 0 0 0 0 0 0 0 R 8 «y O © 7i h 64 6i ii « 44 P G «H d X H P4 3 q Cî 6 1 Cii d 1 w fH: 0 0 ü V u fcï VO >> p VÛ 4» 0 0 h k 4> 3 1 4-> «ri © o vJ AP I I H A VA 6 S VA 5 %» Ï 4 0 H 86

Feeding Experiments

The results of the feeding experiment in which the food intake and frass output of 100 laryjae throughout their lives was weighed and analysed are given in Table 11# From these results the amount of p-carotene assimilated by one larva in its life can be calculated as follows:

Total p-carotene ingested 13,548.95 pg

Total p-carotene defecated 5,770.4? pg

# % total p-carotene assimilated by 100 larvae 7$778#48 pg and total p-carotene assimilated by 1 larva 77*79 VS i.e. 57*41/ of the total ingested is assimilated*

The percentage assimilation of p-carotene started low during the stage but then increased to 88 .85 / at the end of 1^.

During the stage the percentage fell to 53*90"/ and then rose to 70 .15/* More p-carotene was ingested in the 1 ^ ^ stages than at any other time. The amount of p-carotene defecâted by larvae was small, giving rise to a peak of p-carotene assimilation at the end of (pig. 14). When the results are cumulated it can be seen that most of the p-carotene was assimilated after (Table 12,

Fig. 15),

The cumulated amount of p-carotene assimilated per individual was compared with the highest figures for p-carotene content of the individual stages, obtained previously (see Table 7)* Tlie amount of p-carotene assimilated was much greater than the amount of p- carotene in the body (Table 15, Fig. 16), and the difference between p-carotene assimilated and p-carotene retained in the body should represent the amount of p-carotene metabolised. For each instar the percentage metabolised varied from 54.55/ for to

99"25/Iiu 1^* Even if the highest value obtained for p-carotene 87

fo r - CO (\J O O VD ü rvj IT\ co (\j Cu T- 60 lA O -G- s îA

g •H g 1 Xi o o bo lA CO t v co CO d> o c3 0 >. lA K \ P fA lA O - > o 60 §•8 VO ON OJ fA Q LA LA U O p . co V- o ,y- fA -4- VO CO On CO r - tO a lA lA fA o o 1—1 (U VO r - r<\ A - o co co lA fA cî II V- A- O- CO tA ,4* R OJ o to 0 u d 44 03 1 to o o cd c a (d •H G G W 3 44 (0 O td » d u ca-H XI

Oi • o V- O o p o x i OJ VO -d- Ov lA d) X 44 û> t - co co ON Cv o o rH 4> o 44 60 ^ ° OJ VO o o A . VO A - OO rO -ri P (0 p. OJ OO lA -4- co CD co S 8 o c o co OJ A- co ON -4- cd 5 3 o lA lA [N. TA ,4- Eh o 60 a S OJ r- OJ J- lA OJ CO A - co 44 1 d tA LA f\J " R ON ON R ON O A - 0 c a -H hA P LA CO CO ON fA A - o OJ V A - E ÿ 1 -iH c û . q j G O

♦o o - LA CO fA tN +> «H o 60 60 OJ .4 - OJ VO OJ -T-l 8 ° Fg O O OJ J - ON LA VO ON OJ IA VO O O O 60 ^ 44 5 cd ON fA ,4* ON X V g On VO O A- fA >i X C Q . 4 4 ROJ fA ÔJ G cd fA T - Q O g i O

OJ OJ fo IA kT a fT' ^ w V- fA LA [N. ON fA LA A- ON rH A - ON C'J A - ON O I t I 1 > • t I I • ' 44 V- co OJ *4- VO co vi c& OJ -4- VD co S I H 88

Fig. 14

FEEDING EXPERIMENT WITH 100 PBRASSICAE LARVAE

5500^

5 0 0 0 ingested assimilated defecated

O)

3 0 0 0

0 U I CQ

1000

8 10 12 14 16 IB days

L4 L. age 89

Table 12.

3-carotene p-carotene p-carotene ingested defecâted assimilated Stage

VS VS

1- 7 1, 1.2 172.94 142.92 50.02 8 - 9 1,021.04 393.16 627.88 10-11 2,421.16 692.43 1728,63 12-13 5 ,677.02 1056.14 4620.83 14-15 L5 10,851.86 4360.21 6471.65 16-17 13,109.96 5639.45 7470.51 18-19 13,543.95 5770.47 7778.43 90 Fig. 1 5

B-CAROTENE INGESTED. DEFECATED AND ASSIMILATED BY 100 PBRASSICAE LARVAE (cumulated ) 14000 r o Ingested © assimilated A defecated 10000

O) ZI 0 c 0 2 6000 0 (6

2000

2 4 8 10 12 14 16 18 days

Li b Lq L 4 L-5 age 91

Table 13.

p^arotene assimilated per larva (pg) and p-carotene content per larva (pg) (cumulated)

p-carotene p-carotene content p-carotene Day Stage assimilated (see Table 7 metabolised highest values taken) US H %

ova 0.00084 1 4 0*0067 7 0.30 0.107 64.33 8— 9 6.28 96.23 y 0.237 10-11 17.29 0.346 99.25 12-13 H 46.21 14-13 64.72 3.24 16-17 y 74.71 95.83 18-19 77.79 92 F ig . 1 6

6-CAROTENE ASSIMILATED AND BODY CONTENT OF ONE PBRASSICAE LARVA (cumulated)

100

assimilated

/—\

0) s body content

001

0001

ova 2 4 6 8 10 12 14 16 18

Li L' L 4 age 93

content per insect (11*69 vg in pharate pupa, see Table 7) is taken

as the amount in the insect, the larva during its life cycle must

metabolise at least 66*10 pg p-carotene, tliat is 84*97^ of the

amount it assimilated*

It can be seen from Table 11 that the concentration of

p-carotene in the frass dropped during the stage even though

the amount of p-carotene ingested continued to increase.

Frass from larvae kept in the continuous culturing system at

I.e.I*, was analysed for p-carotene (Fig* 17). There was a noticeable decrease in the concentration of p-carotene in the

frass during the stage* The peaks presumably represent periods where little p-carotene is being assimilated and the lows where it is being assimilated at its greatest rate* The second low at 24 days could be the result of some larvae in the ©ample of predominantly mature larvae being still in the stage.

Tills is possible in samples taken from the continuous culture cages at I.C.I.

The p-carotene assimilated by L, •. and larvae was investigated in tliree identically designed experiments* Cabbage leaves from plants of similar age and similar appearance were fed to three different sets of larvae. However, when the plants were analysed for carotenoids it was found that the T/C varied from

229.06 to 800*15 Ig/g (Table 14), The carotenoids of the cabbage with the lowest T/C were all lower relative to the concentration, of the same pigments in the other plants. The intake of carotenoids by the larvae in the throe experiments was therefore different*

An interesting feature of two cf these experiments (1 and 3) was that more p-carotene appeared in the frass during the stage than was ingested (Table 15). Also the buU: of the frass defecated 94 Fig. 17

(3-CAROTENE LOST IN FRASS OF PIERIS BRASSICAE

300

INSTARS

-b 200

O)

Û:

LU

100

4 8 12 16 20 24 26 DAYS OF LARVAL LIFE 95

Table 14.

V£/S

Expt* 1 Expt. 2 Expt. 3 Carotenoids v&/g % ve/s % vs/s % p-carotene 425.77 37.61 800.13 35.79 223.06 56.92 5 t6-raonoepoxy-p-carotene 21.39 1.83 20.91 0.88 19.44 4.91 Violaxanthin 48*00 4.14 292.31 12.34 - Lutein 323.63 27.94 936.82 39.56 62.76 15.87 Neoxanthin 143.23 12.37 297.43 12.36 ~ - Flavoxanthin 149.33 12.89 - 75.11 13.99 Auroxanthin 37.04 3.19 20.36 0.86 13.00 3:29 T/C 1133.41 100 2368.00 100 395.37 100 96

VO LA -d" -d- IN OJ VO CO OJ d 1 -p rA 00 lA VO LA o IN LA LA p - El IN lA OJ

lA cT CO o -d" Q OJ fA -d- ON O IN lA -P p) hA -d" OJ VO lA ON On d O VO ON O OJ o IN LA ■d" OJ s •H d lA VO -d- O O 0 rj IN OJ VO VO fd X p) o O VO O O H lA lA VO OJ

CO d o OJ O LA IN LA VO IN lA pj O VO CO VO ON VO d -H IN -d- O d 1 Ü 10 PU -p -d- VO IN oO I « o 1 -d" VO CO CQ. d «M EH OJ ■d" IN o LA OJ m d OJ v p OJ -d- O CO -d- LA d u lA l A VO OJ OJ -d- -p d •P TJ to -p d lA rA CO OJ O 00 CO o a> d to o B' -d- O m VO CO d "H S' f-i d R -d- 0 to OJ d o h I O -d" O L*A IN VO lA 1 to if\ rH H OJ O -d" OO lA LA AJ d d d 1 ^ S IN •H d -p OJ LA lA CO 0 *H -d- tH o VO VO ON 1 o E EH ON CO CD. t 3 to O •H f-4 OJ CO o Q OJ VO O ON LA O o O LA O O lA d •p p I OJ CN IN lA -p d lA O LA (\J O o o CO VO VO E S S T) r \j CO •H lA - P Q) VO r\j O d ON O ON Q O O -P hO d 0 CO OJ O Ji w d- -d- oo lA o -d" d o IN IN o •p R pl o OJ O fA CO OO o to r r\ o M CO O IN -d* d VO A.: d u 1 LA lA o rj -d- Q CD. ON O -d- CO OJ LA lA Pl O IN O VO o VO -d* -d- LA N- fA VO PI PI PI (p tp (p to 63 o o O •P iÛ to to t_3 60 •H P- to p . p . rH d PL d d> p p P o o O 10 EH Eh EH tJ (0 C O d o o d •fH nd TJ «H tp P 0 0 O 0 d 0T i 0 P 0 P «H rd d •d

during was greater than the weight of food ingested* These

two points suggest that gut contents may be present throughout

ecdysis* some being defecated in the following instar. When the

gut contents of ecdysing larvae of 1^^^, ^3/k H / 5

examined a considerable amount of green material was present.

Larvae do not feed prior to ecdysis and indeed ecdysis only begins after digestion hae finished (Chapman, 19^9)*

If ingested material is always carried over from one instar to

the next the figures for assimilation in each instar in this type of experiment are necessarily not easy to assess.

The percentage p-carotene apparently assimilated by ^ larvae in each experiment varied from 18.52 to The amount assimilated was not correlated with the concentration of {3-carotene in the foodplant. If the results of each instar in each experiment are totalled and compared, the percentage assimilation at is lowest at 47 .52^^ and highest at with 76 .I7 &» This experiment would have to be repeated with the same ten larvae followed through their L^ ^ stages, before the significance of the apparent low assimilation in can be determined. In contrast with this experiment the results for the experiment with 100 larvae showed that the greatest assimilation occurred at L|^*

The amounts of other carotenoids ingested and passed out in the frass were investigated in two of the experiments (1 and 2)

(Table 16). Frequently more of some of the carotenoids were present in the frass than apparently was in the ingested material and so assimilation, if any of each carotenoid could not be determined accurately.

Flavoxantbin, which was detected in the foodplant of experiment 1 was not present in the frass of all three instars. 88

Table l6.

Other carotenoidsi inrrested and lost in frass (vs)

5rd Instar 4th Instar 5th Instar Carotenoid I F I F I F

Experiment 1 5,6-niono epoxy-p- 4.06 14.40 40.85 50.24 carotene 2.25 - Viclaxanthin 5.23 15.39 9,12 54.04 91.68 475:75 Lutein ■ 55.59 15.67 61.49 97.55 618.15 749:66 Leoxanthin 15.75 16.42 27,21 9.68 273.61 219:20 Flavoxanthin 16.42 - 28.57 - 285.22 - Auroxaiithin 4.07 - 7.04 82.96 70.75 31.20

Experiment 2 5 16-monoepoxy-p- 2.71 7.20 14.70 7.94 carotene 8.99 72.55 î'utatochrome - — - - - 13.64 Violaxanthin 58.60 6:08 125.69 - 1011.59 203.20 Lutein 121.79 : 402.85 161.60 5241.39 352.20 Keoxanthin 3S.67 - 127.90 53.00 1029.lS 172.00 Auroxanthin 2.65 6.75 - 70.45 -

I Ingested F Frass yr-eae-nt in the pXsnis cf W l b eXj'tî-rliîtènts %%s

&ba&at La frasa t f 1^ La the fixs't «xX'-erLis^e^it &zL wqia at'Sent frj#

&11 insrtâjrs La t't^ secsaL ex^erLs'e^t. 5 ,&-:'%a-c-epO'.3;y'"T'^-."rQ

&bees.t fr-2# t-V« Ixaa-s € î L^ of 1, &aL Tl,?Laxaiit,bl.s v«îs aiL-siei-i Ltchis tb?© fras^’cX 1.^ La #%^erii;yent 2# ialeia &r,i rejaEanthLa w^ana £b.:ae.a.i Lroa the fra-as *f "L, La the s««ea4 e.xpexiiT'ent»

&'utatochrc»iie was the c&lj carclea^LL ta te fo-uad La the frass %ad mat Lm the pL&at^ s&\^TC &L? jc,.'iL^ 100

vitamin A

Samples of vitamin A potent carctenolds foimd in P. bra^?icae

were chronyritogramod on. TLC with loiown samples of vitamin A

derivatives ond sprayed with antimony trichloride (Table 1?).

With antimony trichloride carotenoids gave a similar colour

reaction to the vitamin A derivatives.

Chromatography of extracts of P. brassj.cae heads and bodies

on TIC and s^iraying with antimony trichloride demonstrated several

spots (Tables 13, 19j fig* 18), Yellow coloured bonds jæcbably

represented carotenoids but they were too weak to elute and

Identify by normal procedures* Six yellow bands were found in one

sample of heads and bodies, while five were foundih the other

sample of heads* ^-carotene, and 5*6—monoepoxy-|3-carotene were

identified from Ef values (Table 17). The spot with the highest

Ef value was present in all samples and was probably p-carotene

with, or without a-csrotene# It was very difficult to separate

these two carotenoids in this solvent system. The xanthophylls

lutein, viclaxanthin and cbrysanthemaxToithin plus flavoxanthin did

not move in the solvent so that the yellow spot near the origin

probably consisted of a mixture cf these carotenoids plus other

polar carotenoids. As the solvent was c ho .sen because it gave &ood

separation of the vitamin A standards, other vitamin A potent

carotenoids found in P. brassicae were cbromatogramed. The Ef

values obtained indicated that possibly mutatociirone and p-seacsrotene were present in the extract of 100 heads (Table 19). However, because the information gained solely from Ef values could not be backed up by spectroscopic evidence more information would have to be obtained to rrvike more positive identifications.

As vitamin A alcohol gives a similar Hf value to 5,6-mon<^p- carotene it was difficult to distinguish the two on the evidence of 101

I 1 &.§ I H :iï I I 44 45 a O o s o © o O O 00 o 5 5 S « S a a k; % % m I g •ë pI O 4> g ON -=»• CN ON fA OJ -d* 8 iS i? R .4 « # # C 0 0 0 0 o o O o B 0 I g H ^m »H ë B ;C p o rrv tr\ iTN r- co 3 p d 0\ 0\ O (A T- 8 8 8 ' S ' ÿ o o * • • « • • • 0 0 0 0 0 o o o o 1 *o H w R PAO E A«o « p < o 0 g K 1 rc

1û.’ •P

Oi r« VD co tN vo 4P s A tr\ ON fA R !X 9 s os PCi 0 0 0 0 0 o o o O O E

q © a ■g © pes p §m 0 S3 Ci I A E c a M «f 1 U o g © R o ë 5? q © M *W B Q © B M *3 a O © © p4 p 0 0 ro © 0 * '5 g § 0 H h ‘45 0 P p P r t © 0 0 5 0 O 0 0 0 E d p S 1 © © N © 1 ië i VO N p p I Ÿ 1 3 EH S > 3 I lA 00. «a 0 5 > > 102

O G

2 d o rH T3 1 O •H c% 4 : O 1 o GN u O X rH O d d O 04 k o C d o d d o .H k E E 3 1 d «H NO 0 ■ri E-lji M P i IfN >

b3 e t S p< a JS>- k I* ü G ® rt C5 44 C Tj fH rH fH (H•H•H m m m cq pq PQ n d 44 h c O fH iH 1—1 3 rJ cj a •H •H •H M o ,d ü -P +> -P ü fH 44 •H H H U (j o •H 3 3 3 1—i H IN ü 5+> eu Ph eu CQ

1 O 1 u rH LJ (4 Ü3 il OA d O ü O o d o d d d d d d d d d a d d rH H rH u rH rH rH rH u rH 1—1 I P3 PQ CP O f*4 03 m P3 m o P3 P3 O o ® U) LO ta fcO d d d d 3 3 d 3 tp (4 Ph P4 k •H 'H O o O O S s S i S 3 S o o o o o O o rH rH rH rH rH rH 1 rH 4-H rH rH 1—1 r-î rH rH ® Q) GO O G II >H N >H >H N X X

XN IfN > . CN Q NO t

c: +> ■y 5 f

o g I •p Ü a a Ÿ 1 § •H I o p I© 2 g I o u p i o p 5 f © i § ■3 '■a I Pu u\ 3 I

I.,W G.H A 1 «.S g I I I I q o A (Q ctf I d •P .2 sa M fH fH © I a pq m A « 3 % ,0 ^ 4> *4 « «5 *© •H U t * ' 0 Ü o cj © iH u O O Ü •H ü i P © O -P © o ON 1 CU M 5% s 3 © o © © © 4-, A a & I 0 P 5 & a $4 rH © a g POO a° I â I rt H a : o ;z) .& 1 P o © * h O 44 A O P S) § © # Q r i i i § s I t I t G © 3 W a a s i a 2 © H a O O *4 •H P rH © •H o« C3 r © t p $ q © © 44 > P Ph H 3 O À (fi fH © © Ao p o © *3 © <\J K\ hC lA VO Cn 00 a g p o g p V S ca. Fig. 18 104

T.L.C. OF PIERIS HEADS AND BODIES WITH KNOWNS using pet.ether, ethyl ether, ( 90^ 10 H ).

s.f.

1

1 500 bodies a reaction with SbClg 2 500 heads a ■ partial " " 3 6-carotene s.f. solvent front 4 vit. A alcohol

5 vit. A acetate 105

these separations only* Hov/ever, 5,6-monoepoxy~p-c&rotene gives a yellow orange colour in white light unlike vitamin A alcohol which is colourless when pure* Both substances give a black reaction with antimony trichloride* 5»6-n3onoepoxy»p-carotene was identified tentatively in both samples of heads and also possibly with vitamin

A alcohol in the sam%)le of bodies*

Further experiments were performed in which vitamin A was detected quantitatively. Ova, heads and bodies of adults, and heads and bodies of pupae were extracted in diethyl ether and a sample of the concentratedfextract tested with TFA, The absorbance recorded on the spectrophotometer for each sample is shown in

Table 20* 11 I*U* of vitamin A was detected in one sample of ova and 2 I*B* was detected in one sample of heads* Because p-carotene and vitamin A alcohol produce the same colour reaction with TFA some difficulty with Identification was caused. However, p-carotene absorbs at a lower wavelength (585 nm) with TFA than vitamin A alcohol which absorbs at 618-620 nm* It is difficult to apply a correction factor to account for the interference of p-carptene because there is no method of removing the carotenoids without the vitamin A compounds.

Separation by TI-0 was used to identify carotenoids qualitatively in haemolymph samples of P. bressicae (Table 21). Known carotenoids, previously extracted, purified and identified from P. brassicae. were run with test haemolymph samples end the Rf values of each compared* p—carotene was found in all stages investigated. Larvae earlier than Lj^ were too small to allow removal of sufficient haemolymph for extraction purposes. lutein, p-carotene and violaxenthln were identified but their presence in all stages was not consistent. The use of this comparative chromatographical method enabled carotenoids to be detected in sarrples of material which were too small to allow identification by standard methods. 106

B

O M I l I I AJ I I •P q O § © b3 I m i €> © •3 o <> ü •ri g i + + I I ♦ -f c: g c- a < k .o a * C-, § c •H X! © 0 4-> o "G c g . c g (4 o K\ VO K\ ON KN ÈH OOP £N VO O O h *i & * • I • • 4= § O 4* m O O o o •H •ri (T\ 3î U g m < c A • C o *H Al E C 43 • -P t3 •p rH *H w 43 > H q co . V\ rj CO r- ITS E-I 4- t— I C7 vo I T- '~0 C j o © vo tA vo VO lA VA C o I •H •p c c T c n o •p *3 V* "M -P % O s6 q q M 8 8 8 8 . 8 o c ©

H P* > e C'4 CO vO O f j r- ^ g p. CO O » C O p' r- f3 «• r c: 5 g 44 w o •ri o -P C:

C'F, « G ® .S ^ ji ; # £• 8 8 f i & =

o

» e 1} O- rs # 15 © -9 « 5 ri vo mrt "5 et ît a o •ri C 0 Xi o c ù t« ë ÎG G* % A îî -p ef ? S xP A. «H 107

Table 21.

values of carotenoids in P, brassicae haemolynmh

Solvent!- n-Kexane / Methanol and Kethylethylketone (1*1) 10 8 1

TLC Polyamide. Egger (1965)

Colour Stage of Violaxanthin Lutein a-carotene p-carotene haeraolymph

H Yellow — 0 .1 7 0 .9 7 h Yellow 0 .0 9 0 .1 5 0 .9 2 0.99 pp Green/ 0 .1 1 0.16 0 .9 2 0 .9 9 yellow p Yellow - - 0 .5 2 0 ,9 9 A Yellow - 0.16 0 .9 6 0 .9 9

Violaxanthin - C.11 - - Lutein - "" 0.16 - a-carotene - - m 0 ,9 5 - p-carotene - - - - 0 .9 9 108

labelling experiments

Before experiments were carried out involving labelled

carotenoids it was necessary to understand how the methods of

extracting and purifying carotenoids affected their behiviour when being counted, and how effective the system was at counting.

The results of investigating the quenching effect of coloured

solutions of p—carotene showed that a deep orange solution of

p-carotene quenched the sample giving low counts, a low ESP. value and a low counting efficiency (Table 22). A pale straw coloured solution of p-carotene counted at efficiency (the highest for tritium sources) g cuve, a relatively high ESH value.

The ESH value of the sample counted could be read off on the calibratiort curve to give an estimation of the percentage efficiency

(Fig. 19)* Pale straw coloured solutions of p-carotene were thus counted at their maximum efficiency. As all samples were bleached so that no colour was left in solution, quenching due to colour was reduced to an absolute minimum.

The method of irradiating samples with UV was used at first to bleach samples but was later replaced by using benzoyl peroxide.

UV was found to destroy the sample| this was shown by a loss in counts. To demonstrate the effect of bleaching by benzoyl peroxide eight samples of labelled haemolymph (coloured yellow) were counted in the usual way, and then they were counted after bleaching

('fable 22). The result was that the counts were increased from

5-100 times. Except in one instance the counts in the unlabelled controls did not increase to the same extent after similar treatment.

Background counts can vary by as much as 5-3 tiaies the minicum count. Benzoyl peroxide (2 ctl) was therefore used to bleach all coloured samples. 109

Table 22.

Colour with , crori*

p-carotene quench

Sample ûpfi EZK Colour of Solution % Efficiency

1 736,000 5.75 Bale Straw 41 2 393,673 4.6$ (3.6 i g/g) 22

3 644,00) 4*11 33 4 485,7%) 2,57 26 5 444,657 2.01 24 6 279,183 0.14 13 7 113,157 O.OD 6*3 a 123,750 c.oo 6.8 9 193,453 0 . 0 0 11.1 ID 103,300 0.00 6 11 157,916 O.OD g Deep Orange 12 53,000 C.GD (37.1 rs/g) 4

Bleiichiag with benzoyl peroxide

Haemolymph eamplss counted Htiemclym■ h i^mples counted before bleaching after bleaching dps d;%

1 1,142 5,091 è 519 10,872 3 202 23,899 4 200 10,769

3 164 2,828 6 154 3,064 7 195 3,594 8 221 3,279

Cclabelled kaemolymrh s K,*®- L’iSLabellsd Imtemolymp'a cam­ pie# CGunted before ples counted after Control# bleaching bleîiCliing érs» dpî5

1 263 397 2 232 2,399 3 249 437 669 4 293 110 Fig. 19

QUENCH CURVE FOR 6-CAROTENE

Deep orange orange pale straw 100

2 3 4 5 ESR Ill

Counting labelled carotenoid sarriples after colucin chromato» graphy and after bleaching means that the sample may also be

Quenched by whatever solvent or mixture of solvents was used for elution* The Quenchang effects of some of the commonly used solvents Used in column chromatography was therefore investigated*

The effects cf increasing concentrations of n-hexane, % diethyl ether in n-hoxans and 5^^ diethyl ether in acetone are sliown in

Table 23» The effects of increasing of silica gel are also shown. In each experiment it was found tliat vdth increasing concen­ trations of the quenching material a decrease in the S32 value occurred* This jraggested that the least wsslble ajîiount of any quonchix-g agent should be counted with any carotenoid sample*

The separate corotanolda were eluted in the fastest time possible without altering their purity. Corotonoid bauds were therefore counted In tho least arro-int of solvent.

The effects cf evaporating the solvent to dryness by applying gentle heat from an hair dryer was investigated. However, preliminary experiments showed tlist up to fOj' of the sample was volatilised so this method was not pursued further.

The list shewn on Table 24 gives the methods which weib tried to get labelled p—cairotene into the rarva. r.etlioufci 1, 9, 10 and 11 were used in a series of exT erif ents to fvluow tiie fate of the label in the plant and in r. hrassTcae after ingestion. Table 23 j[ J_ g

Chemical Quenching n-hexane (approx. 100,000 dpm added to each)

Vial Volume of quenching dpm E3R agent added (ml)

1 None 82,383 5.90 2 0.2 73,536 5.75 3 0.5 75,732 4.64 4 0.7 83,519 5.86 5 1.0 79,481 5.66

6 1.5 . 89,917 5.20 7 2.0 , 71,643 5.25 8 3.0 68,400 4.67 9 4.0 72,804 4.58 10 • 5.0 58,639 4.31

^3 diethyl ether in n-hexane (approx. 1,833,373 dpm added to each)

1 0.5 261,188 10.84 2 1.0 395,083 10.43 3 1.5 387,000 9.68 4 2.0 350,500 9.91 3 2.5 33,400 9.14 6 3.0 335,667 8.82 7 3.5 255,625 8.48 8 4.0 419,667 9.24 9 4.5 327,500 8.22 10 5.0 452,584 8.79

5^3 diethyl ether in acetone (approx. 100,000 dpm added to each)

1 0.1 75,732 5.47 2 0.2 69,083 5.17 3 0.3 70,667 4.88 4 0.4 64,l4i 4.27 5 0.5 67,150 4.4l

Jilica gel G (approx. 100,000 dpm added to each)

1 0.1" 41,410 5.19 2 0.2* 39,019 4.87 3 0.3^ 33,967 5.00 4 0 .4" 31,227 4.47 5 0.5" 31,114 4.43 113

Table 24.

Acceptability of r3H1 S-carotene and C3H1 KVA to P. brassicee larvae.

Methods Tried Besults and Observations

1. p-carotene in n-hexane Larvae ate all the food if evaporated onto square of starved for four hours. cabbage. Offered to starved larvae 2. Injection of p-carotene in Paralysis of injected area, n-hexane • intense watering from mouth# No recovery, dead after 13 mlns. 3* Injection of p-carotene which Paralysis# watering^twitching. had been evaporated from n- Eventual death. hexane into linseed oil. 4. One drop of p-carotene in Paralysis. n-hexane from a Pasteur pipette evaporated onto the larval cuticle. 3. One drop of p-carotene Slight disturbance# no evaporated into linseed oil paralysis, use of spinneretts, and dropped onto the cuticle. locomotion# use of all parts not impaired. 6. p-carotene evaporated into Unacceptable to larvae. linseed oil and evaporated onto a square of cabbage. 7# p-carotene injected into a Unacceptable to larvae. cabbage seedling. 8. p-carotene evaporated into Unacceptable to larvae. linseed oil and injected into cabbage seedling. 9. Roots of 1 week old cabbage Acceptable to larvae. seedlings immersed in an emuls­ ion of C3ÎÎ] p-carotene in water. I"? 10. Seeds of cabbage germinated in Acceptable to larvae. MVA water. 11, Roots of 1 week old cabbage Acceptable to larvae. seedlings immersed in a solution of KVA in water, 114

The results of incorporating [31I] prcarotene and [3H] KVA

into cabbage seedlings by Immersing their roots in an emulsion of

tho labelled compound are shown in Tables 25# 26. [3H] MVA was

also incorporated by germinating seedlings on blotting paper soaked

in a solution of the labelled MVA.

It was interesting to note tliat one hour after administration

of [3:1] p-carotene in one of the experiments (Table 25, Experiment

1) of the label taken up was found in p-carotene. When the

amount of [3 :1] p-carotene given to tJie roots was increased no

incorporation p-carotene was observed. In the final experiment

(3 ) 10/) cf the label taken up was incorporated into p-carotene

after tiii’ee hours. This relatively quick uptake of label from the

roots was also sîiown for roots of seedlings in a solution of [3H'J

î-fifA after 24 hours; p-carotene contained 10^ of the label.

Seeds germinated in [3IÏJ KVA after 4 days showed 10% of the label ia

P-carctene. In all the labelling ex^^eriments with cabbage, incor-

poi'ation of the label cccui’red into both carotenes and xanthophylls

after a short time interval. However# the amount of incorporation

into all the carotenoids was not always the same in each

experiment. In all experiments much incorporation was found into

clear bands# termed blcUiks# which ïj&d no spectral activity in the

spectrophotometer.

beedlings wliich biad been germinated in [3%] p-carotene and

[3H] INA were offered to 1.^ larvae. One of the advantages of this

technique of getting the label inside the Icirvae was that the

larvae did net have to be starved beforehand. They were found to

eat the food almost immediately after it had been offered.

After the food had been consumed the larvae were extracted,

Carotenoids identified in the usual manner and bleached ready for counting. The counts appearing in the crorotenoids are shown in 115

Table 25.

Incorporation of -QH] p-ce;potene Into 1 week old cabbare seedl ■? nas via the roots

Carotenoid Experiment 1 Experiment 2 Experiment 3 After 1 hour After 1 hour After 3 hours dpm added 3pO,OCX) 62,300,000 62,300 dpm % dpm % dpm

Blank 2,302 21.62 1,092 8.17 147 6.46 p-carotene 673 5.83 - - 233 10.32 Blank 3,227 27.89 -- 142 6.23 3 ,6-monoepoxy- - 11.64 p-carotene - 203 1.33 263

Blank 3,672 31.7 'i -- 363 15.94 Mutatcchrcme - - - - 673 29.64 P-zeacorctene - - 307 2.30 - - 3 ,6-fncnoepoxy- - - 2,072 13.30 — - lutein

Violax^'oathin 1,277 11.04 260 1.95 430 19.76 Lutein 217 1.88 1,443 10.81 - - Kecxanthin - - 3,327 39.86 - - Blank - - 2,637 19.88 -

Total Counts 11,370 - 13,363 - 2277 - % incorporation 2.10 - 0.021 - 3.64 - 116

Table 26.

Incorroration of rSHl KVA into cabbage

Carotenoid Experiment 1 Experiment 2 1 week old seedlings Seeds germinated immersed for 24 hours in solution for 4 in approx* days in approx* 50,000,000 dpm 16,666,665 dpm dpm % dpm %

Blanlt - 1,521 16.24 a-carotene - 664 7.09 Blank 26,539 64,62 - - p-carotene 4,132 10.06 933 9.97 Blank 1,033 2.65 177 1.89 5,6-monoepoxy- 1.04 456 P-carotene 426 4.87

Chlorophylls 464 1.13 1,153 12.31 Violaxanthin - - 374 3.99 Cryptoxanthin 4,741 11.54 939 10.03 Blank 1,566 3.81 159 1.70 Lutein 563 1.33 2,987 31.90 Blank 749 1.82 • Auroxanthin 798 1*94 -

Total Counts 41,071 9,363 - % incorporation 0.082 0*056 - 117

Table 27 , Incorporation of the label from cabbage labelled with p H ] p-carotene into p-carotene in the insect differed in the two experiments from 3-36:^, approximately the same difference was found in the incorporation into lutein, 2-31%, In the one experiment in which the cabbage waslabelled with [311] MVA p-carotene in the insect contained 14% of the label taken up and lutein 19%.

In the three experiments ia which cabbage seedlings were given

[3H] p-carotene 0-10% of the label was taken up by p-carotene.

When similarly treated plants were offered to the larvae the p- carotene in the larvae contained in one experiment 3% and in the other 36% of the label y os luéuJtXiM.^.

Suspected p-carcten© and lutein bands, as well as other carotenoid bands, were run on TLC and paper chromatograms to determine their radioactive purity. They gave sindlar Rf values to known samples of carotenoids. Counting of the TLC silica gels and paper chromatograms showed that the carotenoids were practically pure; so pie metabolites Jiaving been left at the origin,

larvae of P, brassicae, wiiich had been previously starved, were fed pieces of cabbage on which had been evaporated [3H] P- carotene, 2,4 and 6 hours after completion of eating the cabbage, the entire gut, haemolymph and fat body of each larva was counted after solubilisation and bloaching. The results appear in Table

2 8 .

The results showed that after 2 hours most of the label was in the gut as might have been expected, and there was significantly less counts in the haemolymph. After 4 and 6 hours the counts in the gut, liaemolymph and fat body did not vary significantly. In a similarly designed experiment in which the gut, haemolymph and fat body of larvae were examined l 4 hours after ingestion of the 118

Tabla 2?.

Incorporation cf label into L larvae of P. brassicae Immediately efter ir>.F:egtion of cabba,g^e~f>e^dlin>’:s labelled with f3H1 0-carotene and f3H1 KVA.

Experiment 1 Experiment 2 Experiment 3 [3H] P-carotene C3H] p-carotene Carotenoid [3H] MVA 25 larvae used 50 larvae used 20 larvae used dpm % dpm % dpm %

Blank -- 422 0,93 162 4,96 p-carotene 30,977 36.41 1,320 2.98 465 14,22

Blank 3,732 4.45 7,582 17.10 - - 5 ,6-monoepoxy- 0,26 4.03 370 p-carotene 217 1,787 11.31

Blanic 145 0,17 390 1.33 - -

Chlorophyll - - - - 333 10.25

Xanthophyll ? 327 0,33 — - -- Blank 4,747 5.33 6,642 14,93 - - lutein 26,353 30,97 962 2.17 617 13.87 Xanthophyll ? 13,535 21.78 - - - -

Beaxanthin -- 24,627 35.34 --

Blank m - - -- -

Auroxanthin - - 412 0,93 - -

Bli-ink - -- — 427 13.06

Violaxanthin — — - - 337 10.31

NeoxantMn •- - - 536 17.01

Total Counts 85,033 — 44,344 - 3,269 - 119

8 CO oo r - <\1 r - -d" VO -d" AJ CO fA CA fA CN- CO A- ON ^ 8 ^ G\ AJ ^ AJ G\ CO oO LA O' lA AJ ON ON OO VO & I# Rl 8 1^0-0 0 - »A Ov ON -d- AJ V- VO AJ JA AI r - V- fA V * V -

I

a 8 K% ITv VO 0 \ T - 0\ JA Q K\ CO ON AJ VO CO -d" A- A- rvj CO j - I N LT\ rA CO fA OO ON r - vo CO fA fA V- g •H 8 -4* V- C\ IT\ V- lA CO CN- -d" N- AJ f A AJ V- V- V- AJ W

I 8 T- VO -d- P'. L A CO g ON ON fA fA O V > AJ P A 9 VO CTv Ov lA S 5 S' S> S S' AJ r - T- T- <\J OO o ITv O f\J -d- T- V- r - CO lA O ^ % 3 r « LA T- fA (A O !>. T- VO VOo

m +> g 3 AJ f A L A VO A - o o ON V - AJ fA -d- LA Ü O VD CN- CO ON O O to u 3 +> 3 o o o jd EH Æ! AJ rH -d" rH U Id k O g ^ Q g +5 3 OJ +> «H (B >

In the fat body (App« Table 2)* However, the standard error was

60 great for each of the regions tliat this indicated that the results were not significantly different.

Two hours afterpreviously starved larvae were fed cabbage squares onto which [3H] p-carotene had been evaporated, the radioactivity in the heads and bodies of each specimen was investigated, (Table 29)* The results ehowed that more of the counts were present in the bodies than heads. As p-carotene in a precursor of vitamin A it was interesting to note whether more labelled p-carotene appeared in the heads than in the body after ingestion of the label. The results showed only one instance when as much as 45% of the label was present in the heads.

The frass of 30 1^ larvae which had been fed labelled cabbage

\j8Ls analysed for radioactivity (Table 29)# Although counts were present in the frass 5 minutes after feeding commenced these counts probably represent only background. The control experiment showed that counts could vary from 150 to 1,302 dpm. Substantially more counts weie present in the frass 1 hour after feeding. This corresponded with a time when nearly ell the larvae were defecating and when all the larvae had finished their food. The majority of the label appeared in the frass 90 after commenaaent of eating. It is likely that the time for passage of material from one end of the gut to the other is between 45 and 60 minutes. 121

Table 29.

Counts In heads and bodies of P. brassicae larvae Immediately after ingestion of C3H]3*carotene -

Counts in Counts in Larva Counts % Counts Heads Bodies in Heads in Bodies dpm dpm

1 227 7,952 2.78 97.22 2 3,355 4,347 43.56 57.44 3 852 5,837 12.74 87.26 k 505 8,050 5.90 94.10 5 1*95 12,502 3.81 96.19

Mesji 1oS6:80 7,737.60 12.32 87.68

Control

1 96 80 54.54 45.45 i 79 75 51.30 43.70 5 85 63 57.43 42.56 4 60 91 35.74 60.26 5 53 74 41.73 5 8 .26

Kean 74.60 76.6 49.34 60.66

Faecal drop by P. bras*^ icae 30 L., laivae after ingestion of [3H] p-carotene. % Time Control dpm finished minutes defecating dpm food

5 262 3 —— 10 — 3 - 173 15 777 3 -- 20 630 16.6 - 150 25 680 23.3 152 30 812 20 0.6 147 35 207 20 16.6 - 45 510 5 3 .3 4 3 .3 232 60 14,325 90 100 - 75 3 ,0 0 5 100 100 - 90 33,657 100 100 925 120 84 100 100 1,302 135 3 4 ,5 6 0 100 100 - 122

Carotene!ds In other species

The total carotenoid concentration of 33 species of lepidoptera

are listed in Table 30 and the individual carotenoids found in 23

cf these species is given in Table 31. The small samples and

differences in age and state of the specimens Eind the lack of

replication means that comparisons between species may not always

be particularly meaningful. However, in the 23 species where

individual carotenoids were identified ^-carotene and lutein were

present in all species. However, f3-carotene was apparently absent

from adult males of one species and lutein was not detected in

cns sample of adult females of another species; but these omissions

are probably net significant. The epoxy-carotenes, zeaxant’nin and

a-caretene were identified in several species.

In 13 out of 23 species lutein was the major cartenoid present

in all samples, while in 2 species p-carotene was the major

carotenoid (Tables 3 I z^nd 32). jVcarotene was the major carotenoid in three cut cf four samples cf A, luna and lutein the major one in three out of four samples of A. tau. However, in both species

only the female adult was different. In female adults cf A . lima lutein was the most abuzidant carotenoid, while in A. tau ^-carotene lutein was the most abundant. The larvae cf A. caja were found to have^as

the most abuiidcnt carotenoid while p-carotene was the most abuiidant in male adults. Tlie most abundant carotenoids in male and female pupae of ?« bncephala were cryptoizmthin and p-carotene respectively. 3»6-Monoepoxy-3-carotene was found to be the most abundant carotenoid in one species in a cingla sample of

H* euphorbiae.

Expressing the results in pg per insect rather than pg/g was

sometimes more useful when comparing species. For example in

A. perry! (9 pupae) and P. ranae (achilt) where the concentration 123 Table 30. Xoi;al carotenoid concentreition (vg/r) and content (iir/ir.aect) in» all epecics analyse!

Total Dry Total Fuicber of Total Weight of Caruteni Species Stage Siieciocns State of Fntorlal Carotenoids 2):tracted Content

PILklBAS P. na;:i A 60 Dried. 2 years R.T. Hot recorded 0.00 0.00 A 23 Freshly caught 0.26 24.4? 0.23 A 27 Dried. 2 years R.T. Hot recorded O.CO 0.00 A 18 Fre silly caught 0.20 66.96 0.74 G. riiamni Acb- 13 Freslily emerged 0.47 E6.64 A M 8 Fresiily emerged 0.28 234.77 A. cardaminea p 10 Living 0.10 54.08* 0.54 0.045 26.66* 0.24 Aoo 5 Freslily caught 4.18 C. pomona A 2 Dried. 1 year R.T. 0.15 55.73* C. crocele rxeen A 2 Dried. 1 year R.T. 0.07 83.12* 2.90 yellow 44,20* A 1 Dried. 1 year R.T. 0.03 3.54 H. calypso A 4 Dried. 2 years R.T. 0.07 179.14* 5.13 F.. ciilorio A 1 Dried. 2 years R.T. Hot recorded trace* -

DAhAIDAE D. plexippua Acb 6 Freshly emerged 0.47 122.53 9.60

SFHI1.GID/1E . L. ponuli A 3 Freshly caught 0.30 17.73' 1.77 24.76 H. euchorbiae fcki 6 Freshly emerged 1.05 141.51

SATUKIIDAE - 279.82 2.36 S. pavonia X Rif 8 living 0.0675 X P M 8 Living 0.171 174.20 X Acb 4 FTeshly emerged 0.03 795.52 Î'M X A9 1 Freshly emerged 0-0827 243.67 20.15 6.42 X.RM' 8 Living 0.34 151.08 7.93 x W 8 living 0.49 130.22 55.94 X f o 1 Freshly emerged 0.197 285.97 65.56- X A9 1 Live. 2/3 days old 0.32 204.88 without eggs Rjü 5 Living 1.4 64.59 18.09 ÏÇ9 5 Living 2.72 61.24 33.31 X A Kot recorded Freciily emerged 640.90 Rx) 5 Living 1.03 96.46 19.87 P M 5 Living 1.16 81.84 18.99 Ac'o 11 Freshly emerged 0.77 117.19 3.20 AOO 5 Freshly emerged 0.83 17.59 3.10 2S.60 A. pcmji PCii 10 Living 4.41 64.86 P99 10 Living 5.60 59.54 33.34 H. cecropla rto- 8 Living 4.19 84.18 44.09 PM a Living 4,43 177.56 98.32

ABCTIIDAE

A.caja XL^ Hot recorded Living 0.12 285.06 - (2 days. Dry ice A J ü 8 IlYesilly emerged 0.65 100.86 8.32 o. Itibricinnda A 21 Freslily caught 0.74 49 .75* 1.75 5. lutcusi A 7 Freshly caught 0.46 14.35* 0.94 T. igcobaeae X L^ Hot recorded living 0.46 427.30 - P" Hot recorded Living 1.11 504.32 - C. donij

aiu=h i p y k d :idas C. trliTommica 42 Freshly caught 1.30 56.62* 1.75 ûXnia'SIDAE A. grossulariata XA9 iïesiily caught 676.00* 6.76 Acb- Fresilly killed 0.02 380.00* 0.53 AM Fresilly killed 0.03 2 76.0 0 * 0 .4l ;.ocruiD.Æ A. eaclsmatiO!0.3 46 Freshly caught 1.64 21.46 0.77

1;. TTcnuba S9 Freshly caught 6.80 25.00 1 . 9 1 I:, eoana 40 Freshly caught 1.15 37.34* 1 . 0 7 Lyi4u;rHiiDAS chrynorrhoea X L„ Hot living 0 .4S 256.58 X Hot Freshly emerged 1.26 91.65 1.10 302.68 T). r.udibunda P 14 Living 23.73 Acb' 8 Freshly emerged 0.13 213.33 3.47 0.415 AM 4 Freshly emerged 209.43 21.73 ;

j(T 2oom Temperature A ,‘idult stage. lixod sexes unlera SjXicificd. P liipa stage. Fixed sexes unless ouocificd. L L'jv.il instar, * Total c.rotenoids eatimated frot crude extract enly. X Insects extracted by lies Loac;.Tary Xumery. Table 31. 124

Carctenoida found in various opecics

C g g Is SP3CIES § & VO 8 il A â g PIcSIIVlE 16.00 p. napl A 8.47 49.28 P. rapae A 17.63 53.77 G. rhami fcxf 5.68 29.19 AÇ9 11.89 222:33 54.08 A. cardanlnes trace

DAîlAIDAE 7.08 37.59 45.48 D. plexippua A d a trace 24.82 7.76 ijPHItiGIDAE 42.32 £6.94 H. euphorbias Aocj- 3.43 45.51 10.42 6.08 7.01

SATURHIDAB 149.92 18.25 6.51 S. pavonia X Tbÿ 5.33 96.97 2.84 104.75 13.35 5.85 2.96 44.87 2.42 - X P99 410.72 38.03 X Ag o 1.59 300.08 29.01 - X AÇ 1.59 87.32 ------125.75 62.83 y ,14.66 3.21 0.87 69.51 _ __ - - A. lima T mb' 9.04 X 0.70 61.03 - 57.59 P99 : 121.42 22.45 - r A d trace "140:10 94.50 27.18 X A9 trace 83.20 ------_ - -- 44.67 5.60 - L. katinka E jO 9.52 4.80 41.02 8.04 P99 9.36 2.82 . - - --- A - 16.43 - - - ~ - 100.59 28.73 - 77.82 - - A tau R±r 18.64 _ - - - 71.92 FÇ9 - 9.92 _ - - t e d 27.53 : 89.66 A99 - 17.59 - - - - " A. pernyi Ebb _ 45.76 2.13 _--- 8.52 8.63 1.77 24.76 2.14 0.71 E9 ? - 29.26 0.89 1.78 trace - -- _ - - H. cecropla Ebb _ 56.07 1.38 trace 9.68 6.73 23.84 4.19 5.96 P29 - 78.01 8.67 6.77 “ 9.10 6.86 - 62.19

AKCTIDAE 182.40 A. caja L 102.66 Aoo 65.46 31.52 trace

T. jacobaeae L 44.60 20.19 17.63 56.34 222.90 55.64 P 15.58 5.29 251.17 32.28 C. doroinula L 64.29 213.66 red A 3.33 20.12 trace "61:50 10.04 yellow A 7.39 6.00 7.20 5537

GE01ETRIDA2 A. grossiilarlata 166.40 510.00

f.'OCTUIDAE A. exclamationls 1.67 0.98 - 18.81 K. pronuba 6.66 3.20 trace 5.26

mUMTHIIDAS E. ch3r/Borrbo«> 2.42 66.50 14.33 10.75 9.00 94.08 59.50 trace 2.89 11.83 3.46 4.42 62.55 6.47 P. pudlbunda P 14.04 21.27 9.93 10.76 234.99 10.89 idd 213.33 AÇ9 9.65 26.31 19.08 149.20 trace 5.26 î;otodoi.-t i d a s P. bucephala Ibb‘ 57.64 84.66 8.86 1.32 P99 69.74 15741 5.97 3.8 ? C. vlntila X P 6.42 42.13 8.00 5.26 ZYGASIÎIDAS Z. filipcod»a.ae 1.04 11.36 3.16 ,1.47 16.86 9 1 . 4 9 13.71

MOTE

A Adult stage, llixed sexes unless specified P Pupal stage. " " " " L Larval Instar. X Insects extracted by Kiss Roscajry Kumnery Kn.ior carotenoids underlined. Table 32. 125

PorcoatanG carotenoids in various srecics

2 g Oo ëo hA Ü SPECIES 11 J ’l ! lÂ,3 PIEKiatE P. napi A - 35 É5 P. rapae A - 26 74

G. rhamni Ado _ 4 62 34 A99 - 5 95 A. cardamines P - trace 100

U/0:aIÛ\3 D. plexlcpus A.O trace 20 31 n. SPnIi.GIOAE H. euohorbiae A d d - 3 32 30 19

SATUWilD^ii: S. -pavonia X V d d 2 35 54 7 _ 2 X £99 2 26 bO 8 - 3 X i d o ^ 1 40 52 5

X A9 < 1 36 52 12 - - A. luna X Rjô <1 46 42 10 ' _ 2 X P99 <1 W 44 7 _ _ X Ad trace 43 8 -- A9 trace ¥T 13 - - L. katinka Rjc - 15 9 - - £99 - 15 b ? 13 A - 12 20 - - A. tau Ibb’ _ 19 81 £99 - 12 - t d o 24 E - -- AÇ? - 100 -- - A. pernyi R b - 63 13 13 3 P?9 - E 42 4 - 1 H. cecropia P CO - 43 trace 12 23 __ 5 POÇ - W 4 5 35 - - 3 ARCTIIDAE A. caja XL _ 36 64 Acb - 61 31 trace T. jacobaeae L - 10 13 13 P - 5 1 10 C. dominula L _ 23 red A _ 4 21 trace m 11 yellow A - 9 8 9 tR GECltCTRIDAS

A. grosculariata A - 25 75 f.'OCTUIDAE A. cxclamationis A - 8 4 N1 pronuba A- 27 13 trace 20 4o E. chrysorrhoea XL 1 25 6 4 4 23 XA- 3 13 4 5 i 7 D. Pudibunda P 5 7 3 A d d - 100 4 A99 - 13 9 71 I.’OTODC'iiTIDAB P. bucephala R b 33 55 6 £99 Z2 15 6 C. vinula XP 10 65 13 ZYGAZNIDAB Z. fllinendulae A 1 8 12 66 10

NOTE

A Adult stage. Mixed sexes unless specified X Insects extracted by Miss Rosenary Munnery P Pupal stage. " " " " I’ajor carotenoids underlined. L Larval instar. 126

of T/C in If g/g vjae approxiTr.ately the same, the T/C per Insect of

A* pernyi was 33.34 pg, while that cf ?> rarae was 0.?4 pg.

Whether carotenoid concentration Is considered in terms of

I’S/S or vg per insect It is difficult to relate differences between

species to other known differences between them. The carotenoid

content per insect yaried between, as wellaas within families. The

Fierids ranged from 0.24 pg in adult, mâles- of A. cerdairdnes to as

much as 18*69 vs in one sample of phsrate pupae of P. brassicae.

The lowest value of any stags of P. brae sic es was 0.0012 vg

of T/C for a sample of ova (Table 7), Arctidda were also within

the same range as Ilerids* Saturnido overlapped at the lower end

of the range with Fierids and Arctids but they had a greater range

within the family. Pany cf the fatumids had T/C contents per

individual in excess:6f l8

A s Saturnids are notorious * assemblers* it was interesting

to note whether there was considerably more T/C in male antennae.

The implication of carotenoids in olfaction has already been

mentioned. Ebthschild, Valadon and Mummery (1973) investigated

the carotenoids in various parts of S. pavonia but they did not find

larger amounts in antennas (3.81/ of T/C of whole body). Greatest

amounts of T / 0 were found in the wings and bodies of male S. ravonia,

16.71 and 72 .57 / respectively. Legs and.-ha:td!5had the smallest

concentration of carotenoids, 5.14/ and 1.73/ respectively.

There was no apparent difference in the T/C content between

the sexes of 11 species examinod. In 4 species, D. r u d ib u n d a ,

G. rhenni. L. katinka, ÏÏ. cecropla), fcniales had more T/C per insect

than males. In P. bucephala greater T/C per insect was found in

males, and in 4 species ( P . brassicae, A . luna, A. pernyi, 0 . brumet a )

approximately equal amounts of T/C wore found in the sexes. In

c. pavonia male adults liad more T/C than females, but the pupae of 127 each eex had approximately equal amounts. In A. tau adult males had more carotenoid than females, while male and female pupae had approximately the same amount.

The presence of more carotenoids in G. rhamni females than

&alas is interesting because it is the male which is yellow coloured, the female is green yellow* Most of the yellow colour in the males is likely therefore to be due tè pteridlnes which have been found in this species. C« brumala^ the newly emerged female of which had many hundreds of greenish eggs in its abdomen, had an higher concentration of T/C in pg/g than males* However, the T/C content per insect showed the sexes to have almost the same amount.

S. luteum, which is reported to mimic g* lubricireda wasffound to have less carotencid por insect than its model.

Pothschild and Feltwell (1972) suggested that some aposematic insects had more T/C in terms cf V g /g than seme cryptic insects.

However, the present work shows tîiat, although the mean T/C of the two groups is approximately eqw;l (163.83 • 107.95 PS/s i%i aposematic and 143.53 - 6 5 .7 9 vs/s cryptic), the T/C content per insect was considerably greater in cryptic Insects (Table 33)* however, the higher content per insect in cryptic species may be due to their predominantly greater dry weights.

The T/G per individual of all species investigated was found to vary from 0.24 pg in A. cardamines to 98*32 pg in R. cecropia

(Table 34). Uhen these results were plotted against the dry weight of insect it is seen that the general tendency is for an increase in T/C with an increase in dry weight (Fig. 20). The correlation coefficient for the two sets of data in Table 34 showed that they wore positively related (p-

Six cut of nine species containing more than 1S pg T/C per insect were Saturnids, the three others were H. eunhorbiae (Acf), 128

73 CO 5 d ' O 73 0 W 1 1 1 1 1 1 1 1 lA G\ rA ^ b c/i r - -cj- P N to

A- A- A- Q A VO lA t>- t>- lA A- T- q <3N tA A -d- LA A t> - 0 \ CA CO l A CA <\J 0 fcj - (A lA CM O q. d: CM cvj ( A OJ ^ t A CM

73 A A rA d fA VD (A CM CM lA d d • VD O' fA 1 'd 0 W t 1 1 t 1 1 1 1 I I VO 0 d d • [> -' ON CA CO 3 d w r - O C\J VO 5 P M - j " r - GO a 01 0 u o O -p M 0 VO rA 0 0 LA T- rvj lA -cK CO l>- O VD SC to d VO E •H p d d E 0 I—1 d (3 rH r5 d A 0 & Ü 0 d d c—1 d EE 0 11 A d 0 0 A d E Ü A KV o to ••H d X 0 d 0 d d c/i P- > Pi 0 A 0 Dd 0 tD A o 0 S < »d 0 %A a Ü E O I to 73 d 3 d * ON •H •d 0 H 1 1 1 1 1 1 1 1 l A I I d d * -p d d to Ü p H 0 CQ w q ON •H d (NJ -d- lA On 0 r - CO ^ O lA 3 rA ON A CO -d- VO L A CM A . I (JN O P, to 0 1 1 o d- % CO 0 CM rA ON NA O O CO ( A (M A CM y 73 ■P d d * 73 73 0 M 1 1 1 1 1 1 1 1 . . d q d d * C7N P, d 3 d to lA P w 8 t : -'•O 0) \ VO t : * A- A 0 VO LA rA ON VO îA 0 rj VO VO vo rA lA 'S M CO On rA A (A A LA 0 V- O ON CO ON d to q I • *rl :A .0 -d* ON ■A CM VO ON -d- co -ci- VO rA A- i cO 0 VO ON -d On 0 0 (M A lA A- OJ CM VO lA VO O d d. rA VO lA V - ^ OE •H p p % « d d 0 d 0 iH Td p -2 P* CM CM

Total carotenoid content per insect of all samples listed in order of decreasing body weight per individual.

T/C content per Species Stage and Sez Dry Weight individual g

A. pernyi P9 0.560 55.54 H. cecropia P9 0.554 93.52 L. katinka P9 0.544 55.51 H. cecropia %" 0.525 44.09 A. pem>-i itr 0.441 28.60 A. luna A9 0.520 65.56 L. katinka Rf 0.280 18.09 P. bucenhala R 5* 0.212 52.28 A. tau P9 0.252 18.99 A, tau Rf 0.206 19.87 A. luna k S 0.197 55.94 A. tau A9 0.176 5.10 H . euphorbiae Acf 0.175 24.76 C . vinula P 0.165 10.17 P. bucephala P9 0.128 16.86 L. populi A 0.100 1.77 A. caja k S 0.085 8.52 S. pavonia A9 0.085 20.15 C. crocale (yellow) A 0.080 5.54 D. pudibunda P 0.079 25.78 D. plexippus k S 0.078 9.60 K . pronuba A 0.076 1.91 C. pomona A 0.075 4.18 A. tau A d 0.070 8.20 S. luteum A 0.066 0.94 A. luna P9 0.061 7.98 Z. filipendulae A 0.054 7.55 A. luna • R>’ 0.045 6.42 P. dominula (yellow) A 0.042 5.41 P. brassicae A d 0.041 7.78 G. crocale (green) A 0.040 2.90 A. cxclamationis A 0.056 0.77 G. rhamni Ac' 0.056 5.15 S. lubricipeda A 0.055 1.75 G. rhamni A9 0.055 8.22 P. dominula (red) A 0.051 2.90 K. comma A 0.029 1.07 £, pavonia P9 0.021 5.72 M. calypso A 0.018 5.15 P. brassicae A9 0.017 2.05 P. napi A 0.011 0.28 P. rapae A 0.011 0.74 A. grosculariata A9 0.010 6.76 A. cardamines P 0.010 0.54 A. cardamines k f 0.009 0.24 S. pavonia Rf 0.003 2.36 S. pavonia A d 0.008 5.65 6. brumata A9 0.0015 0.41 C. brumata Ac* 0.0010 0.58 130 F ig . 2 0

TOTAL CAROTENOID CONTENT AND DRY WEIGHT OF 100 SPECIES ANALYSED POcecropia

A^luna

Ac5luna

PC^cecropia 3 5 L

P pernyi katinka Pcfbucephala

30 .

A adults pdpernyi P pupae O if sex not stated, mixed 25

*^phorbiae POpudibunda

S 2O A jp avonia P cft au P9 tau B C PcTkatinka o Ü P^bucephala O > 1 5 CD o

10 POvinula Acfplexippus brassicae ^ Ao% luna AO AOcaja

rhamni *?iiipendulae A2 i PcT . Acfpavonia grossulariata; luna

pavonia AQ. rh a m n i AOpomona calypso Ao do mi nut a AOcrocalefy) - L ' -AO Ao crocale (gn) AÇ tau Pcf brassier dominula (r) I , Ao pavonia ÇA AO lubricipeda^ronuba AOpopuli brumata

0001 001 10

dry weight of insect (y) 131

D, fuaib'mfa (P ), and P. brce-hala (î>f). Some samples of the

Satiirnids, however, did have considerably loss tlia.n 18 y $ per insect, but st least one sample of most species analysed except

A* remyi and H» cecropia was greater than 1B pg T/C*

A noticeable feature of the graphical distribution of the insects by T/C and weigl.t is that lighter species bad a smaller range of T/C per insect* Above a body weight of 0.08 g the range is considerably greater* However, in comparing these differences it must be remembered that all adult samples were not identical in age, state and quantity. 132

DISCUSSION

This work has demonstrated clearly that there are a number of influencing factors which have to be considered when comparing results for carotenoid content in Insects*

It has been shown that the carotenoids present in P. brassicae are those found in its foodplant, B* oleracea. Whether the relative amounts in the plant influence the relative amounts in the insect is difficult to determine because of the great variability in the plant* It would be worth experimenting to see if cabbage plants with consistent carotenoid content could be produced* The use of a semi synthetic medium as source of food might help to solve the problem. The dried cabbage and any other constituents of the medium which might contain carotenoids could be analysed and therefore a more accurate measurement of carotenoid intake should be possible. Different samples of dried cabbage wouldpiosumably show the range of carotenoid content found in living plants.

Any critical information about the carotenoids present in small quantities in both plant and insect is difficult to obtain*

A large sample of material has to be available before any carotenoid present in very low amounts can be detected* Therefore in samples where the amount of material is limited, the apparent absence of a particular carotenoid may just be a re flection of sample size*

Valid comparisons between different species, or even between different samples of the same insect cannot be made unless the age and state of the material is noted* recorded presence of a carotenoid is of course meaningful, but absence not necessarily so*

Comparisons of the concentrations of T/C and of any particular carotenoid may also be misleading* It has been shown with P. brassicae 133 that dried specimens contain appreciably less T/C than equivalent freshly killed specimens* The use of freshly killed specimens of all insects to be compared would therefore give the most accurate results. It is often helpful to the assessment of the results!if the carotenoid concentration is given in terms of pg per insect as well as ts/c-

The mean carotenoid concentration in terms of vs/s throughout the stages of P, brassicae started high with the ova, loweied,and then levelled off with minor fluctuations during the other stages.

The p-carotene content per insect, however, showed a steady increase from the ova up to the end of the stage* The slight fall in carotene content after the feeding period supports the view that there is no synthesis of carotenoids in tiiis insect.

The high concentration of carotenoids in the eggs gives them a yellow orange colour (Fig. 21). Poultoa (I885 ) noted that the bright yellow colour of most insect eggs was due to the concentration of these pigments (*xanthophylls^) within *a small compass*. An interesting feature of the eggs was that lutein represented 46.27/ of the T/C, higher than at any other stage in the life cycle. A possible explanation of this is that lutein might be associated with the fat and protein reserves found in the eggs. 134

Figure 21.

Ova of P. brassicae (approx. x 10 magnification)

Carotenoids present in the larvae of P. brassicae probably complex with the blue bile pigments to produce the green/yellow colour of the integument. Depending on which background the larva is on it may be either cryptic or aposematic (Baker, 1970), but nevertheless the colouration of the larva in these conditions serves as a protection against predators. Carotenoids in adults of P. brassicae probably do not participate much in the colouration of the wings and bâdies, as the white and black areas of wings and bodies is likely to be due to pteridlnes and melanins respectively.

The yellowness on the undersides of the wings of P. brassicae

(Frontispiece) may be due to pteridlnes or oxidation products of tyrosine, a precursor in melanogenesis (Fuseau-Braeech, I960). 135

It wag expected that the larvae of female P. brassicae adults might have concentrated more total carotenoid than males in order to pass these on to the next generation in the eggs. However, no evidence was obtained to suggest this. Taking the highest value for

T/C per female adult and ova (Table 7) it can be calculated that

0 *0163/ of the female T/G is passed out into each ovum, Fenale

P. brassicae adultscan lay up to 400 ova (Bens, 1970), which in terms of caio tenoid represents 6.52/ of the T/C of the female. In comparison with a crustacean it is interesting that female

P. brassicae adults appear to put less total carotenoid into their ova than Simoce-phalus vetulus C.F. Muller, which puts 5*^/ of tîie total carotenoid of the female into the eggs (Green, I966).

Because of the variability in the foodplant it is difficult to determine whether carotenoids are selectively concentrated by the insect. The mean amount of ps-carotene in cabbage, from the analyses over twelve months, showed that p-caroteae represented 30/ of the total carotenoids present. The mean amount of {3-carotene in all adult samples of P. brassicae v/as 36/. lutein represented in in the plant but was 26/ in the adult insect. Noticeably higher percentages of 5,6-monoepoxy-P-carotene and cryptoxanthin were found in adults of P. brassicae than in the foodplant, while neoxanthin and violaxanthia represented a lower percentage of the carotenoids in the insect than they did in the plant.

In the feeding experiments where cabbage plants with more precisely known amounts of p-carotene were used, p-carotene comprised 3 7 .61/ and 33*79/ of the total carotenoids in two plants.

After larvae fed off these plants p-carotene comprised 30*38/ and 3 5 .26/ of the total carotenoids respectively in the larvae, suggesting that no concentration had talcen place. The considerable variation in carotenoid concentration In cabbage leaves, even from the same plant makes it very difficult to get an accurate estimate of the concentration in the food eaten by the insect. This means that it is not easy to evaluate the significance of any differences in the relative percentages of carotenoids in plant and insect,

ît is interesting to note that Aplin and Eothschild (1972) found that the amount of alkaloids as a percentage of the dry weight of the insect was greater in A, caja and T. .jacobaeae than in their foodplants.

No evidence of keto-carotenoid formation was found in

P. brasaicae although keto-carotenoids have been foundjOn some species of Coleoptera (Leuenbcrger and Thommen, 1970; Valadon and

Kuimmery, 1973) * With the exception of aurochrome and two unknowns all carotenoids found in cabbage were found in at least one sample of P. brassicae.

It is clear that the Variability in the carotenoid content of cabbage plants, such as has been demonstrated, must influence the carotenoids available to P, brassicae at any particular time.

It also makes it impossible to calculate more than approximate estimates of the amount of carotenoid ingested by the insect in feeding experiments whore cabbage is the source of food.

The feeding experiment with 100 larvae and the analyses of frass from the larvae in continuous culture conditions bot&. showed that the greatest assimilation of p-carctene occurred in the stage.

Te the other feeding experiments no correlation between the amount of p-carotene in the foodplant and the amount assimilated by the larvae could be shown, la the experiment where the food- 137

plant had th© raost carotene the greatest assimilation of p— carotene appeared to talie place but in tliis experiment the insects ate considerably more of the plant material than they did in the other experiments. In the other two feeding experiments there was evidence to suggest that ^-carotene and ingested food were carried over from one instar to the next, larvae defecated more frass than the bulk of food eaten by them. So it seems likely that the apparent excess of p-carotene in the frass at this stage is not a real difference between input and output.

It was interesting to note that the T/C content of P. rapae was lower than that of P. brassicae, as the stage of P. rapae feeds entirely witliin the cabbage heart. This adaptation is relatively recent as Bailey (1927) has pointed out the cabbage heart has evolved since primitive Kan (about 2,000 years ago).

When the cabbage heart was investigated no carotenoids were found present from the entire heart, although a very pale yellow coloured solution was extracted. This substantiated Bondi and d

Keyer*s work (1946) that practically no carotenoids were present, end Collison et work (1929) that the vitamin A activity was small.

Be (1936) found only 0,23 VS of 'carotene* per gram of material in the innermost v/hj.te portions.

Vitamin A was identified quantitatively only in small amounts in the heads of P. brassicae and in ova* Relatively small amounts were also found in B. irori (1-14 I.U. per gram) by Franceschini

(1939), It is very likely that vitamin A is metabolised very quickly in the place where it is used, and therefore there would be only small amounts present to be detected at any one time.

The detection of vitamin A in heads and ova of P. brassicae suggests that at least in these sites carotenoids are involved as 138 vitamin A sources. If a more sensitive test for vitamin A could be devised it is likely that vitamin A would be detected in other sites where carotenoids have already been found.

The use of p-carotene and [3H] KVA in labelling experiments, and the subsequent incorporation of the label into many unrelated compounds demonstrated tliat much care has to be given to the interpretation of results. Tritium labelled compounds are known to be very labile. It might have been more satisfactory if

[l4C] labelled p-carotene could hav^been used for all labelling experiments, but this is commercially unavailable and its synthesis is inefficient and very expensive,

Ckita and Spratt (1962) have stated that the purification of labelled compounds to constant specific activity does not preclude the possibility of the tritium atom exchanging with another proton within a biological system, Badiclysis of a tritium source iriay give rise to recoil fragments although this only accounts for 5/ per annum (Ivans and otandford, 1963)* Be la Gardia et al, (1971) have suggested tliat carotenogenesis takes place along a clearly defined pathway in what they described as a multienzyme aggregate*

They proposed tliat any interference to this system brought about by introducing labelled compounds might account for the wide and relatively small incorporation into other carotenoids. Impurities in a purified saiKple are almost impossible to avoid because self­ radiolysis and other processes are continuing all the time

(kadiochemical Centre Review 7), The high incorporation of tritium into colourless bands could be explained by the exchange of tritium atoms with hydrogens of other molecules, _ Specifically labelled tritiated compounds always contain as an impurity some radioactive molecules which are labelled in some other position 139

(Radiochemical Centre Review 7)* A hyperconjugation process could

give rise to proton exchange, so tho.t the tritium label is exclianged

for a proton in the medium. Once released, the tritium could

become associated with other compounds, Tîîis could result in the

loss of C3Hj from the labelled 6-carotene, and its incorporation

into other compounds. Thus on theoretical grounds the label need

not necessarily be associated with carotenoids, making interpre­

tation of results difficult, Tlie use of tritium in labelling

experiments in animals can be very misleading because much incor­

poration into unexpected and unrelated substances can occur

(Gocd/in, 1972),

However, there was some evidence to suggest that C3H] P“*

carotene was sequestered in the larva after ingestion of labelled

cabbage. Experiments in wliich the gut, haemolyrnph and fat body

cf P. brascicae were examined after larvae were fed [3H] p-

carotcne showed that appreciable mmunts of radioactivity appeared

in the fat body after four liours. Although incorporation of the

[3H] label into carotenoids involved in both pathways of carotenoid biosynthesis was shown after ingestion of cabbage labelled with

[3H] p-carotene by P, brassicae larvae, fhis was probably more a reflection cf the mobile tritium atom, tlian conversions from one

pathway to the other.

If experiments could be devised in which specific [14C] labelled carotenoids were fed to insects wliich were subsequently subjected

to electron microscope autoradiographic analysis, a clearer picture

of the fate of ingested carotenoids would be obtained. Such

experiments could lead to a better understanding of the importance of carotenoidsin the plysiology of the insect. 140

The occurrence of all the common plant carotenoids in the various species of lepidoptera examined suggested that the pigments present were a reflection of the Ingested food. The carotenoids were present in the Insects in the same ratios as in plants.

The indication that the greater the dry weight of the insect the more total carotenoid is present per insect supports Eeroults

(1970 ) contentions that carotenoids are not «waste products” of no importance to phytophagous larvae. Perhaps the occurrence of

•defined* amounts of carotenoid in all insects investigated indicates a common and functional significance in all species.

Kany insect eggs are yellow coloured and some may contain carotenoids (Goodwin, 1952b)* Gilchrist and Lee (1972) have suggested that carotenoids play an important part in the reproduction of the female Sand Crab, Fmerita analoga (Stimpson) (C. Crustacea).

They stated that the male and female intake of carotenoids is identical but the female may augment or preferentially take up p- carotene in the breeding season. Although Leuenberger and Thommen

(1970 ) found more p-carotene in the female Colorado beetle,

Leptinotarsa decemlineata Say (O. Coleoptera) than in males, Yoshida

(1955) noted that female B. mori contained less carotenoid than males,

29/ and 34/ of the total carotenoit^respectively.

The results for the 11 species in which the carotenoids of males and females were examined indicated that there were no apparent differences in the T/C content of each sex.

Apart from serving as a means for sexual recognition, colour in insects has mainly evolved with the pressure from vertebrate, particularly bird predation* Most mammals are colour blind, but they can differentiate colour as different shades of black.

Aposematic insects have evolved warning colouration as a means to be seen by the predator and have also added to their defences* 141

distasteful substances which birds learn to associate with th@

particular' colour p&ttoms* In many non—toxic insects similar

colour patterns to those of toxic species have evolved and thus

such insects avoid capture by the process termed mimicry (Wickler,

1506)• Rothschild (I963) has suggested that g, luteum is a mimic

of 3». l^brlclre^a. The latter species is easily observed and

carries a lot cf acotycholine and histaraine which some birds find

distasteful. % l u t e ed-rics à» lubricireda in size toid colour

but cont;d,ns very little of tlw distasteful substances* Its date

cX emergence is usually just after thit cf 3. lubricipeda.

fcthacluld suggested (1971) that carotenoids in insects may

enhance the repellaut natui'e of defensive sprays and secretions by

acting on the vertebrate na^al n:ueo.sa* ghe noted that those

insects that produce deXeûLslvo sprays arid secretions generally

liave yellow haemolysph, and tsra-'.t in some examples carotenoids aro

present. It was therefore interesting to investigate whether there

was any association between carotenoids and toxicity*

Marsh mid Kothscliild (1973) looked at the toxicity to rice of

the various stages of ?, breeaicau and other fieridae after

Intraperitonenl injections* They found tliut the toxicity of

P* bray^icae increased froa the ova, which Imd no apparent effect

oa nice, tlsrough the l^irval stages, which showed increased toxicity

with ago, up to the pupae which had the greatest toxicity* Flee

were killed vitliin 10-13 hours by pupae. A decline in toxicity was appr.trent after the pupal stage. Female adults killed rice after 30 hours and rn^le adults after four days.

The 0-caroton0 content of P. b.rare'*cae larvae up to the end

of the feeding stage increased steadily but thereafter it levelled off. The increase in toxicity of stages up to the pupa shown by 142

Karsh and Eothschild may be correlated with the increase in p- carotene content per insect# However no correlation was found between the greater toxicity of female P. brassicae, and the amount of carotenoids present# Kale and female adults of P. brassicae were found to contain similar amounts of carotenoid per insect# investigated Of the other species of Pieridae/oaly a few specimens of

P. rapae were found to be toxic by Karsh and Eothschild (1973)|

P# rapae being approximately one third as toxic as P# brassicae adults# P. napi was also not very toxic. The total carotenoid content per insect of P. ranae was about ten times less than the highest value for P. brascicae. and P# nani contained about twenty seven times less than P. brassicae.

It has been shown that different insects ere variable in their Pocock W /; toxicity to the same or different species of animal j^Lane, 1957;

Kiarsh and Eothschild, 1973)* Karsh and Rothschild found that the adults of P. brassicae were toxic for mice and hampster but had no effect on the rabbit, rat or guinea pig, whereas A. caja killed the two latter species#

prom a toxicity point of view those species investigated in this work which had large amounts of toxin such as Z. filipendulae#

Cm pomona# C, crocale. did not have larger amounts of p-carotene than non toxic inescts. In terms of defensive mechanisms it could be that p-carotene, by sensitizing the nasal membrane of predators, might be important in insects with email amounts of toxin, but where large amounts of toxin are present the amount of p-carotene or other carotenoids present may not be so important.

Aposematic insects which display bright colours and which may contain distasteful substances, were not found to contain significantly greater amounts of total carotenoid in terms of 143 v s / s P^r insect than cryptic species* Of the species investigated

(see table 33) the range of T/C in pg/g for the two groups was almost the same* However, the larger value of p.g per insect shown for cryptic species appears to be due to their generally greater dry weights than those of aposematic species.

It is interesting to compare the toxic species S* lubricipeda with its Rîiniic S* luteum# S» lubricipeda had the greatest amount cf T/C in terms of vg/g and pg per insect* It was thought that

S. lubricipeda might have greater amounts of carotenoid per insect than S# luteum because it would have more acetylcholine and histamine to detoxify# However, as only one sample was analysed it would be jareferable to analyse several other samples to see whether S. lubricipeda always has larger amounts of T/C than

S. luteum#

The role, if any of carotenoids in toxicity is not a clear one*

Ko evidence was shown that very toxic species had larger amounts of carotenoid than non toxic species# Perhaps the •defined’ amount of carotenoid per insect is sufficient to detoxify any particular toxin present an^ o r enhance the toxicity of any defensive spray or secretion#

The reason wliy carotenoids areopresent in the defensive secretions of some insectsas-part of the haemolyrnph fraction, and in the defensive sprays of other insect species, notably as break­ down products such as sesquiterpenoids, is a problem which deserves more attention* Do carotenoids enhance the toxic nature of the sprays or secretions as Eothschild (1 9 71 ) has suggested, are they present as toxic ingredients themselves, or are they present to draw the attention of the predator to th© coloured defensive secretions produced by many insects when molested? 144

There are many types of toxin ia insects, some sequestered frora the foodplant and others synthesised by the insect, and predators have different reactions to the toxins* Therefore iA any attempt to relate carotenoids and toxicity it would be advisable to study one toxin at a time* Feeding experiments in which amounts of carotenoid and plant derived toxins ingested could be controlled would determine perhaps whether carotenoids are involved either in the detoxification of the toxin In tlie insect and/or in enhancing the repellant nature of the toxin to the insect predator.

The occurrence of so many carotenoids in Nature (up to 300#

Wee don, 1971) is due in part to the number of double bonds of the conjugated chain and also of other modifications of the end rings of the carotenoid molecule. Because carotenoids can easily isomerise in light Wald (I96I) has suggested tiiat they have been selected for by plants for photoreception. The occurrence of many of the different colours in leaves, flowers and fruits is also a result of this facility.

Certain carotenoids are characteristic of certain plant species. The major carotenoids in ©11 green leaved plants ere lutein, neoxanthin and violaxanthin with p-carotene and zeaxanthin also very widespread. Although the plant kingdom has produced so many different carotenoids, it is unlikely that the foodplant of any insect will have more than 20-30 different carotenoids.

Specific carotenoids in the plant function as electron transfer agents in photosynthesis, and others play some part in the colouration of flowers. It is possible that many of the carotenoids botb in plants and insects aid in stability of proteins and other molecules by nature of their physical properties. Certain carotenoids found in the insect are vitamin A precursors wliile the function if any 145 of the other carotenoids in insects has still not been determined#

The involvement and suggested involvement of carotenoids in insect metabolism is summarised in Fig# 22# That at least 8o/ of

0-carotene ingested by P. brassicae larvae is assimilated makes it seem likely that p-carotene plays a part in insect physiology*

Over 90/ of the amount of p-carotene assimilated is metabolised which suggests that processes such as vitamin A metabolism or oxidation of carotencids take place.

It is generally agreed that in plants many different Idinds of secondary plant substances have evolved as protection against many different species of insect posts. The evolutionary response in insects has been the development cf an ability to detoxify the distasteful substances, thus creating a niche, in which other insect species without the ability cannot compete for food#

Carotenoids are involved in wliat Dethier (1970) describes as

’two independently mutating systems’. Insects ha% had some selective influence on the carotenoids present in the plant# For example any carotenoid involved in the plants defensive mechanism against insect pests would be under selective pressure and so too would carotencids involved in attraction cf insect pollinators.

Similarly any insects equipped to use the carotenoids in plants for specialised purposes are lil:ely to have selective advantages in evolution, for example insects which may use carotenoids as detoxification agents. 146

rigure 22#

Fate, functions and suggested functions of carotenoids in animals*

Caro t en d d ■> Colouration o x id a t io n f r e e and bound

Voided' binding witii Stability in exuviae, frass proteins, iats and o r u r in e bile pigmente

b ee

Food'

V is io n yToxicity ^as a toxin in hae-ffiolynph? enhances toxic Growth' ( fa c to r ?

t ho to sen si ti sor?

Gamctogeaesis

Cold-Hardiness-

Temperature control? Enzyme synthesis

Sm ell Detcxlfication'

Invertebrates

— Possibly related functions 147

iUuîTAjrî, K# (1966)# Ph#D# Thesis, Bangor* Studies on the nutrition and feeding of the larvae of the large white butterfly fieris brassicae (L,).

ALLidJ, K.D. and. SSLMAh, I.W. ( 1 9 5 7 )* The response of larvae of the large white butterfly (lieris brassicae L.) to diets of mineral deficient leaves* Bull. eat. Kes*, 48. (1) 229-242. =

A.L, (1932)* The sensitivity of the legs of common butterflies to sugars. J. exp. lool*. 63, ( 1 ) 239-299*

rlPLIa, 2 .T. and kOTIIoJHILD, K. ( 1 9 7 2 )* Poisonous alkaloids in the body tissu e s of the garden tig e r moth (Arc tia caja L*) and the cinnabar moth ( O^rria (=Callimorrha) jacobaeae L. ) (Lepidoptera)* in T oxins of anim al and plant origin* ( ed. by A. de Vries and 1. hochra.) 2 , 3 7 9 - 3 9 3 * dordon and Breach, London. “

APLIh, d.T. and PCZIldHlLD, H. ( 1 9 7 3 ). in preparation.

APLIi:, P .T ., BeMN, K.H. and BCTHBOiiILD, K. ( 1 9 6 3 ). Poisonous allcaloids in the body tissu e s of the cinnabar moth (Callimorpha jacobaeae L.)* Nature, Bond., 2 1 9 . 7 4 7 - 7 4 8 . — —

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Appendix Table 1.

Rf Values of B-carotene end vitamin A alcohol and acetate in various solvents

TLC plates using Silica Gel G

Pet, Ether Benzene Sample Ether Hexane Ethyl Ether Ethyl Ether Acetic Acid (1 t 1) (90 t 10 8 1) (90 t 1)

p-carotene 0,93 0.93 0.99 Vitamin A alcohol 0.48 0.09 0.85 Vitamin A acetate 0.94 0.46 0.93

p-carotene 0.93 0.97 0.99 Vitamin A alcohol 0.49 O.C-6 0.84 Vitamin A acetate 0.93 0.49 0.96

* All tliree cbrcroatogramed together 1, Goodwin and Olson (196?) 2, Kahan (19&7) 3, Targan (1p69) 169

Appendix Table 2.

Incorporation of r3Hl B-carotene label into ^ut. haemolymph and fat body of M JP. brassicae larvae.

After 14 hours.

Coiints in Gut Counts in Eaeroolyraph Counts in Fat Body dpm dpia (dps

1 1548 815 118 2 io 6o 1740 121 3 473 5824 424 4 461 1723 366 5 1028 453 2747 6 - 490 809 7 - 575 3768 8 - 525 716 9 - 237 3527 10 - ~ 4335

Total 4570 10382 Counts 16931 Kean 914.00 1153.60 1693.10

Standard 557.36 878.57 1211.36 Error 170

App Fig. I

ABSORPTION CURVES AND ACID SHIFTS OF MAJOR CAROTENOIDS

acid shift

VIOLAXANTHIN 5.6-MONOEPOXY-LUTEIN

20 20

N\

400 500450 400 450 500

NEOXANTHIN 5.6-MONOEPOXY-3-CAROTENE

20 20

400 450 500 400 450 500 171 A p p Fig. 2

ABSORPTION CURVES

6-CAROTENE AUROXANTHIN

20 20

-e 10 10

400 500450 380 400 425 nm nm

LUTEIN FL AVOXANTHIN

20 20

0

400 450 500 400 450 500 nm nm 172

A p p Fig. 3

WET W I / DRY WT CONVERSION FOR CABBAGE

n

12 to E 10 E CO 8 Î 6 A

2

10 20 30 40 50 60 70 80 90 100 110 120 130

wet weight in grams A p p F ig , 4 173

CALIBRATION CURVE OF VITAMIN A ALCOHOL WITH TRIFLUORO- ACETIC ACID

20

5

S C to 0 ■9 o o after 30 seconds

CO

0 5

100 200 300 400 600500 700 800 International units of vitamin A

CALIBRATION CURVES OF VIT. A ALCOHOL W IT H T F .A .

0 4

o 15 seconds

0 3 A 60

R 0 -2

10 20 30 40 5 0 6 0 70 80

vitamin A (i.u.) A p p Fig. 5 174

CHROMATOGRAPHY OF 6-CAROTENE AND VITAMIN A STANDARDS IN VARIOUS SOLVENTS

A 1 6-carotene 2 vit. A alcohol 3 vit; A acetate 2 3 4 4 all three

A ether hexane (1 = 1)

B B pet.ether ethyl ether ^cejlc , ( 90:10 = I)

C benzene ethyl 2 3 4 ether ( 90:10 )

s.f. solvent front

C

2 3 4 A p p Fig. 6 175

DISINTEGRATION OF VITAMIN A MAXIMA AT 620 nm

VITAMIN A ALCOHOL

30 secs

CO _o (0

0 5

5 5 0 • 600 6 5 0 nm

VITAMIN A ACETATE

2-0

3 5 secs

5

l

_Q 0 ca

0 5

5 5 0 60 0 6 5 0 nm 176

LI3T OF SUPPLIERS

Âmershani/Searle» See Radiochemical Centre.

Beckman Ltd.* Worsley Bridge Road* London* S.S.26.

British Drug Houses Ltd.* Poole, Dorset.

Butterfly Farm, Bexley, Kent.

Griffin and George Ltd.* Ealing Road* Alperton* Wembley* London*

Eoffmann-La Roche & Co., Ltd.* Basle* Switzerland.

Imperial Chemical Industries Ltd., Jealott*s Hill, Bracknell, Berks.

E.î-îil Ltd., Suppliers to Griffin and George Ltd.

Radiochemical Centre* Amersham, Buckinghamshire.

Shell Research Laboratories, Sittingbourne, Kent.

Shandon Sci. Co. Ltd.* 65* Pound Lane* Willesden* London.

Technic Ltd., Suppliers to Griffin and George Ltd.

Thompson and Morgan (Ipswich) Ltd.* Seed Growers* Ipswich* Norfolk.

Tracerlab ^td.. Ship Yard, Weybridge* Surrey.

Volac Ltd., Suppliers to Griffin and George Ltd.

Worldwide Butterflies Ltd.* Over Compton* Sherboume* Dorset. 177

ACKNOWLEDGEMENTS

I would like to express my thanks to Dr. Guy Valadon* my supervisor during cy studies* to Dr. Jan Kerr for her useful criticisms and reading of the manuscripts, and to Kiss Rosemary

Mummery for technical assistance# I would also like to express my thanks to Mr. Devi Davies and Dr. P. Eramley for reading the manuscript on the radioactive studies* and to all the members of the Biological departments of Royal Holloway College* especially

Professor K. Wilson and members of the Botany Department for their useful advice and assistance throughout ny work.

In particular I am very grateful to The Hon. Miriam Rothschild for providing the stimulus and materials for work on the section on toxicity. Also to Dr. Karsden of the Birmingham and Midland Eye

Hospital for providing the unpublished technique for vitamin A determination. My thanks are also due to Dr. M.H. Breese of the

Shell Laboratories, Sittingbourne* Kent; to the staff of the I.C.I. laboratories* Jealott*s Hill* Berks., and to Dr. C.F. Rivers and

Dr. B.O.C. Gardiner, Cambridge, for gifts of material*

For labelled p-carotene I am grateful to Dr. 0. Isler of

HoffmaiRLa Roche & Co. Ltd.* Switzerland. I am very grateful also to Professor T.W. Goodwin of Liverpool* Dr. G,M, Chippendale,

Missouri* and Dr. H. Thoramen of Hoffmann-La Roche & Co. Ltd.* for their useful advice. Finally I Would like to thank Mr. D. Ward and

Mr. M. Colthorpe for their help in preparing the photographs.