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 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10096797 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Pieris brassicae L * 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<xHS d^tr^y^lu^ ikâm, li/oalcL he alim éd ^ Ike pidlic h h& 0/ u ^ e fÀ ayid if^Apcviraui u/oré.:.. fL k^m/lMae cj ilie p^cpedtes^ earu^m ^, propo^aéùoLclm A ii^yfujrt oj^ Ute- lip r (uui c^ise^vcpicvi of -Htepe auinuiLs^ is a U£c6pfxy^ ^ p Iro leaxL as la jow£- yndliPcL of pnevadiAA^ Uiebr depredailcas. 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<!rtu. ~T. Beusle^j L.<mÀ-sity. CONTENTS “ 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.