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ALLELOCHEMICALS FROM SORGHUM BICOLOR 
THAT STIMULATE FEEDING BY THE LARVAE 
OF THE STEM BORER CHILO PARTELLUS 
A thesis submitted to the 
University of Ghana, Legan, 
in fulfilment of the requirements 
for the degree of Doctor of Philosophy. 
by 
BALDWYN TORTO, B.Sc., M.Sc. (GHANA) 
Department of Chemistry AUGUST 1988 
University of Ghana 
LEGaN 
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To Rita, Q'Baka and Nii Sai, 
who put up with so much. 
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II Much of life can be understood in rational terms if 
expressed in the language of chemistry. It is an 
international language, a language for all time, a language 
that explains where we came from, what we are, and where the 
physical world will allow us to go. Chemical language has 
great~sthetic beauty and links the physical sciences to the 
biological sciences. Unfortunately, the full use of this 
language to understand life processes is hindered by a gulf 
that separates chemistry from biology. The gulf is not 
nearly so wide as the one between humanities and sciences. 
Yet, chemistry and biology are two distinct cultures and the 
rift between them is serious, generally unappreciated, and 
counter~roductive ............. " 
Nobel Laureate Arthur Kornberg 
on Chemistry and Biology 
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DECLARATION 
It is hereby declared that the following is the result of 
three years research undertaken by the author under 
supervision at the International Centre of Insect Physiology 
and Ecology (ICIPE), Nairobi, Kenya, and that it has neither 
wholly nor partly been presented elsewhere for another 
degree. 
t1J1vv'() 
.... '.l.-,.~ ......... . 
BALDWYN TORTO 
(candidate) 
.. .Af-!:-:<-..... 
PROF. A. HASSANALI 
(ICIPE supervisor) 
................... 
PROF. K.N. SAXENA 
(ICIPE supervisor) 
... ~: ......  . ~ .... . 
DR. W.R. PHILLIPS 
(University supervisor) 
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(i) 
CONTENTS 
Iv 
Acknowledgements 
Abstract 
1 
Introduction 
CHAPTER 1 
Insect phagostimulants and antifeedants: 
An overview 5 
Allelochemics 7 
Insect behaviour 8 
Insect feeding stimulants 9 
Insect feeding deterrents 25 
Compounds which act both as 
stimulants and deterrents 44 
Bioassay methods 45 
CHAPTER. 2 
Previous feeding allelochemical work on 
sorghum and objectives of the present study 47 
Locusta migratoria L. 48 
Schizaphis graminum (Rondani) 50 
Chilo partellus (Swinhoe) 51 
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(ii) 
Objectives of the present study 52 
CHAPTER 3 
Preliminary investigations 54 
Materials and methods 55 
Plant material 55 
Extraction 55 
Development of a feeding bioassay 55 
Feeding tests 57 
Choice situation 57 
No choice situation 60 
Chromatography 60 
High performance liquid chromatography (HPLC) 61 
Gas liquid chromatography (GLC) 62 
Results 62 
Feeding assays 63 
Choice situation 63 
No choice situation 68 
Discussion 68 
CHAPTER 4 
Experimental details and results 70 
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(iii) 
CHAPTER 5 
Discussion 129 
CHAPTER 6 
Conclusions and suggestions 
for further studies 143 
References 150 
Glossary of special terms 178 
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ACKNOWLEDGEMENTS 
I wish to thank most sincerely Prof. A. Hassanali, my 
immediate supervisor for the deep interest he took in this 
work, for the immense help and guidance he gave me during 
the course of the work, and for critically reading the 
entire manuscript; Prof. K.N. Saxena, Dr. W.R. Phillips, Dr. 
M.E. Smalley and Dr. W. Lwande for their encouragement and 
advice; Dr. S. Nokoe and Mr. O. Okello for biomathematical 
assistance; and Dr. P.G. MCDowell for providing all the MS 
and GC-MS data of the isolated and synthetic samples. 
The part played by technical staff at the ICIPE, 
particularly Mr. H. Amiani, Mr. P.E. Njoroge and Mr. A. 
Majanje, in the care of greenhouse plants and insects is 
gratefully acknowledged. 
I wish also to thank the University of Ghana for 
granting me study leave; the German Academic Exchange 
Program (DAAD) for their generous financial support; and the 
Director of the ICIPE, Prof. T.R. Odhiambo for allowing me 
to use facilities at the ICIPE for the study. 
Finally, I am grateful to Mr. N.K. Mwanga for providing 
the scientific drawings and Miss Jane Mwaniki for typing the 
references and glossary of special terms. 
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ABSTRACT 
Feeding bioassays with cellulose acetate discs 
impregnated with the hexane, ethyl acetate and methanol 
extracts of the leaf-whorls of field grown plants of sorghum 
cultivars IS 18363 (susceptible) and IS 2205 (resistant) 
showed that the methanol extracts were most stimulatory to 
the feeding of the third-ins tar larvae of Chilo partellus. 
Ethyl acetate extracts were intermediate in stimulatory 
activity whilst hexane extracts were the least stimulatory. 
Extracts of the more susceptible cultivar were more 
stimulatory than those of the more resistant cultivar and 
those of the whorls of the 3 week old plants were more 
stimulatory to larvae than those of the 6 week old plants. 
The phagostimulatory compounds in the ethyl acetate 
extracts were phenolic, p-hydroxybenzaldehyde and p-
hydroxybenzoic acid being the major components and ferulic 
and caffeic acids being in minor amounts. p-Coumaric acid 
was also present in minor amounts but was non-stimulatory at 
all the doses tested. p-Hydroxybenzaldehyde was a more 
potent feeding stimulant for the larvae relative to some of 
its possible theoretical biogenetic analogues. Limited 
structure-activity studies with some hydroxybenzoic acids 
and their corresponding cinnamic acids showed that the 
former were more stimulatory to the feeding of the larvae 
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than the latter and that oxygen substitution in the benzene 
ring was crucial for activity. 
The phagostimulatory compounds in the methanol extracts 
were phenolic, identical to those in the ethyl acetate 
extracts, and sugars. The sugars which were identified in 
the extracts comprised sucrose, fructose, glucose and 
xylose. The feeding response of larvae to these sugars 
followed the order sucrose » glucose ~fructosei xylose was 
non-stimulatory. Comparison of the activities of sucrose 
with mixtures of glucose and fructose showed that the high 
activity of the disaccharide is due to its total structure 
and not to a summation of its monosaccharide moieties. 
Sugars synergised with phenolics to give enhanced 
feeding response of the third-instar larvae. 
Chromatographic analyses of the extracts showed that 
stimulatory and non-stimulatory components in the extracts 
differed quantitatively rather than qualitatively in the 
whorls of the two cultivars at the two growth stages. This 
may have implication in resistance screening and breeding 
programmes. 
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INTRODUCTION 
Sorghum bicolor (L.) Moench (Andropogeneae : 
Graminaeae) is a native of Africa and Asia (BrOunk, 1975). 
The plant varies in height from 0.5 to 6 m depending on the 
cultivar. It is an annual with a single stem, usually 
erect, dry or juicy, with a diameter ranging from 0.5-3 cm. 
It is similar to maize, but with only one type of 
inflorescence which is a panicle consisting of spikelets 
with bisexual flowers. The plant is adapted to a wide range 
of ecological conditions, growing in hot and dry conditions 
and in areas with high rainfall in which waterlogging 
occurs. The optimum temperature for growth is 30 °c 
(Purseglove, 1975). 
Sorghum is ranked as the fourth most important world 
cereal, following wheat, rice and maize and it is the staple 
food in the drier parts of tropical Africa, India and China 
(Purseglove, 1975). The grain is used in preparing a 
variety of foods. It is ground into a wholemeal flour and 
made into a thin porridge or a dough by boiling in water. 
The pealed grain is sometimes cooked like rice or ground 
into flour for making biscuits or bread. In certain parts 
of Central, Eastern and Southern Africa, some varieties of 
the grain are used for brewing beer. In the developed 
2 
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countries like the United States, che grain and plant are 
used as food for livestock while syrup is manufactured from 
the varieties called sorgos (Purseglove, 1975). These have 
large juicy stems which have been found to contain up to 10% 
sucrose. It is estimated that 2-3 million gallons of syrup 
are produced per year from sorghum in the United States. 
Sorghum is attacked by a number of insect pests. The 
worst species are the stalk-borers. In Eastern Africa and 
India, Chilo partellus (Swinhoe) has been found to cause the 
severest damage to the plant (Teetes et al., 1983). In 
addition Busseola fusca (Fuller), Locusta migratoria L., 
Eldana saccharina (Walker) and Sesamia calamistis Hmps. are 
important stemborers of the plant, and in some locations one 
of this is found to be its predominant pest (Teetes et al., 
1983; Seshu Reddy, 1985). It is also attacked by the 
sorghum shootfly Atherigona soccata (Rondani) and a variety 
of aphids (Teetes et al., 1983). 
The work reported in this study relates to C. 
partellus. In the field, it attacks all parts of the plant 
except the roots. The first indication that the plant is 
infested by the larvae of this insect is the appearance of 
shot-holes and lesions in the young whorl leaves (Dabrowski 
and Kidiavai, 1983; Teetes et al., 1983). A dead heart and 
a ragged appearance of the leaves indicate severe attack on 
the plant. Larvae also bore into the stem causing extensive 
3 
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tunnelling, which affects the growth and survival of the 
plant. 
The bases of sorghum resistance to its insect pests has 
attracted the attention of a number of researchers allover 
the world. At the International Centre of Insect Physiology 
and Ecology (ICIPE), the focus is on resistance and 
susceptibility manisfestations of different cultivars at 
different stages in the colonisation of the plant by C. 
parteIIus, B. fusca, and E. saccharina. At the 
International Crops Research Institute for the Semi-Arid 
Tropics (ICRISAT), similar studies have been reported for 
the migratory locust L. migratoria (Bernays and Chapman, 
1978; Woodhead and Bernays, 1978; Woodhead, 1982 and 1983). 
The greenbug aphid Schizaphis graminum is the insect of 
interest at the United States Department of Agriculture 
(USDA) (Dreyer et al., 1981). 
Saxena (1985a) described two broad categories of 
factors which govern the interaction between plants and 
their insect pests. These include responses of the insect 
to the plant leading to its establishment, and the 
characters of the plant which control these responses. The 
insect's main responses which include orientation 
(attraction or repulsion), oviposition and feeding are 
determined by the plant's physical and chemical characters. 
It is believed that these plant characters if identified can 
4 
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be used in various pest management strategies such as 
behaviour manipulation with active host chemicals and the 
use of these characters as a basis for screening and 
breeding for resistance. 
As part of this program of the ICIPE, this study was 
undertaken to identify the feeding stimulants in sorghum for 
c. partellus larvae. The report on this study is divided 
into six chapters. Chapter 1 gives an overview on insect 
phagostimulants and antifeedants. Chapter 2 gives a summary 
of the previous allelochemical work on sorghum and the 
detailed objectives of this study. Preliminary 
investigation involving the development of feeding bioassays 
for the third-instar larvae of C. partellus and the 
development of chromatographic separation conditions for 
sorghum extracts are described in Chapter 3. Chapter 4 
describes the experimental details and results of the 
investigations carried out in this study, and Chapter 5 
discusses these results. Some suggestions for future 
studies as a follow up of the present work are given in 
Chapter 6. This is finally followed by a glosSQry of 
special terms and a list of references. 
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CHAPTER 1 
INSECT PHAGOSTIMULANTS AND ANTIFEEDANTS: 
AN OVERVIEW 
n~e~ss~r~ 
Insects are alll.r~y to plants for their pollination 
and thus for their reproduction (Edwards and wratten, 1980). 
Plants have therefore evolved features like alluring colours 
and odours to attract insects for fertilization, and in 
return they provide pollen and nectar to the insects. 
However, research has shown that not all insects are useful 
to the plant (Marini-Bettolo, 1983; Boppre, 1986). Some are 
harmful to it, and these include phytophagous insects which 
solely depend on plants for their food and oviposition (egg-
laying) sites (Edwards and Wratten, 1980). The mechanisms 
by which plants escape from the damaging effects of these 
insects and the adaptation of the insects, in turn, to their 
food supplies have been studied by Erhlich and Raven (1964), 
Jermy (1976) and Feeny (1983). They postulated that plants 
have evolved a great variety of defensive mechanisms, mainly 
production of toxic chemicals to protect themselves from 
these insects. In turn, phytophagous insects have also 
evolved mechanisms enabling them to avoid or detoxify these 
toxins in their food, and in some cases have been able to 
use the chemicals characteristic of certain species of 
plants as cues for the recognition and selection of their 
hosts. These workers described this phenomenon between 
plants and phytophagous insects as coevolution. 
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Before the theory of coevolution was promulgated, 
studies on the role of plant chemicals on the behaviour 
(feeding and oviposition) of phytophagous insects were 
already in progress. The earliest experimental evidence of 
the importance of these chemicals as cues for food selection 
by phytophagous insects was provided by Verschaeffelt (1910) 
working with cruciferous feeding insects. His work showed 
that members of the plant family Cruciferae owed their 
interaction with Pieris species to a single set of 
chemicals, the mustard oils and their glycosides. Though 
this discovery set the direction of research for several 
decades, attention was mainly focussed on plant attractants 
~ 
and stimulants with little emphasis on repell~nts and 
deterrents (Dethier, 1982). However, recent years have 
witnessed intense studies in the establishment of the 
influence of the various classes of plant chemicals on the 
feeding and oviposition of several insect pests, and 
noteworthy reviews on the subject have been provided by 
Dethier (1966), Chapman (1974), Hedin et ai., (1977), 
Schoonhoven (1982) and Staedler (1986). 
In this chapter an attempt has been made to summarise 
the literature on some of the salient features on the 
chemical basis of feeding by phytophagous insects. The 
focus is on studies on well-characterised feeding stimulants 
and deterrents. Tables 1.1 and 1.2 list these compounds, 
their plant sources and the insects they have been 
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demonstrated to affect (see pages 17-23 and 30-43). 
Compounds which play opposite roles, acting as stimulants to 
certain insect species but deterrent to others, are also 
included. Also included in this chapter is a summary of the 
various bioassay methods which have been used in testing for 
phagostimulation and deterrency in insects. 
1.1 Allelochemics 
Chemicals that are produced by individuals of one 
species and which affect the physiology or behaviour of 
individuals of another species are called allelochemics 
(Whittaker and Feeny, 1971). 
In insect-plant relationships, two broad categories of 
allelochemics are recognized: al1omones and kairomones. 
Allomones are chemicals used by the plant to protect itself 
from insect attack. They may act as repellents, deterrents 
or antibiotics which interfere metabolically with normal 
growth and development of the insect. Kairomones are 
chemicals used by the insect for host location and 
recognition, and food detection. They may act as 
attractants, arrestants or stimulants. Reese (1979) found 
that allelochemic effect is both concentration-dependent and 
situation-dependent. Thus a chemical classified as a 
stimulant for one insect species may act as a deterrent to 
8 
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the same insect when present in a high concentration. 
Although plant chemicals are important cues for food 
selection by phytophagous insects, studies have shown that 
they seldom alone determine insect behaviour. The other 
cues used by the insect in discriminating between various 
potential foods include biophysical factors of the plant 
such as shape, colour, texture, water content and 
ultrastructure (Dethier, 1982; Staedler, 1983) 
1.2 Insect Feeding Behaviour 
Insect feeding involves various behavioural responses 
which can be divided into four distinct steps (Dethier, 
1966; Munakata,1977; Schoonhoven, 1982; Miller and 
Strickler, 1984): (1) host plant recognition and 
orientation, comprising locomotion which brings the insect 
to its food and cessation of locomotion on arrival, also 
termed arrest; (2) initiation of feeding (biting, probing or 
sucking); (3) continuation of feeding; and (4) termination 
of feeding as a result of satiation. 
Feeding has been studied with a variety of assays 
reflecting the different behaviours of the insects 
described. From these assays, it has been suggested that 
the acceptance or rejection of a plant as food by an insect 
is determined by a number of factors (Dethier, 1982). These 
9 
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include the intensity of olfactory and gustatory feeding 
stimulants, the intensity of repellents and deterrents, the 
metabolic state of the insect (i.e. the degree of 
deprivation, specific dietary deficiencies, malaise etc.) 
and finally learning acquired as a result of previous 
feeding experience. 
Evidence to support some of these suggestions has been 
provided by electrophysiological studies at the sensory 
level (Schoonhoven and Jermy, 1977; Dethier, 1980; Staedler, 
1982). These have shown that chemoreceptors are the means 
by which the insect detects plant chemicals and is able to 
differentiate between a stimulant and a deterrent compound. 
Thus insects are said to respond to a summation of inputs 
'gestalt' from various chemoreceptors which perceive complex 
mixtures of compounds present in their foods. 
1.3 Insect Feeding Stimulants 
A feeding stimulant enhances the feeding of an insect 
and it can be classified as nutritional or non-nutritional 
(Dethier, 1966). The nutritional compounds are essential 
for the proper development of the insect, and they include 
sugars, amino-acids, inorganic salts, vitamins and 
phospholipids. The non-nutritional chemicals, also 
described as token stimuli (Fraenkel, 1959), are said to 
only guide the insect in discriminating its food, and they 
10 
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comprise secondary plant compounds (Dethier, 1966). Some 
insects are stimulated to feed by a single dominant compound 
while others require several different chemicals in 
combination (Dethier, 1982; Miller and Stricker, 1984; 
Staedler, 1986). 
Of the nutritional compounds, sugars, especially 
sucrose, have been recognized as a feeding stimulant to the 
majority of insects (Thorsteinson, 1960; Dethier, 1966; 
Schoonhoven, 1968; Sutherland, 1977; Staedler, 1983). 
According to Beck (1956), the choice by the European corn 
borer Ostrinia nubilalis (Hubner) of sites on the corn plant 
is based on the highest sugar concentration and is not 
influenced by local attractants. The importance of sucrose 
as a phagostimulant has been demonstrated for a number of 
lepidopteran and coleopteran insects (Dethier, 1966; Hsiao 
and Fraenkel, 1968; Hsiao, 1969; Peacock and Fisk, 1970 ; 
Sutherland, 1971; 1977; Doss and Shanks, 1984; Ladd, 1986 
Shanks and Doss, 1987). Sucrose is also known to synergise 
with other compounds to give enhanced feeding stimulation. 
Sitosterol in combination with sucrose was found to 
stimulate more feeding by obscure root weevil Sciopithes 
obscurus Horn than sucrose alone(Doss, 1983). A similar 
synergism was found by Shanks and Doss (1987) with the same 
mixture with black vine weevil Otiorhynchus sulcatus (F.). 
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Other related sugars have been found to possess 
phagostimulatory activities. These include raffinose for 
the red cotton bug Dsydercus koengii (F.) (Hedin et al., 
1977), fructose for the eastern spruce budworm Choristoneura 
fumiferana Clem. (Albert et al., 1982) and the onion maggot 
Delia (=Hylemya) antiqua (Meigen) (Mochizuki et al., 1985). 
The eastern spruce budworm is stimulated to feed on 
cellulose discs by glucose and inositol (Albert et al., 
1982), and Numata et al., (1979) found that pinitol, 
fructose and myo-inositol stimulated feeding of the larvae 
of the yellow butterfly Eurema hecabe mandarim. Ladd (1986) 
has reported that several Scarabaeidae species are 
stimulated to feed by a vareity of sugars including sucrose, 
maltose, fructose and glucose. However, arabinose, xylose 
and raffinose were rather weak feeding stimulants for these 
species of insects. 
Tests with amino-acids have shown that they are 
generally weak feeding stimulants (Dethier, 1966). However, 
for the brown rice planthopper Nilaparvata lugens (Stal), 
asparigine is the major phagostimulant (Sogawa, 1982). 
Amino-acids have been shown in some studies as chemicals 
which act in combination with other compounds, especially 
sugars, to give enhanced feeding stimulation in a variety of 
insects. An example of this is demonstrated by the study of 
Beck and Hanec (1958). They found synergistic effects 
between serine and glucose for the European corn borer. 
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leucine and methionine each have been shown to synergise 
with sucrose to enhance the feeding of the green peach aphid 
Myzus persicae (Sulzer) (Mittler and Dadd, 1964). In a 
recent study, Albert and Jerrett (1981) found that feeding 
by the eastern spruce budworm is enhanced by a two component 
mixture of sucrose with a variety of amino-acids. These 
amino-acids include L-proline, L-hydroxyproline, L-glutamic 
acid and L-arginine. Protein deprived females of the 
housefly Musca domestica (L.) are stimulated to feed by a 
mixture of L-leucine and sodium phosphate buffer solution 
(Browne and Kerr, 1985). 
In addition to sugars and amino-acids, a number of 
other nutrient components have been reported as insect 
feeding stimulants. Examples of these include betaine, 
ascorbic acid and thiamine for Chorthippus curtipennis 
(Harris) (Dethier, 1966), adenosine for the sweetclover 
weevil Sitona cylindricolis Fahraeus phospholipids for the 
Colorado potato beetle Leptinotarsa decemlineata (Say) 
(Hedin et al., 1977) and sitosterol for the weevils, 
Otiorhynchus sulcatus (F.), SCiopithes obscurus (Horn) and 
Hypera postica (Gyllenhal) (Shanks and Doss, 1987). 
Extensive studies have been carried out on the role 
played by secondary plant (non-nutritional) chemicals in the 
selection of food by phytophagous insects. Table 1.1 shows 
that these compounds range from high molecular weight 
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compounds such as glycosides to low molecular weight ones 
such as simple phenols. 
The well investigated glycosides include mustard oils 
as well as phenolic, cyanogenic and iridoid glycosides. 
Mustard oil chemicals are associated with cruciferous 
plants. Of these sinigrin, glucapparin and glucoiberin have 
been found to trigger on the feeding of the following 
insects: the diamondback moth Plutella maculipennis (L.), 
the mustard beetle Phaedon cochleariae F., Pieris brassicae 
L. and the flea beetles Phyllotreta cruciferae (Goeze) and 
Phyllotreta striolata (F.) (Whittaker and Feeny, 1971). 
According to Wens1er (1962), this class of compounds also 
accounts for the feeding by the cabbage aphid Brevicoryne 
brassicae (L.) on cruciferous plants. Iridoid and 
cyanogenic glycosides are not common insect feeding 
stimulants. However, for the Mexican bean beetle E. 
varivestis feeding is promoted by the cyanogenic glycosides 
phaseolunatin, lotaustrin and linamarin (Hedin et al., 
1977). Bowers (1983 and 1984) in a systematic search 
isolated and identified two iridoid glycosides, catalpol and 
aucubin from the plant Plantago lanceolata which stimulated 
feeding by the checkerspot butterfly Euphydryus challedona 
(Doubleday) and the buckeye butterfly Junonia coenia both of 
which are pests of the plant. 
14 
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Phenolic and flavonoid compounds are well known feeding 
stimulants for certain coleopteran insects. The smaller 
European elm bark beetle Scolytus multistriatus (Marsham) is 
strongly stimulated to feed on pith discs treated with 
p-hydroxybenza1dehyde or some of its analogues (Baker and 
Norris, 1968), (+)-catechin 5-~-D-xylo-pyranoside or its 
aglycone (+)-catechin from Ulmus americana (L.) (Norris, 
1977). The horse-raddish beetle Phyllotreta armoraciae 
(Koch) is stimulated to feed by glucosinolates, kaempferol 
3-0-xylosylgalactoside and quercetin 3-0-xylosylgalactoside 
(Nielsen et al., 19J9). Coleopteran insects have also been 
used in feeding bioassays for tests involving three phenolic 
glycosides: salicin, populin and luteolin-7-glucoside. 
Significant feeding stimulation were found for various 
concentrations of salicin for the willow beetle Gonioctena 
vitallinae (L.) (Hedin et al., 1977) and the salicaceous 
feeding beetle Phyllodecta vitellinae (Hutchinson, 1931). 
When presented on filter paper discs, populin and luteolin-
7-glucoside promoted the feeding of Chrysomelia 
vigontipunctata costella (Marseul), Plagiodera versicolora 
distincta Baly and Lochmaea capreae cribata Solosky (Matsuda 
and Matsuo, 1985). Other active phenolic and flavonoid 
compounds reported to be active insect feeding stimulants 
include chlorogenic acid and cyanidin-3-glucoside (Hedin, et 
al., 1977). 
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Some phytosterols have been reported as potent feeding 
stimulants for certain insect species. An example of this 
is ~-sitosterol which is a feeding and biting stimulant for 
silkworm Bombyx mori (L.) (Hamamura et al., 1962; Ito et 
al., 1964), Colorado potato beetle Leptinotarsa decemlineata 
(Hsiao and Fraenkel, 1968), black vine weevil otiorhynchus 
sulcatus (F.) (DOss and Shanks, 1984) and alfalfa weevil 
Hypera postica (Gyllenhal) (Shanks and Doss, 1987). The 
obscure root weevil Sciopithes obscurus Horn is stimulated 
to feed by sitosterol and stigmasterol (Doss et al., 1982 ; 
Doss, 1983). 
Synergistic effects between secondary plant compounds 
have also been demonstrated. Chromatographic fractions were 
generally found to be less stimulatory than the mother 
extracts, and full activity is often not regenerated by 
recombining the test mixtures (Hedin et al., 1977). The 
major cause of this is the breakdown of some compounds 
during isolation and purification. However, these studies 
go to confirm the suggestions of some workers that adequate 
feeding response from some phytophagous insects is obtained 
from a complex profile of chemicals. 
Studies have also been conducted on a number of insects 
which feed on only a few plant species. Such insects often 
use specific secondary plant metabolites as cues for 
feeding. Smith (1966) found that the change of feeding 
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sites on broom Sarothamus scoparius by adults of the aphid 
Acyrthosiphon spartii is associated with the movement of 
sparteine between the various parts of the plant. A similar 
example was reported by Kogan (1976) that several species of 
Diabrotica and Acalymona show considerable preference for 
certain species of Cucurbitaceae with high cucurbitacin 
concentrations. Similar examples have been found for the 
insect Calpe excavata which feeds on Cocculus trilobus in 
response to the alkaloid isoboldine (Wada and Munakata, 
1968) and the monophagous beetle Chrysolina brunsvicensis 
which lives on Hypericum and requires hypericin in its food 
(Rees, 1969). Kogan (1976) reported that three solanaceous 
feeding insects, Lema trilineata daturaphila Kogan and 
Goeden, L. decemlineata and Manduca sexta (L.), show 
preferences for certain species of solanaceous plants 
characterised by the presence of certain classes of 
alkaloids. Solanum plants characterised by the presence of 
tropane alkaloids are accepted by L. trilineata and those 
characterised by steroidal alkaloids such as solanine and 
chaconine by L. decemlineata but Manduca sexta shows broad 
spectrum tolerance for the various classes of alkaloids. 
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Table 1.1 INSECT FEEDING STIMULANTS. 
!:eediM_ s_tim~!~_~J_ ____ _ P_~I!!l_~_. __ ~_~\l_r_~_e_ _______ ~_~_~~!_ _n~ J!I~_ _ ___ __ . _ __ __RE!f~~_~nC::_I! _____  
ADENOSINE sweet clover Sitona cylindricolis F. Hedin et al.,(1977) 
ALLYL SULFIDE onion Hylemya antiqua (Meigen) Miller et a1., (1984) 
.... 
ANISIC ACID general H. bivittatus (Say) Hedin et al., (1977) ...s 
ANISIC ALDEHYDE anise, citrus P. polyxenes Stoll Hedin et a1., (1977) 
ARGININE pine C. fumiferana Clem. Albert et a1., (1981) 
ASCORBIC ACID general Chorthippus curtipennis Dethier (1966) 
(Harria) 
ASPARIGINE rice Nilaparvata lugens (Stal) Sogawa(1982) 
AUCUBIN P. lanceolata Junonia coenia Bowers (1984) 
BENZOIC ACID general H. bivittatus (Say) Hedin et a1., (1~77) 
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BETAINE general C. curtipennis (Harris) Dethier(1966) 
CARLINOSIDE rice N. 1ugens (Stal) Kim et a1., (1985) 
CARVONE Umbe11iferae P. po1yxenes Stoll Dethier(1966) 
CATALPOL P. 1aceo1ata Euphydryus challedona Bowers (1983) 
(Doubleday) 
(+)-CATECHIN-T~-XYLO­ elm Sco1ytus mu1tistratus Norris (1977) 
PYRANOSIDE (Marsham) 
CHACOLINE potato leaves Leptinotarsa decem1ineata(Say) Kogan(1976) .... 
CD 
CHLOROGENIC ACID potato leaves L. decem1ineata (Say) Hedin et a1., (1977) 
CITRONELLOL cotton Spodoptera 1itora1is (Boisd) Hedin et a1., (1977) 
COUMARIN sweet clover S. cy1indrico1is F. Hedin et a1.,(1977) 
CUCURBITACINS Cucurbitaceae Diabrotica undecimpunctata Hedin et a1., (1977) 
Cruciferae (Barber), Au1acophora 
foveicollis 
FORMIC ACID cotton A.grandis Boheman Hedin et a1.,(1977) 
FRUCTOSE onion Hy1emya antiqua (Meigen) Mochizuki et a1., (1985) 
pine C. fumiferana Clem. Albert et a1., (1981) 
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GLUCOCAPPARIN Cruciferae Pieris brassicae (L.) Whittaker et a1., (1971) 
GLUCOHEIROLIN Brassica spp. P. brassicae (L.) 
Capparidaceae P1ute11a maculipennis (L.) Whittaker et a1., (1971) 
GLUCOIBERIN Cruciferae P. brassicae (L.) Whitakker et a1., (1971) 
GLUCOSE cottonseed Dsydercus koenigii (F.) Hedin et a1., (1977) 
C. fumiferana Clem. Albert et a1.,(1982) 
GLUCOSINALBIN Brassica spp. P. brassicae (L.) Hedin et a1., (1977) 
GLUCOTRICIN rice N. 1ugens (Stal) Sogawa(1982) ~ \D 
GLUTAMIC ACID pine C. fumiferana Clem. Albert & Jerrett(1981) 
GOSSYPOL cotton A. grandis Boheman Hedin et a1.,(1977) 
HOMOINETIN rice N. 1ugens (Stal) Sogawa(1982) 
o-HYDROXYBENZOIC rice N. 1ugens (Stal) Sogawa(1982) 
ACID 
o-HYDROXYBENZYL elm s. mu1tistratus (Marsham) Baker et a1., (1968) 
ALCOHOL 
p-HYDROXYBENZALDEHYDE elm s. mu1tistriatus (Marsham) Baker et a1., (1968) 
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HYPERICIN Hypericium Chrysolina brunsvicensis Rees(1969) 
hirsutum 
INOSITOL C. fumiferana Clem. Albert et al., (1982) 
ISOBOLDINE Cocculus Calpe excavata Wada & Munakata(1968) 
trilobus 
~-KETOGLUTARIC ACID cotton A. grandis Boheman Hedin et al., (1977) 
LACTIC ACID cotton A. grandis Boheman Hedin et al., (1977) 
LECITHIN corn, H. bivittatus (Say), Dethier (1966) N 
o 
soybean Camnula pellucida (Scudder) 
LINAMARIN Phaseolus Epilachna varjvestis Mulsant Hedin et al., (1977) 
spp. 
LOTAUSTRIN Phaseolus E. varivestis Mulsant Hedin et al., (1977) 
spp. 
LUTEOLIN-7-GLUCOSIDE Salicaceous Chrysomelia vigintipunctata Matsuda & Matsuo(1985) 
spp costella (Marseul) 
Plagiodera versicolora 
distincta Baly. 
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MALONIC ACID cotton A. grsndis Boheman Hedin et sl., (1977) 
MONOSODIUM GLUTAMATE c. curtipennis (Harris) Dethier(1966) 
NEOCARLINOSIDE rice N. lugens (Stal) Kim et sl., (1985) 
NEOSCHAFTOSIDE rice N. lugens (Stal) Kim et sl., (1985) 
ORIZATIN rice N. lugens (Stal) Soqawa(1982) 
ORYZANONE rice Chilo suppressslis (Walker) Hedin et sl., (1977) 
PHASEOLUNATIN Phsseolus spp. E. varivestis Mulsant Hedin et al., (J~77) 
POPULIN Salicsceous c. vigintipunctata Matsuda & Matsuo(1985) N... . 
spp. costella (Marseul), 
P. versicolora distincta 
Baly, 
Lochmaea capreae cribata 
Solosky 
PROLINE pine C. fumiferana Clem. Albert & Jerrett(1981) 
QUERCETIN cotton A. grsndis Boheman Hedin et al.,(1977) 
QUERCETIN-3'-GLUCOSIDE cotton A. grandis Boheman 
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QUERCETIN-3-0-XYLOSYL- cotton A. grandis Boheman 
GALACTOSIDE 
RAFFINOSE cottonseed D. koenigii (F.) Hedin et al., (1977) 
SALICIN Salicaceous C. vigintipunctata Matsuda & Matsuo(1985) 
spp. costella (Marseul), 
P. versicolora distincta Matsuda & Matsuo(1985) 
Baly, L. 
Capreae cribata Solosky Matsuda & Matsuo(1985) N 
N 
SCHAFTOSIDE rice N. lugens (Stal) Kim et al., (1985) 
SINALBIN Cruciferae Plutella maculipenis (L.) Whitakker et al., (1971) 
SINIGRIN Cruciferae P. rapae (L.), Vershaeffelt (1910) 
P. brassicae (L.) 
~ -SITOSTEROL cotton, A. grandis Boheman Hedin et al., (1977) 
B. mori (L.) Ito et a1.,(1964) 
Hypera postica (Gyllenhal) Shanks and Doss(1987) 
SOLANINE potato leaves L. decemlineata (Say) Kogan(1976) 
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SPARTEINE Sarothamus Acyrthosiphon spartii Smith(1966) 
scoparius 
STIGMASTEROL Sciopithes obscurus Horn 00ss(1983) 
SUCCINIC ACIO cotton A. grandis Boheman Hedin et al., (1977) 
SUCROSE Ostrinia nubilalis (Hubner) Beck(1956) 
Oulema melanopus (L.), Hedin et al., (1977) 
E. varisvestis Mulsant, 
D. Koenigii (F.) N 
tAl 
C. fumiferana (Clem) Albert & Jerrett(1981) 
'Hylemya antiqua (Meigen) Mochizuki et al., (1985) 
Scarabaeidae spp. Ladd(1986) 
TERPINEOL cotton S. litoralis (Boisd) Hedin et al., (1977) 
THIAMINE C. curtipennis (Harris) Oethier(1966) 
TRICIN-5-GLUCOSIOE rice N. lugens (Stal) Sogawa(1982) 
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Structures of some selected feeding stimulants. 
H~~ -<r.e-t eN 
H I 
H H Me 
H 
Phaseolunatin 
M(l 
Sparteine 
(-)-(R)-carvone 
NOSoj"'K+ 
1/ H 
-C-CJ,.QCH-CHl, H ©H 
H 
Benzoic acid 
Sinigrin Carlinoside 
X =~L-arabinopyranosyl 
25 
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1.4 Insect Feeding Deterrents 
Plant chemicals which possess feeding deterrent 
activities against insects are known as antifeedants 
(Dethier et al., 1960). They prevent or decrease the 
feeding of the insect without killing it, with the insect 
remaining near the treated plant and dying from starvation 
(Dethier, 1960; Munakata, 1977). It has been suggested that 
an antifeedant is concerned with deterring the initiation of 
feeding therefore may act as a resistant factor protecting 
plants against insect attack (Munakata, 1977). However, it 
is plausible that in some cases it is the insect that has 
evolved the means of detecting and avoiding a potentially 
deleterious compound. Many deterrents are also toxic to 
insects. 
Table 1.2 shows that plant deterrent chemicals are 
derived from virtually all classes of natural products, but 
studies to date suggest that the most potent insect 
antifeedants belong to the terpenoid and alkaloid classes 
(Schoonhoven, 1982). Some of the more prominent examples 
from these two classes will be discussed in this section. 
Among the terpenes, the tetranortriterpenoid 
azadirachtin isolated from the neem tree Azadirachta indica 
Meliaceae (Butterworth et al., 1972) is an antifeedant for a 
host of different lepidopteran, coleopteran and orthopteran 
26 
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insects. Other potent related tetranortri~erpenoid 
compounds from A. indica include salanin (Warthen, 1979), 3-
desacetylsalanin, nimbandiol and meliantriol (Warthen, 
1983). Obacunone, harrisonin, pedonin from Harrisonia 
abyssinica and related tetranortriterpenoid compounds, have 
shown antifeedant activities against the African armyworm 
Spodoptera exempta, the cowpea podborer Maruca testuiaiis 
(Geyer) and the sugarcane stalk borer Eidana saccharina 
(HasSanali et ai., 1986; 1987). 
Several diterpenoid compounds function as phago-
deterrents for a number of insects. Among the well known 
ones are the clerodane diterpenes from Ajuga and Verbenaceae 
plants (Munakata, 1977; Belles et ai., 1985). The potent 
ones in this class include clerodendrin-A and B, clerodin, 
caryoptin and some derivatives of caryoptin. Other reported 
antifeedant diterpenes are the grayanoid diterpenes 
grayanotoxin III and kalmitoxin I and II which inhibit 
feeding of the polyphagous gypsy moth Lymantria dispar, 
inflexin and isodomedin from Isodon species which are 
effective antifeedants for S. exempta (Norris, 1986). 
Hydroxygrindelic acid, also a diterpene is, an antifeedant 
for the greenbug aphid Schizaphis graminum (Rondani) (Rose 
et ai., 1981). 
27 
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Antifeedant sesquiterpenoids have been reported for a 
variety of insects. warb~anal, muzigadial are both 
effective antifeedants and cytotoxins to s. exempta (Norris, 
1986). Polygodial and ugandensidial which are both related 
sesquiterpenoids of warbuganal were found to be less active 
than the latter when presented to s. exempta (Kubo et al., 
1976). Studies on the feeding of some species of Neodipron 
sawflies on juvenile and mature needles of the pine tree 
Pinus banksiana, have revealed that larvae do not feed on 
the juvenile ones because they contain relatively high 
concentrations of the sesquiterpene antifeedant, 13-keto-
8(14)-podocarpen-18-oic acid (Norris, 1986). Other potent 
sesquiterpenes include the gerrnacranes shiromodiol di- and 
mono- acetates isolated from the leaves of Parabenzoin 
trilobium (Munakata, 1977), and glaucolide-A (Norris, 1986), 
all of which deter the feeding of a number of lepidopteran 
species. 
Monoterpenes with insect antifeedant properties have 
been tested in most cases against the migratory locust. 
Examples of this include 1,4-cineole, carvone, p-cymene, 
citral, geraniol and some derivatives of these compounds 
(Warthen, 1983). 
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Among the alkaloids, the steroidal alkaloids in the 
Solanaceae have proved to be the most effective antifeedants 
against L. decemlineata, the two-striped grasshopper 
Melanoplus bivittatus (Say) and C. fumiferana (Harley and 
Thorsteinson, 1967; Chapman, 1974; Bentley et al., 1984a). 
The potent ones include tomatine, demissine, tomatidine,~­
solanine, «-chaconine and solanidine. C. fumiferana is also 
inhibited to feed by pyrrolizidine and lupine alkaloids 
(Bentley et al., 1984b and 1984c), and for Dysdercus species 
drinking is inhibited by strychnine, caffeine and nicotine 
(Schoonhoven and Derksen-Koppers, 1973). Broom and lupine 
plants have been shown to be resistant to the aphids, Aphis 
cytisoris (Hartig) and Acyrthosiphon pisum (Harris) 
respectively due to the alkaloids sparteine in broom and 
lupanine in lupine (Wink et al., 1982; Wink and Witte, 
1984). Verma et al., (1986) isolated tylophorine, 
tylophorinine and pergularinine from Tylophora asthmatica 
and bioassays of these compounds showed that they deterred 
feeding of s. litura. Other potent insect antifeedant 
alkaloids include castanospermine and related compounds from 
Castanospermum australe (Dreyer et al., 1985), cocculodine 
and isoboldine from Cocculus trilobus (DC) (Wada and 
Munakata, 1968) and several 9-acridone alkaloids isolated 
from Teclea trichocarpa (Hassanali et al., 1983). 
29 
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Apart from terpenoid and alkaloid classes of compounds 
some furanocoumarins, steroids and acetogenins have been 
found to deter insect feeding. Examples of the furano-
coumarins include isopimpelline, xanthotoxin, imperatorin 
and xanthoxyletin all of which are derived from Rutaceae and 
Umbelliterae plants (Yajima et al., 1979; Berenbaum, 1978; 
Gebreyesus and Chapya, 1983). Steroids are generally not 
well established potent antifeedants, but a few like the 
ergostane-type steroids from Solanaceae, Physalis, Withania 
and Nicandria species deter the feeding of E. varivestis 
(Ascher et al., 1980). The acetogenous aglycone, 6-methoxy-
benzoxazo1inone derived from the glycoside, 2,4-dihydroxy-7-
methoxy-2H-1,4-benzoxazin-3(4H)-one (OlMBDA) in maize, is an 
antifeedant for the larvae of o. nubilalis, and tests with 
various analogues of this compound have shown similar 
effects (Beck, 1960). 
Broad spectrum antifeedants are also known. Apart from 
azadirachtin, the mustard glycoside sinigrin, the 
carbohydrate phlorizin, the flavonoid glycoside rutin, 
several quinones and napthoquinones and some ellagitannins 
have been found to be broad spectrum insect antifeedants 
(Schoonhoven, 1972; Bernays and Chapman, 1977; Hedin et al., 
1977; Norris, 1986; Jones and Klocke, 1987). 
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Table 1.2 INSEG'l' .FEEDING DETERRENTS. 
~~~A~.~g_ ~_E!_terren~_~ ___n ~ru:_._s_Q.\1.:t::~E!. ___. _ _~ _Jl,~E!~.~_.naJ1l_e. ___ ._. .._ _ ._ ._~E!.f_ere_~.e~ __- ..,..-____  
ARBUTIN Locusta migratoria (L.) Adams and Bernays(1978) 
ATROPINE M. bivittatus (Say) Hedin et a1., (1977) 
P. brassicae (L) 
AUCUBIN L. migratoria (L.) Bernays and Chapman(1977)~ 
AZADIRACHTIN Azadirachta Acalymma vittatum (F.) Kubo and Nakanishi(1977) 
indica' D. u. howardii Barber Norris (1986) 
Melia azedarach Earias insulana (Boisd) 
Galleria mellonel1a (L.) 
Heliothis virescens (F.) 
He1iothis zea (Boddie) 
Hypsipyla grande11a (Zeller) 
L. decem1ineata (Say) 
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L. migratoria (L.) 
P. xylostella (L.) 
P. japonica Newman 
S. gregaria Forsk 
S. frugiperda (J. E. Smith) 
S. littoralis (Boisd) 
4-BENZOQUINONE S. multistriatus (Harsham) Norris (1977) 
O. nubilalis (Hubner) w 
~ 
BENZYL ALCOHOL small grains S. graminum (Rondani) Hedin et al., (1977) 
BERBERINE B. mori (L.), P. brassicae (L.) Levinson(1976) 
BETAINE Danaus plexippus (L.) 
BRUCINE Srychnos spp. B. mori (L.), D. plexippus (L.) Levinson (1976) 
P. brassicae (L.) 
CAFFEIC ACID Sorghum bicolor L. migratoria (L.) Adams and Bernays(1978) 
(L.) Hoench 
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CAFFEINE D. plexippus (L.) Schoonhoven et al., (1973) 
H. sexta (L.) 
CARYOPHYLLENE L. m. migratoriodes 
(Reiche &. Fairmaire) 
CASTANOSPERMINE Castanospermum Acyrthosiphon pisum (Harris) Dreyer et a1., (1985) 
australe 
CHLORDIMEFORM Calopilos miranda (Butler) Hirata and Sogawa(1976) 
Hyadaphis erysimi (Kaltenback) W 
N 
Laodelphax striatellus (Fallen) 
Hyzus persicae (Sulzer) 
Nephotettix cincticeps (Uhler) 
N. lugens (Stal) 
Pryeria sinica (F.) 
S. litura (F.) 
CHLOROGENIC ACID Salicaceous spp Lochmaeae capreae cribata Matsuda and Senbo(1986) 
CHOLESTEROL ACETATE corn Diatraea grandiosella (Dyar) 
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CLERODEDRIN Clerodendrum C. miranda (Butler) Hunakata(1977) 
crytophyllum Euprotis subflava (Bremer) 
Clerodendrum o. nubilalis (Hubner) 
trichotomum S. litura (F.) 
COCCULOLIDINE Cocculus Calospi1os miranda (Butler) Wada and Hunakata(1968) 
trilobus Spodoptera littoralis (Boisd.) 
o-COUMARIC ACID Sorghum bicolor L. migratoria (L.) Adams and Bernays(1978) 
S. mu1tistriatus (Harsham) w w 
p-COUMARIC ACID Sorghum bico1or L. migratoria (L.) Adams and Bernays(1978) 
S. multistriatus (Harsham) 
(z)-o-COUMARIC ACID Me1i1otus Epicauta fabricii (Leconte), Hedin et al., (1977) 
GLUCOSIDE infesta Epicauta pestifera Werner, 
Epicauta vittata(F.) 
4-HYDROXYCOUMARIN A. pisum (Harris) Dreyer et a1.,(1987) 
CUCURB ITAC INS Iberis Phyllotreta nemorum (L.) Norris(1986) 
amara Apis me11iferea L. 
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Phaedon coch1eariae F. 
Phyl10treta undu1ata 
Kutschelina 
Epilachna tredecimnotata 
(Latreille) 
~CYMENE Amorpha L. decem1ineata (Say) 
frcticosa L. m. migratoroides 
(Reiche & Fairmaire) •w  
P. brassicae (L.) 
DEMISSINE Solanum L. decem1ineata (Say) Hedin et a1~, (1977) 
demissum 
DHURRIN Sorghum L. migratoria (L.) Woodhead et a1.,(1977) 
bicolor 
DIGITONIN Digitalis M. bivittatus (Say) Hedin et al., (1977) 
purpurea 
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2,4-DIHYDROXY-7-METHOXY- corn O. nubilalis (Hubner) Chapman(1974) 
2H-l,4-BENZOXAZIN-3(4H)-
ONE (DIMBOA) 
ECDYSTERONE P. brassicae (L.) Chapman (1974) 
FERULIC ACID Sorghum L. migratoria (L.) Adams and Bernays(1978) 
bicolor 
FRIDELIN Acokan thera S. littoralis (Boisd.) 
obl ongi foli a w VI 
GENTISIC ACID S. bicolor L. migratoria (L.) Adams and Bernays(1978) 
GLAUCOLIDE-A Vernonia Diacrisia virginica (F.) Norris(i986) 
gigantea Sibine stimulea (Clemens) 
Vernonia S. eridania (Cramer) 
glauca S. frugiperda (J. E. Smith), 
S. ornithogalli (Guenee) 
Trichoplusia ni (Hubner) 
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GOSSYPOL cotton Boarmia (Ascotis) selenaria Norris(1986) 
(Dennis & Schiffermuller) 
L. m. migratorioides Bernays and Chapman(1977) 
(Reiche & Fairmaire) 
Heliothis spp. 
S. littoralis (Boisd) 
HARRISONIN Harrisonia S. exempta (Walker) Hassanali et al., (1986) 
abyssinica w en 
HILDECARPIN Tephrosia Maruca testulalis (Geyer) Lwande et al., (1986) 
hildebrandtii 
p-HYDROXYBENZALDEHYDE S. bicolor L. migratoria (L.) Adams and Bernays(1978) 
m-HYDROXYBENZOIC ACID S. bicolor L. migratoria (L.) Adams and Bernays(1978) 
P-HYDROXYBENZOIC ACID S. bicolor L. migratoria (L.) Adams and Bernays(1978) 
IMPERATONIN Clausena anisata S. exempta (Walker) Gebreysus et sl., (1983) 
INFLEXIN Isodon S. exempta (Walker) Kubo and Nakanishi(1977) 
inflexus 
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ISOBOLDINE C. trilobus S. litura (F.) 
ISOFERULIC ACID S. bicolor L. migratoria (L.) Adams and Bernays(1978) 
ISOHEDIN Isodon S. exempta (Walker) Kubo and Nakanishi(1977) 
shikokianus 
ISOPIHPINELLIN Orixa japonica Blattella germanica (L.) Yajima and Hunakata(1979) 
Neostylopyga rhombifolia (Stoll) 
Periplaneta americana (L.), I..N.,  
S. litura (F.) 
. JUG LONE Carya ovata P. americana (L.) Norris (1977) 
S. multistriatus (Harsham) Norris (1977) 
KAEHPFEROL Robinia S. multistriatus (Harsham) Norris (1977) 
pseudoacacia 
LIHONENE Pinus Dendrolimus pini (L.) 
silvestris L. m. migratorioides Bernays and Chapman(1977) 
(Reiche & Fairmaire) 
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LINAMARIN L. m. migratorioides Bernays and Chapman(1977) 
(Reiche & Fairmaire) 
MELIANTRIOL A. indica S. gregaria Forsk Warthen(1983) 
6-METHOXY-2-BENZOX- Zea mays Heliothis zea (Boddie) Hedin et al., (1977) 
AZOLINONE O. nubilalis (Hubner) 
2-METHYL-1,4-NAPHTHO- Acalymma vittatum (F.) 
QUINONE P. americana (L.) Norris (1977) 
S. multistriatus (Marsham) Norris (1977) w CD 
MONOCROTALINE Senecio spp. A. pisum (Harris) Dreyer et al., (1985) 
MORPHINE P. brassicae (L.)· Hedin et al., (1977) 
MUZIGADIAL Warbugia S. exempta (Walker) Warthen(1983) 
stuhlmanii 
1,4-NAPHTHOQUINONE A. vittatum (F.) Chapman (1974) 
Neodipron rugifrons Middleton 
Neodipron swainei Middleton 
P. americana (L.) Norris (1977) 
S. multistriatus (Harsham) 
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NICOTINE Ni·cotiana Dysdercus spp. Chapman (1974) 
tabacum B. mori Levinson(1976) 
P. brassicae (L.) 
NIMBANDIOL A. indica E. varivestis Mulsant 
OBACUNONE Harrisonia M. testulalis (Geyer) Hassanali et al., (1986) 
abyssinica Eldana saccharina W 
ID 
PEDONIN H. abyssinica M. testulalis (Geyer) Hassanali et al., (1987) 
E. saccharina 
PHASEOLUNATIN Coleopteran sp. Warthen(1983) 
PHLORIZIN Malus sp Amphophora agathonica Hottes Chapman (1974) 
Aphis pomi De Geer 
L. m. migratorioides 
(Reiche & Fairmaire) 
M. persicae (Sulzer) 
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PHYTOL Clerodendron S. li tura (F.) 
fragrans 
PLUMBAGIN Plumbago s. exempta (Walker) 
auriculata s. littoralis (Boisd.) 
POLYGODIAL flarburgia s. exempta (Walker) Kubo et al., (1976) 
stuhlmannii s. littoralis (Boisd.) 
PROTOCATECHUIC ACID S. bicolor L. migratoria (L.) Adams and Bernays(1978) 
QUININE Cinchona B. mori (L. ) .. o 
officinalis L. m. migratorioides 
(Reiche"& Fairmaire) 
P. brassicae (L.) 
RIDELLINE Senecio A. pisum (Harris) Dreyer et al., (1985) 
SALANNIN A. indica A. vittatum (F.) 
Aonidiella aurantii (Maskell) 
D. u. howardii Barber 
Earias insulana (Boisd.) 
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Locusta spp. 
M. domestica L. 
SALICIN B. mori (L.) 
L. m. migratorioides Bernays and Chapman(1977) 
(Reiche & Fairmaire) 
M. sexta (L.) 
SHIROMODIOL DIACETATE Parabenzoin Calospilos miranda (Butler) Norris(1986) 
trilobum S. litura (F.) •....  
SINIGRIN Cruciferae L. migratoria (L.) Adams and Bernays(1977) 
Aphis fabae Scop Chapman (1974) . 
A. pisum (Harris) 
P. p. asterius Stoll Hedin et al., (1977) 
SOLANINE Solanum P. brassicae (L.) Levinson(1976) 
punae C. fumiferana Clem. Bentley et 81.,(1984) 
SPARTEINE 
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SWAINSONINE Astragalus A. pisum (Harris) Dreyer et a1 .. (1985) 
lentiginosus 
STRYCHNINE Strychnos spp. Dysdercus spp Chapman(1974) 
P. brassicae (L.) 
SYRINGIC ACID S. bicolor L. migratoria (L.) Adams and Bernays(1978) 
OC-TERPINEOL Amorpha Dendro1imus pini (L.) 
fructicosa L. decemlineata (Say) 
L. m. migratorioides .. tI.J 
(Reiche & Fairmaire) 
P. brass'icae (L.) 
TOMATINE Nepeta cataria P. brassicae (L.) Chapman(1974) 
L. decemlineata (Say) Levinson(1976) 
C. fumiferana Clem. Bentley et al.,(1984a) 
M. sexta (L.) 
M. bivittatus (Say) 
S. 1i tura (F.) Verma et al., (1986) 
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UGANDENSIOIAL fl. stuhlmanii s. exempta (Walker) Kubo and Nakanishi(1979) 
S. littoralis (Boisd.) 
VANILLIC ACID S. bicolor L. migratoria (L.) Adams and Bernays(1978) 
WARBURGANAL fl. stuhlmanii S. exempta (Walker) Kubo and Nakanishi(1977) 
S. littoralis (Boisd.) 
XANTHOTOXIN Orixa japonica Blattella germanica (L.) Yajima and Munakata(1979) 
Neostylopyga rhombifolia (Stoll) 
P. americana (L.) .. 
IN 
m 
s. li tura (F.) 
XANTHOXYLETIN S. exempta (Walker) Gebreysus et al., (1983) 
XYLOMOLLIN Xylocarpus S. exempta (Walker) Kubo et al., (1976) 
molluccensis 
ZANTHOPHYLLINE Zanthoxylum Hemileuca oliviae Cockerell Warthen(1983) 
monophyllum Hypera postica (Gyllenhal) 
Helanoplus sanguinipes (F.) 
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Structures of some selected feeding deterrents. 
~ M(l.r ) I ft 
H 
Arbutin 
Atropine 
ACO~ 
"0"""  
Azadirachtin 
HQ 
Mq, 
~H~ 
Dhurrin ~ 
Muzigadial 
Isopimpinellin 
44 
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1.5 Compounds which act both as stimulants and deterrents 
A comparison of the list of chemicals in Tables 1.1 and 
1.2 shows that a number of compounds function as anti-
feedants for certain insect species and as phagostimulants 
for others. Gossypol, a dimeric sesquiterpenoid found in 
cottonseed pigment glands, is stimulatory to the bollweevil 
Anthomonus grandis but deters feeding of Heliothis species, 
L. migratoria and S. Iittoralis (Hedin et al., 1977). 
Sinigrin which is a broad spectrum phagostimulant for a 
number of lepidopteran and coleopteran insects (Whittaker 
and Feeny, 1971) is an antifeedant for L. migratoria, Myzus 
persicae and Papilio polyxenes (Chapman, 1974). The 
isoquilonine alkaloid, isoboldine inhibits the feeding of 
CaIospilos miranda, S. Iitura and Prodenia Iitura (Levinson, 
1976) but enhances food intake of Calpe excavata (Wada and 
Munakata, 1968). Salicin, a phenolic glycoside, is 
stimulatory to some salicaceous feeding beetles (Matsuda and 
Matsuo, 1985) but shows antifeedant activity against B. 
mori, L. migratoria and M. sexta (Chapman, 1974). 
p-Hydroxybenzaldehyde, a lignin degradative product is a 
feeding stimulant for S. muitistriatus (Baker and Norris, 
1968) but deterrent to L. migratoria (Woodhead, 1982). 
Chlorogenic acid is a feeding stimulant for the Colorado 
potato beetle L. decemlineata (Hsiao and Fraenkel, 1968) but 
deterrent to the salicaceous feeding beetle Lochmaea capreae 
cribata (Matsuda and Sembo, 1986). Coleopteran insects 
45 
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feeding on Cucurbitaceae species do not all accept 
cucurbitacin in their food (Kogan, 1976). 
1.6 Bioassay Methods 
Phagostimulation or phagodeterrency of chemicals for 
phytophagous insects have been tested by several methods. 
Studies have shown that the ideal bioassay is different for 
each species of insect (Cook, 1976). However, in all 
testing procedures the insect may be exposed to the test 
substances only (no choice situation) or may be offered a 
choice between food with and without the test substance 
(choice situation). 
The main procedure for testing for phagostimulation 
involves the presentation of the test material on an inert 
substrate. Where discs have been used, the test substance 
is impregnated by soaking the discs in a solution of the 
substance or by pipetting a known volume of the test 
solution unto individual discs (Cook, 1976). Where 
agar/cellulose has been used as the substrate, the test 
substance is added to the agar/cellulose before cooling 
(Hsiao and Fraenkel, 1968). The following inert substrates 
have been used in the form of discs: ordinary filter paper 
(Daad, 1960; Wensler and Dadzinski, 1972), elder pith 
(Heron, 1965; Norris and Baker, 1967), styropor (Meisner and 
Ascher, 1968), glass-fibre filter paper (Woodhead and 
46 
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Bernays, 1978) and membrane filters (Doss and Shanks, 1986). 
In some assays, tests substances have been applied directly 
in solution to the mouthparts of the insect (Harley and 
Thorsteinson, 1967) and for drinking insects, the test 
substance has been applied in a polythene film (Kim et al., 
1985). 
Procedures for testing for antifeedants also vary. 
They include adding it to an artificial substrate such as 
agar cellulose which has been made palatable usually with 
sucrose (Schoonhoven, 1972), or mixing it with dried food 
e.g. wheat flour wafers (Bernays and Chapman, 1977), 
spraying of the test chemical on the natural food which 
could be a leaf disc (Kubo and Nakanishi, 1979), or 
cellulosic filter paper or glass fibre discs (Schoonhoven, 
1982). Parafilm sachets have been used for sucking insects 
(Schoonhoven and Derksen-Koppers, 1976; Rose et al., 1981) 
and insects drinking from free water sources have been 
tested by applying antifeedants in their drinking water 
(Schoonhoven and Derksen-Koppers, 1973). 
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CHAPTER 2 
PREVIOUS FEEDING ALLELOCHEMICAL WORK ON SORGHUM 
AND OBJECTIVES OF THE PRESENT STUDY. 
Both physical and chemical characteristics of the 
sorghum plant have been shown to affect feeding and 
oviposition behaviour of some of its insect pests (Blum, 
1968; Chadha and Roome, 1980; Maiti et al., 1980; Jotwani, 
1981; Reddy et al., 1981; Bernays et al., 1983; 1985). 
Insects affected by physical features of the sorghum plant 
include Chilo partellus and the sorghum shootfly Atherigona 
soccata (ROndani). These physical characteristics include 
trichomes, dryness, turgidity, morphology, anatomical 
complexity and colour of the leaves. In addition to these, 
wax on the surface of the leaves and the extent of 
lignification of cells round the vascular bundles and silica 
bodies in the epidermis of the leaves have also been found 
to affect the behaviour of these insects. Chemical 
constituents of the plant have also been shown to be crucial 
in host selection before feeding and oviposition by some of 
the pest insects of sorghum. Insects on which 
allelochemical studies have been conducted include the 
migratory locust Locusta migratoria, the greenbug aphid 
Schizaphis graminum and the spotted stem borer Chilo 
partellus. The insect behaviour which has been the main 
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concern of most researchers in sorghum allelochemical 
studies is feeding. This chapter gives an overview of these 
studies and summarises the objectives of the present study. 
2.1 Locusta migratoria L. (Acrididae Orthoptera) 
Nymphs of the migratory locust feed on a wide variety 
of grasses, but it has been reported that seedlings of 
sorghum are more distasteful to the insect than most grasses 
(Woodhead and Bernays, 1978). This distastefulness has been 
attributed to the presence of a variety of deterrent 
chemicals which override the effects of stimulatory 
compounds in the plant (Bernays and Chapman, 1978; Woodhead 
and Bernays, 1978). These workers identified sugars, amino 
acids and phospholipids as feeding stimulants for the 
nymphs, and different surface wax and tissue chemicals of 
sorghum leaves as playing either deterrent or stimulatory 
roles. 
The surface wax components include n-alkanes, esters 
free fatty acids, alcohols and aldehydes (Atkin and 
Hamilton, 1982; Woodhead, 1982 and 1983). In some 
cultivars, p-hydroxybenzaldehyde is a component in the 
surface wax (Woodhead, 1982; Haskins and Gorz, 1985) and can 
be present at levels deterrent to the migratory locust. 
Woodhead (1983) found that n-alkanes with 19, 21 and 23 
carbon atoms were deterrent, while other n-alkanes between 
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C-18 and C-28 had no effect on feeding. Atkin and Hamilton 
(1982) reported that the major alkanes in the surface waxes 
of some other sorghum cultivars were C-27, C-29 and C-31. 
These did not deter feeding, unlike the shorter chain 
alkanes identified by Woodhead. Woodhead (1983) found that 
a C-32 alkane on the other hand was stimulatory. None of 
the free fatty acids, alcohols, or aldehydes present in the 
wax had any effect on larval feeding. However, an ester 
containing a C-12 fatty acid was deterrent, while that with 
C-22 acid was not. It was concluded from these bioassays 
that differences in surface waxes may contribute to cultivar 
differences in resistance to attack by grasshoppers. 
Deterrent compounds which were identified in the 
tissues of the leaves were mainly phenolic acids, and these 
include p-hydroxybenzoic, vanillic, o-coumaric, p-coumaric, 
gentisic, ferulic and caffeic acids. These phenolic acids 
occur in the leaves of the plant mainly as their 
esterified/glycosidic forms, but following insect attack 
they are released in the free form by enzymes present in the 
plant (Woodhead and Cooper-Driver, 1979). Adams and Bernays 
(1978) have shown that the individual phenolic acids at the 
concentrations occurring in the plant have no effect on the 
feeding of L. migratoria, but in combination they are 
deterrent. 
50 
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Another tissue component is HCN (Woodhead and Bernays, 
1978; Woodhead, 1983), and it is also similarly released 
into the tissues of the leaves from its cyanogenic 
glucoside, dhurrin (Akazawa et al., 1960). Changes in HCN 
and phenolic content with age of the sorghum plant and the 
effects of the changes on the feeding of the migratory 
locust has been studied (Woodhead, 1980). HCN and phenolic 
content of sorghum were found to decrease steadily as plants 
got older but this change varied with the cultivar 
(Woodhead, 1980). It was established that cultivars with 
high phenolic content suffered little damage from the 
insect. Woodhead (1981) also compared the phenolic contents 
of field and laboratory grown plants and found that the 
phenolic content was higher in the field than the laboratory 
grown plants. Woodhead attributed this difference to 
factors such as light intensity and attack by insects and 
pathogens on the field grown plants. 
2.2 Schizaphis graminum (Rondani) (Aphididae Homoptera) 
p-Hydroxybenzaldehyde, dhurrin and procyanidin were 
identified as the major greenbug feeding deterrent isolates 
from sorghum seedlings (Dreyer et al., 1981). Dreyer and 
coworkers carried out tests for synergism between two 
component mixtures of these compounds and found no effect 
for combinations of dhurrin, luteolin-7-g1ucoside, 
p-hydroxybenzaldehyde and p-hydroxybenzoic acid. 
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2.3 Chilo partellus (Swinhoe) (pyralidae Lepidoptera) 
At the ICIPE, Mbita field station in western Kenya and 
the ICRISAT, in India, studies have been conducted both in 
the field and the laboratory on the feeding behaviour of C. 
partellus larvae on sorghum (ROOme and Padgham, 1978; 
Dabrowski and Kidiavai, 1983; Alghali, 1985; Saxena, 1985b). 
It was established that young larvae (first and second 
instars) fed in the whorl and older larvae (fourth and fifth 
instars) in the stern of the plant. The intermediate larval 
stage (third instar) fed in the whorl and gradually moved 
into the stern of the plant. In addition, feeding varied 
with the cultivar and the age of the plant with larvae 
preferentially feeding on the young plant rather than the 
older ones (Alghali, 1985). 
Roome and Padgham (1978) performed bioassay experiments 
which revealed that the feeding responses of Chilo partellus 
larvae to surface wax extracts, stearn distillates and 
homogenates of the whorl leaves of sorghum varied with the 
age of the plant. Larvae preferred artificial diets 
containing leaf extracts of young sorghum to leaf extracts 
of the older plants. However, no detailed chemical 
investigations were performed by these workers to elucidate 
the basis of these observations. In another experiment, 
Roome and Padgham (1978) showed that C. partellus larvae fed 
on artificial diets containing phenolic acids up to three 
52 
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times the concentration normally occurring in sorghum. A 
combined mixture of the phenolic acids which had previously 
been reported to deter feeding by Locusta (Adams and 
Bernays, 1978) did not similarly affect the feeding of C. 
partellus larvae. 
The production of HCN in the whorls of sorghum has been 
shown to be inversely correlated with damage caused by the 
first-instar larvae (Woodhead et al., 1981). However damage 
by larvae did not correlate with phenolic acid production in 
the whorl. 
OBJECTIVES OF THE PRESENT STUDY 
The review of the feeding a11elochemical work on the 
spotted stem borer c. partellus shows that no detailed and 
methodical study has been carried out to identify the 
chemicals from sorghum which affect the feeding of the 
larvae of this insect. Secondly, the literature available 
on this subject suggests that no work has been undertaken to 
demonstrate whether or not there are any allelochemical 
bases for feeding preferences shown by larvae to different 
sorghum cultivars. 
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To fill this gap, this study was undertaken with the 
following objectives:-
(i) to develop a bioassay to monitor the search for 
chemicals in sorghum that affect the feeding of third-ins tar 
c. partellus larvae; 
(ii) to isolate and characterise the active chemicals which 
affect C. partellus larval feeding; 
(iii) to undertake detailed dose-response studies of these 
chemicals and a few selected analogues; 
(iv) to develop chromatographic methods to quantify the 
amounts of the active chemicals in various sorghum cultivars 
and at different plant growth stages. 
Details of the study are described in the chapters that 
follow. 
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CHAPTER 3 
PRELIMINARY INVESTIGATIONS 
The work described in this study was conducted in two 
stages: 
1. preliminary investigation of the whorls of greenhouse 
grown plants of sorghum cultivars IS 18363 (susceptible) and 
IS 2205 (resistant) (Saxena, 1985b) with the purpose of (a) 
developing a suitable feeding bioassay, (b) comparing the 
extractive efficiencies of different solvents and (c) 
comparing the chromatographic profiles and relative 
stimulatory activities of different extracts of the two 
cu1tivars 3 and 6 weeks after plant emergence; 
2. detailed investigation on the extractives of the whorls 
of field grown plants of these two cultivars with the 
purpose of identifying the active allelochemics. 
Only the details of the preliminary investigation will be 
described in this chapter. Details of the second part of 
the study are described in the next chapter. 
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3.1 MATERIALS AND METHODS 
3.1.1 Plant material 
Sorghum cultivars IS 18363 (susceptible) and IS 2205 
(resistant) were grown in plastic pots in a greenhouse at 
28 ± 2 °c at a daylength of 9 h at the ICIPE, Chiromo Campus 
in November, 1985. 
3.1.2 Extraction 
After 3 and 6 weeks of growth, about 250-500 leaf 
whorls of each cultivar were cut off and extracted 
successively with hexane, ethyl acetate and methanol by 
immersion for 30 min in each case. All the extracts were 
concentrated to dryness in vacuo at 40 °c and the residues 
weighed. The methanol extracts were lyophilized after 
removal of the organic solvent by rotary evaporation. 
3.1.3 Development of a feeding bioassay 
The following factors were considered in the 
development of a feeding bioassay: size and type of petri-
dish, larval instar, feeding substrate, moisture retention 
within the dish and the number of larvae in each feeding 
run. The factors were evaluated purely on the basis of 
qualitative observations. 
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Petri-dish: Two types and sizes of petri-dishes , plastic 
and glass pyrex, 60 x 70 mm and 90 x 100 mm, were used to 
determine which would be easier to handle and which would 
allow greater contact between two 12 mm discs used as 
feeding substrates and the test larvae. The 60 x 70 mm 
petri-dish was found to allow greater contact between the 
larvae and the discs. The glass pyrex dish was more 
suitable for the study because it was easy to wash and dry. 
Larval instar: Since the first three stages feed on the 
leaf whorl, feeding tests were conducted using first, second 
and third ins tar larvae to determine the larval stage most 
convenient for use within a short assay period. A high 
mortality was observed for the first instars, but although 
this factor was low for the second instars, they were found 
to be relatively slow feeders. Third ins tar larvae were 
found to feed better, and accordingly were used in 
subsequent bioassays. 
Feeding substrate: Feeding tests on cellulose acetate, 
cellulose nitrate and cellulose discs loaded with whorl 
extracts of sorghum showed that cellulose was the least 
preferred of the three substrates for larval feeding. 
Cellulose acetate and cellulose nitrate were equally 
preferred, but in subsequent assays, cellulose acetate was 
used. Unlike cellulose nitrate, it is insoluble in 
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methanol, the medium used for the transfer of extracts, and 
it is presumed that on hydrolysis in the gut of the larvae, 
acetic acid would be produced which is a cornmon metabolic 
product in the biochemical system of an insect. 
Moisture retention: To prevent larvae from becoming 
dehydrated or drowning, tests were carried out to determine 
the best way of maintaining moisture in the petri-dish for 
an assay period of 24 h. Cellulose and cotton-wool were 
tested for moisture retention but cellulose was found to be 
more suitable. With cotton-wool, larvae got entangled 
within the strands. Filter-paper, 55 rnm diameter soaked 
with 600 ~l of distilled water was found to keep the petri-
dish moist for more than 24 h. A fine wire-gauze (Fig. 3.1) 
was used to stop the larvae from contacting the moist paper 
which was found to irnmobilise the larvae. 
Number of larvae: Three third-ins tar larvae were found to 
give measurable feeding rates on a 12 rnm disc for a period 
of 24 h without any apparent overcrowding. 
3.1.4 Feeding Tests 
3.1.4a Choice situation 
Three third ins tar larvae were given a choice between a 
treated disc and a control d~sc wh~ch were t d b 
~ ~ separa e y a 
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distance of almost 5 mm (Fig. 3.1). Each treated disc was 
loaded with 500 f g of extract, 250 rg on each side of the 
disc. Control discs were treated with test solvent only. 
The control and treated discs were air-dried and weighed 
several times to a constant weight on a Cahn 21 milligram 
balance to ± 0.01 mg before and after the assay. All the 
tests were carried out in the dark in an environmentally 
controlled room (RH = 78 ± 2%, temperature = 29 ± 2 °C), and 
each feeding run was 24 h. Larvae used for the tests were 
starved for 24 h prior to the assay. 
Feeding tests were carried out on the hexane, ethyl 
acetate and methanol extracts of the whorls of the two 
cultivars. Each test was replicated 9 times. 
Feeding data were treated in two ways. First, the 
feeding response in each test was expressed as percentage 
preference calculated from the formula (1 - xo/Xt) x 100 
where Xt is the mean weight of treated disc consumed and Xo 
the mean weight of control disc consumed. A second 
parameter expressing total percentage of substrate consumed 
from the food available in the dish was derived from the 
formula 100 x (Xt + XO)/(Xti + XOi ), where Xt i and xOi are 
the initial weights of treated and untreated discs 
respectively. 
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Apparatus for choice feeding bioassay. 
Fig. 3.1 
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3.1.4b No choice situation 
The same apparatus in Fig. 3.1 was used for feeding 
tests in the no choice situation. However, in this 
experiment, only one test disc (12 mm diameter) loaded with 
the extract was offered in each feeding run to three third 
ins tar larvae. Control discs were similarly tested, and all 
the tests were maintained for 24 h as previously described 
in the choice situation. Each test was replicated 25 times. 
A dose response test was carried out on all the crude 
extracts and the feeding response for each dose was 
expressed in logarithms as the Relative Feeding Response 
(RFR) and was calculated from the formula, RFR = Xt/xo. 
3.1.5 Chromatography 
The main techniques used in the fractionation of 
extracts in this study were high performance liquid 
chromatography (HPLC) and gas liquid chromatography (GLC), 
and the factors considered in the development of 
chromatographic conditions for the separation of the 
extracts were as follows : type of column, solvent system 
(HPLC), type of detector, flow rate and temperature 
depending on the type of chromatographic system used for 
separation of the extracts. 
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3.1.5a High performance liquid chromatography (HPLC) 
Type of column: For analytical work, a Dupont C-18 reverse 
phase column, Zorbax ODS, 25 cm x 4.6 mm was found to be 
suitable for the separation of the ethyl acetate and 
methanol extracts, and for preparative separation of these 
extracts, a Varian MCH-10 C-18 reverse phase column 50 cm x 
8 mm was used. 
Solvent system: The separation of the ethyl acetate and 
methanol extracts was achieved by using a 20% aqueous 
methanol solvent system. 
Flow rate: For analytical studies, a flow rate of 1.0 
ml/min was found to give the best separation of the ethyl 
acetate and methanol extracts. A flow rate of 3.0 ml/min 
was used for preparative separation and this maintained the 
separation profiles of the extracts. 
Type of detector: Two types of detectors were used, a UV 
and a RI detector. The UV detector was sensitive to the 
chromophoric components whilst the refractive index (RI) 
detector was sensitive to the non-chromophoric components. 
Components in the ethyl acetate and methanol extracts were 
easily detected at 240 and at 254 nm. 
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Temperature: Fractionation of the two extracts was 
performed at 25 °e. 
3.1.5b Gas liquid chromatography 
Type of column: A packed column 3 m x 2 mm i.d. of 5% 
silicone OV-17 on chromosorb WHP 80-100 mesh was used as the 
adsorbent for the separation of the hexane extracts and this 
was fitted into a Packard gas chromatograph, model 428. 
Detector: A flame ionisation detector (FlO) was used to 
detect the components in the hexane extracts. 
Temperature: A temperature programme of 220-300 °e at 
3 °e/min gave the best separation of the components in the 
hexane extracts. 
Flow rate: The carrier gas was nitrogen at 20 ml/min. 
3.2 RESULTS 
3.2.1 Yields of extracts from sorghum whorls 
Table 3.1 shows the yields of extracts for sorghum cultivars 
IS 18363 (susceptible) and IS 2205 (resistant) at two growth 
stages about 3 and 6 weeks after plant emergence. 
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Table 3.1 Yields of extracts from sorghum whorls(mg/g whorl) 
Cultivar plant wt (g) wt (mg) extract % total 
age(wks) whorls hexane EtAc MeOH yield 
IS 18363 3 135 123.1 341. 7 279.9 1.1 
IS 2205 3 79 45.8 332.0 236.4 1.8 
IS 18363 6 550 357.5 817.7 2723.2 1.8 
IS 2205 6 485 412.3 742.7 4816.4 4.9 
For both cultivars, the yield of extract from the 
whorls of the 6 week old plants was higher than that from 
the 3 week old plants. At the two growth stages, the 
resistant cultivar IS 2205 yielded more extract than the 
susceptible cultivar IS 18363. 
3.2.2 Feeding Assays 
3.2.2a Choice situation 
The feeding responses of third-instar larvae of C. 
partellus to crude extracts of the whorls of sorghum 
cultivars IS 18363 (susceptible) and IS 2205 (reSistant) at 
the two growth stages are shown in Tables 3.2 and 3.3. 
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Table 3.2 Feeding responses of third ins tar larvae of C. partellus to cellulose acetate discs 
treated with 500~g of leaf whorl extracts of sorghum plants (3 weeks old) in a choice 
bioassay. 
IS 18363 IS 2205 
Feeding response Hexane EtAc MeOH Hexane EtAc MeOH 
m 
~ 
XT ± S.D (mg) 0.41 ± 0.32 2.14 :!: 1.63 3.00 ± 1. 54 0.50 ± 0.19 0.78 ± 0.57 3.35 .± 1. 55 
Xo .± S.D (mg) 0.12 .:t 0.18 0.11 i 0.08 0.03 .:!: 0.03 0.04 :t 0.03 0.04 !. 0.06 0.12 !. 0.25 
Preference Xl-XO 0.29 2.03 2.97 0.46 0.74 3.23 
% Preference 72 95 99 92 96 96 
Total feeding 0.47 2.06 2.74 0.49 0.74 3.13 
*Relative feeding 1 4.4 5.8 1 1.5 6.4 
* Feeding response relative to hexane 
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Table 3.3 Feeding responses of third ins tar larvae of C. partellus to cellulose acetate discs 
treated with 500~9 of leaf whorl extracts of sorghum plants (6 weeks old) in a choice 
bioassay. 
IS 18363 IS 2205 
Feeding response Hexane EtAc MeOH Hexane EtAc MeOH m 
UI 
XT .± S.D (mg) 0.80 :t 0.80 1.12 ± 0.31 4.16 ± 1.15 0.72 ± 0.59 1.70±.1.08 3.34 ± 1.57 
Xo :t S.D (mg) 0.10 ± 0.05 0.08 ± 0.13 0.01 .± 0.01 0.38 :t 0.47 0.22 :t 0.41 0.05 ± 0.12 
Preference XT -Xo 0.70 1. 04 4.15 0.34 1.48 3.29 
% Preference 87 92 99 48 87 98 
Total feeding 0.77 1.08 3.76 0.99 1. 72 3.18 
"Relative feeding 1 1.4 4.9 1 1. 74 3.2 
" Feeding response relative to hexane 
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152205 
2·' 
2·2 
2'() 
11~ 
~ 1.6 
~ 
g'1.4 
-j 1.2 til 1·0 
.~ 
Bo-a 
~ 
.!'O.s 
-l0., 
O· 
o 300 500 0 100 300 500 
Dose pg extract Dose pg extract 
Dose response curves for the crude extracts of the whorls of 
the 3 week old plants of IS 18363 and IS 2205. 
Fig. 3.2 
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IS 18363 IS 2205 
GI 
ecn  
o 
~ 1· 
~ 
o 200 400 600 0 200 400 600 
Dose JJ9 extract 
Dose response CurveL for the crude extracts of the whorls of 
the 6 week old plants of IS 18363 and IS 2205. 
Fig.3.3 
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3.2.2b No Choice situation 
Dose response curves for the extracts are shown in 
Figs. 3.2 and 3.3. The mean feeding on the control disc was 
0.008 ± 0.004 mg 
3.3 DISCUSSION 
In the choice situation, treated discs were more 
preferred by larvae to untreated discs (Tables 3.2 and 3.3) 
indicating that all the extracts contained compounds that 
were stimulatory to the feeding of the larvae. Calculation 
of the degree of preference as defined on page 5, gave 
values 72, 95 and 99 for the hexane, ethyl acetate and 
methanol extracts respectively of the whorls of the 3 week 
old plants of IS 18363 and 92, 96 and 96 for those of IS 
2205. Values obtained for similar extracts of the whorls of 
the 6 week old plants were 87, 92 and 99 for IS 18363 and 
48, 87 and 98 for IS 2205. These values clearly fail to 
differentiate between the stimulatory efficacies of the 
three extracts and between the two different cultivars at 
the two growth stages. Comparison of the total feeding on 
discs treated with the different extracts clearly showed the 
difference. Indeed the methanol extracts of the 3-week-old 
whorls of the two cultivars were about 6-fold more 
stimulatory than the corresponding hexane extracts. The 
ethyl acetate extracts were about 1.5 to 4-fold more 
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stimulatory than the hexane extracts. 
The data on the relative feeding response of larvae to 
treated discs in the no choice situation confirmed that all 
the crude extracts stimulated larval feeding (Figs. 3.2 and 
3.3). The feeding response increased with increasing dose 
of extract, and also with the polarity of the extract. Thus 
of the three sets of extracts, hexane , ethyl acetate and 
methanol of the whorls of the two cultivars tested for 
feeding response, the methanol extracts were most 
stimulatory followed by those of ethyl acetate and hexane. 
It was also clear from the dose response curves that the 
feeding pattern was similar in the two cultivars. However, 
it appeared that larvae responded more to extracts of the 
susceptible cultivar IS 18363 than to extracts of the 
resistant cultivar IS 2205. The feeding pattern did not 
change when extracts of the whorls of the 6 week old plants 
were tested (Fig. 3.3). However, the results suggested that 
there were less stimulatory compounds in the 6 week old 
plants. 
An additional advantage of this method over the choice 
situation is that it simulates more closely the feeding of 
the insect in the natural environment. An improvement of 
the no choice bioassay involved the use of a smaller 
container which allowed greater contact between larvae and 
the test disc. Details of this are described in Chapter 4. 
70 
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Preliminary high performance liquid chromatographic 
analysis (HPLC) of the ethyl acetate and methanol extracts 
of the two cultivars suggested that differences between the 
two cultivars were quantitative rather than qualitative. A 
similar observation was made for the hexane extracts of the 
two cultivars which were analysed by gas chromatography. 
Details of each analysis are described in Chapter 4. 
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CHAPTER 4 
EXPERIMENTAL DETAILS AND RESULTS 
4.1 Plant materials 
Whorls of sorghum cultivars IS 18363 (susceptible) and 
IS 2205 (resistant) were obtained from field grown plants at 
the Mbita point Field Station, ICIPE in Western Kenya. Only 
undamaged (as a result of insect feeding or mutilation) 
whorls were used so as to exclude inducible defensive 
chemicals which are likely to be produced in the plants as a 
result of herbivore feeding (Haukioja, 1982). 
4.2 Insects 
Freshly moulted third-instar larvae of C. partellus 
were obtained for tests from stock cultures maintained on an 
artificial diet described by Ochieng et al., (1985) at the 
ICIPE insectary. 
4.3 Extraction 
500 (3.5 kg) and 800 (3.4 kg) pieces of freshly cut 
whorls of 3 week old plants of sorghum cultivars IS 18363 
and IS 2205 respectively were extracted successively in 
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batches, with hexane and ethyl acetate by immersion for 15 
min in each case to extract surface components. After ethyl 
acetate extraction the plant materials were soaked in 
methanol for 24 h to extract tissue components. All the 
extracts were filtered to remove plant debris and then 
concentrated to dryness in vacuo. The methanol extracts 
were lyophilized after solvent removal by rotary 
evaporation. In another experiment, 80 pieces (3.5 kg and 
2.9 kg) of the whorls of 6 week old plants of each of the 
cultivars IS 18363 and IS 2205 respectively, were similarly 
extracted and the resulting extracts concentrated to 
dryness. 
In view of the different extraction procedures, yields 
of extracts (g/kg whorl) were calculated in two different 
ways. Percentage yield of surface extractives from only the 
hexane and ethyl acetate extracts and percentage total yield 
from the three extracts hexane, ethyl acetate and methanol 
of the whorls of the two cultivars at the two growth stages. 
The results from these calculations are shown in Table 4.1. 
73 
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Table 4.1. Yields of extracts from sorghum whorls 
(g/kg whorl) 
Cultivar Plant wt wt(g) of extract % yield 
age(wks) (kg) Hexane EtAc MeOH surface Total 
IS 18363 3 3.5 1.7 1.1 46.3 0.16 2.8 
IS 2205 3 3.4 1.4 1.7 46.3 0.11 1.8 
IS 18363 6 3.5 1.4 0.8 29.0 0.78 11.1 
IS 2205 6 2.9 0.8 1.3 34.9 0.91 15.9 
For both cultivars the yield of extract increased with 
increasing age of plant reflecting an increase in size of 
the whorls. 
4.4 Chromatography 
Thin layer chromatography (tIc) was performed on MN 
polygram precoated silica gel/uv254 40 x 80 mm plates (0.25 
mm thickness), and preparative tIc on similar plates of 20 x 
20 cm. High performance liquid chromatography (HPLC) was 
performed on a Varian 5000 LC model, equipped with a DUPont 
Zorbax ODS C-18 reverse phase column, 25 cm x 4.6 mm and a 
UV detector at 254 nm. The column was eluted with 20% 
aqueous methanol at a pump pressure of 210 atm and at a flow 
rate of 1.0 ml/min. For preparative separation, a Varian 
MCH-10 C-18 reverse phase column, 50 cm x 8 mm and a UV 
74 
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detector at 240 nrn were used; the column was eluted with 20% 
aqueous methanol at 3.0 ml/min at 25 °C. 
Gas liquid chromatography (GLC) was performed on a 
Packard 42B, equipped with a flame ionisation detector, 
utilising a 3 m x 2 mm i.d. column of 5% silicone OV-17 on 
chromosorb WHP BO-100 mesh. The column temperature was 
programmed from 250-300 °c at 3 o/min. Carrier gas was 
nitrogen at 20 ml/min. 
For profile studies, 5 f9/ra each of the ethyl acetate 
and methanol extracts were prepared in methanol, filtered 
through a wad of cotton wool plugged in a Pasteur pipette 
and 5 ~ aliquot of each preparation were analysed by HPLC 
on the DuPont Zorbax ODS C-1B reverse phase column. All 
solvents used in the analysis were HPLC grade (Aldrich Chern. 
Co. Ltd.). 
Hexane extracts were analysed by GC. 1 tl of solutions 
containing 20 rg/rL of the extracts were used for analysis. 
Figures 4.1a, 4.1b and 4.1c show the chromatographic 
profiles of the hexane, ethyl acetate and methanol extracts 
of the two cultivars, and the most striking feature in the 
chromatograms is quantitative differences between identical 
peaks. 
75 
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3-week-old whorts 
IS 2205 
HeIcanP extract 
I Ub~~--~~~--~---L----~~---~~d-~---L--1 
~ ~~~tract 
b 
J 
6-_ -Old whorls 
IS 2205 
t-\l?xane fttract 
o 10 20 ~ II) 50 60 
.Time (mi~) 
Gas chromatograms of sorghum leaf-whorl extracts. 
Fig·4·1a 
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1518363 152205 
~QH.Q 
At 
Ac Al 
l It 
AI 
At 
Ac 
L ~CL 
o ~ W ~ 0 ro w ~ 
Time (min) 
HPLC profiles of the ethyl acetate extracts of sorghum 
whorls. 
The upper profiles represent extracts of the whorls of the 3 
week old plants and the lower profiles, extracts of the 
whorls of the 6 week old plants of IS 18363 and IS 2205. 
Fig.4.1b 
77 
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1518363 152205 
; 
I 
~l .. 1 
\(1 .~! 
o 10 20 30 0 10 20 30 
Time (min) 
BPLC profiles of the methanol extracts of sorghum whorls. 
The upper profiles represent extracts of the whorls of the 3 
week old plants and the lower profiles, extracts of the 
whorls of the 6 week old plants of IS 18363 and IS 2205. 
Fig. 4·1 c 
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4.5 Spectral analysis 
GC/MS analysis of compounds was carried out on a VG 
Masslab (Chesire, UK) mass spectrometer, model VG12-250 
interfaced with a HP5790 gas chromatograph. Mass spectral 
data were obtained by electron impact at 70 eV and are 
reported as m/e vs relative intensity. Carrier gas for GC 
was helium. 
4.6 Bioassays For Feeding Activity 
Feeding tests were conducted in a no choice situation 
in small glass vials, 23 x 27 mm (4 ml). The vials had 
tight fitting plastic snap-on caps which had holes punched 
with a dissecting pin. Moisture was maintained in each vial 
by soaking a wad of cotton-wool (30 ~ 3 mg) placed inside 
the cap with 200 rl of double distilled water. The cotton-
wool was covered with a fine-wire mesh which fitted tightly 
inside the cap (Fig. 4.1d). 
Test samples in solvents were applied topically unto 
both sides of cellulose acetate discs. One test disc (12 mm 
in diameter), dried in a stream of warm air, was placed at 
the bottom of the vial containing three third-ins tar larvae 
of C. partellus which had been starved for 24 h. All the 
feeding tests were maintained for 24 h in the dark in an 
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environmentally controlled room (RH 78 ± 2%, temperature, 
29 ± 2°C) 
4.7 Solubilisation of extracts and solvent effects on 
feeding 
Crude extracts of hexane were prepared in hexane, ethyl 
acetate extracts dissolved best in methanol and methanol 
extracts in water. Other medium polarity solvents like 
chloroform, acetone and ethyl acetate were found to dissolve 
the feeding substrate and therefore were unsuitable. 
Preliminary tests showed that methanol slightly 
distorted the feeding substrate. Feeding assays were 
therefore carried out on the feeding substrates after they 
had been dipped into the solvents used for solubilisation of 
the extracts. There were two treatments, no water and water 
treatments. In the no water treatment, discs were only 
dipped in the test solvents and air dried. In the water 
treatment, discs dipped into solvent and air dried were 
further loaded with 15 ~ of double distilled water. Each 
test was replicated 8 times. 
The data collected on discs consumed for the different 
solvents were subjected to analysis of variance and the 
means separated by the Duncan's multiple range test. 
80 
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Table 4.2 shows the feeding responses of third-instar larvae 
of c. partellus to cellulose acetate discs treated with 
different solvents. 
Table 4.2. Effect of solvent on the feeding response of 
third-instar larvae of c. partellus in a no choice bioassay. 
Test Solvent Mean feeding Anova model 
performed (mg) F-ratio 
No water no solvent 0.02850 ab 
added hexane 0.02625 b 
methanol 0.00413 c 
water 0.03213 a 
27.02*** 
Water added no solvent 0.02600 b 
(15 rl) hexane 0.02988 ab 
methanol 0.02712 ab 
water 0.03025 ab 
*** Pr>F 0.0001 
Means followed by the same letter are not significantly 
different (P < 0.05; Duncan's multiple range test). 
81 
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There was large variability in the mean weights of 
discs consumed by larvae in the no water treatment, 
particularly in the methanol case, but the addition of 15 ~l 
of water to discs already dipped into solvent eliminated 
this variability. 
4.8 Bioassays of the crude extracts 
Dose response tests were carried out on hexane, ethyl 
acetate and methanol extracts of the whorls of the two 
cultivars. The crude extracts were each tested at 10, 50, 
100, 300 and 500 ~g. Chromatographic fractions of crude 
extracts were tested singly and in combination. Each test 
was replicated 15 times. 
Each test disc was dried thoroughly in a stream of warm 
air and then weighed several times to a constant weight on a 
Cahn 21 milligram balance to ± 0.001 mg before and after the 
assay. The feeding response on each disc was expressed as 
the Relative Feeding Response (RFR) calculated from the 
formula RFR = Xtjxo where Xt and XO are the mean weights of 
treated and control discs consumed respectively. Natural 
logarithms of RFR were plotted against different doses to 
depict the dose-response relationships. 
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These relationships which are shown in Figures 4.2a and 
4.2b were expressed in the regression equation In y = In a + 
b ln x where y = relative feeding response and x = dose in 
~/disc. The intercepts (a) and slopes (b) are compared in 
Tables 4.3a and 4.3b. 
Table 4.3a. Constants for the regressions of the crude 
extracts of the whorls of the 3 week old plants. 
IS 18363 IS 2205 
Extract In a b In a b 
hexane -0.8352 0.5221 -0.7056 0.4427 
ethyl acetate -0.9582 0.6426 -0.5387 0.5272 
methanol -0.0741 0.7486 -0.2656 0.6331 
83 
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x 
4 x 
~ -----.-
0 
0. 
~3 
01 
:Cc  
II 
It 
,.t2 'i  
~, 
z1i i 
0 100 200 300 500 
Dose J.J9/disc 
Dose response curves for the crude extracts of the whorls of 
the 3 week old plants of IS 18363 and IS 2205. 
IS 18363: x methanol, • ethyl acetate, 
IS 2205: • methanol, I:i. ethyl acetate, • hexane o hexane 
Fig."4.2a 
84 
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4 
~ 
8. 
~ 3 
01 
C ..------
x
--------------
 
-~ A -2 
~ ~===~ 
~ 
~'"  
z1 i 
100 200 300 400 500 
Dose JJ9/disc 
Dose response curves for the crude extracts of the whorls of 
the 6 week old plants of IS 18363 and IS 2205. 
IS 18363: x methanol, .ethyl acetate, • hexane 
IS 2205: • methanol, 6 ethyl acetate, o hexane 
Fig.4.2b 
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Table 4.3b. Constants for the regressions of the crude 
extracts of the whorls of the 6 week old plants. 
IS 18363 IS 2205 
Extract In a b In a b 
hexane -1.0000 0.4709 -0.8131 0.4371 
ethyl acetate -1.0073 0.5728 -0.1925 0.3778 
methanol -0.4885 0.6024 -0.3330 0.5124 
A comparison of the data on the feeding responses of 
the third-instar larvae to the extracts of the two cultivars 
showed that they followed the same pattern. Polar extracts 
were more stimulatory to larvae than the nonpolar ones. 
However, the b values indicate that larvae responded more to 
extracts of the more susceptible cultivar, IS 18363 than to 
extracts of cultivar IS 2205. The response was also 
dependent on the age of the plant, with larvae feeding more 
on extracts of the 3 week old whorls than those of the 6 
week old whorls. 
86 
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4.9 Fractionation of extracts 
As a priority the more stimulatory methanol and ethyl 
acetate extracts were fractionated with the purpose of 
identifying the active compounds. unfortunately no similar 
study could be undertaken on the hexane extract in this 
investigation. 
4.10 Fractionation of ethyl acetate ' extract and acetylation 
of organic fraction 
There were four different ethyl acetate extracts 
comprising those of the whorls of 3 and 6 week old plant~ of 
sorghum cultivars IS 18363 and IS 2205. From the 
preliminary study, it was found that differences between the 
extracts were quantitative rather than qualitative. Hence 
detailed fractionation studies were undertaken only on one 
extract (3 week old plants). Other extracts could then be 
analysed quantitatively by comparison of their 
chromatographic profiles. 
Fractionation of the crude ethyl acetate extract was 
undertaken as shown in Scheme 4.1 . 
87 
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d' for the isolation of p-hydroxy-
;~~:~~c4~~idF!~~ P:~~~~~XYbenzaldehyde from ethyl acetate 
extract of 3 week old plants. 
Crude extract 
(108 mg) 
I Partition water/ethyl acetate 
I , I 
Organ~c Aqueous 
(24 mg) (84 mg) 
I dissolved in MeOH 
I filtered 
I I 
Residue Filtrate 
(3 mg) (21 mg) 
I 
I Prep. HPLC 
p-HIB A * unknIo wn p-HIB ALD ** 
* p-hydroxybenzoic acid 
** p-hydroxybenzaldehyde 
The extract was partitioned between water and ethyl 
acetate. The aqueous fraction constituted about 80% of the 
extract and the organic fraction about 20%, 
Preparative HPLC of the organic phase gave p-hydroxy-
benzoic acid and p-hydroxybenzaldehyde as the major 
components (Fig. 4.3a). The two compounds were identified 
by mass spectrometry and coinjection with authentic samples 
on an HPLC reverse phase column. The mass spectrum of 
p-hydroxybenzaldehyde showed peaks at m/e (%), M+-122 (100), 
ss 
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E 
c: COOH 
0 
e~  p - HBA 
"'"" 
6 ¢ 
z  
0 OH 
Q. 
'w"  H a: I 
a: I 
0 I 
~ 
u 
w 4 I 
~ 
0" " I 
II CHO 
¢ p-HBALD 
- OH- - --
0 10 20 30 40 SO 
TIME C MIN ) 
BPLC profiles of the partition phases of the ethyl acetate 
extract of the whorls of the 3 week old plants of is 18363. 
(-----) organic phase, (- - -) aqueous phase. 
Fig.4.3a 
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93 (48), 65 (48), 39 (58) and for p-hydroxybenzoic acid, 
M+-138 (70), 121 (100), 93 (30), 65 (28), 39 (34). The 
retention time of p_hydroxybenzaldehyde on the reverse phase 
column was Rt=20 min and for p-hydroxybenzoic acid Rt=5 min. 
The identities of the two compounds were further established 
by preparing their methylated derivatives with diazomethane 
in peroxide free ether and characterising them by GC-MS and 
GC-coinjection with authentic samples. The mass spectral 
data for the methoxy methyl ester of the acid were mle (%) 
M+-166 (30), 135 (100), 107 (12), 92 (18), 77 (28), 64 (12) 
and for the methoxybenzaldehyde M+-136 (100), 107 (24), 92 
(28), 77 (46), (18), 63 (16), 51 (14), 39 (18). 
Acetylation of the organic fraction with acetic 
anhydride in dry pyridine at 0 °c for 24 h gave a mixture of 
phenolic acetyl derivatives. In addition to the acetyl 
derivatives of p-hydroxybenzaldehyde and p-hydroxybenzoic 
acid, GC/MS analysis of the mixture revealed the presence of 
the acetyl derivatives of caffeic, p-cournaric and ferulic 
acids. The mass spectrum of the acetyl derivative of 
p-coumaric acid showed peaks at M+-206 (4), 163 (6), 119 
(42), 105 (8), 93 (22), 70 (38), 61 (56) and 45 (100); that 
of caffeic acid at M+-264 (4), 221 (100), 178 (8), 143 (60), 
128 (30), 103 (24), 91 (64), 77 (12), 61 (14) and 43 (64); 
and that of ferulic acid at M+-236 (28), 221 (35), 193 (12), 
178 (8), 165 (8), 143 (88), 128 (38), 105 (48), 91 (100), 77 
(45), 65 (15), 51 (28) and 41 (34). These spectra were in 
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agreement with those of authentic samples of these 
compounds. The presence of these cinnamic acids in the 
organic fraction was confirmed by coinjection with authentic 
samples on an HPLC reverse phase column at detector 
wavelength of 280 nm (Fig. 4.3b). The retention times (Rt) 
of caffeic acid, p-coumaric acid, and ferulic acid were 6, 
10 and 13 min respectively. 
No further separation was carried out on the aqueous 
fraction since its HPLC profile was superimposable on that 
of the crude methanol extract of the whorls. Fractionation 
of the latter is described on page 102. 
4.11 Feeding bioassays of fractions of the ethyl acetate 
extract, phenolics identified and their analogues 
The following fractions and phenolics were bioassayed: 
(a) the organic and aqueous fractions of the ethyl 
acetate extract individually and as blend in the proportion 
they were fractionated from the original extract; 
(b) purified synthetic samples of p-hydroxybenzoic 
acid and p-hydroxybenzaldehyde and a mixture of the two in 
the proportion occurring in the crude extract. 
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G 
Ec  
-~ 
o E F 
o 10 20 30 
Ti~ (~). 
HPLC profile of ~he ethyl acetate extract of the whorls of 
the 3 week old plants of IS 18363 (280 nm). 
A-J-l, B-p-hydroxybenzoic acid, C-caffeic acid, D-J-2, 
E-p-coumaric acid, F-ferulic acid, G-p-hydroxybenzaldehyde 
Fig.43b 
91b 
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(c) some selected analogues of the above phenols 
comprising p-hydroxybenzyl alcohol, p-methoxybenzaldehyde, 
p-methoxybenzoic acid and p-methoxybenzyl alcohol. 
(d) purified synthetic samples of p-coumaric acid, 
caffeic acid, ferulic acid, vanillic acid, protocatechuic 
acid, gentisic acid and chlorogenic acid a common 
constituent of plants (Sondheimer, 1964) and quinic acid, a 
degradative product of chlorogenic acid. 
All the phenolic compounds (Fig. 4.4) were obtained 
from Aldrich Chern. Co. Ltd. and the purity of each chemical 
was checked by HPLC and/or GLC. Each phenolic compound was 
tested at 10, 25, 50, 150, 250, 350 and 450 ~g. 
The dose response curves for the fractions of the ethyl 
acetate extract (Fig. 4.5) were also expressed in the 
regression equation In y In a + b In x where y = relative 
feeding response and x dose f9/disc. The equations were 
as follows: organic, In y = -0.9662 + 0.4226 In x; 
aqueous, In y = -0.6881 + 0.5322 In x; organic+aqueous, 
In y -0.9918 + 0.6209 In x and for the crude extract, 
In y -0.9582 + 0.6426 In x. 
A comparison of the regressions showed that 
fractionation lowered the feeding response of the larvae, 
and that the polar fraction retained a higher 
92a 
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b c 
d 
a 
9 h 
e 
Phenolic compounds tested tor larval feeding response. 
(a) p-hydroxybenzaldehyde, (b) p-hydroxybenzoic acid, (c) 
p-coumaric acid, (d) caffeic acid, (e) ferulic acid, (f) 
p-hydroxybenzyl alcohol, (g) p-methoxybenzaldehyde, (h) 
p-methoxybenzoic acid, (i) p-methoxybenzyl alcohol, (j) 
protocatechuic acid, (k) vanillic acid, (1) gentisic acid. 
Fig·4·4 
92b 
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=CH-C
Q... 00. .. H 0 ...  
chlorogenic acid 
Fig- 4·4 
sec 
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4 
Il 
c 
o 
0-
III 
~3 
~ 
1 
• 
100 200 300 400 500 
Dose JJg/disc 
Dose response curves for the partition phases of the ethyl 
acetate extract of the whorls of the 3 week old plants of IS 
18363 • 
• crude, x aqueous+organic, .. aqueous, • organic • 
Fig- 4-5 
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activity than the less polar one (Fig. 4.5). The blend from 
the fractions which was constituted in the proportion 
obtained from the fractionation of the extract, aqueous 
organic (4:1), was almost as active (b = 0.6209) as the 
crude extract (b = 0.6426). This suggested that chemicals 
in the aqueous and organic fractions acted synergistically 
to give an enhanced larval feeding response. 
The dose response curves for the phenolic compounds 
(Figs. 4.6 and 4.7) were expressed in the regression 
equation In y = In a + b In x + x In c where y = relative 
feeding response, x = dose, rmOle/diSC i a, b, and care 
constants. The a, band c values are listed in Table 4.4. 
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2 
x 
-1~---L--~----~--~--~----~--~---
0·5 1·5 2 Z5 3 35 
Dose J.mOIe/disc 
Dose response curves for p-hydroxybenzaldehyde, p-hydroxy-
benzoic acid and some synthetic analogues of the two 
compounds. 
z p-hydroxybenzaldehyde, op-metboxybenEYl alcohol, 
o p-hydroxybenzoic acid, AP-methoxybenzoic acid, 
A p-hydroxybenzyl alcohol, • p-metboxybenzaldehyde. 
Fig. 4.6 
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• 
II 
K • '·5 
~ 
2' 
1, . 
~ 
i 
~ ()'5 
~ 
~ 
0 
()'5 1.0 1-5 2.0 25 30 
Dose J.lmoleldisc 
Dose response curves for some hydroxybenzoic and cinnamic 
acid compounds. 
• protocatechuic acid, x vanillic acid, 0 chlorogenic 
acid, ~ quinic acid, • caffeic acid, C ferulic acid. 
Fig.4·7 
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Table 4.4 Regression constants for phenolics and some 
derivatives. 
Compound In a b In c 
p-hydroxybenzaldehyde -0.1343 0.5575 -0.0060 
p-hydroxybenzoic acid -1.0196 0.6199 -0.0055 
p-hydroxybenzyl alcohol -0.1879 0.3533 -0.0043 
p-methoxybenzaldehyde 1. 9694 -0.5601 0.0018 
p-methoxybenzoic acid -0.6328 0.5043 -0.0048 
p-methoxybenzyl alcohol 1.6133 0.0017 -0.0024 
p-HBALD + p-HBA * -1.2784 0.8347 -0.0051 
vanillic acid 2.1145 0.5945 -0.6074 
protocatechuic acid 2.0369 0.0446 -0.2869 
caffeic acid 0.2679 -0.0624 -0.0524 
ferulic acid 0.1456 -0.0748 0.0029 
chlorogenic acid 2.0330 0.5559 -0.8040 
quinic acid 0.1167 -0.3239 0.0711 
p-coumaric acid not stimulatory 
gentisic acid not stimulatory 
* p-hydroxybenzaldehyde+p-hydroxybenzoic acid 
The data on the phenolics showed that larval feeding 
response varied with the nature of the compound. All the 
phenolic compounds except p-coumaric acid and gentisic acid 
stimulated larval feeding, but became less stimulatory at 
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higher doses. p_Methoxybenzaldehyde was stimulatory at very 
low doses but deterrent at higher doses. The feeding 
response of larvae to a blend of p-hydroxybenzaldehyde and 
p-hydroxybenzoic acid as occurring in the crude extract of 
the whorls of the 3 week old plants of cultivar IS 18363 
showed no synergism between the two compounds (Fig. 4.8). 
4.12 Extent of oxidation of p-hydroxybenzaldehyde during 
bioassays 
Since aldehydes are rapidly oxidised on exposure to 
air, an attempt was made to determine the extent of 
oxidation of p-hydroxybenzaldehyde during the assay period. 
A methanol solution of p-hydroxybenzaldehyde (10 f9/fl) was 
prepared. 10 cellulose acetate discs were each loaded with 
200 ~g of p-hydroxybenzaldehyde followed by 15 ~l of double 
distilled water (to correct any distortion of the discs by 
the methanol) and then air dried in a stream of warm air. 5 
out of these discs were immediately extracted with 1000 ~l 
of methanol, and 10 fl of each extract were analysed on an 
HPLC reverse phase column. The remaining discs were set 
under the bioassay conditions previously described, but 
without larvae. After 24 h the discs were similarly 
extracted with methanol and analysed. The mean peak areas 
corresponding to p-hydroxybenzaldehyde after 0 and 24 h of 
bioassay were calculated and compared. Peaks representing 
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II 
~3 
8. 
~ 
~ 
lfj 
J!2 
~ 
i 
0 
~ 
.8" 
z16  Ii 
100 200 300 500 
Dose JJ9/disc 
Dose response r.urves for p-hydroxybenzaldehyde, p-hydroxy-
benzoic acid and a blend of the two compounds • 
• p-hydroxybenzaldehyde+p-hydroxybenzoic acid (3:2), 
6 p-hydroxybenzaldehyde, Op-hydroxybenzoic acid. 
Fig ·C!.8 
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the absorptions of p_hydroxybenzoic acid were also checked 
for in the analysis. 
The results showed that there was no significant 
difference between peak areas of p-hydroxybenzaldehyde after 
o 2h (2.30 ± 0.05 cm2 ) and 24 h (2.30 ± 0.07 cm ) of bioassay 
indicating that very little oxidation of p-hydroxy-
benzaldehyde occurs under the bioassay conditions. 
4.13 Phenolic levels in the ethyl acetate extracts 
Free phenolics In the extracts were p-hydroxybenzoic 
acid, p-hydroxybenzaldehyde, p-coumaric, ferulic and caffeic 
acids. Preliminary HPLC analysis of the three extracts 
hexane, ethyl acetate and methanol showed that there was 
very little of these compounds in the hexane and methanol 
extracts indicating that the bulk of them were in the ethyl 
acetate extracts. The HPLC data of the ethyl acetate 
extracts showed that ferulic, caffeic, and p-coumaric acids 
were present in trace amounts whilst p-hydroxybenzoic acid 
and p-hydroxybenzaldehyde were in relatively large 
proportions (Fig . 4.3b). Analysis of the free phenolic 
levels in the ethyl acetate extracts was therefore 
determined from the amounts of p-hydroxybenzoic acid and 
p-hydroxybenzaldehyde from a calibration of a standard 
solution containing a mixture of the two. The 
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concentrations of p_hydroxybenzoic acid and p-hydroxy-
benzaldehyde in the standard solution were 0.1,18  rg/tl and 0.24 fg/rl respectively. 1, 2, 5, 10 and 15 aliquots of 
the standard mixture were analysed on a HPLC reverse phase 
column and the effluent detected by UV at 254 nm. 5 r9/rl 
methanol solution of each of these ethyl acetate extracts 
was prepared and 5 ~l each of the extracts were similarly 
analysed. For each volume analysed the peak areas for the 
absorptions of the the two compounds were calculated and 
converted into their corresponding amounts in micrograms. 
From these the amounts in the whorls of the two cultivars at 
the two growth stages were calculated. These data are 
summarised in Tables 4.5 and 4.6. 
Table 4.5 Phenolic levels in 25 t9 of each of the ethyl 
acetate extracts 
Extract Plant p-hydroxy p-hydroxy Total Ratio of 
age benzaldehyde benzoic acid phenolic the two 
(wks) ((9) (t<J) (r9) 
IS 18363 3 1.8 1.2 3.0 1.5 
IS 2205 3 0.9 0.5 1.4 1.8 
IS 18363 6 3.3 0.4 3.7 8.3 
IS 2205 6 1.2 0.3 1.5 4.0 
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Table 4.6 Phenolic levels in the whorls % (fCJ/kg whorl) 
Cultivar Plant wt NQ. of % phenolic % phenolic 
age(wks) (kg) whorls in extract in whorl 
IS 18363 3 3.5 500 30 0.17 
IS 2205 3 3.4 800 14 0.05 
IS 18363 6 3.5 80 37 1. 32 
IS 2205 6 2.9 80 15 0.65 
Several noteworthy results emerge from these data:-
(a) the extracts of the whorls of the resistant 
cultivar IS 2205 contains less p-hydroxybenzaldehyde and 
p-hydroxybenzoic acid than the susceptible one IS 18363 for 
both the 3 and 6 week old plants. Similarly, the total 
phenolic level was less for the extracts and whorls of the 
resistant cultivar at the two growth stages of the plant. 
(b) the total phenolic levels were not significantly 
different for the extracts of the whorls of the 3 and 6 week 
old plants. The whorls of the 6 week old plants contained a 
higher phenolic level. At the two growth stages, the 
susceptible cultivar IS 18363 contained a higher phenolic 
level than the resistant one IS 2205. 
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resistant one IS 2205. 
(c) the level of p-hydroxybenzaldehyde was higher in 
the extracts of the whorls of the 6 week old plants than the 
3 week old ones, whereas the situation for p-hydroxybenzoic 
acid was reverse. 
4.14 Fractionation of methanol extract 
Only the extract of the whorls of the 3 week old plants 
of sorghum cultivar IS 18363 was fractionated for detailed 
study. The composition of the other extracts was determined 
by comparisons of thq:r chromatographic profiles. 
Fractionation of the crude methanol extract was 
undertaken as shown in Scheme 4.2. 
Scheme 4.2 Flow diagram for the fractionation of the 
methanol extract of the whorls of the 3 week old plants 
Crude extract 
(10 g) 
I dissolved in water 
I filtered 
I 
Filtrate I Residue PM-1 
(0.6 g) 
chloroform 
I 
Chloroform PM-2 I Aqueous 
(1.1 g) I 
I ethyl acetate 
I 
Ethyl acetate PM-3 I Aqueous PM-4 
(0.7 g) (7.6 g) 
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The extract (10 g) was dissolved in water (300 ml) and 
filtered by suction. This removed the insoluble materials 
(PM-1, 0.6 g). The resulting filtrate was extracted with 
chloroform (5 X 50 ml). The chloroform extracts were 
combined, dried over anhydrous sodium sulphate and then 
evaporated to dryness in vacuo to give a dark green oily 
mass (PM-2, 1.1 g). The above process was repeated with 
ethyl acetate. The combined ethyl acetate extracts yielded 
a yellowish-green solid after solvent removal in vacuo (PM-3 
0.7 g). The aqueous phase after ethyl acetate extraction 
was concentrated first on a rotary evaporator and then on a 
freeze-drier to give a dark brown solid (PM-4, 7.6 g). 
Fraction PM-1 constituted 6%, fraction PM-2, 11%, 
fraction PM-3, 7%, and fraction PM-4, 76% of the crude 
extract showing that about three-quarters of the components 
in the crude methanol extract of the 3 week old whorls of 
cultivar IS 18363 were water soluble. 
4.15 Bioassays of the fractions 
The fractions PM-1-PM-4 were each bioassayed for 
effects on feeding of C. partellus third-instar larvae. 
Dose response tests were performed for each fraction and for 
a combined mixture of all the four fractions in the 
proportion in which they were obtained from the crude 
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the mixture was tested by elimination. The results of these 
tests are shown in Fig. 4.9 and Table 4.7. 
Fig. 4.9 shows the dose response curves for the 
different fractions expressed in the regression equation 
In y = In a + b In x where y and x are relative feeding 
response and dose in ~g/disc respectively. These equations 
were as follows: PM-I, In y -0.8456 + 0.2234 In x; PM-2, 
In y -1.1611 + 0.4156 In Xi PM-3, In y = -0.7366 + 
0.6613 In Xi PM-4, In y = -1.2695 + 0.5751 In X and for the 
crude extract In y = -0.0741 + 0.7486 In x. 
A comparison of the b values showed that fractionation 
lowered the feeding response of the larvae, with the most 
active fraction being the most polar fraction, PM-4. 
Fractions PM-2 and PM-3 were intermediate while the least 
stimulatory was PM-1. A blend of the fractions 
reconstituted in the ratio in which they were fractionated 
was more stimulatory than any of the individual fractions 
but less active than the crude methanol extract. 
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----------------~--
---------------o~ --
100· 200 300 400 500 
Oose j.Jg/disc 
Dose response curves for the partition phases of the 
methanol extract of the whorls of the 3 week old plants of 
IS 18363. 
x crude, ePM-1+PM-2+PM-3+PM-4, A aqueous (PM-4) 
~ chloroform (PM-2), • ethyl acetate (PM-3), 0 residue 
(PH-l). . 
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Table 4.7 Feeding responses of C. partellus larvae to 
cellulose acetate discs treated with 300 ~ of various 
combinations of t h e fractl.'ons of the methanol extract in a 
no choice bioassay. 
Fraction Mean disc Anova model 
Combinations consumed F-value 
I. Crude extract 1. 6874 a 
II. PM1+PM2+PM3+PM4 1.1986 b 
III. PM2+PM3+PM4 0.9353 c 
IV. PM3+PM4 0.6729 d 
V. PM1+PM2+PM4 0.5838 d 
VI. PM1+PM3+PM4 0.4184 e 156 *** 
VII. PM1+PM4 0.4176 e 
VIII. PM2+PM4 0.4034 e 
IX. PM4 0.2634 f 
X. PM l+PM2 +PM3 0.1206 g 
XI. Control 0.0283 g 
*** Pr>F 0.0001 
Means with the same letter are not significantly different 
(P<0.05j Duncan's multiple range test) 
The results show synergism between components in the 
water soluble fraction PM-4 and components in the organic 
fractions PM-I, PM-2 and PM-3 as evident from I to IX in the 
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table. The combination containing all the fractions was 
most stimulatory but significantly less active than the 
crude extract suggesting that some of the components were 
lost or deactivated during fractionation. The response of 
larvae to the mixture containing the less polar fractions 
was not significantly different from control showing that 
they playa synergistic role in the mixture. Fraction PM-4, 
which forms about 76 percent of the total extract when 
tested alone was significantly more stimulatory than 
control. 
4.16 Chromatographic analysis of the methanolic fractions 
The four methanolic fractions were examined by HPLC on 
a reverse phase analytical column under conditions identical 
to those used for the fractions of the ethyl acetate extract 
using a UV detector at 240 nm. Fraction PM-l did not show 
any well-defined components. Fraction PM-2 contained only 
two major components, one of which corresponded to 
p-hydroxybenzaldehyde and a second highly polar component 
(Rt = 2 min) (J-l) which was also present in a larger 
proportion in fraction PM-4 (Fig. 4.10). PM-3 and PM-4 
contained a common component (J-2) in addition to a series 
of polar and less polar compounds, one of which was 
p-hydroxybenzoic acid (Fig. 4.10). Since the 
chromatographic data of the crude methanol extract (Fig. 
4.1c) did not show the presence of p-hydroxybenzaldehyde and 
'108 
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~ 
\ 9.. 
! 
; 
I t K . I 
o ~ 
o '- 10 20 30 
Tl~ .(mi~ 
HPLC r.rofiles of the partition phases of the _thanal I 
eztract of the whorls of the 3 weelt old plante of IS 18363. 
f~ top to bottom. PX-2, PK-3 and PX-4. 
A-J-l, B-p-hydrozybensoic acid, C-J-2, D-p-hydroxy-
benzaldehyde. -
Fig. ",10 
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p-hydroxybenzoic acid in the extract, it appears that these 
two components might be degradative products of some other 
components in the extract. Since PM-3 and PM-4 between them 
contained all the unidentified compounds they were subjected 
to further analysis as described below. 
(a) Micropreparative HPLC analysis of PM-3 and TLC of PM-4. 
These were carried out to isolate pure samples of J-2. 
A sample of PM-3 (20 mg) was chromatographed on a DuPont 
Zorbax ODS 25 cm x 4.6 rom column. Five fractions were 
collected (PM-3-1 to PM-3-V). Of these PM-3-I1 and PM-3-V 
corresponded to p-hydroxybenzoic acid and p-hydroxy-
benzaldehyde respectively. PM-3-II1 which was obtained as a 
dark yellow solid corresponded to the unidentified compound 
J-2. No significant quantities of materials were obtained 
from fractions PM-3-1 and PM-3-IV. 
Further chromatographic analysis of this sample of J-2 
on an HPLC reverse phase column showed that a proportion of 
it had decomposed into two relatively less polar components 
J-3 and J-4, among others (Fig. 4.11). These results 
confirm the earlier suggestion of the presence of breakdown 
components in some of the fractions of the methanol extract. 
J-3 and J-4 were isolated as a mixture from micropreparative 
HPLC of J-2 on a reverse phase column. GC-MS analysis of 
this mixture showed that J-3 was a major breakdown component 
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- 8 E 
c 
~ 
CLI 
~ 
0 
a. 
en 
~ 
5 
i 
0 
0 40 50 
HPLC profile of J-2 shoving its bre~down components. 
A-unknown, B-J-2, C-p-hydroxybenzaldehyde ~nd D-p-methoxy-
benzyl alcohol. 
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of J-2, and this was identified as p-hydroxybenzaldehyde. 
The minor component J-4 was identified as p-methoxybenzyl 
alcohol. Both components were identified on the basis of 
their mass spectral data and GC coinjection with authentic 
samples. The mass spectrum of p-methoxybenzyl alcohol 
showed peaks at M+-l38 (70), 121 (42), 109 (96), 94 (52), 77 
(100), 65 (35), 51 (52) and 39 (80). The identities of the 
two compounds were further established by HPLC coinjection 
on a reverse phase column with authentic samples (p-hydroxy-
benzaldehyde Rt =20 min, p-methoxybenzyl alcohol Rt =43 min). 
These results suggested that component J-2 had the 
characteristics of a ketal, with p-hydroxybenzaldehyde and 
p-methoxybenzyl alcohol moieties of the molecule. 
Since a pure sample of J-2 could not easily be isolated 
by micropreparative HPLC of PM-3 (Fig. 4.10), an attempt was 
made to isolate it from PM-4 by conventional thin layer 
chromatography. Analysis of PM-4 on silica gel/uv (0.25 
254 
rom thickness) in solvent system methanol/hexane 3:1 showed 
two spots, both of which fluoresced blue in UV light. A 
sample of PM-4 (0.44 g) was dissolved in water and separated 
by preparative tlc in the same solvent system. The two 
fractions were extracted with methanol, the resulting 
extract filtered and concentrated to dryness in vacuo. The 
leading fraction gave a highly hygroscopic pale yellow solid 
(0.17 g) which was identified as J-2 and the trailing 
fraction a dirty white solid (0.03 g) identified as J-1 by 
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comparison of their HPLC profiles with that of a sample of 
PM-4. 
J-1 was recrystallized from methanol to give a white 
solid mp > 250 °C. The mass spectral data for this compound 
were mle (%) M+-502 (100), 347 (60), 303 (14), 229 (18), 191 
(18), 60 (14) and 44 (90). The peaks at mle 60 and 44 were 
suggestive of fragments of a carboxylic acid. Thus the 
physical data of J-1 were suggestive of a high molecular 
weight carboxylic acid. 
Further purification of J-2 was achieved by 
chromatography on silica gel in the same solvent system. 
The mass spectrum of this compound showed that it was a 
mixture of six related compounds (J-2-1 to J-2-6) with a 
common fragmentation pattern. The mle (%) values for peaks 
corresponding to these components in the mixture were as 
follows: 
J-2-1, 236 (8), 193 (8), 177 (18), 165 (100), 151 (18), 101 
(8), 73 (8), 59 (26), 55 (12), 43 (100) and 39 (30) ; 
J-2-2, 193 (6), 177 (6), 163 (12), 150 (12) , 101 (2), 73 
(6), 59 (12), 55 (12), 43 (100), 39 (42); 
J-2-3, 193 (6), 177 (12), 163 (12), 151 (10), 101 (4), 73 
(12), 69 (6), 60 (18), 55 (16), 43 (100), 39 (18); 
J-2-4, 206 (2), 193 (4), 177 (10), 164 (8), 153 (10), 97 
(10), 73 (8), 69 (12), 60 (12), 55 (12), 43 (100), 39 (40); 
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J-2-5 206 (2), 193 (6), 177 (18), 165 (16), 152 (10), 97 
(10), 83 (8), 73 (6), 69 (10), 60 (14), 55 (20), 43 (100), 
39 (30); 
J-2-6 206 (2), 177 (8), 165 (8), 153 (8), 97 (10), 83 (6), 
73 (4), 69 (12), 60 (10), 55 (12), 43 (100), 39 (36). 
Peaks which were common to these components were at the 
following mle values: 193, 177, 165, 151, 101, 73, 60, 43 
and 39. The peaks at mle 193, 177 and 165 were also present 
in the mass spectrum of the acetyl derivative of ferulic 
acid. The peak at mle 60 was suggestive of a carboxylic 
acid moiety in the molecule. These results suggested that 
components J-2-1 to J-2-6 may be phenolic in nature and may 
be related to ferulic acid or some other phenolic compounds 
in the plant. To confirm the presence of these components 
in J-2, the latter was converted into a more volatile 
derivative and the product analysed by GC-MS. Details of 
this are described below. 
(b) GC-MS analysis of trimethylsilyl derivatives of J-2. 
1 mg of a sample of J-2 in 100 fl of dry pyridine was 
derivatized in a 1 ml sample vial with 100 rl of a solution 
of bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) 
containing 1% trimethylchlorosilane (TMCS). The mixture was 
capped and heated to 60 °c for 16 hr. GC-MS (Fig. 4.11b) 
analysis revealed, as expected, the trimethylsilyl 
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derivatives of a mixture of components which appeared to be 
related. Peaks common to these compounds were at the 
following mle values; 44, 57, 73, 103, 117, 129 or 133, 
147, 205, 217. As suggested by Budzikiewicz et al., (1967), 
the peak at mle 73 is due to the trimethylsilyl cation 
(CH )3Si+ but that at mle 147 is an artefact due to the ion 3
(CH3)3Si=~-Si(CH3)~ which is formed by expulsion of a methyl 
group from hexamethyl disiloxane (CH3)3Si~O-Si(CH3)3' a 
condensation product of trimethylsilanol (CH3)3SiOH. Of 
particularly interest was the peak at mle 44 which 
corresponds to the expulsion of CO2 from a trimethylsilyl 
benzoate 
(SChe~4.m3e)· ;(C~ -COZ 
t!J 
Scheme 4.3 
An intense M-CH3 peak which can be used for molecular weight 
determination of trimethylsilyl ethers (Budzikiewicz et al., 
1967) appeared to be present in the spectra of all the 
derivatives in the mixture. These peaks were at mle 243, 
365, 379 and 437 and were found for more than one component 
in the spectra suggesting the presence of isomeric compounds 
in the mixturs. The spectra were compared to those of the 
TMS derivatives of 8 standard phenolic compounds which 
include p-hydroxybenza1dehyde, p-hydroxybenzoic acid, p_ 
coumaric acid, ferulic acid, syringic acid, p-hydroxybenzyl 
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alcohol, gentisic acid and caffeic acid but none was 
identical to any of the components in J-2. However, the 
general fragmentation pattern of the TMS derivatives of 
these compounds was similar to that of the components in J-2 
confirming that components in the latter were phenolic in 
nature. The precise structural identities of the components 
in J-2 were not clear so no further work was carried out on 
it. 
(c) GC-MS analysis of acetylated sample of PM-3. 
Since in addition to J-2, p-hydroxybenzaldehyde and 
p-hydroxybenzoic acid, PM-3 also contained minor quantities 
of polar and less polar components, an attempt was made to 
identify these by GC-MS of their acetylated derivatives. 
Acetylation of PM-3 was carried out in acetic anhydride in 
dry pyridine at 0 °c for 24 h. GC-MS (Fig. 4.12) revealed 
the presence of the acetyl derivatives of p-coumaric, 
caffeic, and ferulic acids and other high molecular weight 
compounds. Some of these showed fragmentation patterns 
charateristic of acetyl derivatives of higher phenols (P1, 
P2 and P3). The rest were characteristic of hydrocarbon 
compounds. The peaks for P1, P2 and P3 were at the 
following mle values: 
P1, M+-324 (20), 309 (100), 147 (10), 133 (15), 119 (36), 
103 (18), 91 (48), 77 (12), 57 (80) and 41 (72). 
P2, M+-330 (35), 315 (100), 253 (8), 237 (22), 178 (8), 165 
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7:42 11:24 2229 26:10 2952 3134 
Gas chromatogram of the trimethylsilyl (THS) derivatives of 
the components in J-2. 
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IP 
HI ~ ~ 
" 
1142 T1:24 2t36 24:47 28:29 3t11 3~52 39:34 
TirM (min) 
Gas chromatogram of the acetylated product of PM-l. 
A,B and C are peaks representing the acetyl derivatives of 
p-coumaric, ferulic and caffeic acids. D,B and F represent 
P1, P2 and Pl described in the text. H1-H8 are peaks 
representing hydrocarbon compounds in the product. 
Fig·4·12 
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(62) and 41 (94). 
P3, M+-386 (28), 371 (62), 293 (28),178 (22),165 (6), 139 
(8), 119 (68), 103 (38), 91 (100), 77 (22), 57 (80) and 41 
(85) . 
(d) Chromatographic analysis of sugars in PM-4 
PM-4 was found to be water soluble, containing both 
chromophoric J-1 and J-2 (UV detection at 240 nm ) and non-
chromophoric compounds (Rr detection). Attempts were made 
to separate all the components of this fraction by various 
chromatographic methods. These methods included column 
chromatography on Pharmacia Sephadex G-10 and Sephadex LH-20 
with a UV monitor at 254 nm and reverse phase thin layer 
chromatography on Merck RP-18 F254S plates. However, these 
methods did not give very satisfactory separation of the 
components in fraction PM-4. A sample of the crude fraction 
was analysed by PMR in 020 on a Varian XL 200 MHz. The PMR 
spectrum showed a concentration of signals between 3 and 4 
ppm which are signals characteristic of the protons of a 
carbohydrate (Lemieux and Stevens, 1966). Fraction PM-4 was 
therefore subjected to carbohydrate analysis on two 
different columns. (a) a Biorad carbohydrate column, Aminex 
HPX-87C, 30 cm x 7.8 rom and (b) a Varian normal phase 
column, Micropak NH2-10, 30 cm x 4 rom. The effluents from 
these columns were monitored by a refractive index detector 
(Varian RI-3). 
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An aqeuous solution of PM-4 (0.3 mg/~) was prepared 
and 40 ~ aliquot of the solution were analysed separately 
on the two columns. The solvent system for the analysis on 
the Biorad column was degassed double distilled water and 
this was maintained at a flow rate of 0.6 ml/min at 80 °C. 
For the normal phase analysis, the Micropak NH2-10 column 
was eluted with 70% aqueous acetonitrile at a flow rate of 
1.4 ml/min at 25 °C. Standard sugars which included 
sucrose, glucose, fructose, galactose, mannose and xylose 
(Sigma Chern., Co.) were similarly analysed both individually 
and in a mixture. The identities of the sugars in fraction 
PM-4 were established by comparison of the chromatograms of 
the standards from the two analytical columns with those of 
PM-4. 
Fig. 4.13 shows the HPLC profiles of the mixture of 
standard sugars and fraction PM-4. The upper and lower 
chromatograms represent the standard sugar mixture and 
fraction PM-4 respectively analysed on (a) Micropak NH -10 
2
and (b) Biorad Arninex HPX-87C columns. The sugars 
identified by comparison of the chromatograms were sucrose, 
fructose, glucose and xylose. The identities of these 
sugars in fraction PM-4 were confirmed by coinjection with 
authentic samples on the two HPLC carbohydrate columns. 
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A • 
g.f • 
! 
'" 
\... -""'-
, 
lSolvent 
9 
9.f 
f 
~l J ~ -~. 1-0  I 
0 10 20 300 10 20 30 
Solvent Time min 
HPLC profiles of a mixture of standard sugars (upper 
profile) and a sample of the aqueous fraction (lower 
profile) of the methanol extract of the whorls of the 3 week 
old plants of IS 18363 analysed on two different columns, 
A-Hicropak NH2 and B-carbohydrate Biorad Aminex HPX-87C. 
s-sucrose, g-glucose, x-xylose, f-fructose, m-maltose 
Fig· "'13 
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4.17 Bioassays of components from methanolic extract 
Feeding bioassays were carried out on all the sugar and 
nonsugar components in fraction PM-4. Each component was 
tested at 10, 25, 50, 150 and 250 f9. 
(a) Nonsugars J-1 and J-2 (chromophoric components). 
Component J-1 was non-stimulatory at all the doses 
tested. 
Fig. 4.14 shows the dose response curve for component 
J-2 expressed in the regression equation In y =0.4435 + 
0.3542 In x where y = relative feeding response and x 
dose, ~g/disc. 
The dose response curve showed that J-2 stimulated 
larval feeding at all the doses tested. 
(b) Sugars (non chromophoric components) 
Fig. 4.15 shows the dose response curves for the 
different sugars which were expressed in the regression 
equation In y = In a + b In x where y = relative feeding 
response and x = dose, rmole/disc. These equations were as 
follows: sucrose, In y = 3.2902 + 0.7539 In Xi glucose, 
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2· • 
~ 
0 
~2. • 
g' 
i 
~ 
~ 
i 
1 1· 
J 
7ii ()'5 
z 
0 
50 100 150 200 250 
Dose JJg/disc 
Dose response curve for the total mixture of components in 
J-2. 
Fig. 4.14 
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Dose IJmoleldisc 
Dose response curves for the sugar components in the . 
methanolic extract of the whorls of the 3 week old plants of 
IS 18363. 
o sucrose, A glucose+fructose, • glucose, )( fructose • 
._. ---·- ~4"·t5 -_. 
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In y = 1.6158 + 0.5108 In Xi fructose, In y = 1.4632 + 
0.4364 In Xi glucose + fructose, In y = 1.8618 + 0.4918 
In x. 
The dose response curves showed that sucrose was most 
stimulatory to the larvae followed by glucose and fructose, 
both of which were similar in their effect on the feeding of 
the larvae. Larvae did not respond to xylose at any of the 
doses tested. A mixture of glucose and fructose in a 1:1 
ratio was more stimulatory to larvae than the individual 
sugars tested alone, but less stimulatory than sucrose. 
(c) A mixture of p-hydroxybenzaldehyde and sucrose 
Tests were performed for synergism between sucrose and 
p-hydroxybenzaldehyde. Cellulose acetate discs were loaded 
with 10 pg of sucrose and air dried. In a previous assay, 
p-hydroxybenzaldehyde was tested at 10, 25, 50, 150, 250, 
350 and 450 rg. In this experiment, the sucrose treated 
discs were loaded with the same doses of p-hydroxy-
benzaldehyde and bioassayed for third-ins tar larval feeding 
response. 
The dose response curves for p-hydroxybenzaldehyde with 
or without sucrose are shown in Fig. 4.16. These were 
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125 
Dose response curves for p-hydroxybenzaldehyde and a blend 
of sucrose and p-hydroxybenzaldehyde. 
• sucrose+p-hydroxybenzaldehyde, 0 sucrose+p-hydroxy_ 
benzaldehyde (theoretical plot), ~ p-hydroxybenzaldehyde, 
(~) activity of 0.03 fIDole sucrose. 
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expressed in the regression equation In y = In a + b In x 
where y = relative feeding response, x = dose of p-hydroxy-
benzaldehyde infmole/discj a,b and C are constants. The 
regression equations were: p-hydroxybenzaldehyde, In y 
2.5288 - 0.7251x + 0.5462 In Xi p-hydroxybenzaldehyde + 
sucrose, In y = 3.8739 - 0.9215x + 0.9023 In 
The dose response curves showed that p-hydroxy-
benzaldehyde alone stimulated feeding at all the doses 
tested in the above experiment but the effect of it 
decreased with increasing dose. Addition of 0.03 rmole 
(10~) sucrose to p-hydroxybenzaldehyde at the doses tested 
increased larval feeding response by an increment which was 
significantly greater than a simple summation of the 
activities of the two compounds would suggest. This 
suggested that p-hydroxybenzaldehyde and sucrose acted 
synergistically to give enhanced feeding response. 
4.18 Sugar and nonsugar levels in the methanol extracts 
The relative amounts of the individual sugars 
identified in the aqueous fractions of the methanol extracts 
of the two cultivars at the two growth stages were 
determined from a calibration of a standard solution 
containing a mixture of the sugars. These sugars were 
sucrose, glucose, fructose and xylose, and the 
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respectively. 5, 10, 15, 20, and 30 rl aliquots of these 
standard solutions were analysed on the Biorad carbohydrate 
column, Aminex HPX-87C and the effluent monitored by a RI 
detector. The methanol extracts of the whorls of the 3 and 
6 week old plants of IS 18363 and IS 2205 were fractionated 
as previously described (page 102) and 40 ~l of each of the 
resulting aqueous fractions similarly analysed. The peak 
areas corresponding to the different sugars in the extracts 
were calculated and in amounts in micrograms. The total 
amount of nonsugar components in each of the fractions was 
calculated by difference. The total amount of sugar in the 
whorls of the two cultivars at the two growth stages were 
also calculated. The data on these calculations are shown 
in Tables 4.8 and 4.9. 
Table 4.8 Sugar and nonsugar levels in 12 mg of each of the 
aqueous fractions of the methanol extracts. 
Aqueous Plant Suc Glu Fru Xyl Total Total 
fraction age (mg) (mg) (mg) (mg) sugar nonsugar 
(wks) (mg) (mg) 
IS 18363 3 5.3 2.5 1.9 0.6 10.3 1.7 
IS 2205 3 4.5 2.5 1.6 0.5 9.1 2.9 
IS 18363 6 4.8 1.8 2.1 0.2 8.9 3.1 
IS 2205 6 5.4 2.4 2.3 0.2 10.3 1.7 
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Table 4.9 Sugar levels in the whorls % (mg/kg whorl) 
Cultivar Plant NQ of wt Total sugar % sugar 
age(wks) whorls (kg) (mg) 
IS 18363 3 500 3.5 10.3 0.6 
IS 2205 3 800 3.4 9.1 0.3 
IS 18363 6 80 3.5 8.9 3.2 
IS 2205 6 80 2.9 10.3 4.4 
The findings from these data were as follows: 
(a) sugars formed between 65 to 85 percent of the mixture 
in the aqueous fractions of the methanol extracts of the 
whorls of the two cultivars at the two growth stages. 
(b) the amounts of the identified sugars in the fractions 
were in the order sucrose > glucose ~ fructose > xylose. 
(C) there was more sugar in the whorls of the 3 week old 
plants of the susceptible cultivar IS 18363 than those of 
the resistant cultivar IS 2205. The sugar levels in the 
whorls of the 6 week old plants of the two cultivars were 
approximately the same, but were higher than the levels 
found for the 3 week old plants. 
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CHAPTER 5 
DISCUSSION 
Previous studies on the effect of plant chemicals on 
the feeding of phytophagous insects have involved bioassays 
employing different feeding substrates such as 
agar/cellulose (Hsiao and Fraenkel, 1968), ordinary filter 
paper (Daad, 1960; Wensler and Dadzinski, 1972), styropor 
(Meisner and Ascher, 1968) and glass-fibre filter paper 
(Staedler and Hanson, 1976; Woodhead and Bernays, 1978). In 
addition, membrane filter discs, which are available in 
sizes within a narrow weight range, have been used with 
advantage (Bristow et al., 1979; Doss et al., 1980; Doss et 
al., 1982; Albert et al., 1982 Capinera et al., 1983; Doss 
and Shanks, 1984; Doss and Shanks, 1986). For bioassays in 
this study, cellulose acetate membrane filter discs were 
tested and found to be well suited for monitoring the 
feeding responses of the third-ins tar larvae to sorghum 
extracts. However, contact with methanol caused slight 
distortion of the discs and were poorly fed on by the larvae 
as shown by the results in Table 4.2. Similar observations 
were made by Doss and Shanks (1986) who found that contact 
with organic solvents caused membrane filters either to 
dissolve or to wrinkle. However, our experimentation showed 
that addition of water to solvent-treated discs restored 
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their palatabilty to the larvae since no significant 
differences were found between the feeding responses of 
larvae to these discs and to control, hexane, and water 
treated discs (Table 4.2). The procedure of adding water 
was thereafter adopted for all discs in the bioassays. 
In no choice tests, third-instar larvae of C. partellus 
were found to exhibit a dose-dependent response to crude 
extracts of sorghum cultivars IS 18363 and IS 2205 applied 
to cellulose acetate discs, confirming the effectiveness of 
the bioassay procedure. The results of the bioassays of the 
crude extracts of hexane, ethyl acetate and methanol showed 
that none of them was deterrent to the feeding of the larvae 
although this did not rule out ~e presence of deterrent 
components in these extracts. The methanol extracts were 
most stimulatory and showed a more linear dose/response 
relationship than the other crude extracts which were tested 
(Figs. 4.2a and 4.2b; Tables 4.3a and 4.3b). Ethyl acetate 
extracts were intermediate in stimulatory activity and these 
were followed by the hexane extracts. The stimulatory 
activities 6f the hexane extracts were consistent with 
previous results (Roome and Padgham, 1978) in that larvae of 
this insect showed little preference for a lipid extract of 
the whorls of sorghum plants. 
Extracts of the whorls of the 3 week old plants were 
more stimulatory to larvae than those of the 6 week old 
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plants. These results were consistent with earlier findings 
(Alghali, 1985) in that leaf feeding and overall plant 
damage by the larvae of C. partellus was more acute at the 
younger than the older vegetative stages. 
The bioassay tests showed that at both growth stages 
(ie. 3 and 6 weeks old plants), larvae responded more to 
extracts of the susceptible cultivar IS 18363 than to those 
of the resistant cultivar IS 2205 (Figs. 4.2a and 4.2b). 
These results are in agreement with observations made 
earlier where larval feeding was found to be high on the 
leaves of IS 18363 but medium on IS 2205 (Saxena, 1985b). 
HPLC analyses showed quantitative rather than qualitative 
differences between the crude extracts of these two 
cu1tivars (Figs. 4.1a, 4.1b and 4.1c). The chromatographic 
data suggested that the methanol and ethyl acetate extracts 
contained some common components (Figs. 4.1b and 4.1c) but 
the bioassay results suggested that the more potent 
phagostimulatory compounds were in the methanol extract. 
Unfortunately, no detailed analytical studies could be 
carried out on the least stimulatory hexane extracts which 
are likely to contain wax components shown in earlier 
studies to comprise n-alkanes, aldehydes, fatty acids and 
esters (Woodhead, 1983; Avato et al., 1984). 
When fractions of the ethyl acetate and methanol 
extracts obtained by partitioning between organic and 
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aqueous phases were tested, it became apparent that these 
fractions were less stimulatory than their mother extracts 
(Fig. 4.5 and 4.9). For both extracts, the more potent 
fractions were the aqueous ones. When the fractions were 
recombined the activities of the crude extracts were 
restored suggesting that several groups of compounds 
combined additively or synergistically to give the enhanced 
feeding activity of the mother extracts. 
Phagostimulatory compounds identified in the ethyl 
acetate extracts of the whorls of sorghum cultivars IS 18363 
and IS 2205 for the third-instar larvae of C. partellus were 
all phenolic. The major ones were p-hydroxybenzaldehyde and 
p-hydroxybenzoic acid previously reported to occur in the 
surface wax of sorghum cultivars studied by Woodhead et al. 
(1982) and Haskins and Gorz (1985). In addition, ferulic, 
caffeic and p-coumaric acids were present as minor 
components. Since hydroxyaromatic acids rarely occur in the 
free form in plants (Harborne, 1964) but occur as soluble 
esters or O-glycosides (Harborne and Corner, 1961), or as 
insoluble esters bound to the cell wall (Hartley and Jones, 
1977), the relatively large proportion of p-hydroxybenzoic 
acid found in the extracts may have been due to formation by 
oxidation of p-hydroxybenzaldehyde. 
Phagostimulatory compounds identified in the methanol 
extracts of the whorls of the two cultivars included the 
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phenolics mentioned earlier and sugars. These sugars 
comprised sucrose, glucose, fructose and xylose, identified 
by comparison of their HPLC retention times and coinjection 
with authentic samples on two columns: (a) carbohydrate 
Biorad Aminex HPX 87C and (b) normal phase Micropak NH2-10. 
Sucrose accounted for the largest proportion by weight of 
the sugars in the methanol extracts followed by either 
glucose or fructose and then xylose (Table 4.8). Components 
J-1 and J-2 were isolated as nonsugar components from the 
aqueous fraction of the methanol extract. J-1 was non-
stimulatory at all the doses which were tested whilst J-2 
stimulated larval feeding (Fig. 4.14). The latter compound 
was found to be a mixture of related phenolic derivatives 
(Fig. 4.10b). The fragmentation patterns of the components 
in this mixture were suggestive of glycosides of some of the 
phenolic compounds identified in the extracts including 
those of p-hydroxybenzaldehyde, ferulic and caffeic acids, 
all of which are known constituents of sorghum seedlings 
(Saunders et al., 1978; Woodhead and Cooper-Driver, 1979). 
It was apparent from its chromatographic performance that 
J-2 was thermally labile, and partially decomposed to 
components two of which were identified as p-hydroxy-
benzaldehyde and p-methoxybenzyl alcohol (Fig. 4.4). It 
appears that J-2 is not the cyanogenic glucoside dhurrin 
present in high concentration in younger than older sorghum 
leaves, and whose hydrolysis products are HCN and p-hydroxy-
benzaldehyde (Woodhead and Bernays, 1978). The exact 
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structural identities of J-2 components are 'not clear. 
However, it is possible that they act as reservoirs for p-
hydroxyb enz aldehyde and other phenolic compounds in the 
plant. Identification of these compounds, and investigation 
of their biological roles in relation to sorghum pests are 
clearly warranted. 
Phenolics have previously been isolated from the leaves 
of sorghum and have been shown to be responsible for 
resistance against L. migratoria and S. graminum feeding 
(Woodhead and Bernays, 1978; Dreyer et al., 1981; Woodhead, 
1982). On the other hand, C. parteIIus larvae have been 
found to feed on artificial diets containing these phenolics 
at levels up to three times the quantities that are normally 
found occurring in sorghum (Roome and Padgham, 1978). A 
similar observation was made by Fisk (1980) working with the 
homopteran insect Peregrinus maidis (Ashun) on a phenolic 
extract from sorghum. Baker et al. (1968) found p-
hydroxybenzaldehyde and some of its derivatives strong 
feeding stimulants for the elm bark beetle Scolytus 
multistriatus. In this study, third-ins tar larvae of C. 
parteIIus were stimulated to feed by p-hydroxybenzoic acid 
and p-hydroxybenzaldehyde and some derivatives of these 
compounds (Fig. 4.6). The feeding responses of larvae to 
increasing doses of these phenolic compounds followed 
broadly the same pattern, reaching an optimum and then 
dropping. However, they varied with the nature of 
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substitution of the phenolic compound suggesting that the 
different functionalities on the benzene ring may play a 
significant role in the food discriminative behaviour of the 
larvae. A comparison of the hydroxy compounds (Fig. 4.6) 
showed that p-hydroxybenzaldehyde was most stimulatory 
followed by p-hydroxybenzoic acid and p-hydroxybenzyl 
alcohol in that order. It may be pointed out that under the 
bioassay conditions of this study, very little of p-hydroxy-
benzaldehyde is oxidised so the activity observed is 
essentially due to the aldehyde. These results follow 
closely that obtained by Baker et al., (1968) for the elm 
bark beetle where p-hydroxybenzaldehyde elicited the 
strongest feeding response from the insect. A comparison of 
the activities of the methoxy derivatives of these 
compounds, showed that larvae responded best to p-methoxy-
benzyl alcohol and poorest to p-methoxybenzaldehyde (Fig. 
4.6), a complete reversal of the trend observed for the 
hydroxy compounds. Comparing the feeding responses of the 
larvae to p-hydroxybenzaldehyde and to its biogenetic 
analogues tested in this study showed that p-hydroxy-
benzaldehyde was the most potent feeding stimulant whilst 
its methoxy derivative was the weakest. These findings 
suggest that p-hydroxybenzaldehyde probably plays a 
prominent role in larval discrimination between potential 
foods. Bioassays of the cinnamic acids present in the ethyl 
acetate and methanol extracts showed that p-coumaric acid 
was non-stimulatory at all the doses tested, whilst ferulic 
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and caffeic acids gave similar feeding rates (Fig. 4.7), 
both being effective at very low doses. Caffeic and ferulic 
acids differ from p-coumaric acid in having an extra oxygen 
function at the 3-position in the benzene ring which may 
thus be important for the activities of this class of 
phenolic compounds. 
Comparison of hydroxybenzoic acid and its analogues 
with cinnamic acids showed that the former were generally 
more stimulatory than the latter. Thus p-hydroxybenzoic 
acid (4-hydroxybenzoic acid) was more stimulatory to larvae 
than p-coumaric acid (4-hydroxycinnamic acid); vanillic acid 
(4-hydroxy-3-methoxybenzoic acid) was more stimulatory than 
ferulic acid (4-hydroxy-3-methoxycinnamic acid); and 
protocatechuic acid (3,4-dihydroxybenzoic acid) was also 
more stimulatory than caffeic acid (3,4-dihydroxy-cinnamic 
acid). Of particular interest was the complete lack of 
response to gentisic acid (2,S-dihydroxybenzoic acid), a 
known phenolic constituent of sorghum seedlings (Woodhead 
and Cooper-Driver, 1979). This shows that the positions of 
the functional moieties in the benzene ring are crucial for 
the activity of the compound. 
Bioassays were also carried out on chlorogenic acid, a 
common plant constituent (Sondheimer, 1964). Its role in 
host plant selection by some insects has been reported. 
Kato and Yamada (1966) reported that it served as a growth 
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factor for silkworm larvae, and Hsiao and Fraenkel (19.68) 
reported that it was a feeding stimulant in potato leaves 
for the colorado potato beetle L. decemiineata. Chawla et 
ai., (1974) found that incorporation of chlorogenic acid in 
an artificial diet improved the growth of the potato aphid 
Macrosiphum euphorbiae. Matsuda and Senbo (1986) reported 
that it deterred feeding by Lochmaeae capreae cribata but 
was stimulatory to Gastrophysa atrocyanea, both of which 
feed on salicaceous plants. In the present study, the 
compound stimulated larval feeding but became less 
stimulatory at higher doses (Fig. 4.7). It was a better 
stimulant than its degradative products, quinic and caffeic 
acids (Fig. 4.7) both of which elicited weak stimulatory 
responses from the larvae at very low doses, although quinic 
acid was a better stimulant than caffeic acid. 
Although only a few phenolic compounds were 
investigated in this study, some tentative inferences can be 
made from the results. The results suggest that there are 
chemoreceptors in the mouthparts of the third-ins tar larvae 
of c. parteiius which perceive different classes of phenolic 
compounds and their derivatives. This may suggest the 
involvement of generalised receptors. However, the 
different structural requirements for the activities of 
benzoic and cinnamic compounds suggest that different groups 
of receptors may be present. Detailed sensory physiological 
studies may help to shed some light on the question. 
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Sugars have been demonstrated as strong phagostimulants 
in a large number of insect species (Thorsteinson, 1960; 
Dethier, 1966; Schoonhoven, 1968; Sutherland, 1977; 
Staedler, 1983). In this study, sucrose, fructose and 
glucose stimulated the feeding of the third-ins tar larvae of 
c. partellus. Sutherland (1977) reported that these sugars 
were ubiquitous nutrient chemicals which function as 
phagostimulants for general plant feeders. The data for C. 
partellus (Fig. 4.5) showed that sucrose was most 
stimulatory and it exhibited a more linear dose/response 
relationship than the other sugars which were tested. These 
results are in agreement with previous reports on the strong 
stimulatory activity of sucrose to a wide variety of insects 
feeding on a diverse range of plants (Dethier, 1966; Hsiao 
and Fraenkel, 1968; Hsiao, 1969; Peacock and Fisk, 1970; 
Sutherland, 1971; 1977; Doss and Shanks, 1984; Ladd, 1986; 
Shanks and Doss, 1987). Glucose and fructose which are 
hexose sugars gave similar feeding rates in our study; but 
xylose, which is a pentose sugar was non-stimulatory at all 
the doses tested. In a recent study, Ladd (1986) found that 
xylose, which is generally unknown as an insect feeding 
stimulant (Chippendale, 1978), elicited a weak stimulatory 
feeding response from the Japanese beetle, Popillia japonica 
Newman, but noted that his results were not conclusive. Our 
results are in agreement with previous reports on the 
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activities of pentose and hexose sugars (Chippendale, 1978; 
Ladd, 1986) in that hexose sugars are more stimulatory to 
phytophagous insects than pentose sugars. In summation, 
apart from phenolic receptors, it appears that the larvae 
have receptors which perceive sugars, but they seem to be 
more receptive to sucrose than to the other sugars which 
were identified in the extracts. 
The feeding tests on blends of some of these 
phagostimulatory compounds revealed interesting results. 
The stimulatory activity of a blend of p-hydroxybenzaldehyde 
and p-hydroxybenzoic acid in a proportion occurring in the 
crude ethyl acetate extract was virtually due to the 
activity of p-hydroxybenzaldehyde suggesting no synergism 
between the two compounds (Fig. 4.8). It appears that the 
absolute amount of p-hydroxybenzaldehyde in the plant may 
playa significant role in the feeding of the larvae. It 
would be interesting to assay a blend of all the phenolic 
components in the proportion they are found occurring in the 
plant to elucidate more precisely the relative importance of 
phenolics in the feeding of the larvae of this insect. 
Bioassays of a blend of glucose and fructose in equal 
ratio (Fig. 4.15) gave a response which appeared to be a 
simple addition of the activities of the two hexoses as 
there was no evidence of synergistic effect. The 
stimulatory activity of this blend was also significantly 
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less than that of sucrose showing clearly that the activity 
of the dissacharide is due to its total structure and not to 
a summation of its component moieties. The results of the 
bioassays of a blend of p-hydroxybenzaldehyde and sucrose, 
on the other hand, indicated synergism between the two 
compounds since the dose/response plot of a summation of the 
activities of the two compounds had a distinctly lower slope 
than that of the experimental one (Fig. 4.16). It would be 
interesting to extend this investigation to studying the 
effect of blending all the phenols and sugars identified in 
sorghum extracts to determine the relative importance of 
each constituent in the total blend. Nevertheless, the 
results obtained so far support the view by Dethier (1982) 
that host acceptance by phytophagous insects is controlled 
by both non-nutritional secondary plant compounds (token 
stimuli), and nutritional chemicals. 
Chromatographic analyses of the levels of the major 
phenolic' and sugar components in the extracts of the whorls 
of the two cultivars showed, as expected, that their amounts 
were dependent on the nature of the cultivar. The results 
of these analyses showed that there was less p-hydroxy-
benzaldehyde and p-hydroxybenzoic acid in the resistant 
cultivar IS 2205 than the susceptible cultivar IS 18363 for 
both the 3 and 6 week old plants (Tables 4.5 and . 4.6) and 
these findings are consistent with the greater palatability 
of the latter cultivar. However, for both cultivars the 
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total amounts of the phenols in the older whorls were 
signif~cantly higher than those in the younger ones. 
Analyses of the sugar components showed that sucrose formed 
about 37-44% of the sugar fraction whilst glucose, fructose 
and xylose formed between 15-21%, 13-19%, and 1-5% 
respectively (Table 4.8). The total nonsugar level in these 
fractions ranged between 14-24%. Comparing the total sugar 
levels in the whorls of the 3 week old plants of the two 
cultivars showed , as expected, that there was less sugar in 
the resistant than the susceptible cultivar (Table 4.9). 
However, the sugar levels in the whorls of 6 week old plants 
of the two cultivars were approximately the same, and~ 
again, significantly higher than the levels found for the 3 
week old plants. 
The higher levels of both phenolics and sugars in the 
older sorghum plants of both IS 18363 and IS 2205 were quite 
unexpected and appeared inconsistent with their lower 
preference by the larvae in the field (Alghali, 1985; 
Saxena, 1985b). Since our crude extracts of 3 week old 
seedlings were also more stimulatory than those of 6 week 
old seedlings of both cultivars, any biophysical differences 
between the younger and older seedlings cannot fully account 
for the difference in preference for the two groups and 
point toward a chemically mediated difference. We propose 
the presence of deterrent compound(s) which increase in 
proportion with the age of the seedlings. The most likely 
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candidate which fits this proposition is the "non-
stimulatory" highly polar component J-l present in both the 
ethyl acetate and methanol extracts. The exact roles of 
this component and other non-stimulatory components 
identified in the extracts in the plant need to be 
reinvestigated further with a modified feeding bioassay. It 
is suggested that these components should be applied to 
sucrose treated discs and assayed for any reduction in 
larval feeding response in both choice and no choice 
situations. It is noteworthy to mention that in this study, 
the complete lack of response by larvae to some of the 
constituents of the sorghum plant support the view by 
Dethier (1982) that host acceptance by phytophagous insects 
is mediated by positive and negative stimuli from plants and 
by the insect's physiological condition. 
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CHAPTER 6 
CONCLUSIONS AND SUGGESTIONS 
FOR FURTHER STUDIES 
It is apparent from this study that feeding behaviour 
of the third-instar larvae of C. partellus is mediated by a 
complex blend of chemicals. These include phenolics, 
sugars, hexane extractables and components which may be 
present in the plant as deterrent compounds. The major 
phenolic compounds were p-hydroxybenzaldehyde and p-hydroxy-
benzoic acid. Ferulic, p-coumaric, and caffeic acids were 
present in minor quantities. All, but p-coumaric acid 
stimulated larval feeding. The major sugar components were 
sucrose, glucose and fructose, xylose being in minor 
amounts. All but xylose were stimulatory to the feeding of 
the larvae. Some components in the extracts could only be 
characterised partially. These include J-l, a highly polar 
high molecular weight carboxylic acid and J-2, a mixture 
composed of related phenolic derivatives. J-l might be the 
deterrent component implicated in the present study. J-2 
appeared to stimulate larval feeding and broke down to give 
p-hydroxybenzaldehyde, among other products. Further study 
to investigate the nature, biological activities and 
degradative studies of the J-2 components would prove useful 
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to the understanding of the feeding behaviour of the larvae 
of this insect. 
It became apparent in this study that the bioassays 
using cellulose acetate were more effective for monitoring 
phagostimulation rather than phagodeterrency of sorghum 
constituents. Thus, it would be interesting to investigate 
further, with a modified bioassay the precise roles of J-1, 
p-coumaric acid, xylose and constituents in the least 
stimulatory hexane extracts (when identified) on the feeding 
of the larvae. This may provide information on the nature 
of deterrency in the plant, and additional insight into the 
bases of food selection by the larvae. However, it is hoped 
that the present work has laid down the groundwork for a 
detailed study of the complex mechanisms underlying the 
chemical basis of food discriminative behaviour of the 
larvae of this insect. 
Blend effects could not be fully investigated in this 
study. However, the study demonstrated that there was an 
additive or synergistic effect between some constituents of 
the plant as a result of reblending of fractions obtained 
from crude extracts. No synergism was found for a blend of 
p-hydroxybenzaldehyde and p-hydroxybenzoic acid. On the 
other hand, bioassays of a blend of p-hydroxybenzaldehyde 
and sucrose clearly showed synergism between the two 
compounds. A blend of glucose and fructose in equal ratio 
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showed no synergism but an effect which appeared to be 
additive. For a thorough understanding of blend effects on 
the feeding of the larvae the following combinations need to 
be assayed: p-hydroxybenzaldehyde with the other phenolic 
constituents of the plant; sucrose with the other sugar 
components; phenolics and sugars; phagostimulatory 
components in the hexane extract (when identified) combined 
with phenolics and sugars; and lastly all the stimulant 
components with any deterrent compounds identified. 
The difference between the two cultivars studied IS 
18363 and IS 2205 appears to be quantitative rather than 
qualitative, with the more potent blends being present in 
the more susceptible cultivar IS 18363. Larval preference 
for extracts of the whorls of IS 18363 corresponded with the 
preference for this cultivar in the field and the preferred 
age for feeding. The levels of phagostimulants per fresh 
weight of whorl increased with plant age but this was not 
directly correlated with larval feeding response to extracts 
containing these phagostimulants. Thus, a study relating 
the levels of the stimulants (phenolics and sugars) to the 
surface areas and dry weights of the plants at the two 
different growth stages which were studied will throw more 
light on this question. In addition, it would be 
interesting to investigate the relationship, if any, between 
phagodeterrency and the age of the sorghum plant, and to 
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extend full allelochemical studies to other sorghum 
cultivars. 
The process of host selection by a phytophagous insect 
is complex and involves physical and morphological factors 
and complex biochemical interactions between the plant and 
the insect. Therefore it will be interesting and 
enlightening to be able to match this work with detailed 
behavioural and electrophysiological studies to determine 
the precise roles played by the different stimulant 
components in the feeding behaviour pattern of the larvae of 
the insect. In addition, it will also be interesting to 
investigate whether the different levels of allelochemicals 
which appear to account for the different levels of 
susceptibility of these two cultivars in the field are 
correlated with those physical characteristics of the plants 
which are associated with pest performance and avoidance. 
The larva of this insect goes through five larval 
ins tars before pupation. Therefore it will be useful to 
investigate whether these phagostimulatory compounds affect 
the feeding of the first, second, fourth and fifth ins tar 
larvae of the insect, and particularly to investigate if 
there are any chemical basis for the shift in the feeding of 
the later ins tars from the leaves to the stem. 
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It has been shown in previous studies that some plant 
chemicals which stimulate larval feeding also stimulate egg-
laying by the adult gravid female of the insect (Hedin et 
al., 1977) Therefore investigations to determine whether 
these stimulants affect oviposition of the adult gravid 
female of this insect need to be undertaken. This will 
provide additional knowledge for the understanding of the 
colonisation of the plant by the insect. 
Although further investigations are needed to 
understand fully the ecochemistry of this insect, several 
potential applications of these phagostimulants in the 
management of this pest may be mentioned. Firstly, they may 
be used as markers in selective screening and breeding 
programmes. Secondly, they may be combined with other 
semiochemicals of the pest in the development of selective 
control technologies. For example, host plant attractants 
might initially be used to attract adult gravid females of 
the insect into traps containing an artificial substrate 
impregnated with oviposition stimulants to stimulate egg-
laying by the female. When the eggs hatch, the larvae can 
be reared on an artificial diet impregnated with these 
feeding stimulants mixed with suitable insect growth 
regulator (IGR) to disturb the normal growth and development 
of the insect. By use of suitable IGR it may be possible to 
have a system which continously produces sterile males and 
females. Adults which emerge from these traps may be 
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released back into the environment where they would mate 
with normal members. The system could thus provide a basis 
for controlling the population of the insect. 
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~ 
Larvae feeding on disc loaded with sorghum extract in a 
choice bioassay (right). Control disc (left) with only 
shot-holes. 
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Adams, C.M. and Bernays, E.A. (1978) The effect of 
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Albert, P.J. and Jerrett, P.A. (1981) Feeding 
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S. (1982) Feeding responses of Eastern spruce 
budworm larvae to sucrose and other carbohydrates. 
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Alghali, A.H. (1985) Insect-Host plant relationships. 
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(Lepidoptera:pyralidae) and its principal host, 
sorghum. Insect SCi App]iC, 6, 315-322. 
Ascher, K.R.S., Hemny, H.E., Eliyahu, H., Kirson, I., 
Abraham, A. and Glotter, E. (1980) Insect 
antifeedant properties of withanolide and related 
steroids from Solanaceae. Experientia, 36, 998-
999. 
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178 
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Glossary of Special Terms 
Allelochemical: A chemical released by one 
species which affects another 
species. 
Allomone: A molecule, ion or free radical 
which is operative as an 
interspecific messenger in chemical 
ecology, and is adaptively 
advantageous to the emitter 
(releaser) of the substance. 
Antifeedant activities: Treatment effects which reduce or 
prevent . feeding. 
Antifeedant: A substance which prevents, or 
reduces, feeding. 
Assay substrate: The material on which, or in which, 
the test substance is presented in 
the bioassay. 
Chemical profile: Species of chemicals contained. 
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Bioassay in which the insect is 
Choice test: 
given a choice between two or more 
treatments. 
Cultivar: Variety of plant. 
Deterrent: A chemical which discourages 
(deters) a given organismal 
behaviour (e.g., feeding). 
Drinking: Ingestion of a liquid. 
Feeding response: Increase or decrease in feeding in 
a bioassay, attributable to 
treatment. 
Feeding: The act of eating. 
Herbivore: An animal that eats plant tissue. 
Host range: Grouping of species or varieties 
which an insect will use for food 
or shelter. 
Host: A live organism that serves as a 
food substance or shelter. 
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Inducible chemical: A chemical produced in an organism 
(e.g., plant) when it is placed 
under environmental stress. 
Inhibition: Reduction of behavioural activity 
level (e.g. feeding). 
Instar: Stage which larva assumes between 
moults. 
Kairomone: A molecule, ion or free radical 
which is operative as an 
interspecific messenger in chemical 
ecology, and is adaptively 
advantageous to the perceiving 
organism. 
Monophagy: Feeding on one species, or a very 
few spe9ies, in one genus. 
Neutral substrate: Substrate which does not influence 
feeding in a bioassay. 
No-choice test: Bioassay in which the insect is 
exposed to only one candidate 
substrate for feeding. 
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Non-host: A live organism that is not serving 
as a food substrate or shelter for 
the insect of interest. 
Oligophagy: Feeding on several species in a few 
genera in one or a very few 
families. 
perception: Direct acquaintance (recognition) 
with anything through the senses. 
Receptor: Macromolecular entity which binds 
(complexes with) a chemical, ion or 
free radical ligand (messenger). 
Repellent: A volatile substance which elicits 
an avoidance behaviour by an 
organism (e.g., insect). 
Semiochemical: Chemicals involved in the 
interaction between living 
organisms. 
Specialist species: A species that practices monophagy. 
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Stimulant: A chemical which alters the 
behaviour of an organism through 
its nervous system. 
Substrate: Any substance ingested in 
conjunction with the act of 
feeding.