THE IDENTIFICATION AND USE OF SEMIOCHEMICALS FOR THE

CONTROL OF THE MAIZE WEEVIL, SITOPHILUS ZEAMAIS

(MOTSCHULSKY) IN NIGERIA.

DONALD A. UKEH

B. Agric. (Hons.) Crop Science M. Sc Crop Protection (Entomology) University of Calabar, Nigeria.

A thesis presented for the degree of Doctor of Philosophy at the University of

Aberdeen, United Kingdom.

2008

In collaboration with the Biological Chemistry Department, Centre for

Sustainable Pest and Disease Management, Rothamsted Research, Harpenden,

United Kingdom.

School of Biological Sciences Biological Chemistry Department University of Aberdeen Rothamsted ResearchAberdeen Harpenden, HertfordshireAB24 2TZ AL5 2JQUnited Kingdom United Kingdom

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DECLARATION

I hereby declare that this thesis has been composed by myself, and has not been

accepted in any previous application for a degree. The work of this thesis is a record

of my work; any collaborative work has been specifically acknowledged as have all

sources of information.

Donald A. Ukeh

2008.

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DEDICATION

To the loving memory of my beloved father, Ukeh Akem (1935-1989); and mother,

Cecilia Bezaunungieye Ukeh-Akem (1937-2008), both of whom inspired me to

become the person I am today.

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ABSTRACT

The maize weevil, Sitophilus zeamais Motschulsky, is the most important insect pest

of stored maize causing considerable damage of economic proportions in the tropics.

In Nigeria, maize production is undertaken by resource-poor farmers with little or no

control measures against S. zeamais during storage. In addition, on a large scale,

there are public concerns over the continuous application of synthetic pesticides in

stored products protection. Both have prompted the search for safer, cheap, easily

biodegradable and readily available plant materials that will not contaminate food

products in acting as grain protectants in small-scale storage systems.

Studies were carried out to evaluate the repellent properties of the seeds of alligator

pepper, Aframomum melegueta and Black pepper, Piper guineense, and ginger,

Zingiber officinale rhizomes against S. zeamais. In 4-way olfactometry bioassays, S.

zeamais adults showed strong attraction to maize and wheat seed volatiles but were

significantly repelled by odours emanating from the seeds of A. melegueta, P.

guineense and Z. officinale rhizomes. In field trials crushed A. melegueta seeds and Z.

officinale rhizomes, significantly repelled S. zeamais from traditional maize granaries

with treated maize cobs giving higher germination than untreated cobs. Laboratory

oviposition studies showed that A. melegueta and Z. officinale powders caused

significant adult mortality and oviposition deterrence against S. zeamais resulting in a

reduction in F1 progeny emergence. Olfactometer bioassays also confirmed that

vacuum distilled A. melegueta and Z. officinale extracts and oleoresins were repellent

towards adult S. zeamais when tested individually, and in combination with maize

grains. Bioassay-guided liquid chromatography of the distillates showed that fractions

containing polar compounds accounted for the repellent activity. Coupled gas

chromatography-mass spectrometry (GC-MS), followed by GC peak enhancement

and enantioselective GC using authentic compounds, identified 3 major compounds in

the behaviourally active A. melegueta fraction to be (S)-2-heptanol, (S)-2-heptyl

acetate and (R)-linalool in the ratio 1:6:3. Z. officinale had 1,8-cineole, neral and

geranial in the ratio of 5.48:1:2.13. The identification of these behaviourally active

compounds provide a scientific basis for the observed repellent properties of A.

melegueta and Z. officinale extracts, and demonstrate the potential for their

development in stored-product protection at the small-scale farmer level in Africa.

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TABLE OF CONTENTS

Declaration ……………………………………………………………………… i

Dedication ………………………………………………………………………. ii

Abstract ………………………………………………………………………… iii

Acknowledgements ……………………………………………………………. vi

CHAPTER 1 GENERAL INTRODUCTION

1.1 Introduction ………………………………………………………………. 2

1.2 General biology and life cycle of Sitophilus zeamais …………………….. 3

1.3 Economic importance of S. zeamais ……………………………………… 5

1.4 Sources of infestation ……………………………………………………… 8

1.5 Control of S. zeamais ……………………………………………………... 10

1.5.1 Sampling and trapping………………………………………………. 11

1.5.2 Manipulation of Storage conditions…………………………………. 13

1.5.3 Contact insecticides …………………………………………………. 15

1.5.4 Synthetic fumigants………………………………………………….. 16

1.5.5 Plant products………………………………………………………… 18

1.6 Conclusions ………………………………………………………………… 20

1.7 Study aim and objectives …………………………………………………. 22

CHAPTER 2 BEHAVIOURAL RESPONSES OF Sitophilus zeamais TO HOST AND NON-HOST PLANT VOLATILES2.1 Introduction………………………………………………………………. 24

2.2 Materials and Methods………………………………………………….. 28

2.2.1 Insect culture………………………………………………………... 28

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2.2.2 Plant materials Collection and Preservation…………………………… 31

2.2.3 Bioassay method…………………………………………………… 33

2.2.4 Statistical analysis…………………………………………………. 38

2.3 Results…………………………………………………………………… 38

2.3.1 Host plant odours………………………………………………….. 38

2.3.2 Non-host plant odours……………………………………………... 42

2.3.3 Host plus Non-host plant odours…………………………………... 45

2.3.4 Dose response bioassays…………………………………………... 54

2.4 Discussion……………………………………………………………….. 66

2.5 Conclusions……………………………………………………………… 71

CHAPTER 3 EXTRACTION AND IDENTIFICATION OF BIOLOGICALLY ACTIVE COMPONENTS FROM THE SEEDS OF Aframomum melegueta AND Zingiber officinale RHIZOME

3.1 Introduction …………………………………………………………….. 73

3.2 Materials and Methods…………………………………………………. 75

3.2.1 Plant materials collection and preservation……………………….. 75

3.2.2 Preparation of essential oils………………………………………. 75

3.2.3 Preparation of Oleoresins…………………………………………. 76

3.2.4 Liquid Chromatography…………………………………………… 78

3.2.5 Gas Chromatography (GC) analysis of vacuum distillates……….. 78

3.2.6 Coupled gas chromatography-mass spectrometry (GC-MS) …….. 79

3.2.7 Preparation of synthetic blends …………………………………… 80

3.3 Results…………………………………………………………………… 82

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3.3.1 Chemical constituents of vacuum distilled essential oils of

A. melegueta and Z. officinale.................................................................... 82

3.3.2 Chemical composition of A. melegueta and Z. officinale Florisil®

diethyl ether essential oil fraction………………………………………… 85

3.4 Discussion………………………………………………………………… 88

3.5 Conclusions………………………………………………………………. 91

CHAPTER 4 BIOACTIVITY OF Aframomum melegueta AND Zingiber officinale EXTRACTS AND SINGLE COMPONENTS AGAINST Sitophilus zeamais

4.1 Introduction……………………………………………………………… 94

4.2 Materials and Methods…………………………………………………. 95

4.2.1 Maize weevils……………………………………………………… 95

4.2.2 Repellency bioassays………………………………………………. 96

4.2.3 Data analysis……………………………………………………….. 96

4.3 RESULTS……………………………………………………………. 97

4.3.1 Vacuum distilled essential oils of A. melegueta and Z. officinale….. 97

4.3.2 Vacuum distilled hexane and diethyl ether fractions of

A. melegueta and Z. officinale essential oils …………………………….. 102

4.3.3 Synthetic blends of vacuum distilled diethyl ether fractions of

A. melegueta and Z. officinale essential oils……………………………… 106

4.3.4 Repellent activity of A. melegueta and Z. officinale oleoresins……. 112

4.3.5 Olfactory responses to A. melegueta and Z. officinale

chemical constituents of essential oils…………………………………… 117

4.3.6 Percentage repellent activity……………………………………….. 19

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4.4 Discussion……………………………………………………………….. 122

4.5 Conclusions……………………………………………………………… 126

CHAPTER 5 FIELD REPELLENT ACTIVITY AND OVIPOSITION DETERRENT EFFECTS OF Aframomum melegueta AND Zingiber officinale AGAINST S. zeamais IN STORED MAIZE 5.1 Introduction……………………………………………………………….. 128

5.2 Fieldwork Materials and Methods………………………………………. 130

5.2.1 Site description and construction of storage barns in Nigeria……….. 130

5.2.2 Seeding the environment…………………………………………….. 134

5.2.3 Repellency trials……………………………………………………. 135

5.2.4 Fieldwork maize seeds germination percentage……………………. 136

5.2.5 Data analysis……………………………………………………….. 139

5.3 Laboratory oviposition deterrence experiments……………………… 139

5.3.1 Materials and Methods…………………………………………….. 139

5.3.2 Data analysis……………………………………………………..... 141

5.4 Results……………………………………………………………………. 141

5.4.1 Repellent effect of A. melegueta and Z. officinale powders against

S. zeamais in storage granaries in Nigeria…………………………………. 141

5.4.2 Effects of Z. officinale and A. melegueta powders on S. zeamais

oviposition and adult emergence in laboratory tests……………………..... 144

5.5 Discussion………………………………………………………………..... 149

5.6 Conclusions……………………………………………………………….. 152

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CHAPTER 6 GENERAL DISCUSSION

6.1 Behavioural responses to host and non-host plant volatiles…………. 154

6.2 Bioactivity of A. melegueta and Z. officinale essential oils and their constituents against S. zeamais………………………………. 156

6.3 Field repellent activity and laboratory oviposition deterrence of A. melegueta and Z. officinale powders against S. zeamais……………… 159

6.4 Potential for application by small-scale farmers………………………. 162

6.5 Conclusions………………………………………………………………. 165

REFERENCES……………………………………………………………….. 167

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ACKNOWLEDGEMENTS

I must first express my utmost gratitude to the Almighty God for His mercy and

Blessing upon me and my family all this while.

I would like to thank my supervisors Professor A. Jennifer Mordue and Dr Alan S.

Bowman for their useful comments, advice and support throughout this study. Thanks

also to Professor John A. Pickett and Dr Michael A. Birkett of Biological Chemistry

Department, Rothamsted Research, Harpenden, for their advice and assistance with

gas chromatography (GC), coupled gas chromatography-mass spectrometry (GC-MS)

and chemistry. Thank you to Professor Bill Mordue for your kind support and

assistance to my young family in the UK. Thanks to Professor Ivara Esu for

nominating me for the Commonwealth Scholarships. My thanks also go to Dr. Alex

Douglas for statistical advice.

Thanks to the Commonwealth Scholarship Commission in the United Kingdom for

funding this study, and to British Council Manchester for the administration of the

funds and other welfare matters. I am very grateful to Rod Weaver and Maureen

Wakefield of Central Science Laboratory, Sand Hutton, York, United Kingdom for

the supply of Sitophilus zeamais used for this research. Thanks to Jamie Sutherland of

AgriSense-BCS Limited, United Kingdom for the supply of delta traps with sticky

bases complete with metal hangers used during field trials in Nigeria.

Thanks to the staff of Biological Science Department, Rothamsted Research,

particularly Toby Bruce, James Logan, Sarah Dewhirst, Barry Pye, Christine

Woodcock, Lynda Ireland, Ben Webster, Lesley Smart and to Duncan, Sarah, Janet,

Amy, Jan, Shaikha, Osei, James, Nneoyi and Anders in the University of Aberdeen.

My love goes to my wife Monica, my children; Elizabeth, Gospel and Donald Jr. for

their patience, help and support over the last three years. Thanks to my siblings Grace,

Justina, Dorathy, Lucy, Mark, Veronica, Jude, Joseph and John. Finally, thanks to my

friends and colleagues in Nigeria especially Idorenyin, Peter, Osai, Umoetok, Oko,

Amalu, Obi Abang, Uwah, Uko, Binang, Shiyam, Nwagwu, Iwo, Ferdinand,

Emmanuel, Vincent, Jerry, Okeme among others for their support and encouragement.

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CHAPTER 1:

GENERAL INTRODUCTION

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1.1 INTRODUCTION

Since the time of the Pharaohs, human beings have stockpiled foodstuffs in times of

plenty for use during leaner periods or for sale at a later date. Equally ancient are the

stored-product insects that infested the Pharaohs’ food storage facilities. Some of the

same pests that infest foods in the late 20th century have been found in the tombs of

the pharaohs (Levinson and Levinson, 1990; 1998). It is thought that between 5000

and 10,000 BC, human society commenced settled agriculture and began to produce

and store large quantities of dried organic materials such as grains, fibres and skins. A

vast new resource then became available which attracted a select band of insects that

feed on dry material of animal and plant origin (Rees, 2004). Before this, storage

insects may have evolved to exploit natural accumulations of seeds in dry, sheltered

habitats; these included those blown into caves, cracks and crevices by the wind or

found in the nests and food hoards of other animals, including birds, rodents and

social insects (Cox and Collins, 2002). More than 600 species of beetles and 70 of

moths among the insects, 355 species of mites, 40 species of rodents and 150 species

of fungi have been reported to be associated with various stored products (Rajendran,

2002). Stored product insects share biological characteristics that are adaptive for

survival under conditions of food handling and storage areas. These characteristics

include; (1) a wide range of tolerance to different environmental conditions such as

temperature and relative humidity; (2) a greater range of food habits than most other

insects; (3) more or less continuous reproductive activity spanning periods of up to

three years; (4) an ability to survive long periods without food; and (5) a capacity to

build up large populations undetected, due to their relatively small size (Throne, 1994;

Cox and Collins, 2002; Rees, 2004).

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The control of the maize weevil, Sitophilus zeamais Motschulsky (Coleoptera:

Curculionidae) is the subject of investigation in this thesis. This introductory chapter

describes the general biology and life cycle, economic importance, sources of

infestation of S. zeamais. Also, an overview of the current control methods of the

weevil with particular reference to West Africa is reported.

1.2 GENERAL BIOLOGY AND LIFE CYCLE OF Sitophilus zeamais

The maize weevil, S. zeamais, is the most important insect pest of stored maize in

tropical and sub-tropical countries. The adults are small brown to black snout weevils,

about 3-6mm long. They are long-lived with a life span of several months to one year.

Females select a spot on the grain surface then chew small holes into grain kernels,

and use their ovipositors to insert one egg per hole. Each hole is then plugged with a

gluey secretion usually referred to as an “egg-plug” (Howe, 1952). Eggs are laid

throughout most of the adult life, but the majority of the eggs are laid within the first 6

weeks, and more than 150 eggs may be laid per female. Eggs are laid at temperatures

between 15 and 35 ºC with an optimum of about 25 °C, and at grain moisture contents

over 10% but not above 32 %. The incubation period of the egg is between 2-6 days at

25 ºC. Larvae are cannibalistic and larger ones may prey upon less developed

individuals should they meet (Rees, 2004). There are four larval instars all of which

bore through the cavity hollowed out in the seed and develop within the grain.

Pupation takes place within the grain, and the newly developed adult may spend

several days within the cavity before chewing its way out leaving a large

characteristic emergence hole called “exit hole”. The total development periods range

from about 35 days under optimal conditions to over 110 days in unfavourable

conditions. The actual duration of life cycle also depends upon the type and quality of

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grain that is infested (Haines, 1991). An index of environmental suitability indicated

that between 25 - 30 C and 65 -75 % temperature and relative humidity, respectively,

are the optimal environmental conditions for growth of maize weevil populations on

stored maize (Throne, 1994; Bekele et al., 1995). Mating in S. zeamais does not occur

before the adults are 3 d old (Walgenbach and Burkholder, 1987) but the insect

continues to feed on grain throughout its life span.

LIFE CYCLE OF S. zeamais

ADULT

PUPAars.usda.gov/npa/gmprc/bru

LARVAE

ars.usda.gov/npa/gmprc/bru

Eggs laid inside grains

Figure 1.1 General life cycle of Sitophilus zeamais.

The food preferences of S. zeamais are maize, rice, wheat and dry cassava. On maize

the weevils infest ripening standing crops immediately prior to harvest and in storage.

They are good fliers and dominant storage pests of these crops in tropical subsistence

agricultural systems. Within grain bulks, S. zeamais populations often aggregate, but

when such aggregations run out of food, adults will disperse, at which stage the

infestation may become visible. However, by this stage, significant damage to the

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quality and quantity of the stored product has been done (Stoll, 2000; Rees, 2004).

Aggregation is made possible by the secretion of a male-released aggregation

pheromone, which attracts members of both sexes resulting in the formation of leks.

The major component of the male aggregation pheromone is sitophinone {(5R, 4S)-5-

hydroxy-4-methyl-3-heptanone} (Walgenbach et al., 1983).

OOH

Figure 1.2 Sitophinone

Pheromones from stored product insects are generally volatile, low molecular weight

organic compounds of various structures (Walgenbach et al., 1983; Phillips, 1997).

1.3 ECONOMIC IMPORTANCE OF S. zeamais

After harvest, growers generally store the products for various purposes including

uniform sully of food and feed throughout the year, future planting and sale at a later

date when product prices might have increased to make a profit (Demissie et al.,

2008). However, during this post-harvest period, stored crop products are usually

liable to infestation and depreciation by various stored-product insect pests. There are

various estimates of losses caused by storage insect pests in the literature. Appert

(1987) reported total post-harvest crop losses of 40% in the hot, humid regions and

more than 10% in dry regions of the world. Other estimates of crop losses have been

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given as 10%-20% world-wide and 25%-40% in tropical regions (Hill and Waller,

1990; Larry, 2000). The maize weevil is one of the most serious pests of farm-stored

grain and basket or bag-stored grain in stores under tropical and sub-tropical

conditions. If left unchecked, infestations of S. zeamais can result in devastating

damage to stored corn. Annual post-harvest losses of over 96 million tonnes of maize

grains by Sitophilus species all over the world have been destroyed (FAO, 1985). The

weevil causes damage to stored maize grain by boring the grains and eating the inner

part which reduces maize weight and quality in terms of consumption and

germination (Adda et al., 2002). Damage caused by S. zeamais on stored cereals can

be extremely high. It is reported that up to 18.3% weight loss occurred due to S.

zeamais infestation when single maize kernels were exposed to ovipositing adults and

kept at 27 ºC and 70% relative humidity for only 37 days (Adams, 1976; Adane et al.,

1996). S. zeamais infestation has also resulted in significant reduction in the viability

of the grains (Okiwelu et al., 1987). Post-harvest crop losses due to storage pests such

as S. zeamais have continued to persist and pose major problems to food security in

Africa (Markham et al., 1994). These problems have increased as traditional crop

varieties have been replaced by improved, high-yielding varieties with shorter growth

cycles but which are generally more susceptible to insect damage. In West Africa for

example, losses caused by storage pests like S. zeamais and the Angoumois grain

moth, Sitotroga cerealella (Olivier), constitute a major constraint to increasing maize

production through the introduction of improved varieties (Markham et al., 1994). In

Nigeria, the loss of maize grains during storage due to insect pests like S. zeamais has

long been a serious problem to the farmers. Inputs in the form of human power and

finances invested in the production of the crop are wasted (Umoetok and Ukeh, 2004;

Law-Ogbomo and Enobakhare, 2006). In Ghana, out of an estimated total annual

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harvest of 250, 000 - 300, 000 tonnes of maize, about 20% was lost to storage pests

like S. zeamais (Obeng-Ofori and Amiteye, 2005). Post-harvest losses caused by

insect infestation and spoilage reduce the availability of maize in Cameroon

throughout the year, and for the western highlands of Cameroon, losses in stored

maize of 12-44% due to S. zeamais during the first 6 months of storage has been

reported (Bouda et al., 2001). Average dry weight losses of farm-stored maize for a

storage period of 6 months caused by S. zeamais and Prostephanus truncatus (Horn.)

(Coleoptera: Bostrichidae) in Togo has been estimated to range between 7% and 30%

(Pantenius, 1988; Richter et al., 1997). In Ethiopia in general, post-harvest losses

caused by S. zeamais ranging from 20-30% are common, and studies in the Bako

areas have shown grain damage levels up to 100% in some samples from farm stores

after 6-8 months (Emana, 1999; Demissie et al., 2008). Insect contaminants such as

excreta (uric acid), exuviate (cast skins) and dead bodies, webbing, and secretions in

food commodities pose a quality-control problem for food industries. Processing and

end-use qualities of food commodities are also affected by insect infestation, as are

cash value and marketability of products. The activity of stored product pests may be

associated with weight losses by direct damage, lowering the nutritional and

economic value of the crop and presence of allergens (Arlian, 2002) or toxinogenic

fungi (Hubert et al., 2002), in the infested stored grain. Food infecting fungi have

been reported to produce many metabolites such as mycotoxin, aflatoxin B1, a

carcinogenic metabolite of Aspergillus flavus which affects the liver (Wild and Hall,

2000), ochratoxin A and citrinin produced by Penicillium verrucosum which are

known to have nephrotoxic effects (Hubert et al., 2004).

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1.4 SOURCES OF INFESTATION

In developing countries like Nigeria, inadequate storage facilities and lack of

economic means constrained resource-poor farmers who form the bulk of food

production, to store their products using traditional storage granaries. Maize, Zea

mays L. (Poaceae) is grown in March/April at the on-set of rains and harvested during

August and September at the state of ripeness. Maize is stored shelled or unshelled in

baskets, jute bags or earthen ware and placed in barns or cribs constructed with wood

or bamboos within the homestead. Generally, the storage period ranges from 4 to 6

months. These storage structures allow air to flow freely through the grain, which

enhances the drying process but also makes the granary vulnerable to infestation by

insect pests such as S. zeamais (Holst et al., 2000). The sources of S. zeamais

infestation of stored commodities in warm climates can be several routes including

standing crops in the fields followed by passive movement with harvested grain into

the store, neighbouring storage facilities containing infested commodities, processing

plants and warehouses or insects migrating from abandoned or old granaries, or those

attracted by the smell of the new grain (Sinha and Watters, 1985; Arbogast and

Throne, 1997; Cox and Collins, 2002; Arthur et al., 2006). In Nigeria, field infestation

of standing crops and store to store infestations appear to be most prevalent. This is

not common in the temperate climate where the majority of infestations commence

after maize is harvested. For example, if the store has not been cleaned properly,

cryptic weevils hidden in the storage structure could be left behind from the previous

stock, ready to infest the new intake commodity (Cox et al., 1990). Also, grain

residues in commercial grain elevators boot pit and tunnel have been reported to

contain large numbers of storage pests including S. zeamais when the bins are empty

and serving as sources of infestation for new grains in the United States (Throne and

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Cline, 1994; Hagstrum, 2001; Arthur, et al., 2006). In elevators, the sources of insects

that infest new grain could be previously infested grain present when the new crop is

received, spills, trucks and railcars used to transport grain (Dowdy and McGaughey,

1998).

It is also very likely that stored product insects like most phytophagous insects, use

chemical cues to find sources of suitable host. Specialist or general pest species

require finding their host plants in a patchy environment, and plant volatiles are

important host location cues. Most host plants release hundreds of volatile organic

compounds, and many of those from grains have been identified as short-chain

alcohols, aldehydes, fatty acids, ketones, esters, terpenes and heterocyclic compounds

(Maarse, 1991; Seitz and Sauer, 1992). For example, wheat germ contains about 15%

lipid and 60% triglyceride (Pomeranz, 1978), and unsaturated triglyceride has been

reported to elicit aggregation responses from the granary weevil, S. granarius (L.)

(Nawrot and Czaplicki, 1982). S. granarius is a sibling species of S. zeamais that is

prevalent in temperate regions. The use of carob pod pieces in combination with

wheat kernels has been reported to enhance trap catches of S. zeamais and its sibling

species S. oryzae (L.) around traditional African granaries (Likhayo and Hodges,

2000). The main volatile compounds of maize seeds, namely hexanoic acid, nonanoic

acid, nonanal, decanal, 2-phenylethanol and vanillin have been reported to be the

main attractants for S. zeamais and S. oryzae (Pike et al., 1994; Hodges et al., 1998).

Infestation of stored maize by S. zeamais can be visualised as a process of invasion,

colonization and population growth. Following initial colonization of stored grain,

weevil population changes may be driven by its response to grain degradation, intra

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and inter-specific interactions, and the arrival of new colonizers (Arbogast and

Throne, 1997). The behaviour of the weevil on stored grain is affected by the inter-

play of different chemical, physical and biotic factors. Factors such as store

temperature, relative humidity, light intensity, grain moisture content, grain size and

variety, pest density and the presence of other insects including parasitoids and

predators, as well as micro-organisms such as fungi will greatly affect the behaviour

of the maize weevil for successful utilization of the store environment (Cox and

Collins, 2002).

1.5 CONTROL OF S. zeamais

When maize is placed in storage, the grains are exposed to a broad range of complex

ecological factors that work against the farmer or merchant’s objective of maintaining

grain quality. Some preventive measures such as seed sanitation, insect resistant

varieties, solar disinfections and weather-proof storage structures, and therapeutic

methods such as the use of insecticides and biological control agents have been in

practice for the protection of stored maize from pest infestation (Oparaeke and

Kuhiep, 2006). In nature, the common parasitoids of Sitophilus species are members

of the order Hymenoptera (Pteromalidae, Bethylidae), including Anisopteromalus

calandrae (Howard), Lariophagus distinguendus (Förster), Choetospila elegans

(Westwood), Theocolax elegans (Westwood), Cephalonomia tarsalis (Ashmead) and

C. waterstoni (Gahan) (Haines, 1991; Lord, 2006; Flinn et al., 2006). These parasitic

wasps lay their eggs into the larvae of the host weevils, and the wasp larvae develop

on host tissue, eventually killing their host as they emerge as mature larvae prior to

pupation or as adults depending on species. The presence of large numbers of these

insects usually indicates that a long-standing infestation is present (Rees, 2004).

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However, for effective control of stored-product insects especially S. zeamais, it is

pertinent to understand the storage environment and monitor pest infestation by

sampling.

1.5.1 Sampling and trapping

The presence of S. zeamais in stored maize is not easily noticeable except when

infestation has become very high, but this disadvantage can be overcome through a

mixture of sampling, inspection and trapping to detect low level infestations. Grains

are sampled at intake, during transportation and use for the presence of insects mainly

for assessment of grade and quality. In the developed world, hand probes which have

been replaced with pneumatic sample probes can be inserted into the back of the truck

using a mechanical arm. Maize moving through a handling system can be sampled

using a diverter system where a small percentage of the grain is continuously diverted

into a sampling system. To check for insects, the sampled seeds are passed over a

mechanical sieving machine which is attended by a specialist. Probes have been

developed to detect noise made by insects when infesting bulk grain, and there are

electronic sensors which detect chemical odours released by stored-product insects.

Hidden infestations are detected by radio photography using soft X-rays and near-

infrared (NIR) spectroscopy (Rees, 2004; Toews et al., 2006; Neethirajan et al.,

2007). The NIR method has been used to identify several coleopteran insects and can

be used to scan 1000 kernels per second, but it cannot detect low levels of infestations

in bulk samples or differentiate between live and dead insects (Dowell et al., 1999). In

addition, the NIR method is very sensitive to moisture content in grains and the

instrument requires frequent calibration, and the cost is prohibitive (Neethirajan et al.,

2007). The soft X-ray method is the only non-destructive, direct method that can

21

detect insect infestations in stores grains (Karunakaran et al., 2003). Also,

Karunakaran et al (2003) reported that with the soft X-ray method, wheat kernels

infested by S. oryzae larvae, pupae and adults were identified correctly by about 97%

and sound kernels with 99% accuracy.

The use of traps has also been employed to monitor and detect insect populations and

distribution in stored products (Dendy et al., 1991). Delta traps and Crevice traps

made from a piece of corrugated cardboard and Pitfall traps made from disposal

plastic drinking cups or used drink cans have been used for detecting insects in

storage structures and empty storage bins (Likhayo and Hodges, 2000; Rees, 2004).

Insect pheromones and other semiochemicals play important roles in the lives of

stored-product insects and hold great potential as tools for pest management (Phillips,

1997). Pheromones have been isolated and lures are commercially available for many

stored-product insects (Chambers, 1990; Phillips et al., 2000). Stored-product insects

can be detected with a variety of traps containing sticky bases with food attractants or

synthetic insect pheromones impregnated in a plastic lure that slowly releases the

active components over a period of several days or weeks (Vick et al., 1990; Mullen,

1992). Three different methods to control stored-product insects have used sex

pheromones of the Pyralid moth and the cigarette beetle, Lasioderma serricorne (L.)

namely: mating disruption where males cannot find females; mass trapping where

males are removed by trapping; or lure-and-kill, where males are lured to a dispenser

that contains a lethal dose of contact insecticide (Phillips et al., 2000; Plare and

Vanderwel, 1999). Similarly, flight traps and refuge traps baited with synthetic

aggregation pheromone (sitophilure) and cracked wheat kernels as food bait were

effective in capturing S. zeamais and S. oryzae, around traditional storage cribs in

22

Kenya (Likhayo and Hodges, 2000). The release rate of the synthetic aggregation

pheromone is dependent on chemical properties of the compound, physical and

chemical properties of the lure matrix, and environmental factors such as temperature

and rainfall, trap type and location influences the number of insect caught (Fields et

al., 1992; Campbell et al., 2002).

1.5.2 Manipulation of storage conditions

Physical control methods such as heat and cold can be manipulated in a stored-

product environment to eliminate pest infestation or slow down their populations.

Low temperatures are commonly used to manage stored-product insects because

between 1 and 5 ºC, depending on acclimation and the species, most stored-product

pests are unable to move and reproduce. Insects are killed at temperatures lower than

0 ºC as the lower the temperature, the faster the insects will succumb to cold injury

(Beckett et al., 2007). Aeration of the grain bulks with ambient air is one of the

methods extensively used after harvest to cool the grain. The air is passed through the

grain at relatively low volume, thereby preserving grain quality by slowing down

population growth, minimizing moisture evaporation and limiting the build-up of hot

spots (Darby, 1998). Aerating the grain bulk with chilled or refrigerated air has also

been reported to be very effective in the prevention of pest build-up in storage (Fields,

1992; Burks et al., 2000). For example, the uses of ambient autumn aeration and

autumn-chilled aeration have been reported to significantly reduce the development of

S. zeamais in maize stored for more than 6 months in Indianapolis. At the end of 12

months storage period, both aeration strategies were reported to produce S. zeamais

population of less than one weevil per 102 kg equivalent to about 97% control, which

23

was far below the Federal Grain Inspection Service threshold of two live insects per

kg of grain (Maier et al., 1996).

The use of elevated temperatures in stored product protection has the advantage of

giving complete disinfection, being rapid and pests are not likely to develop resistance

to it. Various methods of applying heat have been developed that disinfect grain on-

farm and in stores, as well as in processing facilities. Structural heat disinfection

involves raising the temperature of the facility to 50-60 ºC and maintaining these

temperatures for 24-36 h (Dowdy and Fields, 2002; Wright et al., 2002; Dosland et

al., 2006). Heat treatment of structures can be performed using gas, electric or steam

heaters. Depending on the size and nature of the facility, long periods of heating may

be necessary for adequate penetration of wall voids and equipment to kill insects

harbouring in them (Beckett et al., 2007). The ideal environmental conditions for

most stored-product insects are 25-32 ºC and 65-75% relative humidity (Fields, 1992;

Throne, 1994). The first stage of adverse effects of high temperatures up to 40-45 ºC

on stored-product insects include decline and ultimate inability to oviposit, hatching

and eclosion become incomplete, and with declining fecundity and shorter adult life

span, the pest population starts to die out. In the second stage, as temperature

increases to 45-55 ºC, insects could survive for several hours but experiencing severe

water stress. But in the last stage, when the temperature is greater than 55 ºC, there is

rapid mortality and the entire pest population is dead within seconds to minutes

(Beckett et al., 2007). High temperature causes a number of adverse biochemical

changes in insects such as lower ion concentrations, inactivation of major glycolysis

enzymes, disruption of plasma membranes and denaturation of proteins, lipids,

nucleic acids and carbohydrates (Denlinger and Yocum, 1999; Neven, 2000). For

24

example, S. cerealella was controlled by heating the grain to 69 ºC, and Sitophilus

species has been controlled when the temperature of wheat was raised to 57 ºC (Fields

and White, 2002). Also the exposure of red flour beetle, Tribolium castaneum

(Herbst.), pupae to high temperature prevented development to adults (Saxena et al.,

1992), or resulted in adults with separated elytra possibly due to chromosomal

aberrations (Denlinger and Yocum, 1999). While the exposure of T. castaneum pupae

and adults to high temperatures have been reported to reduce fecundity, egg-to-adult

survival and progeny production (Mahroof et al., 2005). The growth and development

of the mealworm, Tenebrio molitor (L.) and confused flour beetle, Tribolium

confusum (Duval) (Coleoptera: Tenebrionidae), were also adversely affected by high

temperature (Adler and Rassmann, 2000).

1.5.3 Contact insecticides

Several contact insecticides such as pirimiphos-methyl, malathion, fenitrothion,

chlorpyrifos-methyl and dichlorvos have been widely used as grain protectants against

stored-product pests (Snelson, 1987), and the continuous application of these

chemicals has led to the development of insect resistance throughout the world

(Subramanyam and Hagstrum, 2000). These insecticides were favoured for stored

grain protection because of their relatively low mammalian toxicity and suitable rates

of degradation, but are currently not considered safe to be on the market based on

toxicological re-evaluation (Fields and White, 2002; Beckett et al., 2007). Synthetic

pyrethroids have also been extensively used for stored-product protection because

they are more toxic to pests than organophosphorus chemicals especially at cool

temperature, and have a fairly low mammalian toxicity (Watters et al., 1983).

Synthetic pyrethroids have been used to control the lesser grain borer, Rhyzopertha

25

dominica (Fab.) and larger grain borer, P. truncatus even though their residues in

treated grain degrade slowly (Snelson, 1987).

In recent years, low toxicity insecticides such as inert dusts including diatomaceous

earth have been used against stored-product insect infestation (Golob, 1997;

Subramanyam and Roesli, 2000). Diatomaceous earth (DE) is composed of minute,

fossilized silicon dioxide remains of diatoms, and each deposit has its own physical

characteristics and toxicity to stored-product insects (Korunic, 1998; Fields and

Korunic, 2000). It has been applied directly to grain and in combination with other

substances to control pests in structures (Dowdy and Fields, 2002; Arthur and Throne,

2003), and it acts by speeding up desiccation of insects through disruption of water

transport through the epicuticle (Glenn et al., 1999; Fields and White, 2002). Arthur

(2002) reported that exposure of 10, 20, and 30 adult S. oryzae of mixed sexes for one

week on wheat treated with 300 ppm of “Protect-It” formulations of diatomaceous

earth resulted in 100% mortality and reduced F1 adults. Similarly, mortality of adults

S. oryzae and S. zeamais emerging from wheat and maize grains respectively treated

with 300 parts per million (ppm) of the “Protect-It” formulations of DE and held at 22

ºC ranged from 56-90%, and greater than 90% at 27 and 32 ºC relative to the controls

(Arthur and Throne, 2003), showing that temperature also had an effect in the efficacy

of DE.

1.5.4 Synthetic fumigants

In many storage systems, fumigation is still one of the most effective methods for the

protection of stored grain and dry food from insect infestation. Fumigation under

vacuum conditions can greatly speed the rate of insect control in storage conditions

26

(Verma, 1991). Methyl bromide (CH3Br) and phosphine (PH3) are the most widely

used fumigants throughout the world for stored-product insect control. Fumigation

may reach all parts of the storage and stored commodity, and usually be effective on

all developmental stages of the pest species while leaving minimal residues. It is often

more convenient than the application of grain protectants because they can be applied

to bulk grains without the need to move the commodity for treatment. The current

method of disinfestation of stored products is carried out by fumigation with methyl

bromide and phosphine (Bond, 1984). The mode of action is thought to be damage to

the membrane of nerve cells and reaction with the sulfhydryl groups in proteins

(Price, 1996). Methyl bromide acts rapidly, controlling insects in less than 48 h in

space fumigations, and it has a broad spectrum of activity, controlling not only insects

but also mites, nematodes and plant-pathogenic microbes. It does not taint the

commodities, is non-corrosive and non-flammable. Phosphine from metal phosphides

such as aluminium phosphide is formulated as solid tablets, pellets, powder in sachets

or used in phosphine generators to control insects in all their developmental stages;

also mites and rodents. The combination of 80-100 ppm phosphine with heat at 30-36

ºC and carbon dioxide at 3-7% concentrations, exhibited 100% insect control in 24-36

h (Fields and White, 2002).

Methyl bromide has been declared an ozone-depleting substance and was banned

completely in 2005 in Western countries. However, methyl bromide is still used in the

fumigation of granaries for the protection of stored product commodities in

developing countries. It was expected that developing countries will follow suit by

drastically reducing their consumption of methyl bromide in 2005, and completely

phase out its application by 2015 (Fields and White, 2002). Control failures and the

27

development of resistance in some stored-product insect pests to phosphine,

flammability above concentrations of 1.8% by volume, corrosion of copper, silver and

gold have been reported. Besides, aluminium phosphide is reported to exhibit toxic

effects to the lungs, heart and blood vessels causing pulmonary oedema, shock and

arrhythmias (Tyler et al., 1983; Bond et al., 1984; Zettler et al., 1989; Singh et al.,

1991; Kholsa et al., 1992; Zettler and Keever, 1994; Abder-Rahman, 1999; Fields and

White, 2002; Faruki et al., 2005; Rajendran and Sriranjini, 2008).

As replacements or alternatives to phosphine and methyl bromide, other fumigants

including carbonyl sulphide and ethyl formate (Desmarchelier et al., 1998), carbon

disulphide (Yonglin and Allen, 1999), and isothiocyanates (Shaaya et al., 2003), have

been studied for the protection of stored grain. Similarly, sulfuryl fluoride a structural

fumigant used for termite control (Bond, 1984), has also been used to control stored-

product pests (Zettler et al., 1989). Rajendran and Muralidharan (2005) reported the

fumigation toxicity of a liquid fumigant, allyl acetate, at doses of 50-150 mg/l against

mixed age cultures of various stored-product pests including S. oryzae which gave

100% mortality of all stages within 24-120 h exposure. Recently, Leelaja et al (2007)

reported that allyl acetate at 5-25 mg/l in combination with carbon dioxide (10% and

20%) significantly increased the mortality of S. oryzae, R. dominica, L. serricorne and

T. castaneum in a 48 h exposure period at room temperature.

1.5.5 Plant products

Some traditional indigenous measures have been taken by small-scale farmers to

protect stored maize from pest infestation (Hassanali et al., 1990; Poswall and Akpa,

1991; Oparaeke and Kuhiep, 2006). Different types of plant materials such as leaves,

28

fruits, seeds, roots, barks and processed as powders or ashes have played an important

role in traditional methods of protection against pest infestation in Africa since time

immemorial, and the protection of stored products has generally involved the

admixture of plant products with the grain (Tapondjou et al., 2002).

The precise processing and application of plant protectants varies from place to place,

and appears to depend on the availability, type and efficacy of suitable plants in

different geographical locations. However, a number of plants have been reported to

possess insecticidal, anti-feedant and insect repellent properties against storage pests

(Subramanyam and Hagstrum, 2000; Tapondjou et al., 2005; Jayasekara et al., 2005).

For example, seed powder and oil bark of black pepper, Piper guineense (L.)

(Piperaceae) and Capsicum frutescens (L.) (Solanaceae) are reported to cause adverse

effects to the biology of Callosobruchus maculatus and high mortality (Ivbijaro and

Agbaje, 1986). The essential oil of Xylopia aethiopica (Dunal) A. Rich (Annonaceae)

applied at 0.3 ml/100 g seed and powder at 1.5 ml/250 g seed reduced the emergence

of F1 adult S. zeamais and achieved 30% mortality of the weevil in 24 h (Okonkwo

and Okoye, 1996). Similarly, admixtures of powders at 20% (w/w) from the stem,

bark and leaf of Erythrophleum guineense (G. Don) (Caesalpiniaceae) and leaf of

Aloe vera (L.) Webb (Aloeaceae) with store maize grains for three months

significantly controlled S. zeamais and suppressed progeny development better than

the untreated maize (Oparaeke and Kuhiep, 2006). Lee et al (2001) also reported

bioactivities of essential oils from various Korean medicinal and spice plants against

S. oryzae.

29

The use of unattractive plant odours to repel insect pests from stored crops has been

the subject of much research. Important sources of repellents are the essential oils

extracted from aromatic plant species commonly used in food flavouring and in

perfumery (Coppen, 1995; Isman, 2000; 2006). Insect repellents are chemical

substances which cause the insect to make oriented movements away from the source

of the substances (Dethier et al., 1960). Repellents in the form of essential oils,

powders or distillates have the potential for exclusion of stored-product pests from

grain, and have been used to prevent insect feeding and oviposition. Chemotaxis

along an odour gradient is probably the most important way for stored product insect

orientation. Thus, it could be useful to avoid the establishment of a gradient by

masking the attractive odours by repellent smells (Adler et al., 2000).

1.6 CONCLUSIONS

The protection of stored products against attack by pests is essential in many

countries, particularly those suffering from inadequate storage facilities. Novel,

environmentally compatible stored product control agents are urgently needed to

replace synthetic pesticides that have been withdrawn for economic or regulatory

reasons or are ineffective, due to the increasing difficulty of managing pesticide

resistance. Control agents that are safe alternatives, and have the potential to replace

these toxic fumigants, yet are simple and convenient to use are required. Furthermore,

there are heightened public concerns over chemical fumigants such as fears about

effects on public health and negative environmental consequences (Duke et al., 2003).

Natural product-based products such as plant extracts offer advantages in that they

can sometimes be specific to the target species, they have local availability, generally

do not persist in the environment, and typically have unique modes of action with

30

little mammalian or ecotoxicity (Liu et al., 2006; Isman, 2006). Thus, it is highly

imperative to continue the search for cheap, less toxic and environmentally friendly

natural products for reducing insect damage in storage.

Plant extracts and essential oils have potential in crop protection. They contain

monoterpenoids, diterpenoids, sesquiterpenoids and other compounds that show

ovicidal, larvicidal, repellent, deterrent, antifeedant and toxic effects in a wide range

of insects (Fields et al., 2001; Pungitore et al., 2003; Boeke et al., 2004; Liu et al.,

2006; Isman, 2000; 2006). In Nigeria, the seeds, roots and leaves of ginger, Zingiber

officinale Roscoe (Zingiberaceae), (Abubakar et al., 2007), West African black

pepper, Piper guineense Thonn and Schum (Piperaceae) (Oyedeji et al., 2005) and

alligator pepper, Aframomum melegueta K. Schum (Zingiberaceae) (Ajaiyeoba and

Ekundayo, 1999) are used in spicing meat, sauces and soups and mixed with other

herbs in traditional medicine for the treatment of body pains, catarrh, congestion,

diarrhoea, sore throat, bronchitis, diabetes mellitus, cancer and rheumatism. Their oil

appears to have antioxidant, antimicrobial, molluscicidal, antischistosomal, anti-

hypertensive and insect repellent properties (Oyedeji, et al., 2005; Adewoyin et al.,

2006; Verspohl et al., 2006; Abubakar et al., 2007; Abo et al., 2008). Such plants

were selected on the basis of their bioactivity as potential candidates for S. zeamais

control in Nigeria. The three plant species have not traditionally been used in maize

protection but are available locally and there is evidence in the literature that their

host plant volatiles are effective against other stored product pests. In order to

properly assess the effectiveness of and understand the role of individual compounds

and mixtures of plant volatiles in such protection, in depth studies of particular stored

product pest and repellent plant systems are required.

31

1.7 AIMS AND OBJECTIVES

The aim of this study was the identification and use of semiochemicals for the control

of the maize weevil S. zeamais in Nigeria. In order to achieve this aim, the following

objectives were envisaged;

1. To investigate the repellent properties of alligator pepper (Aframomum melegueta)

K. Schum, ginger (Zingiber officinale) Roscoe, and West African black pepper (Piper

guineense) Thonn & Schum against S. zeamais.

2. To analyse and identify the active volatile compounds from the plants.

3. To test the plant products and semiochemicals as part of a push-pull strategy

(stimulo deterrent diversionary strategy) for control in storage conditions.

32

CHAPTER 2:

BEHAVIOURAL RESPONSES OF Sitophilus zeamais TO HOST AND NON-

HOST PLANT VOLATILES

33

2.1 INTRODUCTION

Plant semiochemicals have been reported to produce a wide range of behavioural

responses in insects. Interactions between insect pheromones and semiochemicals

from host plant have been known for nearly as long as pheromones have been

recognized as a key communication system within species. Such interactions are

manifested as effects of the host plant on insect physiology and behaviour, reflecting

different types of insect strategies to optimize feeding, mating and reproduction

(Landolt and Phillips, 1997). Some insects sequester or acquire host plant chemicals

to use them as sex pheromones or sex pheromone precursors. One of the best

examples of sequestration of plant chemicals by larvae and their subsequent use by

adult males in sex attraction or courtship interactions is shown in the moth Utetheisa

ornatrix (Lepidoptera: Arctiidae), whose courtship pheromone derives from

pyrrolizidine alkaloids (PAs) ingested at the larval stage from the host plant

Crotalaria spetabilis (Conner, et al., 1990). The larvae of U. ornatrix sequester the

PA monocrotaline and retain the alkaloids through metamorphosis into the adult stage

as hydroxydanaidal, to provide egg protection for the next generation. Females

receive the PAs from males during copulation and transmit the alkaloids together with

their own load to the eggs (Eisner and Meinwald, 1995). PA sequestering species are

found in the Coleoptera (some leaf beetles), Lepidoptera (many butterflies),

Orthoptera (certain grasshoppers) and Homoptera (certain aphids), and are used as

strong feeding deterrents against invertebrate predators such as spiders and ants

(Nishida, 2002).

Other host plant volatiles can induce the production or release of pheromones in

certain species and sometimes synergize or enhance insect responses to sex

34

pheromones. Example, in the Coleoptera, beetle species such as the boll weevil

Anthonomus grandis (Dickens, 1989) are thought to release the pheromone after

feeding on the host plant. In the pine weevil Pissodes nemorensis, the male-produced

pheromones grandisol and grandisal were attractive in the field only when deployed

with odours from a cut pine bolt (Phillips et al., 1984). Also the African oil palm

weevil Rhynchophorus phoenicis (Curculionidae) produces a mixture of volatile

esters from which ethyl acetate induces males to release the pheromone rhyncophorol,

(E)-6-methyl-2-hepten-4-ol (Jaffé et al., 1993). Similarly many Scolytid beetles, e.g.

ambrosia beetles in the genus Gnathotricus, form feeding and mating aggregations on

host plants, as a result of attraction to sex pheromones (Borden et al., 1980). Sex

pheromones of beetles at such feeding and mating sites may be plant-derived due to

adult feeding on plant tissue, passage of chemicals through the gut, and release in the

frass, or production in the gut from plant precursors. This is of particular interest

among the stored-product Coleopterans (S. oryzae, S. zeamais, R. dominica, C.

maculatus and P. truncatus) all of which respond more to male-produced aggregation

pheromones deployed with grain than to either pheromone or grain separately

(Phillips, et al., 1993; Bashir, et al., 2001). Walgenbach et al (1987) reported that S.

zeamais responded significantly more to male-produced aggregation pheromones

deployed with grain than to either pheromone or grain separately. Females of the

cowpea weevil, C. maculatus, are stimulated to release pheromone in the presence of

plant seeds, as evidenced by increased male electroantennogram responses to airflow

from other females provided with such seeds (Lextrait et al., 1995).

Host compounds can also have inhibitory or repellent effects, interrupting the

response of insects to their own pheromone (Reddy and Guerrero, 2004). Example, 4-

allyl anisole, a common compound produced by loblolly pine, Pinus taeda and other

35

conifer species significantly reduced the response of the Southern pine beetles,

Dendroctonus frontalis to their own pheromone when simultaneously released with

the natural attractant in the field (Hayes et al., 1994). A note-worthy example is

shown by the pine shoot beetle Tomicus destruens (Coleoptera: Scolytidae), an

important pine pest widely distributed throughout Europe. Benzyl alcohol, a

semiochemical present in fennel extracts and in the callus of Eucalyptus radiata but

completely absent from pine volatiles of leaves and twigs, induced beetles to bore a

limited number of galleries when the chemical was deposited in the field on cut pine

logs (Guerrero et al., 1997). These results could have important implications for the

control of pine shoot beetle by excluding them from potential hosts or regulating

attack densities to unsuitable levels for tree colonisation.

Host location is frequently the result of chemical and/or visual cues that enable the

insect to recognize its host plant, and pest species are able to recognize and avoid

general volatile signals that are commonly emitted by a range of non-host plant

species. In this way, several species of non-host plants with overlapping blends of

common volatile compounds could be perceived and avoided by pests during host

location processes (Huber and Borden, 2001). For example, eugenol, a major

component of the essential oil of Ocimum suave was highly repellent to the storage

pests Sitophilus zeamais, S. granarius, Tribolium castaneum and Prostephanus

truncatus with overall repellency in the range of 80-100%. The development of eggs

and immature stages inside grain kernels was also completely inhibited by eugenol

treatment (Obeng-Ofori and Reichmuth, 1997). Methanol extract and 2% powder

(w/w) from the rhizome of Cyperus articulatus (Cyperaceae), a plant commonly

found in Nigeria significantly exhibited repellent activity against the red flour beetle,

36

T. castaneum of stored wheat (Abubakar et al., 2000). Filter papers impregnated with

25 µl essential oil extracted from the fruits of Evodia rutaecarpa (Juss.) Benth.

(Rutaceae), a deciduous tree native to China and Korea, was reported to possess toxic,

repellent and feeding deterrent properties toward adult T. castaneum and S. zeamais

and T. castaneum larvae in a 24 h duration (Liu and Ho, 1999). Boeke et al (2004)

also reported that cowpea seeds treated with leaf powders of Momordica charantia L.

(Cucurbitaceae) applied at 25 g kg -1 were protected against weight loss caused by C.

maculatus, those treated with 20 µl essential oil of Ocimum basilicum L. (Lamiaceae)

in 40 g seeds has significantly higher germination percentage than the untreated seeds,

while cowpea treated with Ficus exasperata Vahl (Moraceae) leaf powders had a

decreased in both percentage of infested beans and the number of emerged adults.

In West Africa, infestation by stored-product pests particularly S. zeamais, causes

serious losses in food and feed commodities. Moreover, pest infestations are

responsible for changes in the chemical composition of stored food, reductions in

nutritional values and contamination by harmful compounds and allergens (Rajendran

and Parveen, 2005). Therefore, the search for natural chemicals that are effective,

easily biodegradable and non-toxic to humans will be advantageous in the protection

of stored grains from insect damage. For instance, the resource poor farmers in

Nigeria have access to local ethno botanical plants and have indigenous knowledge

systems that could help increase agricultural productivity with minimal human and

environmental health hazards that are often experienced when synthetic pesticides are

used inappropriately. The seeds, roots and leaves of alligator pepper, Aframomum

melegueta K. Schum (Zingiberaceae), ginger, Zingiber officinale Roscoe

(Zingiberaceae), and West African black pepper, Piper guineense Thonn and Schum

37

(Piperaceae) are used in spicing meat, sauces and soups and mixed with other herbs

are used for the treatment of body pains, catarrh, congestion, diarrhoea, sore throat

and rheumatism. Their oil appears to have antioxidant, antimicrobial, molluscicidal,

antischistosomal, anti-hypertensive and insect repellent properties (Escoubas et al.,

1995; Oyedeji, et al., 2005; Adewoyin et al., 2006; Verspohl et al., 2006).

This chapter evaluates the repellent effects of A. melegueta seeds, Z. officinale

rhizomes, and P. guineense seeds; the attractive effects of white and yellow maize

seeds, and winter wheat seeds; and the dose responses involving three doses of these

plants against S. zeamais in an airflow olfactometer. The implications of these results

in the control of S. zeamais are discussed.

2.2 MATERIALS AND METHODS

2.2.1 Insect culture

Mass rearing of the test insect, Sitophilus zeamais Motschulsky, commenced on

March 23, 2006, the stock obtained from a culture maintained by Central Science

Laboratory, Sand Hutton, York, United Kingdom. In Aberdeen, the insect stock was

divided into two sets; one set was maintained on untreated winter wheat (Triticum

aestivum L.) purchased from food merchants in Aberdeen, and the other either on

untreated Nigerian “local yellow” and “Ikom white” maize (Zea mays L.) seeds

purchased from local foodstuff market in Akim, Calabar-Nigeria in December 2005.

The grains were stored at -20 C until when needed for the experiments. Prior to the

experiment, the grains were removed from the freezer and kept at room temperature

for 1 h. Fifty pairs of adult S. zeamais were introduced into 200 g maize or wheat

seeds in cylindrical transparent plastic containers of 8 cm diameter and Kilner jars

38

(Plate 2.1). The plastic containers had their covers drilled with holes to facilitate air

circulation. The plastic containers were then covered with nylon mesh and their

perforated lids screwed in place. A PC 440 electronic balance (Mettler Instrument

AG, Zurich, Switzerland) was used to weigh the seeds. Cultures of the test insect were

maintained in a standard insect behaviour room at constant temperature of 25 C and

65 % relative humidity on a 12:12 DL photoperiod for the emergence of the first

progeny. The adults were allowed to stay in the containers for 12 days for mating and

oviposition after which they were removed and discarded. After 29 days, the weevils

emerging from each culture were sieved out using mesh number 10 (sieve size 2 mm,

Endecotts LTD, England) and sieved daily afterwards taking into consideration that

mating in S. zeamais does not occur before weevils are 3 d old (Walgenbach and

Burkholder, 1987), and records were kept of their sexes and dates of eclosion. With

the aid of a Nikon binocular microscope (Plate 2.2), the insects were sexed following

the methods of Halstead (1963) and Haines (1991) according to the dimorphic rostral

characteristics in which males have a distinctly shorter, wider and rougher rostrum,

compared to the females with longer, narrower, smooth and shining rostrum.

39

Plate 2.1 Kilner jar and plastic container used to culture S. zeamais

Plate 2.2 Binocular microscope for sexing of newly emerged adult weevils.

40

2.2.2 Plant materials collection and preservation

Three repellent plant materials namely; A. melegueta, Z. officinale and P. guineense

(Plate 2.3-2.5) were collected from fields around Akamkpa (situated between latitude

500 and 515 North and longitude 804 and 825 East) in southern Nigeria in

December 2005. The matured fruits, rhizomes or seeds of these plants were dried in

the shade to approximately 15% moisture content before transportation. These plants

were selected on the basis of their ethnomedical studies and endemicity (Adewoyin et

al., 2006). The plant materials were preserved in Aberdeen at -20 C for 6 months

before they were used for the bioassays.

Plate 2.3 Aframomum melegueta fruits prior to drying

41

Plate 2.4 Zingiber officinale rhizome

Plate 2.5 Fresh Piper guineense leaf and seeds prior to drying

42

2.2.3 Bioassay method

Behavioural bioassays were performed in an olfactometer modified after Pettersson

(1970). The olfactometer consisted of three layers of 6 mm thick transparent Perspex

screwed together. A four-pointed star-shaped exposure chamber was milled into a

circular plate measuring 12 x 12 x 1.2 cm, with a hole (3 mm diameter) drilled into

the walls at each of the four cardinal points. Another plate (10.2 x 10.2 x 0.6 cm)

served as the floor and the third plate of the same size but with a hole (4 mm

diameter) in its centre, served as a cover. Since S. zeamais cannot walk on smooth

surfaces a sheet of Fisherbrand QL 100 filter paper (Springfield Mill, Maidstone,

Kent, England) was used as a floor covering. The olfactometer side arms made of

socket glass were inserted through the holes of the chamber walls. The olfactometer

was housed in the behaviour room running at 25C, 65 % relative humidity, and on a

12: 12 Light and Darkness (LD) cycle illuminated by fluorescent tubes but with no

natural lighting.

The air stream through the olfactometer was supplied by the Air entrainment system

(Plate 2.6) (KNF Neuberger, Germany) through Teflon tubing measuring 3.2 mm i.d.

(Camlab Ltd., UK). Immediately after the pump, the air was divided through 2 carbon

rods to clean it. From each carbon rod, the air stream was then further divided and

pushed through two flow meters (GPE Ltd., Leighton Buzzard, UK) to give a total of

four air flows going into the behaviour chambers. Each air stream then passed through

a glass side arm with a net-covered inlet to prevent insect entry, which contained

either the odour/volatile source presented as plant part or clean filter paper, which

served as a control. From each glass side arm, air was delivered into the bioassay

exposure chamber by the four air-delivery tubes (Plate 2.7). The rate of airflow

43

through each side arm was set at 200 ml min-1. The air streams formed four distinct

zones in the chamber as shown by the smoke tests. The air was pulled from the

chamber at the rate of 800 ml-1 through the central hole in the cover plastic plate.

Plate 2.6 Air entrainment system used for bioassays

The different odour sources (treatments) used to compare the responses of virgin adult

male and female S. zeamais are listed.

(1) Host volatiles emanating from 2 g of whole, sound, grains of yellow or white

maize imported from Nigeria, or winter wheat seeds alone.

(2) Non-host volatile odours from 2 g A. melegueta seeds, Z. officinale rhizome, or P.

guineense seeds alone,

(3) Incorporation of 10% (w/w) (0.2 g non-host + 1.8 g host plant) of each non-host +

host plant volatiles combination;

- A. melegueta + yellow maize

- Z. officinale + yellow maize

44

- P. guineense + yellow maize

- A. melegueta + white maize

- Z. officinale + white maize

- P. guineense + white maize

- A. melegueta + winter wheat

- Z. officinale + winter wheat

- P. guineense + winter wheat

(4) Dose response bioassays using 1%, 10% and 33% non-host plants to yellow

maize (w/w).

- 1% A. melegueta + yellow maize

- 10% A. melegueta + yellow maize

- 33% A. melegueta + yellow maize

- 1% Z. officinale + yellow maize

- 10% Z. officinale + yellow maize

- 33% Z. officinale + yellow maize

- 1% P. guineense + yellow maize

- 10% P. guineense + yellow maize

- 33% P. guineense + yellow maize

Single choice attraction and/or repellent bioassays consisting of three host plants,

three non-host plants and dose response volatiles listed above were conducted in the

constant temperature and humidity (CTH) room. The olfactometer was coded into five

areas: one square shaped central area and four rectangular areas corresponding to the

four arms of the olfactometer, each area was marked with a number between 1 and 5.

In these trials, the arm with the test odour(s) was given number 1; the 3 controls

45

numbered 2, 3, and 4 respectively, while the central area was given number 5 (Plate

2.8). Two g of the seeds of the test material were weighed and placed in arm number

1 while the other 3 arms contained clean filter papers and served as controls. For Z.

officinale, the rhizome was cut to size, weighed and allowed to heal for 1 h before

being used for the bioassays.

The test insects (3 d old virgin adults) were starved for 24 h and kept singly in Petri

dishes prior to the commencement of the bioassays. Each weevil was observed for16

min for all experiments except the dose response experiment in which the weevil was

observed for10 min using a stopwatch, and each trial was replicated 12 times in a

complete randomized design. Test individuals and olfactometers were changed

between replicates, while odour samples or stimulus were replaced after every 2

replications. Assignments of treatments to the olfactometer arms were the same

throughout a test day but the positioning of the arms were rotated 90º the next day. All

experiments were conducted between 9.00 am and 12.00 noon. The experiment

commenced by the release of the insect into the centre of the olfactometer, the insect

was followed visually as it made choices among the different olfactometer arms. Each

of the 4 arms was considered a separate zone when recording the insect positions and

response to test volatiles. The weevil was considered to have entered a given arm

when its entire thorax crossed the zone boundary. A computer programme for

collecting and analysing behavioural data with the four-armed olfactometer

(commonly referred to as OLFA programme) developed by Francesco Nazzi (33100

Udine, Italy) was used to obtain data. The data recorded included the time spent by

the insect in the different areas of the olfactometer and the number of entries or visits

into each area or odour zone.

46

Plate 2.7 Bioassay set-up.

Arm no. 1 Test arm with yellow maize volatilesArm no. 2 Control 1

Arm no. 3 Control 2 Arm no. 4 Control 3

Plate 2.8 Olfactometer

47

2.2.4 Statistical analysis

The time spent in each area and the numbers of entries (visits) by the weevil into the

different odour zones of the olfactometer were the parameters chosen for assessment

of the difference between plant volatiles and the control. The null hypothesis of equal

time spent in each olfactometer arm was tested using one-way analysis of variance

(ANOVA) followed by comparison of means by Tukey’s 95% simultaneous

confidence intervals (MINITAB 15 Statistical Software). The number of visits in the

odour-treated arm was compared with the number of visits in control arms using chi-

square (χ2) tests, where the null hypothesis assumes that the number of visits made by

the insect to the treated arm was be equal to the number of visits to each of the three

control arms. Therefore a 25% frequency of the insect into each arm was tested using

a χ2 goodness-of-fit test (Zar 1999). Data collected from the centre of the olfactometer

were not used in the statistical analysis.

2.3 RESULTS

2.31 Host plant odours

(ii) Blank control

In control experiments, there was no significant difference (p>0.05) in the mean time

spent in the test arm compared to the control arms (hereafter referred to as mean time)

by males (Figure 2.1a) and females (Figure 2.1b), mean number of visits by male (χ 2

= 0.05, df = 3, P = 0.997) and female (χ2 = 1.02, df = 3, P = 0.796) (Table 2.1) S.

zeamais to the 4 arms of the olfactometer.

48

0

0.5

1

1.5

2

2.5

3

Blank Blank Blank Blank

Mean t

ime

spent

(min

)

0

0.5

1

1.5

2

2.5

3

Blank Blank Blank Blank

Mean t

ime s

pent

(min

)(a) (b)

Figure 2.1 Mean time spent out of 16 min by S. zeamais males (a) and females (b) in response to clean air from all 4 control arms in a four way olfactometer, Bars = standard errors (SE) of the means, n = 12.

Table 2.1 Mean number of entries by naive adult Sitophilus zeamais to all blank (control) arms in a 4-way olfactometer

Test stimulusMean no. visits to olfactometer arm n χ2* P*

All 4 blank armsMales 4.39 12 0.05 0.997Females 3.81 12 1.02 0.796

All 4 olfactometer arms contained clean filter paper discs*χ2 analysis was performed on the total number of visits (n =12) into the test, control 1, control 2 and control 3 arms in a 4 way contingency table.

(ii) Yellow maize, white maize and winter wheat seeds

Olfactometer assays showed that for test arm against control arms 1, 2 and 3, males

and females were significantly more attracted to yellow maize (P<0.001) (Figure 2.2a,

b), white maize (P<0.001) (Figure 2.3a, b) and winter wheat seeds (P< 0.001) (Figure

2.4a, b) than the control arms in the mean time spent. A similar behavioural response

was observed in the mean number of visits made to the arm emitting volatiles from

yellow maize by males (χ2 = 14.29, df = 3, P=0.003) and females (χ2 = 27.63, df = 3,

49

P<0.001); white maize by males (χ2 = 15.22, df = 3, P=0.002) and females (χ2 = 13.2,

df = 3, P=0.004), and to winter wheat kernels by males (χ2 = 19.45, df = 3, P=0.001)

and females (χ2 = 27.68, df = 3, P<0.001) (Table 2.2).

0

12

34

5

67

89

10

Yellowmaize

Control1

Control2

Control3

Mea

n tim

e sp

ent

(min

)

a

b bb

0

1

2

3

4

56

7

8

9

10

Yellowmaize

Control1

Control2

Control3

Mea

n tim

e sp

ent

(min

)

b b b

a

(a) (b)

Figure 2.2 Mean time spent out of 16 min by Sitophilus zeamais males (a) and females (b) in response to odours from yellow maize grains in a four way olfactometer. Bars = standard errors (SE) of the mean, n = 12. Bars with the same letter are not significantly different from each other (P>0.05). a-b (males, females), P<0.001.

50

01

23

456

78

910

Whitemaize

Control1

Control2

Control3

Mea

n tim

e sp

ent

(min

)a

b bb

012

34567

89

10

Whitemaize

Control1

Control2

Control3

Mea

n tim

e sp

ent

(min

)

a

b b b

(a) (b)

Figure 2.3 Mean time spent out of 16 min by Sitophilus zeamais males (a) and females (b) in response to odours from white maize grains in a four way olfactometer. Bars = standard errors or the mean, n = 12. Bars with the same letter are not significantly different from each other (P>0.05). a-b (males, females), P<0.001.

0123456789

1011

Winterwheat

Control1

Control2

Control3

Mea

n tim

e sp

ent

(min

)

a

b b b

0123456789

1011

Winterwheat

Control1

Control2

Control3

Mea

n tim

e sp

ent

(min

)

a

b b b

(a) (b)

Figure 2.4 Mean time spent out of 16 min by Sitophilus zeamais males (a) and females (b) in response to odours from winter wheat grains in a four way olfactometer. Bars = standard errors of the means, n = 12. Bars with the same letter are not significantly different from each other (P>0.05). a-b (males, females), P<0.001.

51

Table 2.2 Responses of Sitophilus zeamais to odours of host plant grains in a four wayolfactometer

Test stimulusMean no. visits in olfactometer arm n χ2* P*

i) Yellow maize grains against controls T CMales 6.67 3.97 12 14.29 0.003Females 9.00 4.73 12 27.63 0.001

ii) White maize grains against controlsMales 6.92 4.08 12 15.22 0.002Females 4.17 2.22 12 13.2 0.004

iii) Winter wheat seeds against controlsMales 6.91 3.77 12 19.45 0.001Females 6.17 2.83 12 27.68 0.001

T is the mean value of test armC is the value of the mean of three control arms*χ2 analysis was performed on the total number of visits (n=12) to the test, control 1, control 2 and control 3 arms in a 4 way contingency table.

2.3.2 Non-host plant odours

In each of the three single choice tests, males spent significantly shorter time in the

olfactometer arms emitting volatiles of A. melegueta seeds (P<0.001) (Figure 2.5a), Z.

officinale rhizome (P<0.001) (Figure 2.6a), P. guineense seeds (P<0.006) (Figure

2.7a) than in control arms containing clean filter paper discs and receiving clean air.

The females also preferred control arms to the treated ones for A. melegueta (P<

0.001) (Figure 2.5b), Z. officinale (P<0.001) (Figure 2.6b), and P. guineense (P<

0.001) (Figure 2.7b). Similarly, the insects significantly frequented the control arms

receiving clean air more than the treated arm. The males significantly preferred the

control arm to the arm containing A. melegueta (χ2 = 30.5, df = 3, P<0.001), Z.

officinale (χ2 = 13.88, df = 3, P=0.003), and P. guineense (χ2 = 31.61, df = 3,

P<0.001); as did the females to the arm containing A. melegueta (χ2 = 22.11, df = 3,

52

P<0.001), Z. officinale (χ2 = 16.71, df = 3, P<0.001), and P. guineense (χ2 = 9.12, df =

3, P=0.028) (Table 2.3).

0

1

2

3

4

5

6

7

2g A.melegueta

Control 1 Control 2 Control 3

Mea

n tim

e sp

ent (

min

)

b

b

b

a

0

1

2

3

4

5

6

7

2g A.melegueta

Control 1 Control 2 Control 3

Mea

n tim

e sp

ent (

min

)

b

bb

a

(a) (b)

Figure 2.5 Mean time spent out of 16 min by S. zeamais males (a) and females (b) in response to non-host plant odours from Aframomum melegueta in a four way olfactometer. Bars = standard errors (SE) of the means, n = 12. Bars with the same letter are not significantly different from each other (P>0.05). a-b (males, females), P<0.001.

53

0

1

2

3

4

5

6

7

2g Z.officinale

Control 1

Control 2

Control 3

Mea

n tim

e sp

ent

(min

)

bb b

a

0

1

2

3

4

5

6

7

2g Z.officinale

Control 1

Control 2

Control 3

Mea

n tim

e sp

ent

(min

) b

b

b

a

(a) (b)

Figure 2.6 Mean time spent out of 16 min by S. zeamais males (a) and females (b) in response to non-host plant odours from Zingiber officinale in a four way olfactometer, Bars = standard errors of the means, n = 12. Bars with the same letter are not significantly different from each other (P>0.05). a-b (males, females), P<0.001.

0

1

2

3

4

5

6

7

2g P.guineense

Control 1 Control 2 Control 3

Mea

n tim

e sp

ent (

min

)

b

bb

a

0

1

2

3

4

5

6

7

2g P.guineense

Control 1 Control 2 Control 3

Mea

n tim

e sp

ent (

min

)

b b

b

a

(a) (b)

Figure 2.7 Mean time spent out of 16 min by S. zeamais males (a) and females (b) in response to non-host plant odours from Piper guineense in a four way olfactometer. Bars = standard errors of the means, n = 12. Bars with the same letter are not significantly different from each other (P>0.05). a-b (males), P=0.006; a-b (females), P=0.001.

54

Table 2.3 Responses of Sitophilus zeamais to non-host plant odours in a four way olfactometer

Test stimulusMean no. visits in olfactometer arm n χ2* P*

i) Aframomum melegueta volatiles against controls T CMales 2.08 6.03 12 30.50 0.001Females 2.17 5.33 12 22.11 0.001ii) Zingiber officinale volatiles against controlsMales 1.67 3.81 12 13.88 0.003Females 1.83 4.19 12 16.71 0.001iii) Piper guineense volatiles against controlsMales 2.50 6.61 12 31.61 0.001Females 2.25 3.81 12 9.12 0.028

Control arms contained clean filter paper discsT is the mean value of test armC is the mean value of the mean of three control arms*χ2 analysis was performed on the total number of visits (n=12) to the test, control 1, control 2 and control 3 arms in a 4 way olfactometer

2.3.3 Host and non-host plant odours

(i) Yellow maize

Results of the combined host plant (yellow maize) and non-host plant bioassays are

presented in Figures 2.8 – 2.10 and Table 2.4, in which 0.2 g non-host plants (A.

melegueta, Z. officinale, or P. guineense) were incorporated with 1.8 g yellow maize,

and attraction or repellence was measured against the 3 control arms containing filter

papers only. Males showed significant differences in the mean time spent in the arm

containing A. melegueta plus maize (P=0.015) (Figure 2.8a), Z. officinale plus maize

(P=0.002) (Figure 2.9a), or P. guineense plus maize (P=0.042) (Figure 2.10a) than in

the control arms. Females were equally repelled from the olfactometer arm containing

A. melegueta plus maize (P<0.001) (Figure 2.8b) Z. officinale plus maize (P=0.003)

(Figure 2.9b), and P. guineense plus maize (P<0.001) (Figure 2.10b) in the mean time

spent than in the control arms. For the number of visits, males significantly avoided

the arm containing A. melegueta plus maize (χ2 = 8.24, df = 3, P=0.041) and Z.

55

officinale plus maize (χ2 = 17.08, df = 3, P<0.001), and females A. melegueta plus

maize (χ2 = 15.03, df = 3, P=0.002) and Z. officinale plus maize (χ2 = 8.09, df = 3,

P=0.044), in the number of entries respectively than to the controls (Table 2.4). There

was no significant difference by males (χ2 = 7.24, df = 3, P=0.065) and females (χ2 =

6.7, df = 3, P=0.082) in 10% P. guineense + yellow maize and the controls in the

mean number of entries (Table 2.4).

0

1

2

3

4

5

6

Test Control1

Control2

Control3

Mea

n tim

e sp

ent

(min

) bb b

a

0

1

2

3

4

5

6

Test Control1

Control2

Control3

Mea

n tim

e sp

ent

(min

)

b

b b

a

(a) (b)

Figure 2.8 Mean time spent out of 16 min by S. zeamais males (a) and females (b) in response to 2 g yellow maize seeds + 10% A. melegueta seeds in a four way olfactometer assay. Bars = standard errors of the means, n = 12. Bars with the same letter are not significantly different from each (p>0.05). a-b (males), P=0.015; a-b (females), P=0.001.

56

0

1

2

3

4

5

6

Test Control1

Control2

Control3

Mea

n tim

e sp

ent

(min

) bb b

a

0

1

2

3

4

5

6

Test Control 1

Control 2

Control 3

Mea

n tim

e sp

ent

(min

)

bb

b

a

(a) (b)

Figure 2.9 Mean time spent out of 16 min by S. zeamais males (a) and females (b) in response to 2 g yellow maize seeds + 10% Z. officinale rhizome in a four way olfactometer assay. Bars = standard errors of the means, n = 12. Bars with the same letter are not significantly different from each other (p>0.05). a-b (males), P=0.002; a-b (females), P=0.003.

0

1

2

3

4

5

6

Test Control1

Control2

Control3

Mea

n tim

e sp

ent

(min

)

b

ab ab

a

0

1

2

3

4

5

6

Test Control 1

Control 2

Control 3

Mea

n tim

e sp

ent

(min

)

b bb

a

(a) (b)

Figure 2.10 Mean time spent out of 16 min by S. zeamais males (a) and females (b) in response to 2 g yellow maize seeds + 10% P. guineense seeds in a four way olfactometer assay. Bars = standard errors of the means, n = 12. Bars with the same letter are not significantly different (p>0.05). a-b (males), P=0.042; a-b (females), P=0.001.

57

Table 2.4 Responses of Sitophilus zeamais to a combination of 2 g yellow maize and 10% non-host plant volatiles in a four way olfactometer

Mean no. visits in olfactometer armTest stimulus n

χ2* P*

i) Yellow maize + 10% A. melegueta against controls T CMales 3.27 4.89 12 8.24 0.041Females 2.42 5.06 12 15.03 0.002ii) Yellow maize + 10% Z. officinale against controlsMales 4.00 7.36 12 17.08 0.001Females 3.36 4.97 12 8.09 0.044iii) Yellow maize + 10% P. guineense against controlsMales 4.25 6.08 12 7.24 0.065Females 3.42 5.28 12 6.70 0.082

Control arms contained clean filter paper discsT is the mean value of test armC is the mean value of the mean of three control arms*χ2 analysis was performed on the total number of visits (n=12) to the test, control 1, control 2 and control 3 arms in a 4 way olfactometer

(ii) White maize

With white maize and the 3 repellent plants, males showed significant differences in

the mean time spent in the arm with A. melegueta plus maize (P0.001) (Figure

2.11a), Z. officinale plus maize (P=0.008) (Figure 2.12a), or P. guineense plus maize

(P=0.004) (Figure 2.13a) when compared with the control arms. Females were also

significantly repelled by A. melegueta plus maize (P=0.013) (Figure 2.11b), Z.

officinale plus maize (P<0.001) (Figure 2.12b), and P. guineense plus maize

(P=0.043) (Figure 2.13b) in the time spent in each arm than the control arms. For the

mean number of visits, the males were also significantly repelled from A. melegueta

plus white maize (χ2 = 13.32, df = 3, P=0.004), Z. officinale plus white maize (χ2 =

8.42, df = 3, P=0.038) and P. guineense plus white maize (χ2 = 9.43, df = 3, P=0.024)

respectively than the control arms. The number of visits made by females to A.

58

melegueta plus white maize (χ2 = 8.67, df = 3, P=0.034) or Z. officinale plus white

maize (χ2 = 8.2, df = 3, P=0.042) respectively were also significantly different from

control arms, but was not significantly different to P. guineense plus white maize (χ2

= 6.48, df = 3, P=0.091) arm compared to the control arms (Table 2.5).

0

1

2

3

4

5

6

Test Control1

Control2

Control3

Mea

n tim

e sp

ent

(min

) b

bb

a

0

1

2

3

4

5

6

Test Control1

Control2

Control3

Mea

n tim

e sp

ent

(min

)

b b

ab

a

(a) (b)

Figure 2.11 Mean time spent out of 16 min by S. zeamais males (a) and females (b) in response to 2 g white maize seeds + 10% A. melegueta seeds in a four way olfactometer assay. Bars = standard errors (SE) of the means, n = 12. Bars with the same letter are not significantly different from each other (p>0.05). a-b (males), P=0.001; a-b (females), P=0.013.

59

0

1

2

3

4

5

6

Test Control1

Control2

Control3

Mea

n tim

e sp

ent

(min

)b

ab ab

a

0

1

2

3

4

5

6

Test Control1

Control2

Control3

Mea

n tim

e sp

ent

(min

) bb

b

a

(a) (b)

Figure 2.12 Mean time spent out of 16 min by S. zeamais males (a) and females (b) in response to 2 g white maize seeds + 10% Z. officinale rhizome in a four way olfactometer assay. Bars = standard errors of the means, n = 12. Bars with the same letter are not significantly different from each other (p>0.05). a-b (males), P=0.008; a-b (females), P=0.001.

0

1

2

3

4

5

6

Test Control1

Control2

Control3

Mea

n tim

e sp

ent

(min

) bb

b

a

0

1

2

3

4

5

6

Test Control1

Control2

Control3

Mea

n tim

e sp

ent

(min

) b b b

a

(a) (b)

Figure 2.13 Mean time spent out of 16 min by S. zeamais males (a) and females (b) in response to 2 g white maize seeds + 10% P. guineense seeds in a four way olfactometer assay. Bars = standard errors of the means, n = 12. Bars with the same letter are not significantly different from each other (p>0.05). a-b (males), P=0.004; a-b (females), P=0.043.

60

Table 2.5 Responses of Sitophilus zeamais to 2 g white maize in combination with 10% non-host plant volatiles in a four way olfactometer

Mean no. visits in olfactometer armTest stimulus n χ2* P*

i) White maize + 10% A. melegueta against controls T CMales 2.50 5.06 12 13.32 0.004Females 2.92 4.83 12 8.67 0.034ii) White maize + 10% Z. officinale against controlsMales 2.83 4.83 12 8.42 0.038Females 2.33 4.17 12 8.20 0.042iii) White maize + 10% P. guineense against controlsMales 3.09 5.53 12 9.43 0.024Females 2.92 4.58 12 6.48 0.091

Control arms contained clean filter paper discsT is the mean value of test armC is the mean value of the mean of three control arms*χ2 analysis was performed on the total number of visits (n=12) to the test, control 1, control 2 and control 3 arms in a 4 way olfactometer.

(iii) Winter wheat

With winter wheat combined with 10% non-host plant, the males showed significant

differences (P=0.031) in the mean time spent in test arm with winter wheat plus A.

melegueta (Figure 2.14a), and Z. officinale plus winter wheat seeds (P=0.004) (Figure

2.15a) than the control arms containing filter papers. The females were also

significantly repelled by A. melegueta (P=0.035) (Figure 2.14b), and Z. officinale

(P<0.001) (Figure 2.15b) in the mean time spent than the controls. There was no

significant difference in the mean time spent by males (P=0.170) (Figure 2.16a) and

females (P=0.623) (Figure 2.16b) in the test arm containing P. guineense plus winter

wheat seeds than the control arms. The number of visits made by males to the arm

containing A. melegueta plus winter wheat grains (χ2 = 9.07, df = 3, P=0.028), Z

officinale plus winter wheat grains (χ2 = 8.98, df = 3, P=0.029) and P. guineense plus

61

winter wheat kernels (χ2 = 8.87, df = 3, P=0.031) were significantly different between

the various treatments and control arms. However, females made significantly fewer

visits to A. melegueta plus winter wheat seeds (χ2 = 8.59, df = 3, P=0.035) and Z.

officinale plus winter wheat seeds (χ2 = 8.41, df = 3, P=0.038), but not significantly

different number of visits to P. guineense plus winter wheat seeds (χ2 = 3.29, df = 3,

P=0.349) (Table 2.6).

0

1

2

3

4

5

6

Test Control1

Control2

Control3

Mea

n tim

e sp

ent

(min

) b b b

a

0

1

2

3

4

5

6

Test Control1

Control2

Control3

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Figure 2.14 Mean time spent out of 16 min by S. zeamais males (a) and females (b) in response to 2 g winter wheat seeds + 10% A. melegueta seeds in a four way olfactometer assay. Bars = standard errors of the means, n = 12. Bars with the same letter are not significantly different from each other (p>0.05). a-b (males), P=0.031; a-b (females), P=0.035.

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Figure 2.15 Mean time spent out of 16 min by S. zeamais males (a) and females (b) in response to 2 g winter wheat seeds + 10% Z. officinale rhizome in a four way olfactometer assay. Bars = standard errors of the means, n = 12. Bars with the same letter are not significantly different from each other (p>0.05). a-b (males), P=0.004; a-b (females), P=0.001.

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Figure 2.16 Mean time spent out of 16 min by S. zeamais males (a) and females (b) in response to 2 g winter wheat seeds + 10% P. guineense seeds in a four way olfactometer assay. Bars = standard errors of the means, n = 12. There are no significant differences (P>0.05) between treatments.

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Table 2.6 Responses of Sitophilus zeamais to 2 g winter wheat kernels in combinationwith 10% non-host plant volatiles

Test stimulusMean no. visits in olfactometer arm n χ2* P*

i) Winter wheat + 10% A. melegueta against controls T CMales 3.50 5.91 12 9.07 0.028Females 2.67 4.53 12 8.59 0.035ii) Winter wheat + 10% Z. officinale against controlsMales 3.67 5.69 12 8.98 0.029Females 2.75 4.67 12 8.41 0.038iii) Winter wheat + 10% P. guineense against controlsMales 3.67 5.53 12 8.87 0.031Females 3.42 4.53 12 3.29 0.349

Control arms contained clean filter paper discsT is the mean value of test armC is the mean value of the mean of three control arms*χ2 analysis was performed on the total number of visits (n=12) to the test, control 1, control 2 and control 3 arms in a 4 way olfactometer.

2.3.4 Dose response bioassays of non-host plants with yellow maize as the host

plant.

(i) Yellow maize plus Aframomum melegueta

Yellow maize was selected for the dose response bioassay because the seeds were

more attractive to the male S. zeamais than white maize. Besides, yellow maize

contains carotene which makes it more acceptable for human nutrition compared to

white maize.

At 1% A. melegueta plus yellow maize seeds, both male and female S. zeamais were

not significantly (P>0.05) repelled from the test arm compared to the control arms in

the mean time spent. However, both sexes were significantly (P<0.001) repelled at the

same level from the test arms at 10 and 33% (w/w) of the test stimulus compared to

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the control arms (Table 2.7a). There was no significant difference (P>0.05) in the

mean number of visits by either sex to the test or control arms at 1% test stimulus. But

the males (χ2 = 9.31, df = 3, P=0.025) and females (χ2 = 11.84, df = 3, P=0.008)

significantly preferred the control arms compared to the test arms at 10%, and at 33%

both sexes showed similar behavioural responses and significantly (P<0.001) avoided

the test arms compared to the control olfactometer arms suggesting that the repellent

properties of A. melegueta are stronger at higher concentrations (Table 2.7b).

Table 2.7a Behavioural responses of Sitophilus zeamais measured as mean time spent in the test arm (min ± SE) containing 2 g yellow maize + 3 dosage levels of Aframomum melegueta seed volatiles in a 4-way olfactometer for 10min duration when compared with control arms.

Dosage of A. melegueta (%)Treatments 1 10 33MalesTest 2.31 ± 0.34a 1.00 ± 0.26a 0.70 ± 0.23aControl 1 2.49 ± 0.23a 2.93 ± 0.35b 2.83 ± 0.25bControl 2 2.37 ± 0.29a 3.17 ± 0.30b 3.36 ± 0.30bControl 3 2.28 ± 0.31a 2.50 ± 0.29b 2.74 ± 0.28bP-value 0.964 0.001 0.001FemalesTest 1.82 ± 0.26a 1.18 ± 0.27a 0.69 ± 0.20aControl 1 2.80 ± 0.29a 2.83 ± 0.23b 4.04 ± 0.24bControl 2 2.44 ± 0.27a 2.99 ± 0.27b 3.30 ± 0.25bControl 3 2.41 ± 0.31a 2.55 ± 0.23b 2.73 ± 0.20bP-value 0.112 0.001 0.001

Column means followed by the same letter(s) are not significantly different (P>0.05) after a one-factor analysis of variance and Tukey’s 95% simultaneous confidence intervals.

65

Table 2.7b Mean number of entries made by Sitophilus zeamais in response to 2 gyellow maize + 3 dosage levels of Aframomum melegueta seed volatiles when compared to control arms in a 4-way olfactometer for 10 min bioassay period

Dosage (test stimulus) Mean no. visits in olfactometer arm n χ2* P*

i)Yellow maize + 1% A. melegueta T CMales 3.17 3.19 12 0.12 0.989Females 3.33 3.78 12 0.59 0.899ii) Yellow maize + 10% A. melegueta Males 2.50 4.47 12 9.31 0.025Females 2.00 4.17 12 11.84 0.008iii) Yellow maize + 33% A. melegueta Males 1.67 4.42 12 19.1 0.001Females 1.92 4.44 12 15.71 0.001

Control arms contained clean filter paper discsT is the mean value of test armC is the mean value of the mean of three control arms*χ2 analysis was performed on the total number of visits (n=12) to the treatment, control 1, control 2 and control 3 arms in a 4 way olfactometer.

(ii) Yellow maize plus Zingiber officinale

At the lowest dose of 1% repellent to host plant (w/w), the males were attracted to Z.

officinale (P=0.003) in the mean time spent, but both sexes were significantly repelled

from the test arm at 10 and 33% (P<0.001) respectively when compared with the

control arms. The females did not show any preference for the test or control arms in

mean time spent at 1% Z. officinale (Table 2.8a). The weevils also did not show any

preference to the test or control arms in the mean number of visits at 1% Z. officinale.

However, at 10% Z. officinale, males (χ2 = 8.19, df = 3, P=0.042) and females (χ2 =

8.85, df = 3, P=0.031) significantly made fewer visits to the test arm than the control

arms. The 33% Z. officinale stimulus elicited a similar but stronger behavioural

response by males (χ2 = 18.18, df = 3, P<0.001) and females (χ2 = 21.49, df = 3,

P<0.001) suggesting that Z. officinale is more repellent at a higher concentrations

(Table 2.8b).

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Table 2.8a Behavioural responses of Sitophilus zeamais measured as mean time spent in the test arm (min ± SE) containing 2 g yellow maize + 3 dosage levels of Zingiber officinale in a 4-way olfactometer for 10 min duration when compared with control arms.

Dosage of Z. officinale (%)Treatments 1 10 33MalesTest 3.61 ± 0.39a 1.33 ± 0.18a 1.01 ± 0.33aControl 1 1.88 ± 0.29b 2.89 ± 0.25b 2.89 ± 0.30bControl 2 2.09 ± 0.24b 2.52 ± 0.21b 2.66 ± 0.31bControl 3 2.08 ± 0.26b 2.96 ± 0.26b 2.92 ± 0.30bP-value 0.003 0.001 0.001FemalesTest 3.26 ± 0.37a 1.25 ± 0.24a 0.78 ± 0.26aControl 1 1.90 ± 0.32a 2.52 ± 0.24b 2.60 ± 0.29bControl 2 2.28 ± 0.33a 2.96 ± 0.24b 3.25 ± 0.35bControl 3 2.02 ± 0.29a 2.84 ± 0.29b 2.98 ± 0.33bP-value 0.068 0.001 0.001

Column means followed by the same letter(s) are significantly different (P>0.05) after a one-factor analysis of variance and Tukey’s 95% simultaneous confidence intervals.

Table 2.8b Mean number of visits made by Sitophilus zeamais in response to 2 g yellow maize + 3 levels of Zingiber officinale volatiles in a 4-way olfactometer

Dosage (test stimulus) Mean no. visits in olfactometer arm n χ2* P*

i) 2 g yellow maize + 1% Z. officinale T CMales 3.08 2.61 12 1.06 0.787Females 4.17 2.97 12 4.04 0.257ii) 2 g yellow maize + 10% Z. officinaleMales 2.42 4.24 12 8.19 0.042Females 2.58 4.53 12 8.85 0.031iii) 2 g yellow maize + 33% Z. officinaleMales 1.67 4.39 12 18.18 0.001Females 1.67 4.67 12 21.49 0.001

Control arms contained clean filter paper discsT is the mean value of test armC is the mean value of the mean of three control arms*χ2 analysis was performed on the total number of visits (n=12) to the treatment, control 1, control 2 and control 3 arms in a 4 way olfactometer

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(iii) Yellow maize plus Piper guineense

When a combination of 1% P. guineense and 2 g yellow maize seeds (w/w) was tested

against blanks in a choice test, the mean time spent by males and females was not

significantly different (P>0.05) from the control arms. At 10% (w/w), males

(P<0.001) and females (P<0.001) significantly spent less time in the test than the

control arms. The behavioural response of both sexes to the test stimulus at 33%

(w/w) was similar to the responses at 10% (Table 2.9a). For the mean number of

visits, the weevils did not show any significant preference for either the test or control

arms at 1 and 10% respectively. But at 33% (w/w), males (χ2 = 8.3, df = 3, P=0.040)

and females (χ2 = 8.71, df = 3, P=0.033) made significantly fewer visits to the test

than the control arms (Table 2.9b). This result implies that P. guineense is not

repellent to S. zeamais at 10% (w/w) in the mean number of visit compared to A.

melegueta and Z. officinale.

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Table 2.9a Behavioural responses of Sitophilus zeamais measured as mean timespent in the test arm (min ± SE) containing 2 g yellow maize + 3 dosage levels of Piper guineense volatiles in a 4-way olfactometer for 10 min duration whencompared with control arms.

Dosage of P. guineense (%)

Treatments 1 10 33MalesTest 1.98 ± 0.31a 1.46 ± 0.27a 1.46 ± 0.25aControl 1 2.52 ± 0.24a 2.61 ± 0.23b 2.83 ± 0.29bControl 2 2.28 ± 0.22a 2.88 ± 0.29b 2.65 ± 0.25bControl 3 2.85 ± 0.30a 2.67 ± 0.26b 2.57 ± 0.26bP-value 0.143 0.001 0.001FemalesTest 2.04 ± 0.32a 1.25 ± 0.27a 1.29 ± 0.25aControl 1 2.53 ± 0.24a 2.99 ± 0.28b 2.69 ± 0.23bControl 2 2.57 ± 0.24a 2.74 ± 0.25b 3.09 ± 0.23bControl 3 2.51 ± 0.24a 2.69 ± 0.25b 2.55 ± 0.25bP-value 0.376 0.001 0.001

Column means followed by the same letter(s) are not significantly different (P>0.05) after a one-factor analysis of variance and Tukey’s 95% simultaneous confidence intervals.

Table 2.9b Mean number of visits made by Sitophilus zeamais in response to 2 g yellow maize + 3 dosage levels of Piper guineense volatiles in a 4-way olfactometer for 10 min duration

Dosage (test stimulus)Mean no. visits in olfactometer arm n χ2* P*

i) Yellow maize + 1% P. guineense T CMales 4.42 4.83 12 1.32 0.724Females 3.92 4.25 12 0.40 0.940ii) Yellow maize + 10% P. guineense Males 3.27 4.89 12 7.26 0.064Females 2.58 4.14 12 5.91 0.116iii) Yellow maize + 33% P. guineense Males 2.50 4.38 12 8.30 0.040Females 2.55 4.56 12 8.71 0.033

Control arms contained clean filter paper discsT is the mean value of test armC is the mean value of the mean of three control arms*χ2 analysis was performed on the total number of visits (n=12) to the treatment, control 1, control 2 and control 3 arms in a 4 way olfactometer

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(iv) Dose response percent repellency (PR)

The dose response mean percent repellency (%) showed that with A. melegueta, males

(51.06%) and females (61.69%) were repelled at 1% (w/w) in the mean time spent in

the test arm compared to the controls in olfactometer bioassays. At 10% (w/w), males

(79.17%) and females (75.37%), and at 33% dosage males (85.65%) and females

(87.11%) respectively were repelled from the test arm compared to the control arms

(Figure 2.17a, b). For the number of visits, the mean percent repellency at 1% A.

melegueta (w/w) was males (50.75%), females (54.25%), at 10% it was males

(68.96%), females (71.01%), and at 33%, males (77.39%) and females (74.41%)

(Figure 2.17c, d).

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Figure 2.17 Mean % repellency (PR) values for different dosages of A. melegueta volatile plus yellow maize grains against S. zeamais males (a) and females (b) in the mean time spent in the test arm; and males (c) and females (d) in the mean number of visits to the arm in a 4-way olfactometer bioassays. Bars = standard errors of the means.

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With Z. officinale, olfactometry bioassays revealed that the mean percent repellency

at 1% (w/w) in the mean time spent in the test arm compared to the controls was

males (25.68%) and females (31.65%). At 10% (w/w) dosage, the mean percent

repellency for the males (72.52%) and females (73.95%) was observed, and with 33%

(w/w) dose, males (78.68%) and females (83.56%) were repelled from the test arm

compared to the control arms (Figure 2.18a, b). In the number of visits to the

olfactometer arms, the mean percent repellency for males (40.01%) and females

(36.55%) was observed at 1% Z. officinale. But at 10% (w/w), males (72.52%) and

females (73.95%), and at 33% males (78.68%) and females (83.56%) were repelled

from the test compared with the control arms (Figure 2.18c, d).

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Figure 2.18 Mean % repellency (PR) values for different dosages of Z. officinale volatile plus yellow maize grains against S. zeamais males (a) and females (b) in the mean time spent in the test arm; and males (c) and females (d) in the mean number of visits to the arm in a 4-way olfactometer bioassays. Bars = standard errors of the means.

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The mean percent repellency of 1% P. guineense against males (58.96%) and females

(57.63%) was observed in the mean time spent by the weevil is the test arm compared

to the control arms. But at 10% (w/w), the mean % repellency against males

(69.56%) and females (74.32%) was achieved, and at 33% (w/w) males (69.36%) and

females (73.18%) were repelled from the test arm (Figure 2.19a, b). For the number of

visits, at 1% males (51.77%) and females (54.29%), at 10% males (66.97%) and

females (66.49%), while at 33% males (68.64%) and females (67.47%) were repelled

from the test arm compared to the control olfactometer arms (Figure 2.19c, d).

Generally, olfactometry bioassays showed that the repellency of A. melegueta, Z.

officinale and P. guineense volatiles against S. zeamais increased with dosage.

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Figure 2.19 Mean % repellency (PR) values for different dosages of P. guineense volatile plus yellow maize grains against S. zeamais males (a) and females (b) in the mean time spent in the test arm; and males (c) and females (d) in the mean number of visits to the arm in a 4-way olfactometer bioassays. Bars = standard errors of the means.

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2.4 DISCUSSION

The results of the olfactometer experiments with individuals of S. zeamais showed

that adult S. zeamais demonstrated clear orientation choices between the volatiles

generated by host and non-host plants, and volatiles that had been mixed with both.

Yellow maize, white maize and winter wheat kernels were found to be attractive to S.

zeamais in single choice tests, and this behavioural response was independent of the

sex of the weevil. Overall, the results of this study suggest that a variety of host plant

complexes emit volatile blends that are broadly attractive to S. zeamais adults.

The fact that S. zeamais showed positive behavioural responses to the different host

plant volatiles tested could suggest that this species may use the plant volatiles as cues

during the search for food and oviposition sites. Under natural conditions this

mechanism may occur when the first weevil preferably a male arrives at a food source

and releases aggregation pheromone (4S,5R-sitophinone) which in combination with

food odours contributes to aggregation of small colonies of con-specifics during the

initial phase of weevil infestation (Walgenbach et al., 1987). This result is in

agreement with Pike et al. (1994) who showed that S. zeamais was attracted to maize

volatiles, and identified the main volatile compounds as hexanoic acid, nonanoic acid,

nonanal, decanal, 2-phenylethanol, and vanillin which are common plant volatiles.

These volatiles which are typically present at low concentrations responsible for food

odour, and their sensory relevance is due to considerably lower odour thresholds

(Grosch, 2001). For example, Kaškonienė et al (2008) reported that the composition

of the volatile compounds of honey collected from various floral origins include

nonanoic acid, hexanoic acid, nonanal and decanal. Also the volatile aroma

compounds from different rice flavour types have been reported to include decanal

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and nonanal (Yang et al., 2008). Wakefield et al (2005) also reported the attraction

of S. zeamais and S. oryzae to carob volatiles alone, and significantly greater

attraction of the insects to fresh lures containing 4S,5R-sitophinone and carob

volatiles in pitfall bioassays. Recently, Germinara et al (2008) reported that the

congener, S. granarius adults have the ability to respond behaviourally to wide a

range of cereal volatiles and that response may change as a function of concentration.

In the present study, the attraction of yellow maize to S. zeamais dropped when 1% A.

melegueta and P. guineense were incorporated with yellow maize respectively.

However, with 1% Z. officinale yellow was still attractive to the male but not female

S. zeamais. Edde and Phillips (2006) working with Rhyzopertha dominica (Fab)

another stored-product pest, using a dual-choice, still-air bioassay method, observed

that both sexes of R. dominica were attracted to volatiles from wheat kernels, a plant

species judged to be most suitable to the insect. S. zeamais has a broad host range

including dry cassava tubers, sorghum and rice in addition to the host plants used for

these studies. This could mean that the weevil would require specialized olfactory

receptor neurons (ORN) for the recognition of volatile compounds associated with

each host plant. In many insects, olfactory receptor neurons are found in two

bilaterally symmetrical pairs of olfactory organs, the antennae and the maxillary palps

which functions mainly as contact chemoreceptors. The surfaces of the olfactory

organs are covered with sensory hairs (sensilla) which contain the ORN dendrites

(Hallem et al., 2006). Despite considerable variations in the general morphology of

olfactory organs across insect species, the structure of the olfactory sensillum is

broadly similar and consists of a cuticular wall containing multiple pores through

which odours can enter (Rospars, 1988; Shanbhag et al., 2000). Olfactory sensilla

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contain the dendrites of between one and five ORNs (Vermeulen and Rospars, 2004).

The axons of the ORNs project to functional processing units called glomeruli in the

antennal lobes of the brain (Hildebrand and Shepherd, 1997). In addition to ORNs, the

olfactory organs of several insect species contain smaller numbers of

mechanosensory, thermosensory, hygrosensory, and gustatory neurons (Rospars,

1988). Insect ORNs have been studied extensively by single-unit electrophysiology,

which is an extracellular recording technique used to examine the responses of single

ORNs to odours.

The high attractivity of yellow maize, white maize and winter wheat grains to S.

zeamais in the present study provide a starting point in the understanding of the

importance of volatile cues in host habitat location by the maize weevil. There are

other examples in the literature that demonstrate the attraction of host plant volatiles

to field insects using electrophysiology. Using gas chromatographic-

electroantennographic detection analysis (GC-EAD), Shepherd et al (2008) reported

the antennal responses of the western pine beetle; Dendroctonus brevicomis LeConte

to 42, out of 64 stem volatile compounds of its primary host, Pinus ponderosa Dougl.

et Laws (Pinaceae) and nine sympatric non-host angiosperms and conifers. Also,

nonanal and decanal were reported among the volatile compounds identified from the

shoots of riverbank grape, Vitis riparia that attracts the female grape berry moth,

Paralobesia viteana which attacks the plant (Cha et al., 2008). Van den berg et al

(2008) also showed that three antennally active components of sorghum, Sorghum

bicolor (L. Moench) panicles namely; 2-phenylethanol, benzyl alcohol and linalool in

specific ratios, played a significant role in the host attraction by the pollen beetle,

Astylus atromaculatus Blanchard. Similarly, decanal and nonanal collected from S.

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bicolor and Z. mays by air entrainment have been reported to elicit a large response in

the stemborer, Busseola fusca Fuller, a major pest of cereal crops in Africa (Birkett et

al., 2006). In aphids, Powell et al (2006) reported that host plant selection occurs as a

sequence of several successive behaviours. For instance, landing by flying aphids on

plant has been described to involve phototactic response to plant-reflected

wavelengths (Hardie, 1989), which is modified by plant volatiles (Nottingham and

Hardie, 1993) detected by antennal olfactory sensilla (Hardie et al., 1994). Park et al

(2000) using electroantennogram and linear-track olfactometer, demonstrated that the

male bird cherry-oat aphid, Rhopalosiphum padi, utilises the sex pheromones (-)-

(4aS,7S,7aR)-nepetalactol and (+)-(4aS,7S,7aS)-nepetalactone, and benzaldehyde, a

volatile which is released by the winter host plant, for mate and/or host-plant location

in the autumn. Webster et al (2008) also reported that the winged Aphis fabae Scop.,

was attracted to volatiles from the air entrainment sample of the faba bean, Vicia faba

in olfactometry behavioural and electrophysiological responses, and identified

decanal and methyl salicylate among the 16 electrophysiologically active compounds.

In repellency olfactometry assays, adult S. zeamais that were given the choice

between untreated controls containing clean filter paper discs and 2 g A. melegueta

seeds, Z. officinale rhizome, or P. guineense seeds significantly preferred the control

arms. This suggests that S. zeamais is able to detect these repellent plants through

olfaction and avoid them when given the choice. This could explain, at least in part,

how the application of these plants may protect grain from insect infestation in

storage. It was observed that avoidance of these repellent odours by S. zeamais

increases with dose increment because at 1%, none of these plant volatiles

significantly repelled the weevil. This may suggest that S. zeamais, like many other

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insect species, can potentially tolerate low levels of harmful substances (Rajendran

and Gunasekaran, 2002). However, at the higher doses of 10% and 33%, all the 3

plant products repelled S. zeamais during the behavioural evaluation in the mean time

spent in the test arm compared to the control arms. The results indicate that volatiles

of A. melegueta, Z. officinale, and P. guineense are repellent to S. zeamais at the right

concentrations, and that the presence of these volatiles can inhibit attraction of the

weevil to the host plant, stored maize. It is also possible that non-host plant volatiles

caused a change to the maize and winter wheat seeds by adhering to the surfaces of

the seeds so eliciting repellence, odour masking or other disruption that interfered

with the attraction to the host plant grains. These repellent actions increase the

potential practical value of these plants for grain protection against S. zeamais

infestation. These results are in line with ethnobotanical use by resource-poor farmers

in West Africa of plant powders and ashes to repel insects from stored products. It

was observed that 10% (w/w) P. guineense was not as efficient in repelling the

weevils compared to equal dosages of A. melegueta and Z. officinale in the mean

number of visits especially when combined with yellow maize grains. Notably, both

sexes of the weevil did not show any significant preference to the test or control arms

when 10% P. guineense was tested in combination with white maize and winter wheat

kernels. Considering the olfactometer results, it may be possible that the concentration

of behaviourally active volatiles in A. melegueta and Z. officinale could be higher

compared to P. guineense. However, the fact that insects’ response to particular plant

volatiles can be markedly influenced by the concentration of that volatile is common

in chemical ecology, as it has been reported for other weevil species (Chhabra et al.,

1999; Belmain et al., 2005).

80

The use of tropical plants to control stored-product pests through repellency,

immobilization or deterrent activity has been studied elsewhere. For example, Fields

et al. (2001) reported that wheat treated with Pisum sativum L. protein repelled C.

ferrugineus and S. oryzae in multiple choice laboratory tests. Hassanali et al (1997)

also reported that the bioactivity of materials derived from the leaves and succulent

stems of Ocimum kenyense evoked high repellency against S. zeamais, moderate

repellency against R. dominica and low repellency against S. cerealella in the

laboratory. Belmain et al (2003) corroborated these findings when they reported that

7.5-10.0% (w/w) Tephrosia vogelli Hook repelled 87.5% S. zeamais adults, while T.

vogelli at 2.5 % (w/w) and Lantana camara L. at 10% (w/w) repelled 65 and 62.5%

of the insect respectively.

2.5 CONCLUSIONS

The results reported here are of significance for the management of S. zeamais,

because repellent compounds may be used to mask or alter odours emitted from

stored maize to reduce the ability of the maize weevil to detect the source of food and

oviposition sites. Identification and testing of substances responsible for the repellent

effects of A. melegueta and Z. officinale reported here may yield further candidates

for use in post-harvest crop protection especially at traditional African small-scale

farmer level.

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CHAPTER 3:

EXTRACTION AND IDENTIFICATION OF BIOLOGICALLY ACTIVE

COMPONENTS FROM THE SEEDS OF A. melegueta AND RHIZOMES OF Z.

officinale.

82

3.1 INTRODUCTION

The Zingiberaceae is a tropical monocotyledon family, comprising about 1300

species, of which many produce essential oils, mainly in their rhizomes (Mabberley,

1987). Zingiberaceae species grow naturally in damp, shaded parts of the lowland or

on hill slopes, as scattered plants or thickets in West Africa. The genus Aframomum

comprises more than 10 species, which are endemic to the tropical regions of Africa.

The usual distinguishing features of Aframomum plants are the possession of strong

aromatic and pungent seeds. The most widely distributed and commercially important

of these species is A. melegueta (Roscoe) K. Schum. A. melegueta is a perennial herb,

which grows to about 1 m high. The plant possesses narrow leaves with distinctive

pink or lilac flowers. The reddish-brown seeds commonly referred to as “alligator

pepper” or “grains of paradise”, have a strong aromatic flavour and a pungent taste.

These seeds are widely employed as spices and are also ingredients in numerous West

African ethnomedical practices as a remedy for a number of diseases such as

constipation, rheumatic pains and fever (Ajaiyeoba and Ekundayo, 1999; Fernandez et

al., 2006). A few literatures reports have studied the biological activities of Nigerian-

grown A. melegueta seed oil including anti-microbial activity against a number of

micro-organisms (Oloke and Kolawale, 1988). A number of significant biological

activities in particular anti-inflammatory, antioxidant and antitumour effects

(Tjendraputra et al., 2001; Chung et al., 2001) have been reported from the seed

extracts of A. melegueta elsewhere.

Ginger is the rhizome of Zingiber officinale Roscoe (Zingiberaceae), a herbaceous

perennial species native of tropical Asia where it is rarely found in the wild today. It

is cultivated in most tropical countries, e.g. Australia, Brazil, China, Japan, Mexico,

83

West Africa, the West Indies and parts of the United States. The bulbous rhizome of

the 1-year-old plant is a horizontal creeper and serves as a means of storage,

reproduction and hibernation. The subterranean runners may grow up to 20 cm in

length. When grown, each piece of tuber produces an erect stalk about 1 m tall that

arises from the sheaths of long (about 20 cm) lanceolate leaves. The flowers are

arranged in a dense terminal spike and enveloped by layered bracts, whose axils house

the pale green to yellow, purple-spotted tubular flowers. The taste and pungency of

the harvested rhizome increase with growth and maturity. The completely unscraped

West African variety is reported to have the highest essential oil content and the most

pungent flavour (Langner et al., 1998; Singh et al., 2005). Z. officinale extract is used

to make ginger ale, a non-alcoholic drink (Langner et al., 1998), and confectionary

industries use it in the production of marmalade, pickles, chutney, ginger beer, ginger

wine, ginger biscuits and bakery products (Wohlmuth et al., 2005). Z. officinale and

its constituents have been reported to exhibit a wide range of pharmacological

activities, e.g. antibacterial (Yamada et al., 1992), antioxidant (Jitoe et al., 1992;

Kikuzaki and Nakatani, 1993), analgesic, anti-inflammatory, carminative, diuretic and

stimulating (Tanabe et al., 1993; Langner et al., 1998), and antifungal properties

(Singh et al., 2005) attributed to its pungent principles (Yamahara et al., 1989). For

centuries, Z. officinale has been known in Asia, Africa and other folk medicines as a

most effective remedy for rheumatic diseases, respiratory diseases, nervous diseases,

loss of appetite, vomiting, nausea, and convulsion in children (Langner et al., 1998;

Ali et al., 2008).

84

The aim of this chapter is to analyse and identify the behaviourally active components

of the essential oils of A. melegueta and Z. officinale for repellent activity against the

maize weevil, Sitophilus zeamais.

3.2 MATERIALS AND METHODS

3.2.1 Plant materials collection and preservation

Matured rhizomes of Z. officinale and ripe fruits of A. melegueta were collected from

fields around Akamkpa, while local yellow and white maize seeds were purchased

from Akim foodstuff market in Calabar, all in Cross River State (situated between

latitude 500 and 515 North and longitude 804 and 825 East) in southern Nigeria

in December 2005. The identity of the repellent plant materials was at the department

of Crop Science, University of Calabar, Nigeria. The plant materials were dried in the

shade for 3 days before transportation. In the laboratory, the repellent plant materials

and maize seeds were preserved in the refrigerator at – 20 C until needed for

experiments.

3.2.2 Preparation of essential oils

Approximately 50 g of partially dried Z. officinale rhizomes were chopped into small

pieces in a beaker and extracted with 50 ml of re-distilled diethyl ether. The container

was immersed in an ultrasonic wave device for 5 min to disperse and homogenized

the contents. The contents were transferred to a 100 ml round bottomed flask

connected to a vacuum distillation apparatus. The vacuum distillation apparatus was

then connected to a high vacuum pump (ES50 Vacuum Pump, Edwards, England).

The glass sections of the apparatus were strongly heated with a hot air blower to

remove any less volatile contaminants from its internal surfaces. The U-tube and the

85

pear-shaped vessel for the collection of the distillate were completely submerged in

liquid nitrogen at –196 C and the extract residue was then distilled, under a vacuum

of < 0.05 mm Hg for 24 h. Also, 50 g A. melegueta powder was vacuum distilled as

explained above. The ether distillates of these substances were then pipetted from the

vacuum distillation apparatus through long-drawn Pastuer pipettes into 50 ml

separation funnels to remove water. The extracts were dried using magnesium

sulphate (MgSO4), filtered and concentrated to obtain 3 ml Z. officinale and 4 ml A.

melegueta extracts. Each ether extract vacuum distillate was placed in different

ampoules, sealed under nitrogen and labelled accordingly and stored in the freezer at -

20 ºC until needed for laboratory assays against the maize weevil, S. zeamais or

chemical analysis.

3.2.3 Preparation of Oleoresins

100g seeds from dried fruits of A. melegueta were ground into fine powder using a

laboratory pestle and mortar (Maldenwanger, Berlin). The plant powder was then

extracted with methanol (200 ml) for 24 h at room temperature with additional stirring

using a magnetic stir bar (IKA Labortechnic Staufen, Germany) (Plate 3.1). The

extract was filtered through filter paper and the residue re-extracted for another 24 h

before filtration. Magnesium sulphate was added to the combined filtrate to remove

traces of moisture and then filtered again (Plate 3.2). Methanol was then removed by

evaporation under vacuum using a rotary evaporator (Rotavapor Buchi 461,

Switzerland) at room temperature to obtain the condensing pungent pale yellow

oleoresin. Z. officinale oil was obtained using the same procedure as described above.

Solutions of the oils in redistilled diethyl ether (10 mg in 10 ml) were prepared, sealed

86

under nitrogen and packed for laboratory assays to test for their repellency against the

maize weevil, S. zeamais.

Plate 3.1 Methanolic extraction of A. melegueta (left) and Z. officinale (right) oleoresins with additional stirring using magnetic stir bar.

Plate 3.2 Filtration of extract to obtain plant oils

87

3.2.4 Liquid column chromatography

A portion of the vacuum distilled essential oils of A. melegueta and Z. officinale were

fractionated by small-scale liquid chromatography through Florisil® (100-200 mesh),

using distilled hexane first and then diethyl ether as eluants, to obtain fractions

containing non-polar and polar components respectively.

3.2.5 Gas chromatography (GC) analysis of vacuum distillates

The chemical components of vacuum distilled essential oils of A. melegueta and Z.

officinale, chromatography fractions and oleoresins (1 mg/ml in diethyl ether) were

analysed using a 6890N gas chromatography (GC) system (Agilent Technologies)

equipped with a split-splitless injector (230°C) and flame ionization detector (FID).

Hydrogen was the carrier gas. The GC was equipped with a HP-5 capillary column

(30 m x 0.3 mm id, 0.25m film thickness). The oven temperature programme

comprised of an initial temperature of 30˚C for 0.5 min, a rise to 150˚C at 5 min, a

hold at 150˚C (0.1 min), another rise to 250˚C at 10˚C/min and final hold at 250˚C for

45 min. Results were obtained with an enhanced integrator (HP Chemstation).

The stereochemistry of the chiral compounds was determined by enantioselective gas

chromatography (GC) using authentic compounds using techniques similar to those

described in Birkett et al. (2008). Briefly, enantioselective GC of behaviourally active

A. melegueta and Z. officinale Florisil® diethyl ether fractions was performed on a

5890A GC equipped with a ß-cyclodextrin (Supelco beta-DEXTM 120; 30 m x 0.25

mm i.d., film thickness 0.25 µm) chiral capillary column. Hydrogen was the carrier

gas. The oven temperature programme comprised of an initial 40 ºC for 1 min, then

programmed to rise at 3 °C/min to 150 ºC, and at 5 ºC/min to 180 ºC, and maintained

88

at 180 ºC for 15 min. Initially, a 1-µl aliquot that contained equal quantities of both

enantiomers of the chiral compounds in redistilled hexane was injected onto the chiral

GC, to establish that successful separation of enantiomers took place. This was

followed by co-injections of the vacuum distilled essential oil sample, first with an

authentic standard of one enantiomer and then with the second enantiomer. Peak

enhancement with either enantiomer confirmed the presence of that enantiomer in the

vacuum distilled essential oil sample.

3.2.6 Coupled gas chromatography- mass spectrometry (GC-MS)

The GC-MS analyses of behaviourally active components of A. melegueta and Z.

officinale Florisil® diethyl ether fraction were performed using a fused silica capillary

column (30 m x 0.25 mm i.d., film thickness 0.25 µm, DB-5), fitted with an on-

column injector, which was directly coupled to a magnetic sector mass spectrometer

(Autospec Ultima, Fisons Instruments, Manchester, UK). Ionization was by electron

impact (70 eV, source temperature 250 ºC). Helium was the carrier gas. The oven

temperature was maintained at 30 ºC for 5 min, and then programmed at 5 ºC/min to

250 ºC. Tentative identifications were made by comparison of spectra with mass

spectral databases (NIST, 2005), and confirmed by peak enhancement on GC using

authentic compounds (Pickett, 1990). GC and GC-MS were performed with the

assistance of Dr. M.A. Birkett of Biological Chemistry department, Centre for

Sustainable Pest and Disease Management, Rothamsted Research, Harpenden, UK.

89

3.2.7 Preparation of synthetic blends.

A synthetic blend of the three major components found in the behaviourally active

Florisil® diethyl ether fraction of A. melegueta namely; (S)-2-heptanol, (S)-2-heptyl

acetate and (R)-linalool, was prepared based on their natural ratio of 1:6:3

respectively (Table 3.1). Similarly, a synthetic blend of the major compounds found

in the behaviourally active Florisil® diethyl ether fraction of Z. officinale vacuum

distillates viz: 1,8-cineole, neral and geranial, was prepared based on their natural

ratios of 5.48:1:2.13 respectively (Table 3.1). The synthetic solutions were sealed in

ampoules under nitrogen for storage prior to bioassay.

Table 3.1 Synthetic compounds tested for repellent activity against S. zeamais

Compound SourcePurity by GC (%)

(S)-2-Heptanol Sigma-Aldrich, Gillingham, Kent >99(S)-2-Heptyl acetate Synthesized from (S)-2-heptanol by acetylation >99(R)-Linalool Botanix Ltd, Paddock Wook, Kent >99Citral (neral + geranial) Fluka >951,8-Cineole Fluka >99

A step-by-step diagrammatic representation of the chemical isolation of the

components of A. melegueta and Z. officinale is shown in Figure 3.1.

90

Figure 3.1 Flow diagram of the chemical isolation procedures with accompanying bioassay of behaviourally active components from the seeds of A. melegueta and rhizomes of Z. officinale.

ESSENTIAL OIL PREPARATION

Diethyl ether extraction

Vacuum distillation

Vacuum distilled essential oils(bioassayed)GC and GC-MS analysis

Liquid chromatography

Non-polar hexane Fraction (bioassayed)

Polar diethyl ether fraction (bioassayed)

GC-MS analysis

Synthetic blends(bioassayed)

Single compounds(bioassayed)

OLEORESIN PREPARATION

Methanolic Extraction

Oleoresins(bioassayed)

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3.3 RESULTS

3.3.1 Chemical constituents of vacuum distilled essential oils of A. melegueta and

Z. officinale

The GC-MS analysis of the vacuum distilled essential oil from A. melegueta (Figure

3.2; Table 3.2) and Z. officinale (Figure 3.3; Table 3.3) revealed the presence of 13

and 24 terpenes hydrocarbons respectively. The most abundant constituents from A.

melegueta were tentatively identified as humulene (26.23%), (E)-ocimene (23.22%),

β-caryophyllene (19.17%), (S)-2-heptyl acetate (16.22%), (R)-linalool (8.68%), (S)-2-

heptanol (2.78%), β-pinene (0.64%), linalool oxide (0.59%), (E)-4,8-dimethyl-1,3,7-

nonatriene (0.32%), myrcene (0.21%), α-pinene (0.19%), bisabolene (0.07%) and

germacrene-D (0.06%). Chemical analysis of the volatile essential oil constituents of

Z. officinale consisted mainly of mono- and sesquiterpene hydrocarbons including

1,8-cineole (22.63%), camphene (16.87%), geranial (11.25%), zingiberene (10.63%),

α-pinene (6.39%), neral (5.03%), (E,E)-α-farnesene (3.69%), β-cadinene (3.38%), γ-

cadinene (3.21%) and myrcene (3.20%). Other compounds identified from Z.

officinale were α-muurolene (1.10%), α-curcumene (1%), sabinene (0.84%), (R)-

linalool (0.83%), 3-carene (0.71%), terpinolene (0.66%), borneol (0.63%), α-

terpineole (0.51%), copaene (0.39%), tricyclene (0.33%), β-pinene (0.25%),

cyclosativene (0.20%), borneol acetate (0.15%) and 6-methyl-5-hepten-2-one

(0.05%).

92

DAU/AlligatorpepperVacDist

Time15.00 20.00 25.00 30.00 35.00 40.00

%

0

100

251006MAB01 Magnet EI+ TIC

3.44e6

1

23 4

56

7

8

9 10

11 12

1314

X

Figure 3.2 Total ion chromatogram (TIC) obtained by coupled GC-MS analysis of vacuum distilled essential oil of Aframomum melegueta. Peak numbers correlate to compounds listed in Table 3.2 identified by coupled GC-MS.

Table 3.2 Compounds identified from coupled GC-MS analysis of vacuum distilled essential oil obtained from the seeds of Aframomum meleguetaPeak no. Compound % Peak area by GC

1 (S)-2-Heptanol 2.782 α-Pinene 0.193 β-Pinene 0.644 Myrcene 0.215 (S)-2-Heptyl acetate 16.226 (E)-Ocimene 23.227 Linalool oxide (furan) 0.598 (R)-Linalool 8.689 (E)-4,8-Dimethyl-1,3,7-nonatriene 0.3210 m/z (unidentified) 1.1811 β-Caryophyllene 19.1712 Humulene 26.2313 Germacrene-D 0.0614 Bisabolene 0.07

X = impurities which accounted for 0.44% peak area by GC.

93

DAU/GingerrootVacDist

Time15.00 20.00 25.00 30.00 35.00 40.00

%

0

100

261006MAB01 Magnet EI+ TIC

3.52e6

x 1

2

3

4

56

7

8 10 11 1213

x

9

14

15

16 1718

19

20

21 22

23

24

x

Figure 3.3 Total ion chromatogram (TIC) obtained by coupled GC-MS analysis of vacuum distilled essential oil of Zingiber officinale. Peak numbers correlate to compounds listed in Table 3.3 identified by coupled GC-MS.

94

Table 3.3 Compounds identified from coupled GC-MS analysis ofvacuum distilled essential oil of Zingiber officinale rhizomePeak no. Compound % Peak area by GC

1 Tricyclene 0.332 α-Pinene 6.393 Camphene 16.874 6-Methyl-5-hepten-2-one 0.055 Sabinene 0.846 β-Pinene 0.257 Myrcene 3.208 3-Carene 0.719 1,8-Cineole 22.6310 Terpinolene 0.6611 (R)-Linalool 0.8312 Borneol 0.6313 α-Terpineol 0.5114 Neral 5.0315 Geranial 11.2516 Borneol acetate 0.1517 Cyclosativene 0.2018 Copaene 0.3919 α-Curcumene 1.0020 α-Muurolene 1.1021 Zingiberene 10.6322 (E,E)-α-farnesene 3.6923 γ-Cadinene 3.2124 β-Cadinene 3.38

X=impurities accounting for about 6.01% peak area by GC.

3.3.2 Chemical composition of A. melegueta and Z. officinale Florisil® diethyl

ether essential oil fraction

GC-MS analysis of the behaviourally active Florisil® diethyl ether fractions of A.

melegueta and Z. officinale vacuum distillates showed the presence of 3 major

compounds from each extract (Figure 3.4; Table 3.4). Compounds identified from A.

melegueta were (S)-2-heptanol, (S)-2-heptyl acetate and (R)-linalool. From Z.

officinale, 1,8-cineole, neral and geranial were identified. The stereochemistry of

these compounds was determined by enantioselective gas chromatography (GC) using

authentic samples (Figure 3.5, 3.6).

95

Aframomum melegueta

Zingiber officinale

Retention Time (Min)

Pea

k A

rea

GC/GC-MS Analysis

1

2

3

4

5

6

Figure 3.4. Total ion chromatogram (TIC) obtained by coupled GC-MS analysis of A. melegueta and Z. officinale Florisil® diethyl ether fraction obtained by liquid chromatography. Labels correspond to peak numbers in Table 3.4.

Table 3.4 Compounds identified from coupled GC-MS analysis of A. melegueta and Z. officinale vacuum distilled Florisil® diethyl ether fractions.Peak no. Compound Plant source % Peak area by GC

1 (S)-2-Heptanol A. melegueta 38.262 (S)-2-Heptyl acetate A. melegueta 27.923 (R)-Linalool A. melegueta 33.814 1,8-Cineole Z. officinale 45.945 Neral Z. officinale 14.966 Geranial Z. officinale 34.72

96

O

O

(S)-2-Heptyl acetate

OH

(S)-2-Heptanol

OH

(R)-Linalool

Figure 3.5. Chemical structures of compounds identified from coupled GC-MS analysis of A. melegueta diethyl ether fraction, obtained by liquid chromatography over Florisil®.

O

1,8-Cineole

OH

Neral

O

H

Geranial

Figure 3.6. Chemical structures of compounds identified from coupled GC-MS analysis of Z. officinale diethyl ether fraction obtained by liquid chromatography over Florisil®.

97

3.4 DISCUSSION

The main objective of this chapter was to isolate and identify the major components

with repellent activity against S. zeamais. GC and GC-MS analyses of A. melegueta

seed and Z. officinale rhizome essential oils, which were shown to be repellent,

revealed the presence of a number of components, including monoterpenes,

sesquiterpenes, oxygenated monoterpenes and alcohols. The repellent activity of the

oils was accounted for by diethyl ether fractions that were isolated by liquid column

chromatography. The volatile constituents of A. melegueta with repellent properties

were (S)-2-heptyl acetate, (S)-2-heptanol and (R)-linalool, and from Z. officinale were

1,8-cineole, neral and geranial. There are few reports on the chemical composition of

the essential oil or extracts from the seeds of A. melegueta. In Cameroon, Menut et al

(1991) identified nine compounds from the essential oil composition of A. melegueta

obtained by hydrodistillation with α-humulene and ß-caryophyllene (39.8%) and their

epoxides (45.6%) as main constituents. In 1995, Escoubas et al isolated and identified

four aryldecanones namely gingerdione, paradol, gingerol and shogaols, and eight

minor compounds from n-hexane and methanolic seed extracts of A. melegueta

obtained from south western Nigeria. Ajaiyeoba and Ekundayo (1999) isolated a total

of 27 compounds including linalool from the essential oil of A. melegueta seeds from

Nigeria, but with the quantitative preponderance of α-humulene and ß-caryophyllene

making up 82.6% of the oil. Two other sesquiterpene hydrocarbons, germacrene-D

and δ-cadinene, occurred in trace amounts (0.1%), while 3 oxides namely humulene

oxide I, humulene oxide II and caryophyllene oxide were identified in relatively

substantial amounts (9%) in the volatile oil. Lately, the compositions of absolute and

supercritical CO2 extract of A. melegueta purchased from Côte d’Ivoire have also

been analyzed by GC and GC-MS with 33 components, representing more than 98.3%

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of the absolute, 43 components representing more than 98.2% of the supercritical

fluid extraction (SFE) products identified (Fernandez et al., 2006). The analysis

revealed that absolute contained four oxygenated monoterpenes (0.2%), 11

sesquiterpene hydrocarbons (15.9%), six oxygenated sesquiterpenes (1.6%) and five

phenolic alkenones (75.7%). For the SFE, eight monoterpene hydrocarbons (2.9%),

five oxygenated monoterpenes (0.8%), 11 sesquiterpene hydrocarbons (12.2%), seven

oxygenated sesquiterpenes and five phenolic alkenones (70.5%) were found. The

result from this study is in agreement with Fernandez et al (2006) who identified

heptan-2-ol from SFE, while 2-heptyl acetate and linalool were obtained from both

absolute and SF extraction methods. The volatile constituents responsible for the

characteristic pleasant smell are present in the essential oil, but the essential oil only

contributes partially to the flavour impact. The pungency of the seeds has been

reported to be due to non-volatile phenolic alkenones known as gingerols, shogaols

and paradols with various biological activities (Lee and Surh, 1998; Tjendraputra et

al., 2001; Chung et al., 2001). The oleoresin (when resins are associated with volatile

oils) of A. melegueta obtained by solvent extraction is a combination of both the

important characteristics such as aroma and pungency compounds in the same extract.

Several methods for the determination of aroma and pungent compounds in Z.

officinale have been described, and the constituents of the rhizome have been

examined for quality and quantity. Depending on the place of origin, the powdered

rhizome contains 3-6% fatty oil, 9% protein, 60-70% carbohydrates, 3-8% raw fibre,

up to 8% ash, 9-12% water, and 2-3% volatile oil (Langner et al., 1998). The volatile

oil consists mainly of mono- and sesquiterpenes namely; camphene, ß-phellandrene,

curcumene, cineole, geranyl acetate, terpineol, terpenes, borneol, geraniol, neral, α-

99

pinene, 1,8-cineole, limonene, linalool, myrcene, alpha-(-)zingiberene, ß-

sesquiphellandrene, ß-bisabolene and (E)(E)-α-farnesene (Harvey, 1981; MacLeod

and Pieris, 1984; Langner et al., 1998 ). The results from this study confirmed the

report of Ekundayo et al (1988) who isolated 54 components including geranial, neral,

1,8-cineole, zingiberene, ß-sesquiphellandrene and ß-bisabolene from the essential

oils of Nigerian Z. officinale rhizomes. Using solid-phase microextraction (SPME),

combined with comprehensive two-dimensional (2D) gas chromatography (GC X

GC), Shao et al (2003) reliably identified 36 compounds including neral, geranial, ß-

sesquiphellandrene, ß-phellandrene, camphene, α-muurolene, α-farnesene, α-

zingiberene, Z-α-bisabolene, α-pinene, myrcene and γ-curcumene from the volatile

fraction of fresh ginger. The results are also in agreement with the findings of Singh et

al (2005) who reported that the major components of GC-MS analysis of fresh Z.

officinale essential oil were α-zingiberene (28.62%), camphene (9.32%), ar-

curcumene (9.09%), ß-sesquiphellandrene (8.24%), ß-phellandrene (7.97%), E,E-α-

farnesene (5.52%), ß-bisabolene (5.40%), α-pinene (2.57%), geranial (2.08%), endo-

borneol (2.04%), neral (1.72%), valencene (1.42%), 1,8-cineole (1.20%) and

germacrene D (1.03%). Similarly, Nishimura (1995) identified various components

from the fresh rhizomes of ginger having high flavour dilution factor including

linalool, geraniol, geranial, neral, isoborneol, borneol, 1,8-cineole, 2-pinen-5-ol,

geranyl acetate, (E)-2-octenal, (E)-2-decenal and (E)-2-dodecenal. The flavour

dilution factor (FD) for a compound is the ratio of its concentration in the initial

extract to its concentration in the most diluted extract in which the odour was detected

by gas chromatography-olfactometry (GCO). The odour of Z. officinale is not

characterized by one particular compound, but depends mainly on its volatile oil

which is composed of a mixture of various terpenoids as well as some non-terpenoids

100

(Nishimura, 1995; Ali et al., 2008). Some of the aroma-defining components

including geranial, neral and limonene are converted into less odour-defining

metabolites on drying (Sakamura, 1987). Wohlmuth et al (2005) reported that the

pungency of fresh ginger was due primarily to the gingerols, which are a homologous

series of phenols and the most abundant is (6)-gingerol. While, the pungency of dry

ginger mainly results from shogaols, e.g. (6)-shogaol which are dehydrated forms of

gingerols. Shogaols are therefore formed from corresponding gingerol during thermal

processing. Degradation rates of (6)-gingerol to (6)-shogaol were also found to be pH

dependent, with greatest stability at pH 4, whereas at 100 °C and pH 1, the reversible

degradation was relatively rapid (Bhattarai et al., 2001). Singh et al (2005) also

reported that the oleoresin of Z. officinale accounted for 88.63% of the total oil, with

trans-6-shogaol (26.32%), trans-10-shogaol (13.0%), α-zingiberene (9.66%), trans-8-

shogaol 7.72%), 10-gingerdione (6.80%), cis-6-shogaol (3.31%), ß-

sesquiphellandrene (2.94%), ar-curcumene (2.76%), 6-gingerdiol diacetate (2.00%),

β-bisabolene (1.98%), 6-gingerol (1.87%), E,E-α-farnesene (1.63%), 6-paradiol

(1.50%) and cis-8-shogaol (1.21%) as the major components. Many of these

compounds and crude extractives are known for their medicinal importance (Denyer

et al., 1994; Katiyar et al., 1996).

3.5 CONCLUSIONS

GC and GC-MS analysis showed that the essential oil of A. melegueta from Nigeria

contains 40.48% monoterpenes, 9.59% oxygenated monoterpenes, 45.53%

sesquiterpene hydrocarbons and 2.78% alcohol. (S)-2-Heptyl acetate (16.22%) and

(E)-ocimene (23.22%) were the main monoterpenes, (R)-linalool (8.68%) the major

oxygenated monoterpene, and β-caryophyllene (19.17%) and humulene (26.23%) the

101

main sesquiterpene hydrocarbon fractions. Z. officinale essential oil contain 29.30%

monoterpenes with camphene (16.87%) as the major and sulcatone (6-methyl-5-

hepten-2-one) as minor monoterpenes respectively, 41.03% oxygenated

monoterpenes, and 23.60% sesquiterpene hydrocarbons. The major oxygenated

monoterpenes were 1,8-cineole (22.63%), geranial (11.25%) and neral (5.03%), and

zingiberene (10.63%) the main sesquiterpenes hydrocarbon compound. There are

major differences in chemical composition between samples of essential oils obtained

from aromatic plants of the same species or genus, which may be due to genetic,

environmental, developmental or other factors. The chemical composition of the

oleoresins also depends on whether the plant product is fresh or dried, as well as the

nature of solvents used for the extraction. This study shows that using analytical

techniques such as liquid chromatography and GC-MS, it was possible to identify

biologically active compounds that could eventually be used as repellents against S.

zeamais in stored-product protection.

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CHAPTER 4:

BIOACTIVITY OF Aframomum melegueta AND Zingiber officinale EXTRACTS

AND SINGLE COMPONENTS AGAINST Sitophilus zeamais

103

4.1 INTRODUCTION

Insect damage in stored food grains may amount to 10% - 40% in countries where

modern storage technologies have not been introduced (Shaaya et al., 1997) or are

inadequate. Among the stored-product Coleoptera, the maize weevil, Sitophilus

zeamais is an important primary pest of stored maize in the tropics (Wakefield et al.,

2005). It is an internal feeder and causes considerable loss to stored maize affecting

the quantity and quality of the grains. In many storage systems, applications of

contact chemical insecticides have been used for the control of this pest. More

frequently, fumigation with appropriate chemical substances such as methyl bromide

and aluminium phosphide are the most economical and convenient tools for managing

the maize weevil because of their ease of penetration into the commodity while

leaving minimal residues (Bond, 1984). But safety and environmental impact

concerns about the continuous application of these chemical substances have

prompted the search for more environmentally sound and novel methods for the

control of storage pests. Globally, the management of stored product pests using plant

substances has been the subject of much research (Isman, 2006), and there has been a

growing interest in research concerning the possible use of plant extracts as

alternatives to synthetic insecticides in stored-product protection (Obeng-Ofori and

Reichmuth, 1997; Shaaya et al., 2003; Sahaf, et al., 2007). A large number of plant

species used traditionally as medicines have been reported to possess bioactivities

against several insect species (Ivbijaro and Agbaje, 1986; Singh and Upadhyay, 1993;

Bekele et al., 1995). Among possible strategies aimed at reducing the use of synthetic

insecticides and fumigants, natural repellents produced by edible plants represent a

vital approach for ecochemical control of stored-product insect pests (Adler et al.,

2000; Rozman et al., 2007). Plant essential oils may act as fumigants, contact

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insecticides, repellents, deterrents and antifeedants to storage insect species

(Hassanali et al., 1997; Isman, 2000; 2006; Bekele and Hassanali, 2001; Huang et al.,

2000; Stamopoulos, 1991; Shakarami et al., 2004; Rajendran and Sriranjini, 2008).

The chemical compositions of Aframomum melegueta and Zingiber officinale (both

Zingiberaceae) essential oils have been reported (Ekundayo et al., 1988; Ajaiyeoba

and Ekundayo, 1999; Fernandez et al., 2006; Ali et al., 2008). These studies show that

the essential oils of these plants comprise mainly of monoterpenoid and

sesquiterpenoid hydrocarbons some of which have been extracted from other plants

and which have been reported to possess repellent properties against some stored-

product pests (Tapondjou et al., 2005; Stamopoulos et al., 2007; Sahaf et al., 2007).

Z. officinale has been reported to possess antimicrobial properties (Yamada et al.,

1992; Singh et al., 2005), and Escoubas et al (1995) indicated that A. melegueta

essential oils exhibited termite antifeedant activity. The objective of this chapter was

to determine the repellent activity of A. melegueta and Z. officinale vacuum distilled

essential oils, their synthetic blends, oleoresins and identified bioactive compounds

against S. zeamais in olfactometry behaviour experiments.

4.2 MATERIALS AND METHODS

4.2.1 Maize weevils

The test insects were obtained from my laboratory colony reared in medium-sized

heating laboratory incubator (Gallenkamp, UK) maintained at 25 ºC on untreated

yellow maize grains purchased from Akim foodstuff market in Calabar, Cross River

State, Nigeria.

105

4.2.2 Repellency bioassays

All bioassays were carried out using a 4-way airflow olfactometer using the same

procedure described in Chapter 2 but the duration of each replicate was 10 min. The

test stimuli or odour sources tested for repellent activity against 3 d old virgin adult

male and female S. zeamais were; A. melegueta and Z. officinale vacuum distilled

essential oils tested individually and in combination with 2 g yellow maize grains,

diethyl ether and hexane fractions of each vacuum distillate, synthetic blend of diethyl

ether fraction of each vacuum distillate tested singly and in combination with maize

grains, oleoresin of A. melegueta and Z. officinale tested singly and in combination

with maize grains, single compounds identified from the diethyl ether fraction of A.

melegueta and Z. officinale vacuum distillates, as well as comparison of all bioactive

fractions for repellent activity against S. zeamais. In each assay the odour source was

10 µl of test substance impregnated onto filter paper discs and tested singly or in

combination with 2 g yellow maize grains, while 10 µl of the solvent used for

extraction of the substance served as control.

4.2.3 Data analysis

The time spent in each olfactometer arm was tested using a one-way analysis of

variance (ANOVA) followed by comparison of means by Tukey’s 95% simultaneous

confidence intervals (MINITAB 15 Statistical Software). For data on the number of

visits, the null hypothesis was that the weevils behaved randomly and choose each

olfactometer arm with a 25% frequency. The number of visits to the odour-treated

arm was compared with the number of visits in control arms using a “global” chi-

square contingency table (Zar, 1999). Upon rejection of that hypothesis, data were

analysed by targeted pairwise comparisons using a 2 x 2 χ2 contingency table (Zar

106

1999). Comparison of percent repellency (PR) values for all tests were computed as

PR = [(Nc-Nt)/(Nc+Nt)] x 100. Where Nc is the time spent or number of visits to the

control arm, and Nt is the time spent or number of visits to the treated arm.

4.3 RESULTS

4.3.1 Vacuum distilled essential oils of A. melegueta and Z. officinale

In control olfactometry assays, both male (Figure 4.1a) and female (Figure 4.1b) S.

zeamais did not show any significant preference (P>0.05) to either the 10 µl diethyl

ether treated arm or blank arms in the mean time spent and mean number of visits in

the arms (Table 4.1).

0

1

2

3

Diethylether

Control1

Control2

Control3

Mea

n tim

e sp

ent

(min

)

0

1

2

3

Diethylether

Control1

Control2

Control3

Mea

n tim

e sp

ent

(min

)

(a) (b)

Figure 4.1 Mean time spent in the arm out of 10 min by S. zeamais males (a) and females (b) in response to 10 µl diethyl ether and three control arms in a four way olfactometer, Bars = standard errors (SE) of the means, n = 12.

107

Table 4.1 Behavioural responses of Sitophilus zeamais to 10 µl redistilled diethyl ether and blank control arms in a four way olfactometer.

Test stimulusMean no. visits in olfactometer arm n χ2* P*

10 µl diethyl ether against blanks T CMales 4.67 4.83 12 0.99 0.8037Females 4.42 4.39 12 0.24 0.9709

The test olfactometer arm contained 10 µl diethyl ether loaded onto filter paper disc while the three control arms contained blank filter paper discs.*χ2 analysis was performed on the total number of visits (n =12) into the test, control 1, control 2 and control 3 arms in a 4 way contingency table.

The essential oil of A. melegueta obtained by vacuum distillation showed significant

repellency to both the male (Figure 4.2a) and female (P<0.001) (Figure 4.2b) weevils

in the mean time spent in the test arm when tested individually when compared with

the control arms. In combination with maize grains, A. melegueta essential oil was

still repellent to the males (P<0.001) (Figure 4.2c) and females (P<0.001) (Figure

4.2d) when compared with the control arms. In the mean number of visits, both males

(χ2 =14.53, df =3, P=0.002) and females (χ2 = 13.2, df =3, P=0.004) significantly

preferred control arms to the test arm in response to 10 µl A. melegueta essential oil.

With maize, males (χ2 = 7.62, df =3, P=0.055) and females (χ2 = 11.68, df =3,

P=0.009) still showed preference for the control arms compared to the test arm in the

mean number of visits (Table 4.2).

108

0

1

2

3

4

A.melegueta

Diethylether

Diethylether

Diethylether

Mea

n tim

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min

) b

bb

a

0

1

2

3

4

A.melegueta

Diethylether

Diethylether

Diethylether

Mea

n tim

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ent (

min

)

bb b

a

(a) (b)

0

1

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4

Test Diethylether

Diethylether

Diethylether

Mea

n tim

e sp

ent (

min

) b b b

a

0

1

2

3

4

Test Diethylether

Diethylether

Diethylether

Mea

n tim

e sp

ent (

min

) bb

b

a

(c) (d)

Figure 4.2 Mean time spent in the arm out of 10 min by male S. zeamais in response to 10 µl Aframomum melegueta vacuum distillate tested individually (a) and in combination with 2 g maize grains (c), and mean time spent by females in response to 10 µl A. melegueta vacuum distillate tested individually (b) and in combination with 2 g maize grains (d) in a four way olfactometer. Bars = standard errors of the means, n = 12. Bars followed by the same letter are not significantly different from each other (P>0.05). a-d: (males, females), P<0.001

109

Table 4.2 Responses of Sitophilus zeamais to volatiles from 10 µl Aframomum melegueta vacuum distillates and in combination with maize grains in a four way olfactometer.

Test stimulusMean no. visits in olfactometer arm n χ2* P*

i) 10 µl A. melegueta T CMales 2.5 5.14 12 14.53 0.002Females 2.67 5.19 12 13.2 0.004ii) 10 µl A. melegueta + 2 g maizeMales 2.67 4.44 12 7.62 0.055Females 2.5 4.81 12 11.68 0.009

Each control arm contained 10 µl diethyl ether loaded on clean filter paper discT is the mean value of test armC is the mean value of the mean of three control arms*χ2 analysis was performed on the total number of visits (n =12) into the test, control 1, control 2 and control 3 arms in a 4 way contingency table.

Similarly, air passing over 10 µl Z. officinale vacuum distilled essential oil elicited

significant (P<0.001) repellent activity to both males (Figure 4.3a) and females

(Figure 4.3b) when tested individually, and to males (P<0.001) (Figure 4.3c) and

females (P<0.001) (Figure 4.3d) in combination with maize grains, in the mean time

spent in the arms. Z. officinale essential oil also significant repelled males (χ2 = 12.5,

df =3, P=0.006) and females (χ2 = 12.99, df =3, P=0.005) when tested alone, and

males (χ2 = 12.93, df =3, P=0.005), females (χ2 = 8.48, df =3, P=0.037) in

combination with maize grains in the mean number of visits to the test arm (Table

4.3).

110

0

1

2

3

4

Z.officinale

Diethylether

Diethylether

Diethylether

Mea

n tim

e sp

ent (

min

)b

b b

a

0

1

2

3

4

Z.officinale

Diethylether

Diethylether

Diethylether

Mea

n tim

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ent (

min

)

bb b

a

(a) (b)

0

1

2

3

4

Test Diethylether

Diethylether

Diethylether

Mea

n tim

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ent (

min

)

bb

b

a

0

1

2

3

4

Test Diethylether

Diethylether

Diethylether

Mea

n tim

e sp

ent (

min

)

b b b

a

(c) (d)

Figure 4.3 Mean time spent in the arm out of 10 min by male S. zeamais in response to 10 µl Zingiber officinale vacuum distillate tested individually (a) and in combination with 2 g maize grains (c), and mean time spent by females in response to 10 µl Z. officinale vacuum distillate tested individually (b) and in combination with 2 g maize grains (d) in a four way olfactometer. Bars = standard errors of the means, n = 12. Bars followed by the same letter are not significantly different from each other (P>0.05). a-d: (males, females), P<0.001.

111

Table 4.3 Responses of Sitophilus zeamais to volatiles from 10 µl Zingiber officinale vacuum distillate tested alone and in combination with maize grains in a four way olfactometer.

Test stimulusMean no. visits in olfactometer arm n χ2* P*

i) 10 µl Z. officinale T CMales 2.5 4.94 12 12.5 0.006Females 2.5 4.97 12 12.99 0.005ii) 10 µl Z. officinale + 2 g maizeMales 2.33 4.69 12 12.93 0.005Females 2.83 4.75 12 8.48 0.037

Each control arm contained 10 µl diethyl ether loaded on clean filter paper discT is the mean value of test armC is the mean value of the mean of three control arms*χ2 analysis was performed on the total number of visits (n =12) into the test, control 1, control 2 and control 3 arms in a 4 way contingency table.

4.3.2 Vacuum distilled Hexane and Diethyl ether fractions of A. melegueta and Z.

officinale essential oils.

Olfactometry bioassay results also showed that both male and female weevils did not

respond significantly to the hexane fraction of A. melegueta in the mean time spent in

the test arm (Figure 4.4a, b) compared to the control arms. The hexane fraction of Z.

officinale elicited significant differences in the mean time spent in the arm with

overall P=0.042 (Figure 4.4c), but there was no significant difference between the test

arm and control arms 1 and 2. In the mean number of visits, the weevils did not show

any significant preference to the test or the control arms when the hexane fractions of

A. melegueta and Z. officinale vacuum distillates were tested (Table 4.4).

112

0

1

2

3

4

Aframomummelegueta

Hexane Hexane Hexane

Mea

n tim

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ent (

min

)

0

1

2

3

4

Aframomummelegueta

Hexane Hexane Hexane

Mea

n tim

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ent (

min

)

(a) (b)

0

1

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Zingiberofficinale

Hexane Hexane Hexane

Mea

n tim

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ent (

min

)

b

a a a

0

1

2

3

4

Zingiberofficinale

Hexane Hexane Hexane

Mea

n tim

e sp

ent (

min

)

(c) (d)

Figure 4.4 Mean time spent out of 10 min by S. zeamais males (a) and females (b) in response to 10 µl Aframomum melegueta hexane fraction vacuum distillate, and mean time spent by males (c) and females (d) in response to 10 µl Zingiber officinale hexane fraction vacuum distillate in a four way olfactometer. Bars = standard errors of the means, n = 12. Bars followed by the same letter are not significantly different from each other (P>0.05). a-d: a (males), P=0.982; b (females), P=0.688; c (males), P=0.042; d (females), P=0.625.

113

Table 4.4 Mean number of visits into the arms of the olfactometer made by Sitophilus zeamais in response to 10 µl hexane fractions of vacuum distilled Aframomum melegueta and Zingiber officinale extracts.

Test stimulusMean no. visits in olfactometer arm n χ2* P*

i) A. melegueta hexane fraction T CMales 3.83 3.73 12 0.37 0.946Females 2.75 2.78 12 0.38 0.944ii) Z. officinale hexane fractionMales 4.33 4.58 12 2.93 0.403Females 2.92 3.03 12 0.28 0.964

Each control arm contained 10 µl hexane loaded on clean filter paper discT is the mean value of test armC is the mean value of the mean of three control arms*χ2 analysis was performed on the total number of visits (n =12) into the test, control 1, control 2 and control 3 arms in a 4 way contingency table.

However, there were significant differences between the diethyl ether fractions of A.

melegueta and Z. officinale vacuum distilled essential oils and the control arms. In the

mean time spent, males (P=0.006) and females (P=0.002) were significantly repelled

by 10 µl A. melegueta (Figure 4.5a, b), and males (P=0.005), and females (P=0.004)

by 10 µl Z. officinale respectively (Figure 4.5c, d). But in the mean number of visits,

behavioural responses from both sexes were not statistically different from the control

and neither of the two treatments (Table 4.5).

114

0

1

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3

4

Aframomummelegueta

Diethylether

Diethylether

Diethylether

Mea

n tim

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ent (

min

)

bb

ab

a

0

1

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Aframomummelegueta

Diethylether

Diethylether

Diethylether

Mea

n tim

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min

)

b

b

b

a

(a) (b)

0

1

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Zingiberofficinale

Diethylether

Diethylether

Diethylether

Mea

n tim

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ent (

min

)

bb

ab

a

0

1

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Zingiberofficinale

Diethylether

Diethylether

Diethylether

Mea

n tim

e sp

ent (

min

)

bb

b

a

(c) (d)

Figure 4.5 Mean time spent in the arm out of 10 min by S. zeamais males (a) and females (b) in response to 10 µl Aframomum melegueta diethyl ether fraction vacuum distillate, and mean time spent by males (c) and females (d) in response to 10 µl Zingiber officinale diethyl ether fraction vacuum distillate in a four way olfactometer. Bars = standard errors of the means, n = 12. Bars followed by the same letter are not significantly different from each other (P>0.05). a-d: a (males), P=0.006; b (females), P=0.002; c (males), P=0.005; d (females), P=0.004.

115

Table 4.5 Mean number of entries made by Sitophilus zeamais in response to 10 µl Diethyl ether fractions of vacuum distilled Aframomum melegueta and Zingiber officinale extracts.

Test stimulusMean no. visits to olfactometer arm n χ2* P*

i) A. melegueta diethyl ether fraction T CMales 2.83 4.22 12 4.75 0.191Females 2.17 3.47 12 5.53 0.137ii) Z. officinale diethyl ether fractionMales 2.83 4.33 12 5.66 0.129Females 3.5 4.42 12 1.85 0.604

Each control arm contained 10 µl diethyl ether loaded on clean filter paper discT is the mean value of test armC is the mean value of the mean of three control arms*χ2 analysis was performed on the total number of visits (n =12) into the test, control 1, control 2 and control 3 arms in a 4 way contingency table.

4.3.3 Synthetic blends of vacuum distilled Diethyl ether fractions of A. melegueta

and Z. officinale essential oils.

Results from bioassays using a synthetic blend of the identified bioactive compounds

from the diethyl ether fractions of A. melegueta and Z. officinale vacuum distilled

essential oils showed that, in control experiments, virgin adults of S. zeamais exposed

to 10 µl hexane and blank control arms did not show significant differences in the

mean time spent (Figure 4.6) and in the mean number of visits in the arms of the

olfactometer (Table 4.6) respectively.

116

0

1

2

3

4

Hexane Blank Blank Blank

Mea

n tim

e sp

ent

(min

)

0

1

2

3

4

Hexane Blank Blank Blank

Mea

n tim

e sp

ent

(min

)

(a) (b)

Figure 4.6 Mean time spent in the arm out of 10 min by S. zeamais males (a) and females (b) in response to 10 µl hexane and three control arms in a four way olfactometer. Bars = standard errors of the means, n = 12. a-b (males), P=0.479; a-b (females), P=0.238.

Table 4.6 Mean number of visits made by Sitophilus zeamais in response to10 µl redistilled hexane and blank control arms in a four way olfactometer

Test stimulusMean no. visits in olfactometer arm n χ2* P*

i) 10 µl Hexane T CMales 2.33 2.50 12 0.17 0.982Females 2.16 2.06 12 0.16 0.984

The test olfactometer arm contained 10 µl hexane loaded in filter disc while the three control arms contained blank filter paper discs.*χ2 analysis was performed on the total number of visits (n =12) into the test, control 1, control 2 and control 3 arms in a 4 way contingency table.

In contrast to the control experiments, the synthetic blend of the identified volatile

constituents of A. melegueta and Z. officinale diethyl ether fractions of vacuum

distilled essential oils prepared in their natural ratios (see chapter 3), showed

significant pest repellent activity against S. zeamais. The synthetic blend of A.

melegueta essential oil was prepared using (S)-2-heptanol, (S)-2-heptyl acetate and

117

(R)-linalool in the ratio 1:6:3, and Z. officinale synthetic blend was prepared from 1,8-

cineole, neral and geranial in the ratio 5.48:1:2.13. In the mean time spent, both males

and females were significantly repelled (P<0.001) by 10 µl A. melegueta synthetic

blend tested individually (Figure 4.7a, b), and males (P=0.002) (Figure 4.7c), females

(P=0.022) (Figure 4.7d) were significantly repelled by 10 µl A. melegueta + 2 g maize

grains when compared with the control arms. For the number of visits, male responses

were not significantly different from the control when A. melegueta was tested

individually and in combination with maize grains. Females were significantly

repelled (χ2 = 8.95, df =3, P=0.03) in response to the synthetic blend individually and

not in combination with maize grains (Table 4.7).

118

0

1

2

3

4

Test Control1

Control2

Control3

Mea

n tim

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(min

) b bb

a

0

1

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Test Control1

Control2

Control3

Mea

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(min

) bb

b

a

(a) (b)

0

1

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Test Control1

Control2

Control3

Mea

n tim

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(min

) b b

ab

a

0

1

2

3

4

Test Control1

Control2

Control3

Mea

n tim

e sp

ent

(min

) bab ab

a

(c) (d)

Figure 4.7 Mean time spent in the arm out of 10 min by male S. zeamais in response to 10 µl Aframomum melegueta diethyl ether fraction synthetic blend tested individually (a) and in combination with 2 g maize grains (c), and mean time spent by females in response to 10 µl A. melegueta synthetic blend tested individually (b) and in combination with 2 g yellow maize grains (d) in a four way olfactometer. Bars = standard errors of the means, n = 12. Bars followed by the same letter are not significantly different from each other (P>0.05). a-d: a (males), P<0.001; b (females), P<0.001; c (males), P=0.002; d (females), P=0.022.

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Table 4.7 Responses of Sitophilus zeamais to volatiles from 10 µl Aframomum meleguetadiethyl ether fraction synthetic blend and in combination with maize grains in a four way olfactometer

Test stimulus

Mean no. visits in olfactometer arm n χ2* P*

i) 10 µl Aframomum melegueta synthetic blend T CMales 1.58 2.67 12 5.03 0.169Females 0.67 1.58 12 8.95 0.03ii) 10 µl A. melegueta synthetic blend + 2 g maizeMales 2.08 2.89 12 2.19 0.534Females 1.75 2.78 12 4.39 0.222

Each control arm contained 10 µl hexane loaded onto a clean filter paper discT is the mean value of test armC is the mean value of the mean of three control arms*χ2 analysis was performed on the total number of visits (n =12) into the test, control 1, control 2 and control 3 arms in a 4 way contingency table.

Z. officinale essential oil synthetic blend also elicited significant repellent activity

(P<0.001) against males and females (Figure 4.8a, b) when tested alone, and against

males (P<0.001), and females (P=0.008) in combination with 2 g yellow maize

kernels in the mean time spent when compared with the control arms (Figure 4.8c, d).

In the mean number of visits, males significantly (χ2 = 9.19, df =3, P=0.027) preferred

control arms to the test arm, and females did not show a preference to the test or

control arms when tested individually. In combination with yellow maize grains,

adults of S. zeamais showed no significant repellence to the test and control arms in

the mean number of visits (Table 4.8).

120

0

1

2

3

4

Test Control1

Control2

Control3

Mea

n tim

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(min

)

b bb

a

0

1

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Test Control1

Control2

Control3

Mea

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(min

)

b

b b

a

(a) (b)

0

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Test Control1

Control2

Control3

Mea

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ent

(min

)

b b b

a

0

1

2

3

4

Test Control1

Control2

Control3

Mea

n tim

e sp

ent

(min

) bb

ab

a

(c) (d)

Figure 4.8 Mean time spent in the arm out of 10 min by male S. zeamais in response to 10 µl Zingiber officinale diethyl ether fraction synthetic blend tested individually (a) and in combination with 2 g maize grains (c), and mean time spent by females in response to 10 µl Z. officinale synthetic blend tested individually (b) and in combination with 2 g maize grains (d) in a four way olfactometer. Bars = standard errors of the means, n = 12. Bars followed by the same letter are not significantly different from each other (P>0.05). a-d: a (males), P<0.001; b (females), P<0.001; c (males), P<0.001; d (females), P=0.008.

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Table 4.8 Behavioural responses of Sitophilus zeamais to odours from 10 µl Zingiber officinale diethyl ether fraction synthetic blend and in combination with maize grains in a four way olfactometer

Test stimulus

Mean no. visits in olfactometer arm n χ2* P*

i) 10 µl Zingiber officinale synthetic blend T CMales 2.17 4.03 12 9.19 0.027Females 2.17 3.72 12 7.6 0.055ii) 10 µl Z. officinale synthetic blend + 2g maizeMales 2.25 2.61 12 0.55 0.908Females 2.5 3.61 12 3.8 0.284

Each control arm contained 10 µl hexane loaded on clean filter paper discT is the mean value of test armC is the mean value of the mean of three control arms*χ2 analysis was performed on the total number of visits (n =12) into the test, control 1, control 2 and control 3 arms in a 4 way contingency table.

4.3.4 Repellent activity of A. melegueta and Z. officinale oleoresins.

When solutions of A. melegueta and Z. officinale oleoresins were tested for bioactivity

against S. zeamais, no significant activity was observed in the control experiments

involving the solvent, 10 µl diethyl ether and blank control arms in the mean time

spent (Figure 4.9) and mean number of visits (Table 4.9).

However, 10 µl A. melegueta oleoresin showed significant repellent (P<0.001)

activity against male (Figure 4.10a) and female (Figure 4.10b) S. zeamais

individually, and against males (P=0.026) and females (P=0.029) in combination with

2 g yellow maize seeds in the mean time spent when compared with the control arms

(Figure 4.10a, b). In the number of visits, the males (χ2 = 8.04, df =3, P=0.045) and

females (χ2 = 10.15, df =3, P=0.017) significantly preferred control arms to the test

arm when tested alone. But both sexes failed to make any significant choice between

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the test and control arms when equal amounts of A. melegueta oleoresin was tested in

combination with 2 g yellow maize kernels (Table 4.10).

0

1

2

3

Diethylether

Blank Blank Blank

Mea

n tim

e sp

ent

(min

)

0

1

2

3

Diethylether

Blank Blank Blank

Mea

n tim

e sp

ent

(min

)

(a) (b)

Figure 4.9 Mean time spent out of 10 min by S. zeamais males (a) and females (b) in response to 10 µl diethyl ether and three control arms in a four way olfactometer, Bars = standard errors of the means, n = 12.

Table 4.9 Mean number of visits made by adult Sitophilus zeamais in response to 10 µl redistilled diethyl ether and blank control arms in a four way olfactometer assay

Test stimulusMean no. visits in olfactometer arm n χ2* P*

10 µl Diethyl ether against blanks T CMales 2.25 2.08 12 0.2 0.978Females 2.33 2.56 12 0.33 0.954

The test olfactometer arm contained 10 µl diethyl ether loaded onto filter paper disc while the three control arms contained blank filter paper discs.*χ2 analysis was performed on the total number of visits (n =12) into the test, control 1, control 2 and control 3 arms in a 4 way contingency table.

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0

1

2

3

4

A.melegueta

Control 1 Control 2 Control 3

Mea

n tim

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min

)

b b

b

a

0

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2

3

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A.melegueta

Control 1 Control 2 Control 3

Mea

n tim

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min

)

b

b b

a

(a) (b)

0

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Test Control1

Control2

Control3

Mea

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(min

) bab ab

a

0

1

2

3

4

Test Control1

Control2

Control3

Mea

n tim

e sp

ent

(min

) bab ab

a

(c) (d)

Figure 4.10 Mean time spent out of 10 min by male S. zeamais in response to 10 µl Aframomum melegueta oleoresin tested individually (a) and in combination with 2 g maize grains (c), and mean time spent by females in response to 10 µl A. melegueta oleoresin tested individually (b) and in combination with 2 g maize grains (d) in a four way olfactometer. Bars = standard errors of the means, n = 12. Bars followed by the same letter are not significantly different from each other (P>0.05). a-d: a (males), P<0.001; b (females), P<0.001; c (males), P=0.026; d (females), P=0.029.

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Table 4.10 Mean number of visits made by adult Sitophilus zeamais in response tovolatiles from 10 µl Aframomum melegueta oleoresin tested alone and in combination with maize grains in a four way olfactometer.

Test stimulus

Mean no. visits in olfactometer arm n χ2* P*

i) 10 µl Aframomum melegueta oleoresin T CMales 1.33 2.72 12 8.04 0.045Females 1.17 2.56 12 10.15 0.017ii) 10 µl A. melegueta oleoresin + 2 g maizeMales 2.08 2.89 12 2.32 0.509Females 1.5 2.28 12 2.8 0.424

Each control arm contained 10 µl diethyl ether loaded on clean filter paper discT is the mean value of test armC is the mean value of the mean of three control arms*χ2 analysis was performed on the total number of visits (n =12) into the test, control 1, control 2 and control 3 arms in a 4 way contingency table.

Olfactometer assays also showed that 10 µl Z. officinale oleoresin presented on filter

paper discs was repellent to the male (P<0.001) and female (P<0.001) weevils

individually, and to the male (P<0.001) and female (P<0.001) in combination with 2 g

yellow maize seeds in the mean time spent in the arms (Figure 4.11a, b, c, d). For the

number of visits, the males significantly (χ2 = 8.39, df =3, P=0.039) preferred control

arms to the test, but females (χ2 = 7.52, df =3, P=0.057) did not show any significant

preference to any olfactometer arm in response to Z. officinale oleoresin individually.

Both sexes also failed to show significant choice of test or control arms when Z.

officinale oleoresin was presented in combination with 2 g yellow maize seeds (Table

4.11). Summarily, the weevils spent less time in the olfactometer arm with yellow

maize plus a repellent but not fewer numbers of visits.

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0

1

2

3

4

Z.officinale

Control 1

Control 2

Control 3

Mea

n tim

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min

) b

b b

a

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Z.officinale

Control 1

Control 2

Control 3

Mea

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min

)

b

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(a) (b)

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Test Control1

Control2

Control3

Mea

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(min

)

b

bb

a

0

1

2

3

4

Test Control1

Control2

Control3

Mea

n tim

e sp

ent

(min

) b b b

a

(c) (d)

Figure 4.11 Mean time spent in the arm out of 10 min by male S. zeamais in response to 10 µl Zingiber officinale oleoresin tested individually (a) and in combination with 2 g maize grains (c), and mean time spent by females in response to 10 µl Z. officinale oleoresin tested individually (b) and in combination with 2 g maize grains (d) in a four way olfactometer. Bars = standard errors of the means, n = 12. Bars followed by the same letter are not significantly different from each other (P>0.05). a-d: a (males), P<0.001; b (females), P<0.001; c (males0, P<0.001; d (females), P<0.001.

126

Table 4.11 Mean number of visits made by adult Sitophilus zeamais in response tovolatiles from 10 µl Zingiber officinale oleoresin tested alone and in combination with maize grains in a four way olfactometer.

Test stimulus

Mean no. visits in olfactometer arm n χ2* P*

i) 10 µl Zingiber officinale oleoresin T CMales 2.25 4.08 12 8.39 0.039Females 1.42 2.75 12 7.52 0.057ii) 10 µl Z. officinale oleoresin + 2 g maizeMales 2.83 3.81 12 2.5 0.475Females 1.67 2.81 12 4.92 0.178

Each control arm contained 10 µl diethyl ether loaded on clean filter paper discT is the mean value of test armC is the mean value of the mean of three control arms*χ2 analysis was performed on the total number of visits (n =12) into the test, control 1, control 2 and control 3 arms in a 4 way contingency table.

4.3.5 Olfactory responses to A. melegueta and Z. officinale chemical constituents

of essential oils.

The responses of adult S. zeamais to the identified bioactive chemical constituents of

A. melegueta and Z. officinale vacuum distilled essential oils in the mean time spent

are shown in Table 4.12a and mean number of visits in Table 4.12b. Equal amounts

(10 µl) of the solvent and each synthetic compound were impregnated into filter

papers in 4-way olfactometer semiochemical bioassays. In control experiments, there

was no significant (P>0.05) bioactivity by the weevils in the mean time spent and

mean number of visits to the arms. In contrast, males were significantly repelled by

(R)-linalool (P<0.001), (S)-2-heptyl acetate (P=0.039), (S)-2-heptanol (P<0.001), and

citral (P<0.001) but not by 1,8-cineole (P=0.207) when compared with the control

arms in the mean time spent in the arms. Females were significantly repelled by all

the synthetic organic compounds tested with (R)-linalool (P<0.001), (S)-2-heptyl

acetate (P<0.001), (S)-2-heptanol (P=0.002), citral (P=0.047) and 1,8-cineole

(P=0.011) causing the repellency when compared with the control arms (Table 4.12a).

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Table 4.12a Behavioural responses of Sitophilus zeamais measured as mean time spent in each arm (min ± SE) to bioactive compounds extracted from Aframomum melegueta and Zingiber officinale in a 4-way olfactometer

TreatmentsStimulus presentation Males Females

Control Hexane 2.36 ± 0.15a 2.34 ± 0.15aBlank 2.58 ± 0.23a 2.42 ± 0.15aBlank 2.27 ± 0.17a 2.48 ± 0.16aBlank 2.21 ± 0.21a 2.24 ± 0.21a

(R)-linalool Test 1.22 ± 0.22a 0.89 ± 0.28aControl 1 2.47 ± 0.20b 2.58 ± 0.26bControl 2 3.05 ± 0.21b 3.35 ± 0.30bControl 3 2.81 ± 0.24b 2.68 ± 0.23b

(S)-2-heptyl acetate Test 1.81 ± 0.28a 1.57 ± 0.27aControl 1 2.65 ± 0.29b 2.61 ± 0.25bControl 2 2.62 ± 0.22b 2.69 ± 0.26bControl 3 2.46 ± 0.21ab 2.80 ± 0.25b

(S)-2-heptanol Test 1.23 ± 0.27a 1.61 ± 0.24aControl 1 2.51 ± 0.19b 2.73 ± 0.23bControl 2 3.02 ± 0.25b 2.55 ± 0.19bControl 3 2.92 ± 0.24b 2.80 ± 0.28b

Citral Test 1.18 ± 0.24a 1.84 ± 0.29aControl 1 2.75 ± 0.22b 2.38 ± 0.24bControl 2 2.92 ± 0.23b 2.65 ± 0.25bControl 3 2.75 ± 0.24b 2.71 ± 0.26b

1,8-Cineole Test 2.09 ± 0.24a 1.89 ± 0.26aControl 1 2.42 ± 0.22a 2.33 ± 0.22abControl 2 2.57 ± 0.22a 2.72 ± 0.22bControl 3 2.48 ± 0.21a 2.67 ± 0.23b

Means for each treatment followed by the same letter in the same column are not significantly different at the 0.05 level as determined by Tukey’s 95% Simultaneous Confidence Intervals.

In the mean number of visits, females significantly preferred the control arms to (R)-

linalool (χ2 = 10.1, df =3, P=0.018) and (S)-2-heptanol (χ2 = 8.42, df =3, P=0.038)

treated arms only. For all other bioassays, both males and females did not show any

significant (P>0.05) preference to the test or control arms (Table 4.12b).

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Table 4.12b Mean number of visits to the olfactometer arms made by Sitophilus zeamais in response to bioactive compounds from Aframomum melegueta and Zingiber officinale in a 4-way olfactometer

Test stimulusMean no. visits in olfactometer arm n χ2* P*

i) Hexane T CMales 4.08 3.72 12 1.02 0.796Females 4.33 4.42 12 0.02 0.999ii) (R)-linaloolMales 2.00 3.36 12 6.09 0.107Females 1.58 3.00 12 10.1 0.018iii) (S)-2-heptyl acetateMales 2.17 2.86 12 2.19 0.534Females 2.08 2.44 12 0.66 0.883iv) (S)-2-heptanolMales 3.00 2.67 12 3.98 0.264Females 2.33 4.14 12 8.42 0.038v) CitralMales 2.33 3.69 12 5.48 0.139Females 2.75 3.42 12 1.38 0.710vi) 1,8-CineoleMales 2.50 3.11 12 1.27 0.736Females 2.17 2.89 12 2.06 0.56

The test arm contained 10 µl of a bioactive compound; while each control arm contained 10 µl hexane loaded on clean filter paper discsT is the mean value of test armC is the mean value of the mean of three control arms*X2 analysis was performed on the total number of visits (n =12) into the test, control 1, control 2 and control 3 arms in a 4 way contingency table.

4.3.6 Percentage repellent activity

A comparison of the repellent activity of three different treatment levels of A.

melegueta essential oils and their synthetic blends against S. zeamais in olfactometry

bioassays in the mean time spent by the weevils is presented in Table 4.13a, and mean

number of visits in Table 4.13b. The overall percentage repellency of A. melegueta

vacuum distillate alone was 76.96%, A. melegueta vacuum distillate + 2 g maize

grains (76.74%) and A. melegueta synthetic blend + 2 g maize grains was 66.96% in

the mean time spent in the test arm when compared with control arms. In mean

number of visits, A. melegueta vacuum distillate gave the highest overall % repellency

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of 72.01%, A. melegueta vacuum distillate + 2 g yellow maize gave 65.43% and A.

melegueta synthetic blend + 2 g yellow maize 62.39% compared to the controls

respectively.

Table 4.13a Comparison of percent repellency values (Mean ± SE) for different treatments of Aframomum melegueta against Sitophilus zeamais in the mean time spent in a 4-way olfactometer

Mean % repellency

Treatments Males FemalesOverall % repellency

Control (Hexane) 0.00 ± 0.00 0.00 ± 0.00 0.00A. melegueta vacuum distillate 77.86 ± 2.63 76.05 ± 2.96 76.96A. melegueta vacuum distillate + maize 76.92 ± 1.24 76.55 ± 3.78 76.74A. melegueta synthetic blend + maize 71.36 ± 4.51 62.55 ± 6.02 66.96

Table 4.13b Comparison of percent repellency values (Mean ± SE) for different treatments of Aframomum melegueta and Zingiber officinale against Sitophilus zeamais expressed in the mean number of visits in a 4-way olfactometer

Mean % repellency

Treatments Males FemalesOverall % repellency

Control (Hexane) 0.00 ± 0.00 0.00 ± 0.00 0.00 A. melegueta vacuum distillate 72.16 ± 2.21 71.86 ± 2.05 72.01A. melegueta vacuum distillate + maize 62.16 ± 1.80 68.51 ± 4.28 65.34A. melegueta synthetic blend + maize 63.43 ± 3.39 61.36 ± 4.19 62.39

Z. officinale vacuum distillate gave an overall % repellency of 79.52%, Z. officinale

vacuum distillate + 2 g yellow maize grains (72.29%) and Z. officinale synthetic blend

+ 2 g yellow maize grains (68.55%) in mean time spent in the test arm when

compared with the controls respectively. In the mean number of visits, Z. officinale

vacuum distillate gave an overall 70.65% repellency, and Z. officinale vacuum

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distillate recorded 64.72%. The least overall % repellency in the number of visits was

recorded in Z. officinale synthetic blend (61.82%) when compared with control arms.

Table 4.14a Comparison of percent repellency values (Mean ± SE) for different treatments of Zingiber officinale against Sitophilus zeamais in the mean time spent in a 4-way olfactometer

Mean % repellency

Treatments Males FemalesOverall % repellency

Control (Hexane) 0.00 ± 0.00 0.00 ± 0.00 0.00Z. officinale vacuum distillate 77.41 ± 3.29 81.63 ± 2.34 79.52Z. officinale vacuum distillate + maize 71.67 ± 2.03 72.92 ± 3.28 72.29Z. officinale synthetic blend + maize 68.18 ± 4.85 68.91 ± 3.12 68.55

Table 4.14b Comparison of percent repellency values (Mean ± SE) for different treatments of Zingiber officinale against Sitophilus zeamais expressed in the mean number of visits in a 4-way olfactometer

Mean % repellency

Treatments Males FemalesOverall % repellency

Control (Hexane) 0.00 ± 0.00 0.00 ± 0.00 0.00 Z. officinale vacuum distillate 70.33 ± 3.34 70.97 ± 2.69 70.65Z. officinale vacuum distillate + maize 65.65 ± 2.57 63.79 ± 3.81 64.72Z. officinale synthetic blend + maize 61.06 ± 4.05 62.58 ± 3.07 61.82

The vacuum distilled essential oils of both plants showed higher repellency against S.

zeamais when tested individually, than in combination with maize grains. A.

melegueta vacuum distillate gave 76.96% compared with Z. officinale vacuum

distillate which had 79.52% weevil repellency. Within plants, higher percent

repellency was observed in vacuum distillates plus yellow maize grains compared

with synthetic blend plus maize grains. The repellency dropped slightly with synthetic

blends for both plant extracts but was significantly different compared to control

arms.

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4.4 DISCUSSION

Research has been undertaken in order to evaluate the potential of many local plant

species to control insect pests during grain storage in Africa (Keita et al., 2000;

Bekele and Hassanali, 2001; Kouninki et al., 2005; 2007; Tapondjou et al., 2005).

Bekele and Hassanali (2001) reported the contact toxicity of the essential oils of

Ocimum kilimandscharicum and O. kenyense, and blend of the essential oil

constituents against S. zeamais and R. dominica within 24 h. Similarly, Xylopia

aethiopica essential oil at a concentration of 1 ml per 100 g maize seeds has been

reported to cause 100% S. zeamais mortality in 24 h, while 4 of the major components

namely α-pinene, β-pinene, δ-3-carene and terpinen-4-ol according to their proportion

in the essential oil were responsible for 50% mortality (Kouninki et al., 2005: 2007).

Keita et al (2000) reported the fumigant activity of the essential oils extracted from

four West African plant species namely, Tagetes minuta (Compositae), Hyptis

suaveolens (Labiatae), O. canum and P. guineense against C. maculatus of stored

cowpea in 24 h. O. canum caused the highest mortality of 94% followed by P.

guineense, T. minuta and H. suaveolens in that order. Tapondjou et al (2005) reported

the toxic and repellent activity of essential oils extracted from Eucalyptus saligna

(Myrtaceae) and Cupressus sempervirens (Cupressaceae) against S. zeamais and T.

confusum. The local plants are a part of an indigenous ancestral knowledge which

exhibit interesting bioactivities against various pest species and are currently

available. In the present study, the repellent effects of vacuum distilled essential oils

and major constituents of the oils of A. melegueta and Z. officinale as well as their

synthetic blends against S. zeamais were evaluated in 4-way olfactometry bioassays.

The aim of this chapter was to elucidate the roles and relative importance of the

bioactive components of the two oils in conferring some of the observed bioactivity of

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the plants, and to use this information in the future for effective deployment of these

Zingiberaceae plant oils in small-scale farmer level post-harvest grain protection in

Africa.

Of the vacuum distilled essential oils, both A. melegueta and Z. officinale oils

consistently elicited repellent activity against adult S. zeamais when tested

individually and in combination with maize grains. The repellent effects of the

phytochemicals on S. zeamais depend on several factors among which are the

chemical composition of the crude oils, the part of the plant extracted, geographical

location and insect susceptibility (Casida, 1990). The results show that the constituent

hydrocarbons responsible for the repellent activity of the essential oils were present in

the diethyl ether fraction, as indicated by the repellent activity of the diethyl ether

fraction isolated by bioassay guided column chromatography. The repellent activity

was accounted for by the synthetic blends of A. melegueta containing (S)-2-heptanol,

(S)-2-heptyl acetate and (R)-linalool in their natural ratios of 1:6:3, and from Z.

officinale, 1,8-cineole, neral and geranial in the ratio of 5.48:1:2.13 in the essential oil

(see chapter 3). On the other hand, the hexane fractions of the essential oils were not

repellent to S. zeamais adults. (R)-linalool, (S)-2-heptanol, (S)-2-heptyl acetate, neral

and geranial (citral) were very repellent to males and female S. zeamais in

olfactometry experiments, while 1,8-cineole was marginally repellent to the weevils.

The toxicity, fumigant and repellent effects of some of these main constituents of

essential oils have been demonstrated by other researchers. Five monoterpenoids

namely, terpinen-4-ol, 1,8-cineole, linalool, R-(+)-limonene and geraniol have bee

reported to elicit direct toxicity and fumigant activity against 3-day-old eggs, third-

instar larvae and pupae of T. confusum (Stamopoulos et al., 2007). Obeng-Ofori et al

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(1997) found 1,8-cineole to be highly repellent and toxic to S. granarius, S. zeamais,

T. castaneum and P. truncatus. Bioactivities of some of the identified bioactive

components of A.melegueta and Z. officinale essential oils have been reported. For

instance, linalool (Kessler and Baldwin, 2001), linalool, limonene, myrcene, (E)-β-

ocimene (monoterpenes), (E)-β-farnesene, β-bisabolene and (E)-β-caryophyllene

(sesquiterpenes) (Degenhardt et al., 2003) have been shown as components of the

volatile blends released after herbivore attack in some plant species to repel

herbivores and attract parasitoids and predators of the herbivores. In choice field tests,

Myzus persicae (Sulzer) was repelled from Arabidopsis thaliana (L.) plants that

constitutively produce high levels of linalool (Aharoni et al., 2003). (R)-linalool

proved to the major component of the essential oils conferring repellent activity,

linalool is known to act as a reversible competitive inhibitor of acetylcholinesterase

(Ryan and Byrne, 1988).

According to the percent repellency (PR) classes of Juliana and Su (1983) from 0 to

V: class 0 (PR < 0.15), class I (PR = 0.1-20%), class II (PR = 20.1-40%), class III (PR

= 40.1-60%), class IV (PR = 60.1-80%) and class V (PR = 80.1-100%), the mean

repellency values of A. melegueta and Z. officinale essential oils fall within class IV

which may be recommended for stored-product protection in a small scale. In recent

times, plant extracts have been the focus of research to reduce the amount of crude

material that must be mixed with stored grain to achieve effective insect pest control

(Bouda et al., 2001). Sahaf et al (2007) indicated the bioactivity of the medicinal

plant Carum copticum (Apiaceae) against S. oryzae and T. castaneum with 100%

mortality of the insects at concentrations higher than 185.2 µl /L and 12 h exposure

time. The strong repellent and fumigant activity of the essential oil of mugwort,

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Artemisia vulgaris at 0.6 µl/mL essential oil in 1 ml acetone (v/v) against T.

castaneum has been reported by Wang et al (2006). Koona et al (2007) also reported

the repellent effect of the essential oil of the perennial herb, Tephrosia vogelii

(Leguminosae) against S. zeamais in Cameroon.

The repellent action of A. melegueta and Z. officinale essential oils may be caused by

an additive effect of most of the compositionally significant constituents with

different levels of repellency because none of the bioactive compounds exhibited total

repellent activity against S. zeamais when tested individually. The implication of this

result in practice is that the blend as whole rather than specific components could

constitute a control agent with sufficient broad spectrum of bioactivity to be deployed

in stored-product pest control. The effectiveness of blend repellents is in agreement

with Ndungu et al (1995) who reported the repellent action of the shrub Cleome

monophylla (Capparidaceae) essential oil against S. zeamais in a Y-tube olfactometer

as an additive effect of the component compounds. Also, Bekele and Hassanali (2001)

reported blend effects as responsible for bioactivity of the essential oil constituents of

O. kilimandscharicum and O. kenyense against S. zeamais and R. dominica in Kenya.

Tapondjou et al (2005) attributed the repellent effects of crude oil extracts of C.

sempervirens and E. saligna from the Western highlands of Cameroon against S.

zeamais and R. dominica to an enhancing effect of some other minor constituents of

the essential oils. Indeed, there is accumulating research evidence of the adaptive

value of phytochemical diversity in ecological interactions among plants and their

associated herbivores (Cates, 1996). Secondary plant compounds are therefore

recognised important components of plant defence system against herbivores and

pathogens, as well as shaping the diet of herbivores. Terpenes are the most

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widespread and important secondary plant compounds, and can exert toxic, deterrent,

antifeedant and repellent effects on insect herbivores. They are the dominant

components of many natural volatile blends and responsible for many of the

characteristic smells of plant oils, resins, fruits and flowers (Paré and Tumlinson,

1999). Terpenoid chemistry may vary among plants due to many factors which may

include environmental and genetic influences (Langenheim, 1994; Powell and Raffa,

1999; Wang and Lincoln, 2004).

4.5 CONCLUSIONS

The results obtained in the present study are encouraging given the on-going search

for environmentally safe and non-toxic natural products for the protection of stored

grains globally. The results stressed the importance of evaluating plant essential oils

or components in blends to elucidate their full potency in a given bioactivity. In the

future, with proper formulation and production technology, these essential oils could

be exploited for use against insect infestation of stored-products at the small scale

farmer’s level especially in the developing world for a more sustainable food security.

The effects of these essential oils are not particularly dangerous to human health and

the environment (Isman, 2006) since the plants are edible and often incorporated in

the diet as spices in soups, meat and stew.

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CHAPTER 5:

FIELD REPELLENT ACTIVITY AND OVIPOSITION DETERRENT

EFFECTS OF A. melegueta AND Z. officinale AGAINST S. zeamais IN

STORED MAIZE.

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5.1 INTRODUCTION

Maize or corn, Zea mays L. (Poaceae) is one of the most important cereal crops in the

world (Ishaya et al., 2008). In Nigeria, agriculture is one of the most important

sectors of the economy, because it contributes more than 30% of the total annual

gross domestic products (GDP), employs about 70% of the labour force, accounts for

over 70% of the non-oil exports and, perhaps provides well over 80% of the food

needs of the country (Babatunde et al., 2007). Maize is an important crop widely

grown across the different ecological zones of the country ranging from the rain forest

belt in the south to the northern Guinea savannah as a major source for food, feed and

raw material for agro-allied industries (Agboola and Fayemi, 1999). Its annual

production is estimated at 5.4 million metric tonnes from about 3.4 million hectares of

land (FAO, 2004). Nutritionally, maize contains 80% starch, 13% water, 10% protein,

4% oil, 2% sugar and 3% fibre. Yellow maize contains some vitamins notably vitamin

A in addition (Purseglove, 1974). There is, however, an increasing concern about the

sustainability of large-scale maize production in Nigeria, as a result of field to store

insect pests’ infestation. After harvest, inadequate infrastructure and lack of economic

means constrains smallholder farmers to store grain using traditional storage

structures and procedures (Markham et al., 1994), such as cribs, baskets, jute bags,

and earthen ware or in the open. Of all these methods, the open storage system allows

air to flow freely through the grain, which enhances the drying process but also makes

the store vulnerable to attack by insect pests (Holst, et al., 2000). One of the major

pests of maize is the maize weevil, Sitophilus zeamais Motschulsky, an internal feeder

of grains. S. zeamais is a very serious pest in Nigeria because environmental

conditions are suitable for population growth for long periods of time as maize

harvest and storage occur early in the year. Grain spillages and residues in machinery

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and storage containers, as well as empty granaries frequently support residual

infestations of stored-grain insects which can find their way into grain destined for

local and international markets (Smith and Barker, 1987; Reed et al., 2003; Daglish,

2006). Also the ability of grain insects to withstand periods of starvation influences

residual pest populations (Daglish, 2006).

Maize production in Nigeria is undertaken by resource-poor farmers with little or no

control measures during storage. But merchants and large-scale producers use

chemical insecticides to protect stored maize from attack by stored-product pests such

as S. zeamais. However, there have been heightened public concerns over the

continuous application of synthetic pesticides in stored products protection, including

fears about lethal effects on human health, non-target organisms, food residues, high

cost of importation, unavailability at critical periods and negative environmental

consequences (Fields and White, 2002; Tapondjou et al., 2002; Duke et al., 2003;

Umoetok and Ukeh, 2004; Beckett et al., 2007). There is therefore an urgent need to

search for cheap, easily biodegradable and readily available plant materials that will

not contaminate food products in acting as grain protectants in small-scale storage

systems. There have been increasing research efforts to understand indigenous pest

control strategies, with a view to reviving and modernizing their use (Belmain et al.,

2001). Bio-rational products such as natural plant derived volatile organic compounds

(VOCs) that can offer compatible control efficiency plus the benefit of reduced

hazards to the environment, have been found to be effective against a wide range of

insect pests (Bekele, 2002; Isman, 2006). Ethno-botany has played a very important

role in traditional methods of protection against storage pests in Africa and Asia

(Hassanali et al., 1990; Tiwari, 1994). In view of the potential of natural plant

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products in small-scale farm holdings typical of bulk grain production in sub-Sahara

Africa, there has been growing interest in evaluating their efficacies and elucidating

the basis of their protective action (Bekele et al., 1995; Bekele et al., 1997; Obeng-

Ofori et al., 1997; Bekele and Hassanali, 2001; Tapondjou et al., 2002). This

alternative strategy aimed at decreasing the use of classical insecticides; sometimes

referred to as ecochemical control based on plant-insect relationships is currently

attracting considerable research attention. Plant allelochemicals exert a wide range of

effects on insects as repellents, deterrents and antifeedants (Isman, 2006). They may

inhibit digestion, enhance pollination and capture the insect with their attractive

properties; they may increase oviposition or, contrarily, decrease reproduction by

ovicidal and larvicidal effects. Most of these chemicals are secondary plant

metabolites and have chemical structures that classify them such as terpenes for

example monoterpenes and sesquiterpenes (Regnault-Roger, 1997; Rajendran and

Sriranjini, 2008).

This chapter was designed to evaluate the repellent activity of powders of alligator

pepper Aframomum melegueta and ginger Zingiber officinale implicated from ethno-

botanical considerations as having insect controlling properties for the control of S.

zeamais in traditional storage facilities in southern Nigeria, and oviposition deterrent

effect in the laboratory.

5.2 FIELDWORK MATERIALS AND METHODS

5.2.1 Site description and construction of traditional storage barns in Nigeria.

Nigeria lies between 3° and 14º longitude and 4° and 140° latitude, and covers an area

of 923,768 km2 with more than 140 million people (FRN Gazette, 2007). Much of the

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country is covered by productive rivers. In the low-lying coastal region, mangroves

line the brackish lagoons and creeks, and swamp forest grows where the water is

fresh. Further inland, this vegetation gives way to tropical forest, with its many

species of tropical hardwoods. Immediately north of the forest is the Guinea, or moist

savannah, a region of tall grasses and trees. Beyond the Guinea savannah lies the drier

Sudan savannah, a region of shorter grasses and more scattered, drought-resistant

trees. In Nigeria’s very dry north-eastern corner, the semi-desert Sahel savannah

persists (Ajose, 2007).

Experiments were carried out in the rural farming district of Bebuatsuan in Obudu

Local Government Area of Cross River State, Nigeria (located within latitude 500

and 540 North and longitude 804 and 862 East) in November 2006 – February,

2007 (Plates 1 and 2). Cross River has a humid tropical climate with total annual

rainfall of 1500-3000 mm, humidity of 65-90%, ambient temperatures of 22.2 C -

23.8 C minimum, and 27 C - 40 C maximum.

Four storage barns were constructed and each barn measured 2.5 m in diameter, 3 m

in height and was set 20 m apart. All barns were constructed using wooden poles,

thatch roof and bamboo walls in accordance with the local storage pattern (Plate 3).

Up to 70% of local farmers store their products in this manner. Four flight traps baited

with synthetic multi-attractant pheromone lure capsule, sitophilure manufactured by

Insects Limited, Inc., USA, were set-up in four cardinal positions (north, south, east

and west) outside each storage barn where shelled maize was stored for 2 weeks. Each

trap was set-up 3 m away from the wall of the barn, suspended from wooden posts at

1.4 m above the ground (Plate 4). The flight traps used were the white delta traps

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complete with sticky bases and metal hangers supplied by AgriSense-BCS Limited,

UK. The traps were inspected daily for 2 weeks before final assessment for insect

catch was done.

Niger Republic

Cameroon

Atlantic Ocean

Benin Republic

Obudu

Calabar

Plate1. Map of Nigeria showing Cross River State where the experiment was conducted from November 2006 to February 2007.

142

Field study site

Calabar

Plate 2. Map of Cross River State where the experiment was carried out.

143

Adult insects captured by the pheromone-baited sticky traps were removed and placed

in labelled vials containing ethanol before classification and identification.

5.2.2 Seeding the environment.

At each storage barn, the environment was seeded with the local strain of S. zeamais

cultured on white maize collected from the department of Crop Science, University of

Calabar, because catches by the pheromone-baited traps were poor. 500 unsexed S.

zeamais were sprinkled around each barn at a distance of 3 m away from the wall.

Seeding was done at 6.00 pm local time when birds (fowls) kept by the local people

had returned to the huts for the day. It was reasoned that at dusk all the insects could

have found their way to the stored maize following odour cues emanating from the

storage bins.

Plate 3. Traditional storage barn in Bebuatsuan village, Obudu, Nigeria.

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Plate 4. Setting up Delta traps for S. zeamais in Bebuatsuan village, Obudu, Nigeria.

5.2.3 Repellency trials.

Fresh maize cobs were bought from the Cassava and maize growers’ cooperative

society in Obudu Local Government Area of Cross River State, Nigeria from

November 23 – 30, 2006. The ripe maize cobs (Plate 5) were dehusked (Plate 6) and

sun-dried for 2-3 days before used for the experiment. Before setting up the trials, a

total of 10 whole dehusked cobs of maize were randomly selected from the samples

for the repellency trials, shelled and assessed for S. zeamais infestation, to obtain

baseline data.

In each storage barn, 4 baskets containing 18 kg of shelled maize cobs and 10%

repellent plant powders were set up (Plate 7) as shown below:

Treatment 1: 18 kg of maize + 2 kg Aframomum melegueta seed powder.

Treatment 2: 18 kg of maize + 2 kg Zingiber officinale rhizome powder.

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Treatment 3: 18 kg of maize + 1 kg A. melegueta seed powder + 1 kg Z. officinale

rhizome powder.

Treatment 4: Control, 18 kg of maize alone.

A. melegueta fruits and Z. officinale rhizomes were collected from fields around

Akamkpa Local Government Area and some supplied by local farmers in Obudu

Local Government Area of Cross River State, Nigeria. A. melegueta fruits were sun

dried for 2-3 days after which the seeds were extracted and ground . Z. officinale

rhizomes were sliced, dried in the shade for 5 days and ground to a powder as well.

The plant products were pounded to powdered form using the local wooden mortar

and pestle, and sieved through a mesh of < 2mm diameters. The required quantities of

each plant powders were mixed with the maize manually in baskets. All the baskets

were covered with dried banana leaves in order to protect them from dust (Plate 8).

Treatments were sampled weekly and at each sampling occasion 2 ears were

randomly taken from each basket, making sure that sampled ears were not touching

each other. Immediately after removal from the basket each ear was shelled, and the

grain sieved and all S. zeamais counted. Other insect species and dead insects were

also counted, classified and recorded.

5.2.4 Fieldwork maize seeds germination percentage

After 3 months (12 weeks) at the end of the experiment, germination tests were

carried out in Petri dishes and Fisherbrand QL 100 filter papers. Two cobs were

randomly selected from each replicate, shelled and 50 seeds from each replicate were

again randomly picked for the germination test. The grains were soaked in distilled

water for around 30 min after which time the grains were removed and placed in

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labelled Petri dishes lined with filter paper and then covered and moistened daily with

distilled water. Four days later, germination was assessed by calculating the number

of seeds germinated out of the total of 50 in each Petri dish. Percentage germination

was calculated as: (number of seeds germinated/total number of seeds) x 100.

Plate 5.Ripening maize cobs soon ready for harvest in Obudu, Cross River State, Nigeria. November 2006.

147

Plate 6. Maize is stored on the cob with the husk removed or intact in this part of Nigeria. November 2006.

Plate 7. Setting up repellency trials using Z. officinale and A. melegueta powders, December 02, 2006.

148

Plate 8. After the application of plant powders, the treatments were covered with dried banana leaves. December 02, 2006.

5.2.5 Data Analysis.

Differences in the mean number of S. zeamais per 2 cobs sampled per treatment was

determined by analysis of variance (ANOVA) using the statistical software Minitab

15.The germination percentage was calculated using simple percentage method.

5.3 LABORATORY OVIPOSITION DETERRENCE EXPERIMENTS

5.3.1 Materials and Methods

Fifty grams of yellow maize were weighed out into 8 x 8.5 x 8.5 cm transparent

plastic containers for each replicate. The dried seeds of A. melegueta and rhizomes of

Z. officinale were ground into powders and applied by direct admixtures to the maize

grains calculated on the weight of plant material/weight of grain (w/w) basis. Each

plant powder was applied at three dosage rates of 1%, 5% and 10% in transparent

plastic containers, while the controls received no plant powders. Fifteen pairs of 3 d

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old S. zeamais adults were introduced into each plastic container containing

treatments for mating and oviposition for 6 days. The plastic containers had their

covers drilled with holes to facilitate air circulation. They were then covered with

nylon mesh and their perforated lids screwed in place to facilitate confinement of the

weevils. Each treatment was replicated 4 times and laid out in a Completely

Randomized Design (CRD) on the laboratory bench for 10 weeks. The experiments

were conducted in a CTH room maintained at 25 ºC and 65% rh on a 12:12 L.D

photoperiod. A mortality count was done every 24 h for 72 h after treatment by

sieving out the contents into a clean white tray and counting the number of dead

insects. Each time a count was done; dead individuals were discarded while live ones

were returned to their respective treatments. After 6 days all live and dead weevils

were removed and discarded and the seeds kept aside for F1 progeny emergence. After

five weeks, the weevils emerging from each jar were counted to give a measure of

productivity and sieved off subsequently every 2 days to prevent mating and

subsequent oviposition by F1 as mating in S. zeamais does not occur before weevils

are 3 d old (Walgenbach and Burkholder, 1987). The numbers of F1 progeny from

each treatment were counted, weighed and recorded. Five weevils were randomly

selected from each treatment and weighed on the first week of emergence (week 5

after treatment) using Sartorius weighing balance. The oviposition

deterrence/inhibition experiment was terminated after 10 weeks, and germination tests

were carried out. Thirty seeds were randomly selected from each replicate and soaked

in water for about 30 min after which time the grains were removed and placed in

labelled Petri dishes lined with filter paper which were covered and moistened daily

with water. Germination was assessed after 4 days by calculating the number of seeds

germinated out of the total of 30 in each Petri dish. Percentage oviposition deterrence

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of treatments to S. zeamais was also calculated using the formula: Mean No. of

emerged adults in control – Mean observed adults in treatment)/Mean No. of emerged

adults in control x 100.

5.3.2 Data analysis

The experiment was laid out in Completely Randomized Block Design (CRBD) and

data obtained analysed using analysis of variance (ANOVA) in MINITAB 15, and all

treatment means were compared using Tukey’s Test.

5.4 RESULTS.

5.4.1 Repellent effects of A. melegueta and Z. officinale against S. zeamais in

traditional storage granaries in Nigeria.

The results of field trials in traditional granaries showed that 10% powders (w/w) of

the 2 plant products (A. melegueta and Z. officinale) and a combination of the two

(5% each) significantly repelled (p<0.001) S. zeamais from stored maize cobs when

compared with the untreated control at 12 weeks post treatment. The prevalence of

weevil associated with the combination of A. melegueta and Z. officinale was not

significantly (p>0.05) different than either of A. melegueta or Z. officinale alone

(Table 5.1) showing no synergistic or additive effects of the combination. Z. officinale

alone appeared a poorer repellent than A. melegueta or the two together although not

significantly different (p>0.05) from the other treatments.

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Table 5.1.Mean number of S. zeamais counted 12 weeks post treatment with A. melegueta and Z. officinale powders in traditional storage barns in Nigeria. Means in the same column followed by the same letter superscript are not significantly different at the 0.05 level as determined by Tukey’s test. N=4, a-b, P=0.01.

TreatmentsMean no. of S. zeamais/2cobs

A. melegueta 1.33 ± 0.49b

Z. officinale 1.56 ± 0.51b

A. melegueta + Z.officinale 1.08 ± 0.48b

Untreated (Control) 4.46 ± 0.62a

The weevil population in the storage barns crashed immediately after treatment with

plant powders, but increased gradually after this first week up to the 10 th week (Figure

5.1), after which it surged upwards suggesting that the plant volatiles could lose their

repellency potential with time probably by volatilization. There was a significant (p<

0.001) difference in the mean weekly weevil population between the treatments and

control throughout the trials. It was observed that maize cobs treated with powders of

Z. officinale rhizomes and A. melegueta seeds were protected from insect attack for up

to 10 weeks.

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0

1

2

3

4

5

6

7

0 1 2 3 4 5 6 7 8 9 10 11 12

Time (weeks)

Me

an

no

. of

we

ev

ils/ 2

co

bs

A.meleguetaZ.officinaleA.melegueta + Z.officinaleControl

Figure 1.Mean weekly population responses of S. zeamais to A. melegueta and Z. officinale powders in traditional storage facilities in Nigeria. Error bars represent standard errors of the means. n=4.

The percentage germination in the various treatments (Figure 5.2) was significantly

higher (p< 0.01) in the treated than untreated cobs.

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01020304050

60708090

100

A.mel

egue

ta

Z.offi

cinale

A.mel

egue

ta+Z

.offi

cinale

Contro

l

Treatments

Me

an

(%

) g

erm

ina

tio

n

Figure 5. 2. Effects of powders of A. melegueta seeds and Z. officinale rhizomes on the germination (± SE) of maize seeds, p< 0.01).

5.4.2 Effect of A. melegueta and Z. officinale powders on S. zeamais oviposition

and adult emergence in laboratory tests.

There was no significant difference (P>0.05) in S. zeamais mortality at 24, 48 and 72

h between 1% of both plant powder treatments and the untreated control. However,

there were significant differences (p<0.05) within plant powder treatments, and

between 5% and 10% plant powders and the untreated control (Table 5.2). The

cumulative mortality values at 72 h post treatment showed that Z. officinale at 5 and

10% rates of plant powders caused higher weevil mortality compared to other

154

treatments, and weevil mortality appeared to increase with concentration and days of

exposure for all treatments.

Table 5.2 Effect of A. melegueta and Z. officinale powders on adult S. zeamais mortality at 24, 48 and 72 h post-treatment in the laboratory. Means in the same column followed by the same letter are not significantly different at the 0.05 level as determined by Tukey’s test. n=4.

TreatmentsCumulative mean % mortality (± SE) of S. zeamais (h) 24 48 73

Control (Untreated) 0.83 ± 0.65d 6.67 ± 0.83c 10.83 ± 1.36c

Z. officinale (1%) 1.67 ± 0.69d 7.50 ± 1.36c 13.33 ± 1.34cZ. officinale (5%) 9.17 ± 1.36ab 19.17 ± 1.31b 42.50 ± 2.32aZ. officinale (10%) 12.50 ± 1.12a 28.33 ± 1.04a 48.33 ± 1.26a

A.melegueta (1%) 0.00 ± 0.00d 2.49 ± 0.65d 8.34 ± 0.69cA.melegueta (5%) 4.17 ± 0.65c 9.17 ± 0.89c 23.33 ± 1.47bA.melegueta (10%) 7.50 ± 1.02b 18.33 ± 1.26b 30.83 ± 1.66ab

The results also showed significant differences (p<0.001) between the treatments and

the control in the mean number of F1 adult emergence, as the mean number of

emerged adults decreased with increase in concentration of each plant powder. Z.

officinale (10%) appeared to be more effective in deterring oviposition by S. zeamais

and suppressing adult emergence than other treatments and the control (Table 5.3).

This was evident because the highest percentage oviposition deterrence and the least

number of emerged progeny were obtained in Z. officinale (10%) followed by A.

melegueta (10%) respectively. It may be interesting to note that although Z. officinale

(5%) showed higher weevil mortality than A. melegueta (10%), it exhibited lower

oviposition deterrent effect compared to the later. The highest mean adult emergence

occurred in the control, followed by A. melegueta (1%), Z. officinale (1%), A.

melegueta (5%) and Z. officinale (5%) respectively. However, there were no

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significant differences among the treatments regarding the weight of the emerged

adults (Table 5.3).

Table 5.3. Percentage oviposition deterrent effect of A. melegueta and Z. officinale powders as measured by adult S. zeamais emergence and body weight 10 weeks post treatment. Means in the same column followed by the same letter are not significantly different at the 0.05 level as determined by Tukey’s test. n=4

Mean adult Mean body weight OvipositionTreatments emergence at 5 weeks (mg) deterrence (%)Control (Untreated) 41.75 ± 1.06a 3.13 ± 0.23a 0.00 ± 0.00d

Z. officinale (1%) 26.5 ± 1.09b 3.09 ± 0.60a 35.58 ± 1.99cZ. officinale (5%) 16.75 ± 1.07bc 3.23 ± 0.49a 59.35 ± 1.78bZ. officinale (10%) 6.25 ± 0.99c 3.33 ± 0.54a 84.22 ± 1.69a

A.melegueta (1%) 32.5 ± 1.58ab 3.10 ± 0.49a 21.01 ± 2.35cA.melegueta (5%) 23.25 ± 1.33b 3.13 ± 0.51a 43.65 ± 2.02bcA.melegueta (10%) 14.25 ± 1.07bc 3.30 ± 0.53a 64.76 ± 1.77b

The mean weekly adult S. zeamais emergence from maize grains treated with A.

melegueta and Z. officinale powders (Figures 5.3 and 5.4) showed that F1 emerged

from the 5th week in all treatments and peaked at about the 9th week. No adults

emerged from the treatments and the control after the 10 th week. It therefore meant

that all viable eggs laid by the weevils had hatched by the 10th week post treatment.

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0

5

10

15

20

25

30

35

40

45

Week 5 Week 6 Week 7 Week 8 Week 9 Week 10Time

Me

an

ad

ult

em

erg

en

ce

Control A. melegueta 1%A. melegueta 5% A. melegueta 10%

Figure 5.3. Weekly S. zeamais F1 progeny emergence from maize grains treated with A. melegueta powders at 3 dosages. Bars= standard errors of the means, n=12.

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0

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Week 5 Week 6 Week 7 Week 8 Week 9 Week 10

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Me

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Control Z. officinale 1%Z. officinale 5% Z. officinale 10%

Figure 5.4. Weekly S. zeamais progeny emergence from maize grains treated with Z. officinale powders at 3 dosages. Bars=standard errors of the means, n=4.

The percentage germination of yellow maize seeds in the various treatments (Figure

5.5) ranged from 59.17% in the control to 86.67% in A. melegueta (10%) and 94.17%

in Z. officinale (10%) treated grains respectively. The germination percentage in

Z.officinale (5%) compared favourably with A. melegueta (5%) and A. melegueta

(10%), but the control showed the lowest percentage germination and this differed

significantly (p<0.001) from the other treatments.

158

0

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50

60

70

80

90

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A. mele

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a (1

%)

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a (5

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a (1

0 %

)

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Me

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Figure 5.5. Effect of A. melegueta and Z. officinale powders on germination (± SE) of maize grains.

5.5 DISCUSSION

Results reported in this chapter show the toxicity, oviposition deterrent and repellent

effect of the powders from two plant species against S. zeamais in southern Nigeria,

and in laboratory studies. The powders from the seeds of A. melegueta and rhizomes

of Z. officinale were effective in repelling the insect population from stored maize

when compared with the control at 10 % (w/w) and at a combination of 5 % (w/w) of

each plant powder treatments after 12 weeks in traditional storage environment in

Nigeria. A combination of the two plants did not produce any significant synergistic

or additive effect on their repellency against S. zeamais. It was observed that the mean

number of visiting weevils to treated maize cobs fell sharply one week after the

admixture with plant powders, while the number of insects in the untreated maize

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cobs continues to increase week by week. Maize cobs were protected from pest

infestation up to 10 weeks in traditional storage granaries. Laboratory studies

confirmed the results from field trials and showed that the powders of A. melegueta

and Z. officinale are toxic and repellent to S. zeamais and can suppress oviposition as

measured by progeny development of the weevils resulting in better protection of

stored maize grains from pest infestation and damage. The mean number of progeny

produced by S. zeamais in the untreated control was significantly higher than the one

treated with 5% and 10% (w/w) concentration of plant powders. The efficacy of the

powders was dose-dependent with higher doses providing greater protection with

significantly fewer emergent adults. However, the treatments did not influence the

weight of emergent adults. This finding is in agreement with Danho et al (2002) who

reported that emergent S. zeamais adult weight was not affected by competition on

different quantities of host grains.

Although the mode of action of these plant powders is not clearly understood, it was

observed that the repellent and pungent odours from these plants caused the insects to

climb to the walls of the containers soon after introduction thereby limiting adequate

feeding and oviposition. Also the physical abrasion of the insect cuticle with the

resultant loss of body haemolymph or partial blockage of the spiracles (Ogunwolu et

al., 1998; Oparaeke and Kuhiep, 2006) may have contributed to mortalities in

suffocation and death. The observation that high dosages of A. melegueta and Z.

officinale powders caused significant adult mortality and a reduction in F1 progeny

emergence could be due either to repellent, feeding or oviposition deterrence effects

on the weevils or to a combination of all the three. This could also be due to plant

material having toxic effects on the larvae hatching from eggs laid on grains resulting

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in reduced progeny emergence (Tapondjou et al., 2002, 2005; Akob and Ewete,

2007), however it was probably not likely that the active ingredients of the powders

entered the seeds as after washing the maize seeds treated in this fashion tasted

normal. Similar results have been reported elsewhere. For example, Bekele (2002)

reported that seed powder of Milletia ferruginea (Hochest) Baker applied at 10% w/w

to maize seeds was toxic to S. zeamais and caused significant reduction in F1 progeny

production, and attributed the toxicity to rotenone. Powders and essential oil obtained

from dry ground leaves of wormseed, Chenopodium ambrosioides (L.)

(Chenopodiaceae) at a dosage of 6.4% (w/w) have been reported to induce total

mortality of S. granarius and S. zeamais two days after treatment, and inhibited F1

progeny production and adult emergence under laboratory conditions. The toxicity of

C. ambrosioides was attributed to major constituents such as ascaridole, cymol and α-

terpinen (Tapondjou et al., 2002). Bekele et al (1995) also reported the repellent

effect of dried ground leaves (25 g/250 g of maize seeds) and essential oil (0.3

mg/250 g of maize seeds) of Ocimum kilimandscharicum (Labiatae) against S.

zeamais, R. dominica and S. cerealella resulting in lower weight loss and number of

damaged maize seeds compared with untreated grains. Similarly, Silva et al (2005)

reported that 2% concentration (w/w) powdered leaves of C. ambrosioides and boldo,

Peumus boldus Mol. (Monimiaceae) exhibited 90.1% and 98.8% mortality of S.

zeamais after 24 h exposure, and suppressed progeny production by 13% in Chile.

While in Tanzania, 10% (w/w) leaf powders of eucalyptus, Eucalyptus macrorhyncha

(F. Muell) (Myrtaceae), pawpaw, Carica papaya (L.) (Caricaceae), neem,

Azadirachta indica (A. Juss) (Meliaceae) and lantana, Lantana camara (L.)

(Verbenaceae) were toxic to S. zeamais and significantly reduced grain damage and

weight loss (Mulungu et al., 2007). Under small-scale farmer conditions as is the case

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in this study, powder treatments may protect stored grains for some time (Bekele,

2002). It is quite obvious that phytochemicals have potential for use in modern stored

product protection. As attractants or repellents, they may be used to modify insect

behaviour. Repellent compounds could also be effective as control agents and some of

these compounds may even be used as fumigants (Adler et al., 2000).

5.6 CONCLUSIONS

The application of plant powders may minimize insecticide usage thereby reducing

health hazards to applicators and the amount of toxic residues to the environment.

Treatment of grains with repellents could also have important practical applications in

the parts of the world where insecticides are expensive, in short supply or where these

repellent plants are cheap and readily available. The results from this study indicate a

possible scientific rationale for the traditional use of plant powders as grain

protectants by resource poor farmers. The implication of this in practice is that A.

melegueta and Z. officinale powders could constitute agents with sufficient broad

spectrum of repellent activity for use as general purpose stored product insects

repellent in developing countries.

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CHAPTER 6:

GENERAL DISCUSSION

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The main aim of this study was the identification and use of semiochemicals for the

control of the maize weevil, Sitophilus zeamais in Nigeria. The use of semiochemicals

has stimulated much interest because they may be used to improve current monitoring

as well as direct means of controlling stored-product insect pest species (Cox, 2004;

Germinara et al., 2008). Laboratory olfactometry, oviposition deterrence and field

experiments were designed to investigate the repellent properties of alligator pepper,

Aframomum melegueta, ginger Zingiber officinale and the West African black pepper,

Piper guineense against S. zeamais of stored maize. Gas chromatography (GC),

coupled gas chromatography mass spectrometry (GC-MS), and liquid column

chromatographic separation of A. melegueta and Z. officinale vacuum distilled

essential oils were carried out to identify the chemical constituents that confer

repellency to these plants. The aim was to investigate the possibility of using these

non-host plants as sources of repellent signals for S. zeamais in a novel,

environmentally sound, small-scale crop protection strategy in Africa.

6.1 BEHAVIOURAL RESPONSES TO HOST AND NON-HOST PLANT

VOLATILES

Insect host location frequently involves the detection of volatile chemical cues by

olfactory receptors located on the antenna (Bruce et al., 2005). This implies that insect

species are able to detect a suitable host while walking or in flight, and also that host

selection can depend on a lack of repellency. Single choice olfactometer experiments

were conducted with virgin male and female S. zeamais adults to study odour

responses to their host and non-host plant volatiles. S. zeamais responded with

positive anemotaxis to air plumes passed over 2 g white maize, yellow maize and

winter wheat kernels, but negatively to air that passed over host plant plus 10% non-

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host plants (w/w), namely A. melegueta, Z. officinale and P. guineense respectively

when compared to the control arms emitting clean air. Both males and females

showed a general attraction to all three host plants tested, and were able to distinguish

between host plants and non-host plants. Odours from a combination of host and non-

host plants, and non-host plants individually, repellent to the weevils in olfactometer

bioassays. The repellency of non-host plant volatiles was dose-dependent. These

results confirm similar reports that phytophagous insects employ volatiles from plant

materials to locate suitable substrates for food, mating and aggregation (Visser, 1986).

It has been shown that extracts from carob pods (Collins et al., 2004) and specific

cereal volatiles attract the granary weevil S. granarius, a sibling species of S. zeamais

(Collins et al., 2007; Germinara et al., 2008). S. zeamais adults oriented to a volatile

blend emitted by grains of white maize, yellow maize and winter wheat kernels in

olfactometry bioassays. The presence of phagostimulatory compounds have been

considered crucial in the infestation process by this weevil (Kanaujia and Levinson,

1981). The use of host volatiles for host finding and colonisation is not limited to

stored-product insects. Couty et al (2006) reported that the diamondback moth,

Plutella xylostella L. (Lepidoptera: Plutellidae) the major insect pest of cruciferous

crops such as cabbages, broccoli and cauliflower, uses volatile plant chemical cues to

locate and to promote landing on their hosts, even in a complex mixed-crop

environment in large cages. Amarawardana et al (2007) studied the odour-mediated

effects of leek, Allium porum and chives, Allium schoenoprasum (Alliaceae), on the

host searching behaviour of the peach-potato aphid, Myzus persicae Sulzer

(Homoptera; Aphididae) in 4-way olfactometry experiments. They reported that odour

of the host plant sweet pepper, Capsicum annum L. (Solanaceae), was significantly

attractive, whereas odour of chives was significantly repellent. The combined odour

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of sweet pepper and chives was neither attractive nor repellent, but when sweet

pepper plants were exposed to volatiles from chives for five days, their odour became

repellent to M. persicae. An extract of A. porum plants was significantly repellent to

aphids in the olfactometer, as were C. annum plants that were sprayed with this

extract. Viewing this analogous report, it may be possible to mask host plant odours

with non-host plants in order to disrupt host finding by insect pest species thereby

protecting the desired stored product.

6.2 BIOACTIVITY OF A. melegueta AND Z. officinale ESSENTIAL OILS AND

THEIR CONSTITUENTS AGAINST S. zeamais.

The use of unattractive plant odours to repel insect pest species from stored

commodities has resulted in some commercial pest control products in recent years

(Isman, 2000; 2006). One of the most important sources of repellents is the essential

oil extract from aromatic plants commonly used to flavour foods and in perfumery

(Coppen, 1995). Plant essential oils or their constituents have been valued in crop

protection due to their broad spectrum of biological activity. They consist of complex

mixtures of mono and sesquiterpene hydrocarbons, aliphatics as well as aromatic

compounds with a few major constituents (Rosell et al., 2008). In olfactometry

experiments, vacuum distilled essential oils extracted from A. melegueta and Z.

officinale were repellent to adult S. zeamais when tested individually and in

combination with seeds of the host plant, Z. mays. GC-MS analysis of the vacuum

distilled essential oil from A. melegueta and Z. officinale identified 13 and 24

compounds respectively. The repellency of the essential oils was accounted for by the

Florisil® diethyl ether fractions. GC-MS analysis of the behaviourally active

Florisil® diethyl ether fractions of A. melegueta identified (S)-2-heptanol, (S)-2-

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heptyl acetate and (R)-linalool as major components in a 1:6:3 ratio, and for Z.

officinale vacuum distillates, 1,8-cineole, neral and geranial in a 5.48:1:2.13 ratio. To

my knowledge, this is the first time (S)-2-heptyl acetate has been identified from A.

melegueta seeds. Apart from (R)-linalool, the synthetic preparations of these

compounds individually could not produce total protection of the maize grains, but

the blend of each plant essential oil in their natural ratios gave significantly higher

repellent activity against S. zeamais in laboratory studies than individual compounds.

Both blends of essential oils produced percent repellency (PR) equivalent to a class

IV repellent with between 60-80% repellency of the pest species. According to the

percent repellency (PR) classes of Juliana and Su (1983) from 0 to V: class 0 (PR <

0.15), class I (PR = 0.1-20%), class II (PR = 20.1-40%), class III (PR = 40.1-60%),

class IV (PR = 60.1-80%) and class V (PR = 80.1-100%). The implication of this

result in practice is that the blend as whole rather than specific components could

constitute a control agent with sufficient broad spectrum of bioactivity to be deployed

in stored-product pest control. (R)-Linalool is a volatile oxygenated monoterpene

compound found ubiquitously in the plant and even human odours (Logan, 2006), and

different concentrations could result in either attractancy or repellency for a variety of

insect species (Mauchline et al., 2008). Insects can determine ratios of volatile

chemicals by comparing the stimulation of one type of receptor cell with that of

another (Blight et al., 1995; Bruce et al., 2005). For example, using single cell

recordings (SCR), Wadhams (1990) reported 166 responding olfactory cells on the

antenna of the cabbage seed weevil, Ceutorhynchus assimilis Paykull, and most of

these cells exhibited high specificity in their response profiles. Many insects use a

specific blend and ratio of volatiles to find their host plant (Städler, 1992), a negative

selection by the insect for specific odours associated with unsuitable host plants

167

within the complex of odours plays a role in selection or avoidance of plant species

(Dickens et al., 1992; Hayes et al., 1994; Poland et al., 1998). This avoidance is not

always based on selection and learning of the insect species alone but can also involve

an induced defence mechanism by plants after attack of the insect herbivore which

makes them unattractive for the insect (Franceschi et al., 2005) and to attract natural

enemies (Takabayashi and Dicke, 1996; van Tol et al., 2007). Specific ratios of all the

behaviourally active compounds from Florisil® diethyl ether fractions of A.

melegueta and Z. officinale may therefore be responsible for the repellent activity of

their essential oils against the maize weevil. These results identified individual

chemical constituents that were likely to be involved in the repellent activity of blends

of A. melegueta and Z. officinale essential oils against S. zeamais in olfactometry

bioassays.

Direct toxicity of essential oils to pest species appear to result from interaction with

the insect nervous system, either by acetylcholinesterase inhibition or antagonism of

the octopamine receptors (Rosell et al., 2008). The lack of octopamine receptors in

vertebrates likely accounts for the profound mammalian selectivity of essential oils as

insecticides and repellents because the octopaminergic system of insects represents a

biorational target for insect pest management (Isman, 2000; Enan, 2001). Effects with

the essential oil of A. melegueta and Z. officinale against S. zeamais such as

repellency and feeding deterrence may be consistent with this mode of action.

Applications of other essential oils such as citronella oil in stored-product protection,

mosquito repellency and domestic pest control (cockroaches, ants and fleas),

cinnamon oil in mite and urban pest control have been reported (Wong et al., 2005;

Isman, 2006). This study presents a rational approach to investigating the chemical

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basis of repellent non-host plant volatiles, for assessing the behavioural effects of

such repellent volatiles on stored-product pests such as S. zeamais in small-scale

farmer conditions in Nigeria.

6.3 FIELD REPELLENT ACTIVITY AND LABORATORY OVIPOSITION

DETERRENCE OF A. melegueta AND Z. officinale POWDERS AGAINST S.

zeamais.

The post-harvest system in Nigeria presents particular problems for pest control. The

traditional storage facilities are varied and include sacks, baskets, earthenware pots,

farmers’ houses, cribs, and sacks in small communal stores, wooden barns, and

trader’s stores and in the open. Another problem is that the stored-product and its

associated insect pests are constantly being moved through different stores as it goes

through processing, marketing and distribution systems. Such post-harvest systems in

which stored grain is being moved around with the commodity batches being split or

combined are logistically problematic even for conventional pesticide application

(Haines, 1991). However, research findings from several countries confirm that some

plant powders, essential oils or their constituents not only repel insects, but also have

contact and fumigant insecticidal actions against specific stored-product pests (Isman,

2000; Rajendran and Sriranjini, 2008). As a part of an effort aimed at the development

of reduced-risk stored-product protection based on repellent plant products, 10%

powders of A. melegueta and Z. officinale, and a combination of both (5% each) were

evaluated for repellent activity against S. zeamais under traditional storage conditions

in Obudu, southern Nigeria for 12 weeks. Results showed that maize cobs treated with

powders of A. melegueta seeds and Z. officinale rhizomes were protected from insect

attack for up to 10 weeks and gave significantly higher germination rates than

169

untreated cobs. A combination of the two plants did not produce any significant

synergistic or additive effect on their repellency against S. zeamais. It was also

observed that the mean weekly number of S. zeamais on treated maize cobs fell

sharply one week after the application of plant powders, whereas the number of

weevils in the untreated maize cobs continued to increase weekly. Adults of S.

zeamais showed their susceptibilities to the repellent properties of A. melegueta and Z.

officinale which are generally used as spices. Spices and their extracts have been

reported to exhibit various bioactivities against stored-product insects (Shaaya et al.,

1991). Spices have characteristic odours and flavours due to the presence of volatile

essential oils acting as allelopathic agents, or as irritants and repellents that protect

plants from insect herbivores (Simpson, 1995). Insect repellents are chemical

substances which cause the insect to make oriented movements away from the source

of the substances (Dethier et al., 1960). In insect-plant interactions, Visser (1986)

reported that phytophagous insects use species-specific volatiles and ratio-specific

odour recognition of host and non-host olfactory cues in host location. As repellents,

A. melegueta and Z. officinale volatiles may have disrupted the odour plumes from

treated maize cobs thereby protecting them from recognition by the visiting maize

weevils and resulting in discrimination between treated and untreated maize cobs.

Discrimination between plants is a product of the central nervous system processing

whereby blends of volatiles in specific ratios from a host plant are detected by insects

within a complex background of volatiles from non-host plants. This could be

facilitated by paired or groups of olfactory receptor neurons that permit fine scale

resolution of such complex signals (Bruce et al., 2005). Results from laboratory

studies confirmed the results from field experiments and showed that the powders of

A. melegueta and Z. officinale were toxic and repellent to S. zeamais and can suppress

170

oviposition as measured by progeny development of the weevils, resulting in better

protection of stored maize grains from pest infestation and damage.

Reproduction inhibition could imply that the powders also affected developmental

stages of the insect. In this study, the effect of A. melegueta and Z. officinale volatiles

on the developmental stages of S. zeamais was not demonstrated. However, Huang et

al (2000) reported that the essential oil from the seeds of cardamom, Elletaria

cardamomum (L.) Maton applied to filter papers in the concentration of 1.04-2.34 mg

cm-2 significantly reduced the hatching of T. castaneum eggs and survival rate of the

larvae. The adults of S. zeamais and T. castaneum, and larvae of T. castaneum were

equally susceptible to contact toxicity of the essential oil at the LD50 level, with LD50

values of 56 and 52 µg mg-1 insect respectively. As repellents, they produced a

vapour layer that has an offensive odour or taste to the weevils. Results from this

study demonstrate practically that the repellent effects of A. melegueta and Z.

officinale could be used to prevent S. zeamais infestation of stored maize by masking

the odours from grain in order to make the weevils unable to detect the presence of

food and oviposition sites. The use of locally available plant materials for stored-

product protection is a common practice, and has more potential in subsistence and

traditional farm storage conditions, in developing and under-developed countries

(Weaver and Subramanyam, 2000; Nikpay, 2007). Under small-scale farmer

conditions, protecting grains with indigenous repellent plants could lead to the

development of a sustainable crop protection method.

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6.4 POTENTIAL FOR APPLICATION BY SMALL-SCALE FARMERS

The ultimate goal of this study is the development of a push-pull strategy for the

control of S. zeamais in small-scale traditional storage facilities in Africa. Push-pull or

stimulo-deterrent diversionary strategies (S.D.D.S.) involve a combination of various

behaviour-modifying stimuli to manipulate the prevalence and distribution of pest

species and beneficial organisms for pest management. The principles of the push-pull

strategy are to maximize pest control efficacy, efficiency, sustainability, and output,

while minimizing negative environmental effects. The ‘push’ could be a repellent,

antifeedant or an oviposition deterrent natural agent, and the ‘pull’ a kairomone,

aggregation, sex and oviposition pheromones or a selective control agent (Pickett et

al., 1997; Cook et al., 2007). There appears to have been no successful application of

the push-pull strategy to protect stored grain from insect attack, although the literature

is rich in reports of plant-derived repellents in stored-product protection (Obeng-Ofori

and Reichmuth, 1997; Bekele et al., 1997; Isman, 2000; 2006; Huang et al., 2000;

Nikpay, 2007). Findings from this study could be applied practically by small-scale

farmers in Nigeria by direct administration of the seeds of A. melegueta and Z.

officinale rhizomes powders with maize cobs for protection against S. zeamais.

However, there may be a need to re-apply the repellent plant powders after 9-10

weeks to boost their efficacies. Framers could also be encouraged to expand the

existing cultivation of A. melegueta and Z. officinale thereby boosting agriculture and

more cash in their pockets. This study provides the underpinning science for use of

the repellent plant materials in stored-product pest control, and provides chemical

markers for quality assurance and control if the envisaged push-pull system breaks

down.

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Monitoring is a vital component of all integrated pest management programmes. The

foundation of any successful agricultural production programme is an effective pest

monitoring system that supplies information on not only the number and type of pests

present in the agro-ecosystem, but also detects changes in pest populations over time

and locates foci of infestation and routes of entry (Campbell et al., 2002). The use of

attractants in insect traps can lead to earlier detection of infestations, more accurate

monitoring of pest population levels and development of proper control measures.

Several trap designs specific for stored-product pests have been developed and are

commercially available (Mullen and Dowdy, 2001). Since insects recognize suitable

hosts by using key volatiles that are most often present in specific ratios (Bruce et al.,

2005), host volatiles used in host location by Sitophilus species such as kibbled pods

of the carob tree Ceratonia siliqua L. or their extracts (Wakefield et al., 2005), (E)-2-

nonenal and 4-ethylacetophenone reported to be attractive to S. granarius, O.

surinamensis and C. ferrugineus (Collins et al., 2007) can be used to bait traps for

monitoring, mass-trapping, or in attracticide strategies. Insect sex and aggregation

pheromones have also been isolated and lures are commercially available for many

stored-product pests including S. zeamais (Chambers, 1990; Phillips et al., 2000). For

efficiency, host volatiles can be deployed with the synthetic aggregation pheromone

sitophilure encapsulated in delta traps with sticky bases placed at strategic positions

outside the granary could serve to “pull” the visiting pest species as the trap. The

deployment of host volatiles in combination with aggregation pheromone could

produce improve attraction, because synergistic or additive effects of cracked wheat

and sitophilure have been reported for S. zeamais (Walgenbach et al., 1987). Three

grain volatiles, valeraldehyde, maltol and vanillin, plus sitophilure were more

attractive to S. oryzae than either the pheromone or the grain volatile mixture alone

173

(Phillips et al., 1993). Likhayo and Hodges (2000) also found a significant increase in

catch of S. zeamais in refuge trap and flight traps baited with sitophinone and cracked

wheat compared to either component alone, and some olfactory cells may respond to

both host-plant volatiles and pheromones (Ansebo et al., 2005). The trapped weevils

could then be selectively destroyed. The application of the push-pull strategy in

stored-product protection may reduce pest damage by feeding and oviposition and

result in a more environmentally sound crop protection practice by small-scale

farmers in Africa.

In future, the push-pull strategy could be extended to larger granaries. This future

work could combine this identified ‘push’ component with a ‘pull’ component

perhaps comprising of aggregation pheromone with host plant volatiles in traps to

capture pest species. The identified biologically active compounds from A. melegueta

and Z. officinale can be used as repellents or “push” against S. zeamais away from

stored maize. Blends of (S)-2-heptanol, (S)-2-heptyl acetate and (R)-linalool in their

natural ratios of 1:6:3 from A. melegueta, and blends of 1,8-cineole, neral and geranial

in the ratio 5.48:1:2.13 from Z. officinale essential oil can be prepared and applied as

protective bands around grain bulks or incorporated into packaging materials, such as

sacking and paper, to mask odours from stored maize or evoke non-host avoidance

and repellent behaviours in the weevils. The synthetic blends could also be used to

treat the structure of an empty store to flush out hidden infestation before fresh grain

is introduced.

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6.5 CONCLUSIONS

An understanding of insect-plant interactions and chemical ecology is needed for the

successful management practices relying on semiochemicals (Pickett et al., 1997;

Piesik et al., 2008). The current study has identified components that provide the

“push” side of a push-pull control strategy. This study has also shown that blends of

volatile compounds from the essential oils of A. melegueta and Z. officinale in their

natural ratios were repellent to S. zeamais, inhibited its feeding and oviposition and

suppressed progeny emergence. The identified repellent compounds from A.

melegueta were (S)-2-heptanol, (S)-2-heptyl acetate and (R)-linalool, and from Z.

officinale, 1,8-cineole, neral and geranial. Repellence of S. zeamais adults was

demonstrated in olfactometer tests with walking weevils, laboratory oviposition tests

and under natural tropical storage conditions in southern Nigeria. The findings

suggest that odour is the main stimulus for oriented movement of S. zeamais to the

cue for available food substrate. On the “pull” side, the result of attractiveness based

on the walking behaviour of S. zeamais in olfactometer bioassays confirmed that

white maize; yellow maize and winter wheat kernels were attractive to the weevils.

This could lead to the development of synthetically-baited lures to be used in delta

traps, possibly in combination with its synthetic aggregation pheromone sitophilure.

The results obtained in this study are encouraging given the ongoing search for

attractants for maize weevils. Further research is needed to identify biologically active

compounds and other potential semiochemicals released by host plants (maize, wheat,

sorghum) that attract the maize weevils. When all active compounds in the volatile

collections from maize and wheat grains are identified, detailed behavioural studies

should be undertaken with blends containing varying amounts of behaviourally active

compounds in their natural ratios. The experiments will determine the optimal blend

175

of the synthetic plant compounds for use in applied research targeting semiochemical

based stored-product pest management. Also, application of slow release formulations

of a blend of attractive chemicals may be used to concentrate S. zeamais to traps from

where they will be selectively destroyed.

The present study will contribute to the development of management tactics that rely

on the exploitation of semiochemicals to manipulate oviposition behaviour of the

maize weevils such as application of repellent blends of essential oils from A.

melegueta and/or Z. officinale to the packaging materials of stored maize and mass

trapping of visiting weevils with host volatiles deployed with sitophilure outside the

granaries. Therefore, the use of a stimulo-deterrent diversionary strategy that takes

advantage of naturally-occurring semiochemicals in traditional maize storage

granaries in Africa appears to be feasible in the short term.

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REFERENCES

Abder-Rahman, H. (1999) Effect of aluminium phosphide on blood glucose level. Veterinary and Human Toxicology, 41: 31-32.

Abo, K.A., Fred-Jaiyesimi, A.A. and Jaiyesimi, A.E.A. (2008) Ethnobotanical studies of medicinal plants used in the management of diabetes mellitus in South Western Nigeria. Journal of Ethnopharmacology, 115: 67-71.

Abubakar, M.S., Abdurahman, E.M. and Haruna, A.K. (2000) The repellent and antifeedant properties of Cyperus articulatus against Tribolium castaneum Hbst. Phytotherapy Research, 14: 281-283.

Abubakar, M.S., Musa, A.M., Ahmed, A. and Hussaini, I.M. (2007) The perception and practice of traditional medicine in the treatment of cancers and inflammations by the Hausa and Fulani tribes of Northern Nigeria. Journal of Ethnopharmacology, 111: 625-629.

Adams, J.M. (1976) Weight loss caused by development of Sitophilus zeamais Motsch. in maize . Journal of Stored Products Research, 12: 269-272.

Adane, K., Moore, D. and Archer, S.A. (1996) Preliminary studies on the use of Beauveria bassiana to control Sitophilus zeamais (Coleoptera: Curculionidae) in the laboratory. Journal of Stored Products Research, 32: 105-113.

Adda, C., Borgemeister, C., Biliwa, A., Meikle, W.G., Markham, R.H. and Poehling, H.-M. (2002) Integrated pest management in post-harvest maize: a case study from the Republic of Togo (West Africa). Agriculture, Ecosystems and Environment, 93: 305-321.

Adewoyin, F.B., Odaibo, A.B. and Adewunmi, C.O. (2006) Mosquito repellent activity of Piper guineense and Xylopia aethiopica fruits oils on Aedes aegypti. African Journal of Traditional, Complementary and Alternative Medicines, 3: 79-83.

Adler, C., Ojimelukwe, P. and Leon, T.A. (2000) Utilisation of phytochemicals against stored product insects. Integrated Protection of Stored Products, IOBC Bulletin, 23: 169-175.

Adler, C. and Rassmann, W. (2000) Utilization of extreme temperatures in stored product protection. Integrated Protection of Stored Products, IOBC Bulletin, 23: 257-262.

Agboola, A.A. and Fayemi, A.A. (1999) Preliminary trials on intercropping of maize with different tropical legumes in western Nigeria. Nigeria Journal of Agricultural Science, 71: 219-225.

177

Aharoni, A., Giri, A.P., Deuerlein, S., Griepink, F., de Kogel, W.J., Verstappen, F.W.A., Verhoeven, H.A., Jongsma, M.A., Schwab, W. and Bouwmeester, H.J. (2003) Terpenoid metabolism in wild and transgenic Arabidopsis plants. The Plant Cell, 115: 2866-2884.

Ajaiyeoba, E.O. and Ekundayo, O. (1999) Essential oil constituents of Aframomum melegueta (Roscoe) K. Schum seeds (alligator pepper) from Nigeria. Flavour and Fragrance Journal, 14: 109-111.

Ajose, F.O.A. (2007) Some Nigerian plants of dermatologic importance. International Journal of Dermatology, 46: 48-55.

Akob, C.A. and Ewete, F.K. (2007) The efficacy of ashes of four locally used plant materials against Sitophilus zeamais (Coleoptera: Curculionidae) in Cameroon. International Journal of Tropical Insect Science, 27: 21-26.

Ali, B.H., Blunden, G., Tanira, M.O. and Nemmar, A. (2008) Some phytochemical, pharmacological and toxicological properties of ginger (Zingiber officinale Roscoe): A review of recent research. Food and Chemical Toxicology, 46: 409-420.

Amarawardana, L., Bandara, P., Kumar, V., Pettersson, J., Ninkovic, V. and Glinwood, R. (2007) Olfactory response of Myzus persicae (Homoptera: Aphididae) to volatiles from leek and chive: Potential for intercropping with sweet pepper. Acta Agriculturae Scandinavica Section B – Soil and Plant Science, 57: 87-91.

Ansebo, L., Ignell, J. and Hansson, B. (2005) Responses to sex pheromone and plant odours by olfactory receptor neurons housed in sensilla auricillica of the codling moth, Cydia pomonella (Lepidoptera: Tortricidae). Journal of Insect Physiology, 51: 1066-1074.

Appert, J. (1987) The storage of Food: Grains and Seeds. Macmillan Publishers Ltd., London, 146p.

Arbogast, R.T. and Throne, J.E. (1997) Insect infestation of farm-stored maize in South Carolina: Towards characterization of a habitat. Journal of Stored Products Research, 33: 187-198.

Arlian, L.G. (2002) Arthropod allergies and human health. Annual Review of Entomology, 47: 395-434.

Arthur, F.H. (2002) Survival of Sitophilus oryzae (L.) on wheat treated with diatomaceous earth: impact of biological and environmental parameters on product efficacy. Journal of Stored Products Research, 38: 305-313.

Arthur, F.H. and Throne, J.E. (2003) Efficacy of diatomaceous earth to control internal infestations of rice weevil and maize weevil (Coleoptera: Curculionidae). Journal of Economic Entomology, 96: 510-518.

178

Arthur, F.H., Hagstrum, D.W., Flinn, P.W., Reed, C.R. and Phillips, T.W. (2006) Insect populations in grain residues associated with commercial Kansas’s grain elevators. Journal of Stored Products Research, 42:226-236.

Babatunde, R.O., Omotesho, O.A. and Sholotan, O.S. (2007) Socio-economics characteristics and food security status of farming households in Kwara State, North-Central Nigeria. Pakistan Journal of Nutrition, 6: 49-58.

Bashir, T., Birkinshaw, L.A., Hall, D.R. and Hodges, R.J. (2001) Host odours enhance the responses of adult Rhyzopertha dominica male-produced aggregation pheromone. Entomologia Experimentalis et Applicata, 101: 273-280.

Beckett, S.J., Fields, P.G. and Subramanyam, Bh. (2007) Disinfestation of stored products and associated structures using heat. In: Tang, J., Mitcham, E., Wang, S. and Lurie, S. (eds.), Heat treatments for post harvest pest control: Theory and practice. Wallingford, UK, CAB International, pp. 182-965.

Bekele, J. (2002) Evaluation of the toxicity potential of Milletia ferruginea (Hochest) Baker against Sitophilus zeamais (Motsch.). International Journal of Pest Management, 48(1): 29-32.

Bekele, J. and Hassanali, A. (2001) Blend effects in the toxicity of the essential oil constituents of Ocimum kilimandscharicum and Ocimum kenyense (Labiateae) on two post-harvest insect pests. Phytochemistry, 57: 385-391.

Bekele, J., Obeng-Ofori, D. and Hassanali, A. (1995) Products derived from the leaves of Ocimum kilimandscharicum (Labiatae) as post-harvest grain protectants against the infestation of three major stored product insect pests. Bulletin of Entomological Research, 85: 361-367.

Bekele, J.A., Obeng-Ofori, D. and A. Hassanali (1997) Evaluation of Ocimum kenyense (Ayobangira) as repellents, toxicants and protectants in storage against three stored product insect pests. Journal of Applied Entomology, 121: 169-173.

Belmain, S.R., Neal, G.E., Ray, D.E. and Golop, P. (2001) Insecticidal and vertebrate toxicity associated with ethnobotanicals used as post-harvest protectants in Ghana. Food and Chemical Toxicology, 39: 287-291.

Belmain, S.R., Ogendo, J.O., Deng, A.L. and Walker, D.J. (2003) Comparison of toxic and repellent effects of Lantana camara L. with Tephrosia vogelli Hook and a synthetic pesticide against Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) in stored grain. Insect Science and its Application, 23(2): 127-135.

Belmain, S.R., Jayasekara, T.K., Stevenson, P.C. and Hall, D.R. (2005) Effect of volatile constituents from Securidaca longepedunculata on insect pests of stored grain. Journal of Chemical Ecology, 31(2): 303-313.

Bhattarai, S., Tran, V.H. and Duke, C.C. (2001) The stability of gingerol and shogaol in aqueous solution. Journal of Pharmaceutical Sciences, 90: 1658-1664.

179

Birkett, M.A., Al Abassi, S., Kröber, T., Chamberlain, K., Hooper, A.M., Guerin, P. A., Pettersson, J., Pickett, J.A., Slade, R. and Wadhams, L.J. (2008) Antiectoparasitic activity of the gum resin, gum haggar, from the East African plant, Commiphora holtziana. Phytochemistry, 69: 1710-1715.

Birkett, M.A., Chamberlain, K., Khan, Z.R., Pickett, J.A., Toshova, T., Wadhams, L.J. and Woodcock, C.M. (2006) Electrophysiological responses of the Lepidopterous stemborers Chilo partellus and Busseola fusca to volatiles from wild and cultivated host plants. Journal of Chemical Ecology, 32: 2475-2487.

Blight, M.M., Pickett, J.A., Wadhams, L.J. and Woodcock, C.M. (1995) Antennal perception of oilseed rape, Brassica napus (Brassicaceae), volatiles by the cabbage seed weevil Ceutorhynchus assimilis (Coleoptera: Curculionidae). Journal of Chemical Ecology, 21: 1649-1664.

Boeke, S.J., Baumgart, I.R., Van Loon, J.J.A., Van Huis, A., Dicke, M. and Kossou, D.K. (2004) Toxicity and repellence of African plants traditionally used for the protection of stored cowpea against Callosobruchus maculatus. Journal of Stored Products Research, 40: 423-438.

Bond, E.J. (1984) Manual for fumigation for insect control. Rome, FAO, 432pp.

Bond, E.J., Dumas, T. and Hobbs, S. (1984) Corrosion of metals by the fumigant phosphine. Journal of Stored Products Research, 20: 57-63.

Borden, J.H., Lindergren, B.S. and Chong, L. (1980) Ethanol and alpha pinene as synergists for the aggregation pheromones of two Gnathotrichus species. Canadian Journal of Forestry Research, 10: 290-292.

Bouda, H., Tapondjou, L.A., Fontem, D.A. and Gumedzoe, M.Y.D. (2001) Effects of essential oils from leaves of Ageratum conyzoides, Lantana camara and Chromolaena odorata on the mortality of Sitophilus zeamais (Coleoptera: Curculionidae). Journal of Stored Products Research, 37: 103-109.

Bruce, T.J.A., Wadhams, L.J. and Woodcock, C.M. (2005) Insect host location: a volatile situation. Trends in Plant Science, 10: 269-274.

Burks, C.S., Johnson, J. A., Maier, D.E. and Heaps, J.W. (2000) Temperature. In: Subramanyam, Bh. and Hagstrum, D.W. (eds.), Alternatives to Pesticides in Stored-product IPM. Boston, Massachusetts, Kluwer Academic Publishers, pp. 73-104.

Campbell, J.F., Mullen, M.A. and Dowdy, A.K. (2002) Monitoring stored-product pests in food processing plants with pheromone trapping, contour mapping and mark-recapture. Journal of Economic Entomology, 95: 1089-1101.

Casida, J.H. (1990) Pesticide mode of action, evidence for implications of a finite number of biochemical targets. In: Casida, J.E. (ed.), Pesticides and Alternatives. Innovative Chemical and Biological Approaches to Pest Control, Amsterdam, Elsevier, pp. 11-22.

180

Cates, R.G. (1996) The role of mixtures and variations in the production of terpenoids in conifer-insect-pathogen interactions. Recent Advances in Phytochemistry, 30: 179-216.

Cha, D.H., Nojima, S., Hesler, S.P., Zhang, A., Linn, C.E., Roelofs, W.L. and Loeb, G.M. (2008) Identification and field evaluation of grape shoot volatiles attractive to female grape berry moth (Paralobesia viteana). Journal of Chemical Ecology, 34: 1180-1189.

Chambers, J. (1990) Overview on stored-product insect pheromones and food attractants. Journal of the Kansas Entomological Society, 63: 490-499.

Chhabra, S.C., Ndungu, M.W. and Lwande, W. (1999) Cleome hirta essential oil as livestock tick (Rhipicephalus appendiculatus) and maize weevil (Sitophilus zeamais) repellent. Fitoterapia, 70: 514-516.

Chung, W-Y, Jung, Y-J, Surh, Y-J, Lee, S-S, and Park, K-K. (2001) Antioxidative and antitumour promoting effects of (6)-paradol and its homologs. Mutation Research, 496: 199-206.

Collins, L.E., Wakefield, M.E., Chambers, J. and Cox, P.D. (2004) Progress towards a multi-species lure: comparison of behavioural bioassay methods for multi-species attractants against three pests of stored grain. Journal of stored products research, 40: 341-353.

Collins, L.E., Bryning, G.P., Wakefield, M.E., Chambers, J. and Cox, P.D. (2007) Progress towards a multi-species lure: identification of components of food volatiles as attractants for three storage beetles. Journal of stored products research, 43: 53-63.

Conner, W.F., Roach, B., Benedict, E., Meinwald. J. and Eisner, T. (1990) Courtship pheromone production and body size as correlates of larval diet in males of the arctiid moth, Utetheisa ornatrix. Journal of Chemical Ecology, 16: 543-552.

Cook, S.M., Khan, Z.R. and Pickett, J.A. (2007) The use of Push-Pull strategies in integrated pest management. Annual Review of Entomology, 52: 375-400.

Coppen, J.J.W. (1995) Flacours and Fragrances of plant origin. Food and Agricultural Organisation (FAO) Report, Rome, Italy.

Couty, A., Van Emden, H., Perry, J.N., Hardie, J., Pickett, J.A. and Wadhams, L.J. (2006) The roles of olfaction and vision in host-plant finding by the diamondback moth, Plutella xylostella. Physiological Entomology, 31: 134-145.

Cox, P.D. (2004) Potential for using semiochemicals to protect stored products from insect infestation. Journal of stored products research, 40: 1-25.

181

Cox, P.D and Collins, L.E. (2002) Factors affecting the behaviour of beetle pests in stored grain, with particular reference to the development of lures. Journal of stored products research, 38: 95-115.

Cox, P.D., Parish, W.E. and Ledson, M. (1990) Factors affecting the refuge-seeking behaviour of Cryptolestes ferrugineus. Journal of Stored Products Research, 26: 169-174.

Daglish, G.J. (2006) Survival and reproduction of Tribolium castaneam (Herbst), Rhyzopertha dominica (F.) and Sitophilus oryzae (L.) following periods of starvation. Journal of Stored Products Research, 42: 328-338.

Danho, M., Gaspar, C. and Haubruge, E. (2001) The impact of grain quantity on the biology of Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae): oviposition, distribution of eggs, adult emergence, body weight and sex ratio. Journal of Stored Products Research, 38: 259-266.

Darby, J. (1998) Putting grain aeration in order with generalized aeration categories. In: Banks, H.J., Wright, E.J. and Damcevski, K.A. (eds.), Stored grain in Australia. Proceedings of the Australian Postharvest Technical Conference, Canberra, 26-29 May 1998. CSIRO, Canberra. Pp. 203-208.

Degenhardt, J., Gershenzon, J., Baldwin, I.T. and Kessler, A. (2003) Attracting friends to feast on foes: engineering terpene emission to make crop plants more attractive to herbivore enemies. Current Opinion in Biotechnology, 14: 169-176.

Demissie, G., Teshome, A., Abakemal, D. and Tadesse, A. (2008). Cooking oils and “Triplex” in the control of Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) in farm-stored maize. Journal of Stored Products Research, 44: 173-178.

Dendy, J., Dobie, P., Smith, J.A. and Urono, J.L. (1991) Trials to assess the effectives of new synthetic pheromone mixtures for trapping Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae) in maize stores. Journal of Stored Products Research, 27: 69-74.

Denlinger, D.L. and Yocum, G.D. (1999) Physiology of heat sensitivity. In: Hallman, G. and Denlinger, D. (Eds.), Temperature sensitivity in insects and application in IPM. Colorado, Westview Press, Boulder, pp. 7-53.

Denyer, C.V., Jackson, P., Loakes, D.M., Ellis, M.R. and Young, D.A.B. (1994) Isolation of antirhinoviral sesquiterpenes from ginger (Zingiber officinale). Journal of Natural Products, 57: 658-662.

Desmarchelier, J.M., Allen, S.E., Yonglin, R., Moss, R. and Vu, L.T. (1998) Commercial-scale trials on the application of ethyl formate, carbonyl sulphide and carbon disulphide to wheat. CSIRO Entomology Technical Report No. 75.

182

Dethier, V.B., Browne, B.L. and Smith, C.N. (1960) The designation of chemicals in terms of the responses they elicit from insects. Journal of Economic Entomology, 53: 134-136.

Dickens, J.C. (1989) Green leaf volatiles enhance aggregation pheromone of the boll weevil Anthonomus grandis. Entomologia Experimentalis et Applicata, 52: 191-203.

Dickens, J.C., Billings, R.F. and Payne, T.L. (1992) Green leaf volatiles interrupt aggregation pheromone response in bark infesting pines. Experientia, 48: 523-524.

Dosland, O., Subramanyam, Bh., Sheppard, K. and Mahroof, R. (2006) Temperature modification for insect control. In: Heaps, J. (ed.), Insect Management for food storage and processing, 2nd edition. Minnesota, American Association for Clinical Chemistry, St Paul, pp. 89-103.

Dowdy, A.K. and Fields, A.K. (2002) Heat combined with diatomaceous earth to control the confused flour beetle (Coleoptera: Tenebrionidae) in a flourmill. Journal of Stored Products Research, 38: 11-22.

Dowdy, A.K. and McGaughey, W.H. (1998) Stored-product insect activity outside of grain masses in commercial grain elevators in the Midwestern United States. Journal of Stored Products Research, 34: 129-140.

Dowell, F.E., Throne, J.E., Wang, D. and Baker, J.E. (1999) Identifying stored-grain insects using near-infrared spectroscopy. Journal of Economic Entomology, 92: 165-169.

Duke, S.O., Baerson, S.R., Dayan, F.E., Rimando, A.M., Scheffler, B.E., Tellez, M.R., Wedge, D.E., Schrader, K.K., Ahey, D.H., Arthur, F.H., De Lucca, A.J., Gibson, D.M., Harrison, H.F., Peterson, J.K., Gealy, D.R., Tworkoski, T., Wilson, C.L. and Morris, J.B. (2003) United States Department of Agriculture – Agricultural Research Service research on natural products for pest management. Pest Management Science, 59: 708-717.

Edde, P.A. and Phillips, T.W. (2006) Potential host affinities for the lesser grain borer, Rhyzopertha dominica: behavioural responses to host odours and pheromones and reproductive ability on non-grain hosts. Entomologia Experimentalis et Applicata, 119: 255-263.

Eisner, T. and Meinwald, J. (1995) The chemistry of sexual selection. Proceedings of the National Academy of Science, U.S.A., 92: 50-55.

Ekundayo, O., Laakso, I. and Hiltunen, R. (1988) Composition of ginger (Zingiber officinale Roscoe) volatile oils from Nigeria. Flavour and Fragrance Journal, 3: 85-90.

183

Emana, G. (1999) Use of botanical plants in the control of stored maize grain insect pests in Ethiopia. In: CIMMYT and EARO (Eds.), Maize Production Technology for the future: Challenges and Opportunities. Proceedings of the sixth Eastern and Southern Africa Regional Maize Conference, 21-25 September, 1998, Addis Ababa, Ethiopia, IMMYT and EARO, pp. 105-108.

Enan, E. (2001) Insecticidal activity of essential oils: Octopaminergic sites of action. Comparative Biochemistry and Physiology, 130C: 325-337.

Escoubas, P., Lajide, L. and Mizutani, J. (1995) Termite antifeedant activity in Aframomum melegueta. Phytochemistry, 40: 1097-1099.

Food and Agricultural Organisation of the United Nations (FAO). (1985) Prevention of post-harvest food losses. Training Series No 10 (122), Food and Agricultural Organisation of United Nations, Rome, Italy, pp. 120.

Food and Agricultural Organisation of the United Nations (FAO). (2004) FAOSTAT database (http://apps.fao.org/faostat).

Faruki, S.I., Miyanoshita, A., Takahashi, K., Misumi, T., Imamura, T., Naito, H., Goto, M. and Soma, Y. (2005) Susceptibility of various developmental stages of the maize weevil, Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) to methyl iodide in brown rice. Journal of Applied Entomology, 129: 12-16.

Federal Republic of Nigeria Extraordinary Gazette No. 4. (2007) Abuja, Nigeria.

Fernandez, X., Pintaric, C., Lizzani-Cuvelier, L., Loiseau, A-M, Morello, A. and Pellerin, P. (2006) Chemical composition of absolute and supercritical carbon dioxide extract of Aframomum melegueta. Flavour and Fragrance Journal, 21: 162-165.

Fields, P.G. (1992) The control of stored-product insects and mites with extreme temperatures. Journal of Stored Products Research, 28: 89-118.

Fields, P.G. and Korunic, Z. (2000) The effect of grain moisture content and temperature on the efficacy of diatomaceous earths from different geographical locations against stored-product beetles. Journal of Stored Products Research, 36: 1-13. Fields, P.G. and White, N.D.G. (2002) Alternatives to methyl bromide treatments for stored-product and quarantine insects. Annual Review of Entomology, 47: 331-359.

Fields, P.G., Van Loon, I., Dolinski, N., Harris, L. and Burkholder, W.E. (1992) The distribution of Rhyzopertha dominica in Western Canada. The Canadian Entomologist, 125: 317-328.

Fields, P.G., Xie, Y.S. and Hou, X. (2001) Repellent effect of pea (Pisum sativum) fractions against stored-product insects. Journal of Stored Products Research, 37: 359-370.

184

Flinn, P.W., Kramer, K. J., Throne, J.E. and Morgan, T.D. (2006) Protection of stored maize from insect pests using a two-component biological control method consisting of a hymenopteran parasitoid, Theocolax elegans, and transgenic avidin maize powder. Journal of Stored Products Research, 42: 218-225.

Franceschi, V.R., Krokene, P., Christiansen, E. and Krekling, T. (2005) Anatomical and chemical defences of conifer bark against bark beetles and other pests. New Phytologist, 167: 353-376.

Germinara, G.S., De Cristofaro, A. and Rotundo, G. (2008) Behavioural responses of adult Sitophilus granarius to individual cereal volatiles. Journal of Chemical Ecology, 34: 523-529.

Glenn, D.M., Puterka, G.J., Vanderwet, T., Byers, R.E. and Feldhake, C. (1999) Hydrophobic particle films, a new paradigm for suppression of arthropod pests and plant diseases. Journal of Economic Entomology, 92: 759-771.

Golob, P. (1997) Current status and future perspectives for inert dusts for control of stored product insects. Journal of Stored Products Research, 33: 69-79.

Grosch, W. (2001) Evaluation of the key odorants of foods by dilution experiments, aroma models and omission. Chemical Senses, 26: 533-545.

Guerrero, A., Feixas, J., Pajares, J., Wadhams, L.J., Pickett, J.A. and Woodcock, C.M. (1997) Semiochemically induced inhibition of behaviour of Tomicus destruens (Woll.) (Coleoptera: Scolytidae). Naturwissenschaften, 84: 155-157.

Hagstrum, D.W. (2001) Immigration of insects into bins storing newly harvested wheat on 12 Kansa farms. Journal of Stored Products Research, 37: 221-229.

Haines, C.P. (1991) Insects and Arachnids of Tropical stored products: Their biology and identification (A training manual). Natural Resources Institute, Chatham, UK, 246pp.

Hallem, E.A., Dahanukar, A. and Carlson, J.R. (2006) Insect odour and taste receptors. Annual Review of Entomology, 51: 113-135.

Halstead, D.G.H. (1963). External sex differences in stored products Coleoptera. Bulletin of Entomological Research, 54: 119-134.

Hardie, J. (1989) Spectral specificity for targeted flight in the black bean aphid, Aphis fabae. Journal of Insect Physiology, 35: 619-626.

Hardie, J., Visser, J.H. and Piron, P.G.M. (1994) Perception of volatiles associated with sex and food by different adult forms of the black bean aphid, Aphis fabae. Physiological Entomology, 19: 278-284.

Harvey, D.J. (1981) Gas chromatographic and mass spectrometric studies of ginger constituents. Identification of gingerdiones and new hexa hydrocurcumin analogues. Journal of Chromatography A, 212: 75-84.

185

Hassanali, A., Lwande, W., Sitayo, O., Moreka, L., Nokoe, S. and Chayapa, A. (1990) Weevil repellent constituents of Ocimum suave leaves and Eugenia caryophyllata cloves used as grain protectants in parts of East Africa. Discovery and Innovation, 2: 91-95.

Hassanali, A., Bekele, A.J. and Obeng-Ofori, D. (1997) Evaluation of Ocimum kenyense (Ayobangira) as source of repellents, toxicants and protectants in storage against three major stored product insect pests. Journal of Applied Entomology, 121: 169-173.

Hayes, J.L., Strom, B.L., Roton, L.M. and Ingram, L.L. (1994) Repellent properties of the host compound 4-allylanisole to the southern pine beetle. Journal of Chemical Ecology, 20: 1595-1615.

Hildebrand, J.G. and Shepherd, G.M. (1997) Mechanism of olfactory discrimination: converging evidence for common principles across phyla. Annual Review of Neuroscience, 20: 595-631.

Hill, D.S. and Waller, J.M. (1990) Pests and Diseases of Tropical Crops. Field Handbook, Longman Scientists and Technical, UK, 432p.

Hodges, R.J., Hall, D.R., Mbugua, J.N. and Likhayo, P.W. (1998) The responses of Prostephanus truncatus (Coleoptera: Bostrichidae) to pheromone and synthetic maize volatiles as lures in crevices or flight traps. Bulletin of Entomological Research 88:131-139.

Holst, N., Meikle, W.G. and Markham, R.H. (2000) Grain injury models for Prostephanus truncatus (Coleoptera: Bostrichidae) and Sitophilus zeamais (Coleoptera: Curculionidae) in rural maize stores in West Africa. Journal of Economic Entomology, 93:1338-1346.

Howe, R.W. (1952) The biology of the rice weevil, Calandra oryzae (L.). Annals of Applied Biology, 39: 168-180.

Huang, Y., Lam, S.L., Ho, S.H. (2000) Bioactivities of essential oil from Elletaria cardamomum (L.) Maton. to Sitophilus zeamais Motschulsky and Tribolium castaneum (Herbst.). Journal of Stored Products Research, 36: 107-117.

Huber, D.P.W. and Borden, J.H. (2001) Angiosperm bark volatiles disrupt response of Douglas-fir beetle, Dendroctonus pseudotsugae, to attractant-baited traps. Journal of Chemical Ecology, 27: 217-233.

Hubert, J., Stejskal, V., Kubatova, A., Münzbergová , Z., Vanova, M. and Zdarkova, E. (2002) Mites as selective fungal carriers in stored grain. Experimental and Applied Acarology, 29: 69-87.

Hubert, J., Stejskal, V., Münzbergová, Z., Kubatova, A., Vanova, M. and Zdarkova, E. (2004) Mites and fungi in heavily infested crops in the Czech Republic. Journal of Economic Entomology, 97: 2144-2153.

186

Ishaya, D.B., Tunku, P. and Kuchinda, N.C. (2008) Evaluation of some weed control treatments for long season weed control in maize (Zea mays L.) under zero and minimum tillage at Samaru, in Nigeria. Crop Protection, 27: 1047-1051.

Isman, M.B. (2000) Plant essential oils for pest and disease management. Crop Protection, 19: 603-608.

Isman, M.B. (2006) Botanical Insecticides, Deterrents, and Repellents in Modern Agriculture and an increasingly regulated World. Annual Review of Entomology 51: 45-66.

Ivbijaro, M.F. and Agbaje, M. (1986) Insecticidal activities of Piper guineense and Capsicum species on the cowpea bruchid, Callosobruchus maculatus F. Insect Science and its Application, 7: 353-357.

Jaffé, K., Sánchez, P., Cerda, H., Hernández, J.V., Jaffé, R., Urdaneta, N., Guerra, G., Martínez, R. and Miras, B. (1993) Chemical ecology of the palm weevil Rhynchophorus palmarum (L.) (Coleoptera: Curculionidae): Attraction to host plants and to a male-produced aggregation pheromone. Journal of Chemical Ecology, 19: 1703-1720.

Jayasekara, T.K., Stevenson, P.C., Hall, D.R. and Belmain, S.R. (2005) Effects of volatile constituent from Securidaca longepedunculata on insect pests of stored grain. Journal of Chemical Ecology 31(2): 303-312.

Jitoe, A., Masuda, T., Tengah, G. P., Suprapta, D.N., Gara, I.W. and Nakatani, N. (1992) Antioxidant activity of tropical ginger extracts and analysis of the contained curcuminoids. Journal of Agricultural and Food Chemistry, 40: 1337-1340.

Juliana, G. and Su, H.C.F. (1983) Laboratory studies on several plant materials as insect repellents for protection of cereal grains. Journal of Economic Entomology, 76: 154-157.

Kanaujia, K.R. and Levinson, H.Z. (1981) Phagostimulatory responses and oviposition behaviour of Sitophilus granarius L. to newly harvested and stored wheat grains. Zeitschrift fuer Angewandte Entomologie, 91: 417-424.

Karunakaran, C., Jayas, D.S. and White, N.D.G. (2003) Soft X-ray inspection of wheat kernels infested by Sitophilus oryzae. Transactions of the American Society of Agricultural and Biological Engineers, 46: 739-745.

Kaškonienė, V., Venskutonis, P.R. and Čeksterytė, V. (2008) Composition of volatile compounds of honey of various floral origin and beebread collected in Lithuania. Food Chemistry, 111 :988-997.

Katiyah, S.K., Agarwal, R and Mukhtar, H. (1996) Inhibition of tumour promotion in sencar mouse skin by ethanol extract of Zingiber officinale rhizome. Cancer Research, 56: 1023-1030.

187

Keita, S.M., Vincen, C., Schmit, J.P., Ramaswamy, S. And Belanger, A. (2000) Effect of various essential oils on Callosobruchus maculatus (F.) (Coleoptera: Bruchidae). Journal of Stored Products Research, 36: 355-364.

Kessler, A. and Baldwin, I.T. (2001) Defensive function of herbivore-induced plant volatile emissions in nature. Science, 291: 2141-2144.

Kholsa, S.N., Handa, P. and Kholsa, P. (1992). Aluminium phosphide poisoning. Tropical Doctor, 22: 155-157.

Kikuzaki, H. and Nakatani, N. (1993) Antioxidant effects of some ginger constituents. Journal of Food Science, 58: 1407-1410.

Koona, P., Malaa, D. and Koona, O.E.S. (2007) Hexane extracts from Tephrosia vogelii Hook. F. protect stored maize against the weevil Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae). Entomological Science, 10: 107-111.

Korunic, Z. (1998) Diatomaceous earths, a group of natural insecticides. Journal of Stored Products Research, 34: 87-97.

Kouninki, H., Hance, T., Noudjou, F.A., Lognay, G., Malaisse, F., Ngassoum, M.B., Mapongmetsem, P.M., Ngamo, L.S.T. and Haubruge, E. (2007) Toxicity of some terpenoids of essential oils of Xylopia aethiopica from Cameroon against Sitophilus zeamais Motschulsky. Journal of Applied Entomology, 131(4): 269-274.

Kouninki, H., Haubruge, E., Noudjou, F.A., Lognay, G., Malaisse, F., Ngassoum, M.B., Ngamo, L.S.T. and Hance, T. (2005) Potential use of essential oils from Cameroon applied as fumigant or contact insecticides against Sitophilus zeamais Motsch. (Coleoptera: Curculionidae). Communications in Agricultural and Applied Sciences, 70: 787-792.

Landolt, P.J. and Phillips, T.W. (1997) Host plant influences on sex pheromone behaviour of phytophagous insects. Annual Review of Entomology, 42: 371-391.

Langenheim, J.H. (1994). Higher plant terpenoids: a phytocentric overview of their ecological roles. Journal of Chemical Ecology, 20: 1223-1280.

Langner, E., Greifenberg, S. and Gruenwald, J. (1998) Ginger: History and uses. Advances in Natural Therapy, 15: 25-44.

Larry, P.P. (2000) Entomology and Pest Management, fourth edition. Printice-Hall, India.

Law-Ogbomo, K.E. and Enobakhare, D.A. (2006) Efficacy of rubber seed oil, Palm oil and Palm kernel oil as grain protectants against Sitophilus zeamais (Motschulsky) (Coleoptera: Curculionidae) in three maize varieties. Journal of Entomology, 3: 40-47.

Lee, E. and Surh, Y-J. (1998) Induction of apoptosis in HL-60 cells by pungent vanilloids, (6)-gingerol and (6)-paradol. Cancer Letters, 134: 163-168.

188

Lee, S.E., Lee, B.H., Choi, W.S., Park, B.S., Kim, J.G. and Campbell, B.C. (2001) Fumigant toxicity of volatile natural products from Korean spices and medicinal plants towards the rice weevil, Sitophilus oryzae (L.). Pest Management Science, 57: 548-553. Leelaja, B.C., Rajashekar, Y., Reddy, P.V., Begum, K. and Rajendran, S. (2007) Enhanced fumigant toxicity of allyl acetate to stored-product beetles in the presence of carbon dioxide. Journal of Stored Products Research, 43: 45-48.

Levinson, H.Z. and Levinson, A.R. (1990) The Egyptian plagues and the beginnings of pest control in the ancient Oricent. Anzeiger für Schädlinskunde Pflanzenschutz Umweltschutz, 63: 81-96.

Levinson, H.Z. and Levinson, A.R. (1998) Control of stored food pests in the ancient Orient and classical antiquity. Journal of Applied Entomology, 122: 137-144.

Lextrait P., Biemont, J-C and Pouzat, J. (1995) Pheromone release by the two forms of Callosobruchus maculatus females: effects of age, temperature, and host plant. Physiological Entomology, 20: 309-317.

Likhayo, P.W and Hodges, R.J. (2000) Field monitoring Sitophilus zeamais and Sitophilus oryzae (Coleoptera: Curculionidae) using refuge flight traps baited with synthetic pheromone and cracked wheat. Journal of Stored Products Research, 36: 341 – 353.

Liu, C.H., Mishra, A.K. and Tan, R.X. (2006) Repellent, insecticidal and phytotoxic activities of isoalantolactone from Inula racemosa. Crop Protection, 25: 508-511.Liu, Z.L. and Ho, S.H. (1999) Bioactivity of the essential oil extracted from Evodia rutaecarpa Hook f. et Thomas against the grain storage insects, Sitophilus zeamais Motsch. and Tribolium castaneum (Herbst). Journal of Stored Products Research, 35: 317-328.

Logan, J.G. (2006) Differential responses of the yellow fever mosquito, Aedes aegypti and the Scottish biting midge, Culicoides impunctatus, to human host odours. PhD Thesis, University of Aberdeen, United Kingdom, pp. 282.

Lord, J.C. (2006) Interaction of Mattesia oryzaephili (Neogregarinorida: Lipotrophidae) with Cephalonomia species (Hymenoptera: Bethylidae) and their hosts Cryptolestes ferrugineus (Coleoptera: Laemophloeidae) and Oryzaephilus surinamensis (Coleoptera: Silvanidae). Biological Control, 37: 167-172.

Maarse, H. (1991) Volatile compounds from in foods and beverages. Marcel Dekker Inc., New York.

Mabberley, D.J. (1987) The plant book, A portable dictionary of higher plants. Cambridge University Press, Cambridge, UK.

MacLeod, A.J. and Pieris, N.M. (1984) Volatile aroma constituents of Sri Lanka ginger. Phytochemistry, 23: 353-359.

189

Mahroof, R., Subramanyam, Bh. and Flinn, P. (2005) Reproductive performance of Tribolium castaneum (Herbst.) (Coleoptera: Tenebrionidae) exposed to the minimum heat treatment temperature as pupae and adults. Journal of Economic Entomology, 98: 626-633.

Maier, D.E., Adams, W.H., Throne, J.E. and Mason, L.J. (1996) Temperature management of the maize weevil, Sitophilus zeamais Motsch. (Coleoptera: Curculionidae), in three locations in the United States. Journal of Stored Products Research, 32: 255-273.

Markham, R.H., Bosque-Perez, N., Borgemeister, C. and Meikle, W.G. (1994) Developing pest management strategies for the maize weevil, Sitophilus zeamais, and the Larger grain borer, Prostephanus truncatus, in the humid and sub humid tropics. FAO Plant Protection Bulletin, 42: 97-116.

Mauchline, A.L., Birkett, M.A., Woodcock, C.M., Pickett, J.A., Osborne, J.L. and Powell, W. (2008) Electrophysiological and behavioural responses of the pollen beetle, Meligethes aeneus, to volatiles from a non-host plant, lavender, Lavandula angustifolia (Lamiaceae). Arthropod-Plant Interactions, 2: 109-115.

Menut, C., Lamaty, G., Amvam Zollo, O.H., Atogho, B.M., Abondo, R. and Bessière, J.M. (1991) Aromatic plants of tropical central Africa V. volatile components of three Zingiberaceae from Cameroon: Aframomum melegueta (Roscoe) K. Schum., A. daniellii (Hoof. F) K. Schum. and A. sulcatum (Oliv. and Hanb.) K. Schum. Flavour and Fragrance Journal, 6: 183-186.

Mullen, M.A. (1992) Development of pheromone trap for monitoring Tribolium castaneum. Journal of Stored Products Research, 28: 245-249.

Mullen, M.A. and Dowdy, A.K. (2001) A pheromone-baited trap for monitoring the Indianmeal moth, Plodia interpunctella (Hubner) (Lepidoptera: Pyralidae). Journal of Stored Products Research, 37: 231-235.

Mulungu, L.S., Lupenza, G., Reuben, S.O.W.M. and Misangu, R.N. (2007) Evaluation of botanical products as stored grain protectants against maize weevil, Sitophilus zeamais on maize. Journal of Entomology, 4: 258-262.

Nawrot, J. and Czaplicki, E. (1982) Behaviour of granary weevil beetle towards some substances extracted from natural products. Zeszyty Problemowe Postepow Nauk Rolniczych, 202: 183-191.

Ndungu, M.W, Lwande, W., Hassanali, A., Moreka, L. and Chhabra, S.C. (1995) Cleome monophylla essential oil and its constituents as tick (Rhipicephalus appendiculatus) and maize weevil (Sitophilus zeamais) repellents. Entomologia Experimentalis et Applicata, 76: 217-222.

Neethirajan, S., Karunakaran, C., Jayas, D.S. and White, N.D.G. (2007) Detection techniques for stored-product insects in grain. Food Control, 18: 157-162.

190

Neven, L.G. (2000) Physiological responses of insects to heat. Postharvest Biology and Technology, 21: 103-111.

Nikpay, A. (2007) Insecticidal efficacy of three vegetable oils as post-harvest grain protectants of stored wheat against Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae). Insect Science, 14: 145-150.

Nishida, R. (2002) Sequestration of defence substances from plants by Lepidoptera. Annual Review of Entomology, 47: 57-92.

Nishimura, O. (1995) Identification of the characteristic odorants in fresh rhizomes of ginger (Zingiber officinale Roscoe) using aroma extract dilution analysis and modified multidimensional gas chromatography-mass spectrometry. Journal of Agricultural and Food Chemistry, 43: 2941-2945.

NIST (2005) NIST mass spectral search for the NIST/EPA/NIH mass spectral library version 2.0. Office of the Standard Reference Data Base, National Institute of Standards and Technology, Gaithersburg, Maryland.

Nottingham, S.F. and Hardie, J. (1993) Flight behaviour of the black bean aphid, Aphis fabae, and the cabbage aphid, Brevicoryne brassicae, in host and non-host plant odour. Physiological Entomology, 18: 389-394.

Obeng-Ofori, D. and Amiteye, S. (2005) Efficacy of mixing vegetable oils with pirimiphos-methyl against the maize weevil, Sitophilus zeamais Motschulsky in stored maize. Journal of Stored Products Research, 41: 57-66.

Obeng-Ofori, D. and Reichmuth, Ch. (1997) Bioactivity of eugenol, a major component of essential oil of Ocimum suave (Wild.) against four species of stored-product Coleoptera. International Journal of Pest Management, 43: 89-94.

Obeng-Ofori, D., Reichmuth, Ch., Bekele, J. and Hassanali, A. (1997) Biological activity of 1,8-cineole, a major component of essential oil of Ocimum kenyense (Ayobangira) against stored product beetles. Journal of Applied Entomology, 121: 237-243.

Ogunwolu, E.O., Igoli, J.O. and Longe, N.N. (1998) Reduction in reproductive fitness of Callosobruchus maculatus F. exposed to Zanthoxylum zanthoxyloides (Lam.) Watern. Journal of Herbs, Spices and Medicinal Plants, 6: 19-27.

Okiwelu, S.S., Adu, O.O. and Okonkwo, V.N. (1987) The effect of Sitophilus zeamais (Mots.) (Coleoptera: Curculionidae) on the quality and viability of stored maize in Nigeria. Insect Science and its Application, 8: 379-384.

Okonkwo, E.U. and Okoye, W.I. (1996) The efficacy of four seed powders and the essential oils as protectants of cowpea and maize grains against infestation by Callosobruchus maculatus (Fabricius) (Coleoptera: Bruchidae) and Sitophilus zeamais (Motschulsky) (Coleoptera: Curculionidae) in Nigeria. International Journal of Tropical Pest Management, 42: 143-146.

191

Oloke, J.K. and Kolawale, D.O. (1988) The antibacterial and antifungal activities of certain components of Aframomum melegueta fruits. Fitoterapia, 59: 384-388.

Oparaeke, A.M. and Kuhiep, G.C. (2006) Toxicity of powders from indigenous plants against Sitophilus zeamais Motsch on Stored Grains. Journal of Entomology, 3: 216-221.

Oyedeji, O.A, Adeniji, B.A., Ajayi, O. and Konig, W.A. (2005) Essential oil composition of Piper guineense and its antimicrobial activity: Another chemotype from Nigeria. Phytotherapy Research, 19: 362-364.

Panthenius, C.U. (1988) Storage losses in traditional maize granaries in Togo. Insect Science and its Application, 9:725-735.

Paré, P.W. and Tumlinson, J.H. (1999) Plant volatiles as a defence against insect herbivores. Plant Physiology, 121: 325-331.

Park, K.C., Elias, D., Donato, B. and Hardie, J. (2000) Electroantennogram and behavioural responses of different forms of the bird cherry-oat aphid, Rhopalosiphum padi, to sex pheromone and a plant volatile. Journal of Insect Physiology, 46: 597-604.

Pettersson, J. (1970) An aphid sex attractant 1. Biological studies. Entomologica Scandinavica, 1: 63-73.

Phillips, T.W. (1997) Semiochemicals of stored product insects: Research and applications. Journal of Stored Products Research, 33: 17-30.

Phillips, T.W., Cogan, P.M. and Fadamiro, H.Y. (2000) Pheromones. In: Subramanyam, Bh and Hagstrum, D.W. (eds.), Alternatives to pesticides in stored-product IPM. Boston, Kluwer Academic Publishers, pp. 273-302.

Phillips, T.W., Jiang, X.L., Burkholder, W.E., Phillips, J.K. and Tran, H.Q. (1993) Behavioural responses to food volatiles by two species of stored-product Coleoptera, Sitophilus oryzae (Curculionidae) and Tribolium castaneum (Tenebrionidae). Journal of Chemical Ecology, 19:723-734.

Phillips, T.W., West, J.R., Foltz, R.M., Silverstein, R.M. and Lanier, G.N. (1984) Aggregation pheromone of the deodar weevil, Pissodes nemorensis (Coleoptera: Curculionidae): isolation and activity of grandisol and grandisal. Journal of Chemical Ecology, 10: 1417-1423.

Pickett, J.A. (1990) Gas chromatography – mass spectrometry in insect pheromone identification: three extreme cases. In: McCaffery, A.R. and Wilson I.D. (eds.), Chromatography and isolation of insect pheromones and hormones. New York, Plenum Press, pp. 209-309.

192

Pickett, J.A., Wadhams, L.J. and Woodcock, C.M. (1997) Developing sustainable pest control from chemical ecology. Agriculture, Ecosystems and Environment, 64: 149-156.

Piesik, D., Weaver, D.K., Runyon, J.B., Buteler, M., Peck, G.E. and Morrill, W.L. (2008) Behavioural responses of wheat stem sawflies to wheat volatiles. Agricultural and Forest Entomology, 10: 245-253.

Pike, V., Smith, J.L., White, R.D. and Hall, D.R. (1994) Studies of responses of stored-products pests, Prostephanus truncatus (Horn) and Sitophilus zeamais (Motsch.), to food volatiles. In: Highley, E., Wright, E.J., Banks, H.J. and Champ, B.R. (eds.), Proceedings of the 6th International Working Conference on Stored-Product Protection, Canberra, Australia. Wallingford, UK, CAB International, pp. 566-569.

Plarre, R. and Vanderwel, D.C. (1999) Stored-product beetles. In: Hardie, J. and Minks, A.K. (eds.). Pheromones of Non-Lepidopteran insects associated with Agricultural plants. Wallingford, UK, CABI International, pp. 149-198.

Poland, T.M., Borden, J.H., Stock, A.J. and Chong, L.J. (1998) Green leaf volatiles disrupt responses by the spruce beetle, Dendroctonus brevicomis (Coleoptera: Scolytidae) to attractant-baited traps. Journal of the Entomological Society of British Colombia, 95: 17-24.

Pomeranz, T. (1978) Cereals ’78: Better nutrition for the world’s millions. In Proceedings of the Sixth International Cereal and Bread Congress. American Association of Cereal Chemists Inc., USA, 272 pp.

Poswall, M.A.T. and Akpa, A.D. (1991) Current trends in the use of traditional and organic methods for the control of crop pests and diseases in Nigeria. Tropical Pest Management, 37: 329-333.

Powell, G., Tosh, C.R. and Hardie, J. (2006) Host plant selection by aphids: behavioural, evolutionary, and applied perspectives. Annual Review of Entomology, 51: 309-330.

Powell, J.S. and Raffa, K.F. (1999) Sources of variation in concentration and composition of foliar monoterpenes in Tamarack (Larix laricina) seedlings: roles of nutrient availability, time of season, and plant architecture. Journal of Chemical Ecology, 25: 1771-1797.

Price, N. (1996) Methyl bromide in Perspective. In: Bell, C. H., Price, N. and Chabrabarti, B. (eds.), The methyl bromide issue. New York, John Wiley and Sons, pp. 1-26.

193

Pungitore, C.R., Garcia, M., Gianello, J.C., Sosa, M.E. and Tonn, C.E. (2003) Insecticidal and antifeedant effects of Junellia aspera (Verbernaceae) triterpenes and derivatives on Sitophilus oryzae (Coleoptera: Curculionidae). Journal of Stored Products Research, 41: 433-443.

Purseglove, J. W. (1974) Tropical Crops: Dicotyledons, Longman, London, 719pp.

Rajendran, S. (2002) Post harvest pest losses. In D. Pimentel (ed.), Encyclopaedia of Pest Management. New York, Marcel Dekker Inc., pp. 654-656.

Rajendran, S. and Gunasekaran, N. (2002) The response of phosphine-resistant lesser grain borer Rhyzopertha dominica and rice weevil Sitophilus oryzae in mixed-age cultures to varying concentrations of phosphine. Pesticide Management Science, 58: 277-281.

Rajendran, S. and Muralidharan, N. (2005) Effectiveness of allyl acetate as a fumigant against five stored grain beetle pests. Pest Management Science, 61: 97-101.

Rajendran, S. and Parveen, H.K.M. (2005) Insect infestation in stored animal products. Journal of Stored Products Research, 41: 1-30.

Rajendran, S. and Sriranjini, V. (2008) Plant products as fumigants for stored-product insect control. Journal of Stored Products Research, 44: 126-135.

Reddy, G.V.P. and Guerrero, A. (2004) Interactions of insect pheromones and plant semiochemicals. Trends in Plant Science, 9: 253-261.

Reed, C.R., Hagstrum, D.W., Flinn, P.W. and Allen, R.F. (2003) Wheat in bins and discharge spouts, and grain residues on floors of empty bins in concrete grain elevators as habitats for stored grain beetles and their natural enemies. Journal of Economic Entomology, 96: 996-1004.

Rees, D. (2004) Insects of stored products. CSIRO Publishing, Collingwood, Australia, 181p.

Regnault-Roger, C. (1997) The potential of botanical essential oils for insect pest control. Integrated Pest Management Reviews, 2: 25-34.

Richter, J., Biliwa, A. and Henning-Helbig, S. (1997) Losses and pest infestation in different maize storage systems with particular emphasis on Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae) in Togo. Anzeiger für Schadlingskunde-Pflanzenschutz-Umweltschutz, 70: 112-116.

Rosell, G., Quero, C., Coll, J. and Guerrero, A. (2008) Biorational insecticides in pest management. Journal of Pesticide Science, 33: 103-121.

Rospars, J.P. (1988) Structure and development of the insect antennodeutocerebral system. International Journal of Insect Morphology and Embryology, 17: 243-294.

194

Rozman, V., Kalinovic, I. and Korunic, Z. (2007) Toxicity of naturally occurring compounds of Lamiaceae and Lauraceae to three stored products insects. Journal of Stored Products Research, 43: 349-355.

Ryan, M.F. and Byne, O. (1988) Plant-insect coevolution and inhibition of acetylcholinesterase. Journal of Chemical Ecology, 14: 1965-1975.

Sahaf, B.Z., Moharramipour, S. and Meshkatalsadat, M.H. (2007) Chemical constituents and fumigant toxicity of essential oil from Carum copticum against two stored product beetles. Insect Science, 14: 213-218.

Sakamura, F. (1987) Changes in volatile constituents of Zingiber officinale during storage and cultivation. Phytochemistry, 26: 2207-2212.

Saxena, B.P., Sharma, P.R., Thappa, R.K. and Tikku, K. (1992) Temperature-induced sterilization for control of three stored grain beetles. Journal of Stored Products Research, 28: 67-70.

Seitz, L.M. and Sauer, D.B. (1992) Off-odours in grains. In: Charalambous, G. (ed.), Off-Flavours in Food and Beverages. Amsterdam, Elsevier, pp. 17-35.

Shaaya, E., Kostjukovsky, M., Eilberg, J. and Sukprakarn, C. (1997) Plant oils as fumigants and contact insecticides for the control of stored-product insects. Journal of Stored Products Research, 33: 7-15.

Shaaya, E., Kostjukovsky, M. and Demchenko, N. (2003) Alternative to fumigants for the control of stored-product insects. In: Credland, P.F., Armitage, D.M., Bell, C.H., Cogan, P.M. and Highley, E. (eds.), Advances in Stored-product Protection. Proceedings of the eighth International Working Conference on Stored-product Protection, York, UK, 22-26 July 2002. CAB International, Wallingford, UK, pp. 556-560.

Shaaya, E., Ravid, U., Paster, N., Juven, B., Zisman, U. and Pissarev, V. (1991) Fumigant toxicity of essential oils against four major stored-product insects. Journal of Chemical Ecology, 17: 499-504.

Shakarami, J., Kamali, K., Moharramipour, S. and Meshkatalsadat, M. (2004) Effects of three plant essential oils on biological activity of Callosobruchus maculatus F. (Coleoptera: Bruchidae). Iranian Journal of Agricultural Sciences, 35: 965-972.

Shanbhag, S.R., Muller, B. and Steinbrecht, R.A. (2000) Atlas of olfactory organs of Drosophila melanogaster 2. Internal organisation and cellular architecture of olfactory sensilla. Arthropod Structure and Development, 29: 211-229.

Shao, Y., Marriot, P., Shellie, R. and Hügel, H. (2003) Solid-phase micro-extraction – comprehensive two-dimensional gas chromatography of ginger (Zingiber officinale) volatiles. Flavour and Fragrance Journal, 18: 5-12.

195

Shepherd, W.P., Huber, D.P.W., Seybold, S.J. and Fettig, C.J. (2008) Antennal responses of the western pine beetle, Dendroctonus brevicomis (Coleoptera: Curculionidae), to stem volatiles of its primary host, Pinus ponderosa, and nine sympatric non-host angiosperms and conifers. Chemoecology, 17: 209-221.

Silva, G., Orrego, O., Hepp, R. and Tapia, M. (2005) Search for plants with insecticidal properties for Sitophilus zeamais control in stored corn. Pesquisa Agropecuaria Brasileira, 40: 11-17.

Simpson, B.B. (1995) Spices, herbs and perfumes. In: Simpson, B.B. and Ogorzaly, M.C. (eds.), Economic Botany: Plants in our world. McGraw-Hill, New York, pp. 278-301.

Singh, G. and Upadhyay, R.K. (1993) Essential oils: a potent source of natural pesticides. Journal of Scientific and Industrial Research, 52: 676-683.

Singh, G., Maurya, S., Catalan, C. and De Lampasona, M.P. (2005) Studies on essential oils, Part 42: chemical, antifungal, antioxidant and sprout suppressant studies on ginger essential oil and its oleoresin. Flavour and Fragrance Journal, 20: 1-6.

Singh, R.B., Singh, R.G. and Singh, U. (1991) Hypermagnesemia following aluminium phosphide poisoning. International Journal of Clinical Pharmacology and Therapeutic Toxicology, 29: 82-85.

Sinha, R.N. and Watters, F.L. (1985) Insect Pests of Flour Mills, Grain Elevators, and Feed Mills and their Control. Canada Government Publishing Centre, Agricultural Canada Publication No. 1776, Ottawa, Canada.

Smith, L.B. and Barker, P.S. (1987) Distribution of insects found in granary residues in the Canada Prairies. Canadian Entomologist, 119: 873-880.

Snelson, J.T. (1987) Grain Protectants. Monograph No. 3, Australian Centre for International Agricultural Research, Canberra, Australia, 448pp.

Städler, E. (1992) Behavioural responses of insects to plant secondary compounds. In G.A. Rosenthal and M.R. Berenbaum (eds.), Herbivores: their interactions with secondary plant metabolites, volume II. London, Academic Press, pp. 45-88.

Stamopoulos, D.C. (1991) Effects of four essential oil vapours on the oviposition and fecundity of Acanthoscelides obtectus (Say) (Coleoptera: Bruchidae): laboratory evaluation. Journal of Stored Products Research, 27: 199-203.

Stamopoulos, D.C., Damos, P. and Karagianidou, G. (2007) Bioactivity of five monoterpenoid vapours to Tribolium confusum (du Val) (Coleoptera: Tenebrionidae). Journal of Stored Products Research, 43: 571-577.

Stoll, G. (2000) Natural crop protection in the tropics: letting information come to life. Margraf Verlag, Weikersheim.

196

Subramanyam, Bh and Hagstrum, D.W. (2000) Botanicals. In: Subramanyam, Bh and Hagstrum, D.W. (eds.), Alternatives to Pesticides in Stored Product IPM. Boston, Kluwer Academic Publishers, pp. 303-320.

Subramanyam, Bh and Roesli, R. (2000) Inert Dusts. In: Subramanyam, Bh and Hagstrum, D.W. (eds.), Alternatives to Pesticides in Stored Product IPM. Boston, Kluwer Academic Publishers, pp. 321-380.

Takabayashi, J. and Dicke, M. (1996) Plant-carnivore mutualism through herbivore-induced carnivore attractants. Trends in Plant Science, 1: 109-113.

Tanabe, M., Chen, Y.D., Saito, K. and Kano, Y. (1993) Cholesterolbiosynthesis inhibitory component from Zingiber officinale Roscoe. Chemical and Pharmaceutical Bulletin, 41: 710-713.

Tapondjou, A.L., Adler, C., Bouda, H. and Fontem, D.A. (2002) Efficacy of powder and essential oil from Chenopodium ambrosioides leaves as post-harvest grain protectants against six-stored product beetles. Journal of Stored Products Research, 38: 395-402.

Tapondjou, A.L., Adler, C., Fontem, D.A., Bouda, H. and Reichmuth, C. (2005) Bioactivities of cymol and essential oils of Cupressus sempervirens and Eucalyptus saligna against Sitophilus zeamais Motschulsky and Tribolium confusum du Val. Journal of Stored Products Research, 41: 91 – 102.

Throne, J.E. (1994) Life history of Immature maize weevils (Coleoptera: Curculionidae) on Corn stored at constant temperatures and relative humidities in the Laboratory. Environmental Entomology, 23: 1459-1471.

Throne, J.E. and Cline, L.D. (1994) Seasonal flight activity and seasonal abundance of selected stored-product insects around grain storage in South Carolina. Journal of Agricultural Entomology, 11: 321-338.

Tiwari, S.N. (1994) Efficacy of some plant products as grain protectants against Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae). International Journal of Pest Management, 40: 94-97.

Tjendraputra, E., Tran, V.H., Liu-Brennan, D., Roufogalis, B.D. and Duke, C.C. (2001) Effect of ginger constituents and synthetic analogues on cyclooxygenase-2 enzyme in intact cells. Bioorganic Chemistry, 29: 156-163.

Toews, M.D., Pearson, T.C. and Campbell, J.F. (2006) Imaging and automated detection of Sitophilus oryzae (Coleoptera: Curculionidae) pupae in hard red winter wheat. Journal of Economic Entomology, 99: 583-592.

Tyler, P.S., Taylor, R.W. and Rees, D.P. (1983) Insect resistance to phosphine fumigation in food warehouses in Bangladesh. International Pest Control, 25: 10-21.

197

Umoetok, S.B.A. and Ukeh, D.A. (2004) Comparison of Aframomum melegueta and Piper nigrum crude oil extracts in the control of Callosobruchus maculatus (F.) and Sitophilus zeamais (Motsc.) on storage products in South Eastern Nigeria. Journal of Applied Sciences, 7: 3885-3892.

Van den Berg, J., Torto, B., Pickett, J.A., Smart, L.E., Wadhams, L.J. and Woodcock, C.M. (2008) Influence of visual and olfactory cues on field trapping of the pollen beetle, Astylus atromaculatus (Coleoptera: Melyridae). Journal of Applied Entomology, 132: 490-496.

Van Tol, R.W.H.M., James, D.E., de Kogel, J.W. and Teulon, D.A.J. (2007) Plant odours with potential for a push-pull strategy to control the onion thrips, Thrips tabaci. Entomologia Experimentalis et Applicata, 122: 69-76.

Verma, B.R. (1991) Vacuum fumigation schedules for seed inhabiting chalcidoids. Journal of Entomological Research, 15: 229-232.

Vermeulen, A. and Rospars, J.P. (2004) Why are insect olfactory receptor neurons grouped into sensilla? The teachings of a model investigating the effects of the electrical interaction between neurons on the transepithelial potential and the neuronal transmembrane potential. European Biophysics Journal, 33: 633-643.

Verspohl, E.J., Abdel-Aziz, H., Windeck, T. and Ploch, M. (2006) Mode of action of gingerols and shogaols on 5-HT3 receptors: Binding studies, cation uptake by the receptor channel and contraction of isolated guinea-pig ileum. European Journal of Pharmacology, 530: 136-143.

Vick, K.W., Mankin, R.W., Cogburn, R.R., Mullen, M., Throne, J. E. and Wright, V. F. (1990) Review of pheromone-baited sticky traps for detection of stored-product insects. Journal of Kansas Entomological Society, 63: 526-532.

Visser, J.H.(1986) Host odour reception in phytophagous insects. Annual Review of Entomology, 31: 121-144.

Wadhams, L.J. (1990) The use of coupled gas chromatography: electrophysiological techniques in the identification of insect pheromones. In: McCaffery, A.R. and Wilson, I.D. (eds.), Chromatography and the isolation of insect hormones. New York, Plenum Press, pp.289-298.

Wakefield, M.E., Bryning, G.P. and Chambers, J. (2005) Progress towards a lure to attract three stored product weevils, Sitophilus zeamais Motschulsky, S. oryzae (L.) and S. granarius (L.) (Coleoptera: Curculionidae). Journal of Stored Products Research, 41: 145-161.

Walgenbach, C.A. and Burkholder, W.E. (1987) Mating behaviour of the maize weevil Sitophilus zeamais (Coleoptera: Curculionidae). Annals of the Entomological Society of America, 80: 578-583.

198

Walgenbach, C.A., Burkholder, W.E., Curtis, M.J. and Khan, Z.A. (1987) Laboratory trapping studies with Sitophilus zeamais (Coleoptera: Curculionidae). Journal of Economic Entomology, 80: 763-767.

Walgenbach, C.A., Phillips, J.K., Faustini, D.L. and Burkholder, W.E. (1983) Male-produced aggregation pheromone of the maize weevil, Sitophilus zeamais, and interspecific attraction between three Sitophilus species. Journal of Chemical Ecology, 9: 831-841.

Wang, M. and Lincoln, D. (2004) Effect of light intensity and artificial wounding on monoterpenes production in Myrica cerifera from two different ecological habitats. Canadian Journal of Botany, 82: 1501-1508.

Wang, J., Zhu, F., Zhou, X.M., Niu, C.Y. and Lei, C.L. (2006) Repellent and fumigant activity of essential oil from Artemisia vulgaris to Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). Journal of Stored Products Research, 42: 339-347.

Watters, F.L., White, N.D.G. and Coté, D. (1983) Effects of temperature on toxicity and persistence of three pyrethroid insecticides applied to fir plywood for control of the red flour beetle (Coleoptera: Tenebrionidae). Journal of Economic Entomology, 76: 11-16.

Weaver, D.K. and Subramanyam, Bh. (2000) Botanicals. In: Subramanyam, Bh and Hagstrum, D.W. (eds.), Alternatives to Pesticides in Stored-product IPM, Boston, Kluwer Academic Publishers, pp. 303-320.

Webster, B., Bruce, T.J.A., Dufour, S., Birkemeyer, C., Birkett, M.A., Hardie, J. and Pickett, J.A. (2008) Identification of volatile compounds used in host location by the black bean aphid, Aphis fabae. Journal of Chemical Ecology, 34: 1153-1161.

Wild, P.C. and Hall, A.J. (2000) Primary prevention of hepatocellular carcinoma in developing countries. Mutation Research, 462: 381-393.

Wohlmuth, H., Leach, D.N., Smith, M.K. and Myers, S.P. (2005) Gingerol content of diploid and tetraploid clones of ginger (Zingiber officinale Roscoe). Journal of Agricultural and Food Chemistry, 53: 5772-5778.

Wong, K.K.Y., Signal, F.A., Campion, S.H. and Motion, R.L. (2005) Citronella as an insect repellent in food packaging. Journal of Agricultural and Food Chemistry, 53: 4633-4636.

Wright, E.J., Sinclair, E.A. and Annis, P.C. (2002) Laboratory determination of the requirements for control of Trogoderma variabile (Coleoptera: Dermestidae) by heat. Journal of Stored Products Research, 38: 147-155.

Yamada, Y., Kikuzaki, H. and Nakatani, N. (1992) Identification of antimicrobial gingerols from ginger (Zingiber officinale Roscoe). Journal of Antibacterial and Antifungal Agents, 20: 309-311.

199

Yamahara, J., Rong, H.Q., Iwamoto, M., Kobayashi, H., Matsuda, H. and Fujimura, H. (1989) Active components of ginger exhibiting anti-serotonergic action. Phytotherapy Research, 3: 70-71.

Yang, D.S., Shewfelt, R.L., Lee, K-S. and Kays, S.J. (2008) Comparison of odour-active compounds from six distinctly different rice flavour types. Journal of Agricultural and Food Chemistry, 56: 2780-2787.

Yonglin, R. and Allen, S.E. (1999) A reappraisal of an old fumigant, carbon disulphide, under modern farm storage conditions. In: Zurun, J., Quan, L., Yongsheng, L., Xianchang, T. and Lianghua, G. (Eds.), Stored Poduct Protection. Proceedings of the Seventh International Working Conference on Stored-product Protection, 14-19 October 1998, Beijing, China. Sichuan Publishing House of Science and Technology, Chengdu, China, pp.516-525.

Zar, J.H. (1999) Biostatistical analysis. 4th edition, Prenctice Hall, Upper Saddle River, New Jersey.

Zettler, J.L. and Keever, D.W. (1994) Phosphine resistance to cigarette beetle (Coleoptera: Anobidae) associated with tobacco storages in the southern United States. Journal of Economic Entomology, 87: 546-550.

Zettler, J.L., Halliday, W.R. and Arthur, F.H. (1989) Phosphine resistance in insects infesting stored peanuts in the southern United States. Journal of Economic Entomology, 82: 1508-1511.

200