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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
1
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
viii
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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
13
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
14
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
15
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
16
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).
17
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
18
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
19
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).
20
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
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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.
63
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
64
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).
66
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
67
(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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0
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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
76
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
79
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.
81
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%
98
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.
102
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
104
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
e sp
ent (
min
) b
bb
a
0
1
2
3
4
A.melegueta
Diethylether
Diethylether
Diethylether
Mea
n tim
e sp
ent (
min
)
bb b
a
(a) (b)
0
1
2
3
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
e sp
ent (
min
)
bb b
a
(a) (b)
0
1
2
3
4
Test Diethylether
Diethylether
Diethylether
Mea
n tim
e sp
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
e sp
ent (
min
)
0
1
2
3
4
Aframomummelegueta
Hexane Hexane Hexane
Mea
n tim
e sp
ent (
min
)
(a) (b)
0
1
2
3
4
Zingiberofficinale
Hexane Hexane Hexane
Mea
n tim
e sp
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
2
3
4
Aframomummelegueta
Diethylether
Diethylether
Diethylether
Mea
n tim
e sp
ent (
min
)
bb
ab
a
0
1
2
3
4
Aframomummelegueta
Diethylether
Diethylether
Diethylether
Mea
n tim
e sp
ent (
min
)
b
b
b
a
(a) (b)
0
1
2
3
4
Zingiberofficinale
Diethylether
Diethylether
Diethylether
Mea
n tim
e sp
ent (
min
)
bb
ab
a
0
1
2
3
4
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
e sp
ent
(min
) b bb
a
0
1
2
3
4
Test Control1
Control2
Control3
Mea
n tim
e sp
ent
(min
) bb
b
a
(a) (b)
0
1
2
3
4
Test Control1
Control2
Control3
Mea
n tim
e sp
ent
(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.
119
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
e sp
ent
(min
)
b bb
a
0
1
2
3
4
Test Control1
Control2
Control3
Mea
n tim
e sp
ent
(min
)
b
b b
a
(a) (b)
0
1
2
3
4
Test Control1
Control2
Control3
Mea
n tim
e sp
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
e sp
ent (
min
)
b b
b
a
0
1
2
3
4
A.melegueta
Control 1 Control 2 Control 3
Mea
n tim
e sp
ent (
min
)
b
b b
a
(a) (b)
0
1
2
3
4
Test Control1
Control2
Control3
Mea
n tim
e sp
ent
(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
e sp
ent (
min
) b
b b
a
0
1
2
3
4
Z.officinale
Control 1
Control 2
Control 3
Mea
n tim
e sp
ent (
min
)
b
b
b
a
(a) (b)
0
1
2
3
4
Test Control1
Control2
Control3
Mea
n tim
e sp
ent
(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.
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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.
137
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
150
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.
153
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.
157
0
5
10
15
20
25
30
35
40
45
Week 5 Week 6 Week 7 Week 8 Week 9 Week 10
Time
Me
an
ad
ult
em
erg
en
ce
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
10
20
30
40
50
60
70
80
90
100
Contro
l
Z. ofiic
inale
(1 %
)
Z. ofiic
inale
(5 %
)
Z. ofiic
inale
(10
%)
A. mele
guet
a (1
%)
A. mele
guet
a (5
%)
A. mele
guet
a (1
0 %
)
Treatments
Me
an
(%
) G
erm
ina
tio
n
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
160
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
163
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-
164
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
165
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-
166
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
168
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.
171
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.
172
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.
176
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