Final Thesis Copy

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 University of Aberdeen Aberdeen AB24 2TZ United Kingdom

Biological Chemistry Department Rothamsted Research Harpenden, Hertfordshire AL5 2JQ United Kingdom

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DECLARATION

I hereby declare that this thesis has been composed by myself, and has not been accepted in any previous application for a degree. The work of this thesis is a record of my work; any collaborative work has been specifically acknowledged as have all sources of information.

Donald A. Ukeh 2008.

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DEDICATION To the loving memory of my beloved father, Ukeh Akem (1935-1989); and mother, Cecilia Bezaunungieye Ukeh-Akem (1937-2008), both of whom inspired me to become the person I am today.

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ABSTRACT The maize weevil, Sitophilus zeamais Motschulsky, is the most important insect pest of stored maize causing considerable damage of economic proportions in the tropics. In Nigeria, maize production is undertaken by resource-poor farmers with little or no control measures against S. zeamais during storage. In addition, on a large scale, there are public concerns over the continuous application of synthetic pesticides in stored products protection. Both have prompted the search for safer, cheap, easily biodegradable and readily available plant materials that will not contaminate food products in acting as grain protectants in small-scale storage systems. Studies were carried out to evaluate the repellent properties of the seeds of alligator pepper, Aframomum melegueta and Black pepper, Piper guineense, and ginger, Zingiber officinale rhizomes against S. zeamais. In 4-way olfactometry bioassays, S. zeamais adults showed strong attraction to maize and wheat seed volatiles but were significantly repelled by odours emanating from the seeds of A. melegueta, P. guineense and Z. officinale rhizomes. In field trials crushed A. melegueta seeds and Z. officinale rhizomes, significantly repelled S. zeamais from traditional maize granaries with treated maize cobs giving higher germination than untreated cobs. Laboratory oviposition studies showed that A. melegueta and Z. officinale powders caused significant adult mortality and oviposition deterrence against S. zeamais resulting in a reduction in F1 progeny emergence. Olfactometer bioassays also confirmed that vacuum distilled A. melegueta and Z. officinale extracts and oleoresins were repellent towards adult S. zeamais when tested individually, and in combination with maize grains. Bioassay-guided liquid chromatography of the distillates showed that fractions containing polar compounds accounted for the repellent activity. Coupled gas chromatography-mass spectrometry (GC-MS), followed by GC peak enhancement and enantioselective GC using authentic compounds, identified 3 major compounds in the behaviourally active A. melegueta fraction to be (S)-2-heptanol, (S)-2-heptyl acetate and (R)-linalool in the ratio 1:6:3. Z. officinale had 1,8-cineole, neral and geranial in the ratio of 5.48:1:2.13. The identification of these behaviourally active compounds provide a scientific basis for the observed repellent properties of A. melegueta and Z. officinale extracts, and demonstrate the potential for their development in stored-product protection at the small-scale farmer level in Africa. iii 4

TABLE OF CONTENTS Declaration ………………………………………………………………………

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Dedication ……………………………………………………………………….

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Abstract …………………………………………………………………………

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Acknowledgements …………………………………………………………….

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CHAPTER 1

GENERAL INTRODUCTION

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

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

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

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

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

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

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

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

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

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

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1.6 Conclusions …………………………………………………………………

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

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CHAPTER 2

BEHAVIOURAL RESPONSES OF Sitophilus zeamais TO HOST AND NON-HOST PLANT VOLATILES 2.1 Introduction……………………………………………………………….

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

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

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

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

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

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2.3 Results……………………………………………………………………

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

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

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

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

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

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2.5 Conclusions………………………………………………………………

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CHAPTER 3

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

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

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

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

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

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

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

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

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

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

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3.3 Results……………………………………………………………………

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3.3.1 Chemical constituents of vacuum distilled essential oils of A. melegueta and Z. officinale....................................................................

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3.3.2 Chemical composition of A. melegueta and Z. officinale Florisil® diethyl ether essential oil fraction…………………………………………

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3.4 Discussion…………………………………………………………………

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

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CHAPTER 4 BIOACTIVITY OF Aframomum melegueta AND Zingiber officinale EXTRACTS AND SINGLE COMPONENTS AGAINST Sitophilus zeamais 4.1 Introduction………………………………………………………………

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

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

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

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

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4.3 RESULTS…………………………………………………………….

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

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4.3.2 Vacuum distilled hexane and diethyl ether fractions of A. melegueta and Z. officinale essential oils ……………………………..

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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…….

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4.3.5 Olfactory responses to A. melegueta and Z. officinale chemical constituents of essential oils……………………………………

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

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

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4.5 Conclusions………………………………………………………………

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

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

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

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

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

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

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

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

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

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

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

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5.4.1 Repellent effect of A. melegueta and Z. officinale powders against S. zeamais in storage granaries in Nigeria………………………………….

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5.4.2 Effects of Z. officinale and A. melegueta powders on S. zeamais oviposition and adult emergence in laboratory tests…………………….....

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

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

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CHAPTER 6

GENERAL DISCUSSION

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

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

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

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

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

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REFERENCES………………………………………………………………..

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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. ix 10

CHAPTER 1: GENERAL INTRODUCTION

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1.1 INTRODUCTION Since the time of the Pharaohs, human beings have stockpiled foodstuffs in times of plenty for use during leaner periods or for sale at a later date. Equally ancient are the stored-product insects that infested the Pharaohs’ food storage facilities. Some of the same pests that infest foods in the late 20th century have been found in the tombs of the pharaohs (Levinson and Levinson, 1990; 1998). It is thought that between 5000 and 10,000 BC, human society commenced settled agriculture and began to produce and store large quantities of dried organic materials such as grains, fibres and skins. A vast new resource then became available which attracted a select band of insects that feed on dry material of animal and plant origin (Rees, 2004). Before this, storage insects may have evolved to exploit natural accumulations of seeds in dry, sheltered habitats; these included those blown into caves, cracks and crevices by the wind or found in the nests and food hoards of other animals, including birds, rodents and social insects (Cox and Collins, 2002). More than 600 species of beetles and 70 of moths among the insects, 355 species of mites, 40 species of rodents and 150 species of fungi have been reported to be associated with various stored products (Rajendran, 2002). Stored product insects share biological characteristics that are adaptive for survival under conditions of food handling and storage areas. These characteristics include; (1) a wide range of tolerance to different environmental conditions such as temperature and relative humidity; (2) a greater range of food habits than most other insects; (3) more or less continuous reproductive activity spanning periods of up to three years; (4) an ability to survive long periods without food; and (5) a capacity to build up large populations undetected, due to their relatively small size (Throne, 1994; Cox and Collins, 2002; Rees, 2004).

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The control of the maize weevil, Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) is the subject of investigation in this thesis. This introductory chapter describes the general biology and life cycle, economic importance, sources of infestation of S. zeamais. Also, an overview of the current control methods of the weevil with particular reference to West Africa is reported.

1.2 GENERAL BIOLOGY AND LIFE CYCLE OF Sitophilus zeamais The maize weevil, S. zeamais, is the most important insect pest of stored maize in tropical and sub-tropical countries. The adults are small brown to black snout weevils, about 3-6mm long. They are long-lived with a life span of several months to one year. Females select a spot on the grain surface then chew small holes into grain kernels, and use their ovipositors to insert one egg per hole. Each hole is then plugged with a gluey secretion usually referred to as an “egg-plug” (Howe, 1952). Eggs are laid throughout most of the adult life, but the majority of the eggs are laid within the first 6 weeks, and more than 150 eggs may be laid per female. Eggs are laid at temperatures between 15 and 35 ºC with an optimum of about 25 °C, and at grain moisture contents over 10% but not above 32 %. The incubation period of the egg is between 2-6 days at 25 ºC. Larvae are cannibalistic and larger ones may prey upon less developed individuals should they meet (Rees, 2004). There are four larval instars all of which bore through the cavity hollowed out in the seed and develop within the grain. Pupation takes place within the grain, and the newly developed adult may spend several days within the cavity before chewing its way out leaving a large characteristic emergence hole called “exit hole”. The total development periods range from about 35 days under optimal conditions to over 110 days in unfavourable conditions. The actual duration of life cycle also depends upon the type and quality of

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grain that is infested (Haines, 1991). An index of environmental suitability indicated that between 25 - 30 °C and 65 -75 % temperature and relative humidity, respectively, are the optimal environmental conditions for growth of maize weevil populations on stored maize (Throne, 1994; Bekele et al., 1995). Mating in S. zeamais does not occur before the adults are 3 d old (Walgenbach and Burkholder, 1987) but the insect continues to feed on grain throughout its life span.

LIF Figure 1.1 General life cycle of Sitophilus zeamais.

The food preferences of S. zeamais are maize, rice, wheat and dry cassava. On maize the weevils infest ripening standing crops immediately prior to harvest and in storage. They are good fliers and dominant storage pests of these crops in tropical subsistence agricultural systems. Within grain bulks, S. zeamais populations often aggregate, but when such aggregations run out of food, adults will disperse, at which stage the infestation may become visible. However, by this stage, significant damage to the

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quality and quantity of the stored product has been done (Stoll, 2000; Rees, 2004). Aggregation is made possible by the secretion of a male-released aggregation pheromone, which attracts members of both sexes resulting in the formation of leks. The major component of the male aggregation pheromone is sitophinone {(5R, 4S)-5hydroxy-4-methyl-3-heptanone} (Walgenbach et al., 1983).

OH

O

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

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harvest of 250, 000 - 300, 000 tonnes of maize, about 20% was lost to storage pests like S. zeamais (Obeng-Ofori and Amiteye, 2005). Post-harvest losses caused by insect infestation and spoilage reduce the availability of maize in Cameroon throughout the year, and for the western highlands of Cameroon, losses in stored maize of 12-44% due to S. zeamais during the first 6 months of storage has been reported (Bouda et al., 2001). Average dry weight losses of farm-stored maize for a storage period of 6 months caused by S. zeamais and Prostephanus truncatus (Horn.) (Coleoptera: Bostrichidae) in Togo has been estimated to range between 7% and 30% (Pantenius, 1988; Richter et al., 1997). In Ethiopia in general, post-harvest losses caused by S. zeamais ranging from 20-30% are common, and studies in the Bako areas have shown grain damage levels up to 100% in some samples from farm stores after 6-8 months (Emana, 1999; Demissie et al., 2008). Insect contaminants such as excreta (uric acid), exuviate (cast skins) and dead bodies, webbing, and secretions in food commodities pose a quality-control problem for food industries. Processing and end-use qualities of food commodities are also affected by insect infestation, as are cash value and marketability of products. The activity of stored product pests may be associated with weight losses by direct damage, lowering the nutritional and economic value of the crop and presence of allergens (Arlian, 2002) or toxinogenic fungi (Hubert et al., 2002), in the infested stored grain. Food infecting fungi have been reported to produce many metabolites such as mycotoxin, aflatoxin B1, a carcinogenic metabolite of Aspergillus flavus which affects the liver (Wild and Hall, 2000), ochratoxin A and citrinin produced by Penicillium verrucosum which are known to have nephrotoxic effects (Hubert et al., 2004).

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1.4 SOURCES OF INFESTATION In developing countries like Nigeria, inadequate storage facilities and lack of economic means constrained resource-poor farmers who form the bulk of food production, to store their products using traditional storage granaries. Maize, Zea mays L. (Poaceae) is grown in March/April at the on-set of rains and harvested during August and September at the state of ripeness. Maize is stored shelled or unshelled in baskets, jute bags or earthen ware and placed in barns or cribs constructed with wood or bamboos within the homestead. Generally, the storage period ranges from 4 to 6 months. These storage structures allow air to flow freely through the grain, which enhances the drying process but also makes the granary vulnerable to infestation by insect pests such as S. zeamais (Holst et al., 2000). The sources of S. zeamais infestation of stored commodities in warm climates can be several routes including standing crops in the fields followed by passive movement with harvested grain into the store, neighbouring storage facilities containing infested commodities, processing plants and warehouses or insects migrating from abandoned or old granaries, or those attracted by the smell of the new grain (Sinha and Watters, 1985; Arbogast and Throne, 1997; Cox and Collins, 2002; Arthur et al., 2006). In Nigeria, field infestation of standing crops and store to store infestations appear to be most prevalent. This is not common in the temperate climate where the majority of infestations commence after maize is harvested. For example, if the store has not been cleaned properly, cryptic weevils hidden in the storage structure could be left behind from the previous stock, ready to infest the new intake commodity (Cox et al., 1990). Also, grain residues in commercial grain elevators boot pit and tunnel have been reported to contain large numbers of storage pests including S. zeamais when the bins are empty and serving as sources of infestation for new grains in the United States (Throne and

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Cline, 1994; Hagstrum, 2001; Arthur, et al., 2006). In elevators, the sources of insects that infest new grain could be previously infested grain present when the new crop is received, spills, trucks and railcars used to transport grain (Dowdy and McGaughey, 1998).

It is also very likely that stored product insects like most phytophagous insects, use chemical cues to find sources of suitable host. Specialist or general pest species require finding their host plants in a patchy environment, and plant volatiles are important host location cues. Most host plants release hundreds of volatile organic compounds, and many of those from grains have been identified as short-chain alcohols, aldehydes, fatty acids, ketones, esters, terpenes and heterocyclic compounds (Maarse, 1991; Seitz and Sauer, 1992). For example, wheat germ contains about 15% lipid and 60% triglyceride (Pomeranz, 1978), and unsaturated triglyceride has been reported to elicit aggregation responses from the granary weevil, S. granarius (L.) (Nawrot and Czaplicki, 1982). S. granarius is a sibling species of S. zeamais that is prevalent in temperate regions. The use of carob pod pieces in combination with wheat kernels has been reported to enhance trap catches of S. zeamais and its sibling species S. oryzae (L.) around traditional African granaries (Likhayo and Hodges, 2000). The main volatile compounds of maize seeds, namely hexanoic acid, nonanoic acid, nonanal, decanal, 2-phenylethanol and vanillin have been reported to be the main attractants for S. zeamais and S. oryzae (Pike et al., 1994; Hodges et al., 1998).

Infestation of stored maize by S. zeamais can be visualised as a process of invasion, colonization and population growth. Following initial colonization of stored grain, weevil population changes may be driven by its response to grain degradation, intra

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and inter-specific interactions, and the arrival of new colonizers (Arbogast and Throne, 1997). The behaviour of the weevil on stored grain is affected by the interplay of different chemical, physical and biotic factors. Factors such as store temperature, relative humidity, light intensity, grain moisture content, grain size and variety, pest density and the presence of other insects including parasitoids and predators, as well as micro-organisms such as fungi will greatly affect the behaviour of the maize weevil for successful utilization of the store environment (Cox and Collins, 2002).

1.5 CONTROL OF S. zeamais When maize is placed in storage, the grains are exposed to a broad range of complex ecological factors that work against the farmer or merchant’s objective of maintaining grain quality. Some preventive measures such as seed sanitation, insect resistant varieties, solar disinfections and weather-proof storage structures, and therapeutic methods such as the use of insecticides and biological control agents have been in practice for the protection of stored maize from pest infestation (Oparaeke and Kuhiep, 2006). In nature, the common parasitoids of Sitophilus species are members of the order Hymenoptera (Pteromalidae, Bethylidae), including Anisopteromalus calandrae (Howard), Lariophagus distinguendus (Förster), Choetospila elegans (Westwood), Theocolax elegans (Westwood), Cephalonomia tarsalis (Ashmead) and C. waterstoni (Gahan) (Haines, 1991; Lord, 2006; Flinn et al., 2006). These parasitic wasps lay their eggs into the larvae of the host weevils, and the wasp larvae develop on host tissue, eventually killing their host as they emerge as mature larvae prior to pupation or as adults depending on species. The presence of large numbers of these insects usually indicates that a long-standing infestation is present (Rees, 2004).

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However, for effective control of stored-product insects especially S. zeamais, it is pertinent to understand the storage environment and monitor pest infestation by sampling.

1.5.1 Sampling and trapping The presence of S. zeamais in stored maize is not easily noticeable except when infestation has become very high, but this disadvantage can be overcome through a mixture of sampling, inspection and trapping to detect low level infestations. Grains are sampled at intake, during transportation and use for the presence of insects mainly for assessment of grade and quality. In the developed world, hand probes which have been replaced with pneumatic sample probes can be inserted into the back of the truck using a mechanical arm. Maize moving through a handling system can be sampled using a diverter system where a small percentage of the grain is continuously diverted into a sampling system. To check for insects, the sampled seeds are passed over a mechanical sieving machine which is attended by a specialist. Probes have been developed to detect noise made by insects when infesting bulk grain, and there are electronic sensors which detect chemical odours released by stored-product insects. Hidden infestations are detected by radio photography using soft X-rays and nearinfrared (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

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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

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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 storedproduct 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

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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 onfarm 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

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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

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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 storedproduct 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,

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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.

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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, antihypertensive 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.

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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.

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CHAPTER 2: BEHAVIOURAL RESPONSES OF Sitophilus zeamais TO HOST AND NONHOST 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

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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, 4allyl 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,

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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

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(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

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(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.

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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.

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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 5°00′ and 5°15′ North and longitude 8°04′ and 8°25′ 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

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Plate 2.4 Zingiber officinale rhizome

Plate 2.5 Fresh Piper guineense leaf and seeds prior to drying

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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 25°C, 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

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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.

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Plate 2.7 Bioassay set-up.

Plate 2.8 Olfactometer

47

Arm no. 2

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 chisquare (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 (χ 2 = 1.02, df = 3, P = 0.796) (Table 2.1) S. zeamais to the 4 arms of the olfactometer.

48

3

2.5

Mean time spent (min)

Mean time spent (min)

3

2 1.5 1 0.5

2.5 2 1.5 1 0.5 0

0

Blank Blank Blank Blank

Blank Blank Blank Blank

(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 Mean no. visits to Test stimulus olfactometer arm n χ2* P* All 4 blank arms Males 4.39 12 0.05 0.997 Females 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)

10 9 8 7 6 5 4 3 2 1 0

a

b

b b

Mean time spent (min)

Mean time spent (min)

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

Yellow Control Control Control maize 1 2 3

10 9 8 7 6 5 4 3 2 1 0

a

b

b

b

Yellow Control Control Control maize 1 2 3

(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

a

b

b

b

Mean time spent (min)

Mean time spent (min)

10 9 8 7 6 5 4 3 2 1 0

10 9 8 7 6 5 4 3 2 1 0

a

b

b

b

White Control Control Control maize 1 2 3

White Control Control Control maize 1 2 3

(a)

(b)

11 10 9 8 7 6 5 4 3 2 1 0

a

b

b

b

Mean time spent (min)

Mean time spent (min)

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.

Winter Control Control Control wheat 1 2 3

11 10 9 8 7 6 5 4 3 2 1 0

a

b

b

b

Winter Control Control Control wheat 1 2 3

(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 way olfactometer Mean no. visits in Test stimulus olfactometer arm n χ2* P* i) Yellow maize grains against controls T C Males 6.67 3.97 12 14.29 0.003 Females 9.00 4.73 12 27.63 0.001 ii) White maize grains against controls Males Females

6.92 4.17

4.08 2.22

12 12

15.22 13.2

0.002 0.004

iii) Winter wheat seeds against controls Males Females

6.91 6.17

3.77 2.83

12 12

19.45 27.68

0.001 0.001

T is the mean value of test arm C 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).

7

7 b

6

b

b Mean time spent (min)

Mean time spent (min)

5 4 3 2

b

6

a

1 0

b

5

b

4 3 2

a

1 0

2 g A . C o n t ro l 1 C o n trol 2 C o n t ro l 3 m e le g u e ta

2 g A . C o n tro l 1 C o n tro l 2 C on tro l 3 m e le gu e ta

(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

7

7 b

b

5 4 3 2 1

b

6 Mean time spent (min)

Mean time spent (min)

6

b

a

0

b b

5 4 3 2

a

1 0

2g Z. Control officinale 1

Control 2

Control 3

2g Z. Control Control Control officinale 1 2 3

(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.

7

7 b b

5 4 3 2

a

1

b

6

b Mean time spent (min)

Mean time spent (min)

6

0

b b

5 4 3 2

a

1 0

2 g P . C o ntrol 1C o nt ro l 2C o nt ro l 3 gu in ee n s e

2g P . Control 1 C ontrol 2 Control 3 guineens e

(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 Mean no. visits in Test stimulus olfactometer arm n χ2* i) Aframomum melegueta volatiles against controls T C 30.5 Males 2.08 6.03 12 0 22.1 Females 2.17 5.33 12 1 ii) Zingiber officinale volatiles against controls 13.8 Males 1.67 3.81 12 8 16.7 Females 1.83 4.19 12 1 iii) Piper guineense volatiles against controls 31.6 Males 2.50 6.61 12 1 Females 2.25 3.81 12 9.12

P*

0.001 0.001 0.003 0.001 0.001 0.028

Control arms contained clean filter paper discs T is the mean value of test arm C 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)

55

(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. 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 (χ 2 = 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).

6

6 b

b

b

4 3

a

2 1

b

5 Mean time spent (min)

Mean time spent (min)

5

b

b

4 3 2

a

1 0

0 Test

Test

Control Control Control 1 2 3

(a)

Control Control Control 1 2 3

(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

6 b

5

b

b

Mean time spent (min)

Mean time spent (min)

6

4 3

a

2 1

b

b

5

b

4 3

a

2 1 0

0 Test

Test

Control Control Control 1 2 3

Control Control Control 1 2 3

(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.

6 b

5 4

ab

ab

Mean time spent (min)

Mean time spent (min)

6

a

3 2 1 0

b

b

5

b

4 3 2

a

1 0

Test

Control Control Control 1 2 3

Test

(a)

Control Control Control 1 2 3

(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 Test stimulus arm n χ2* P* i) Yellow maize + 10% A. melegueta against controls T C Males 3.27 4.89 12 8.24 0.041 15.0 Females 2.42 5.06 12 3 0.002 ii) Yellow maize + 10% Z. officinale against controls 17.0 Males 4.00 7.36 12 8 0.001 Females 3.36 4.97 12 8.09 0.044 iii) Yellow maize + 10% P. guineense against controls Males 4.25 6.08 12 7.24 0.065 Females 3.42 5.28 12 6.70 0.082 Control arms contained clean filter paper discs T is the mean value of test arm C 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 (P<0.001) (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) 58

respectively than the control arms. The number of visits made by females to A. 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).

b b

5

6 b Mean time spent (min)

Mean time spent (min)

6

4 3 2

a

1

b

5 ab

4 3

b

a

2 1 0

0 Test

Test

Control Control Control 1 2 3

(a)

Control Control Control 1 2 3

(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

6

6 5

ab

ab

4 3

a

2 1

b

b

5 Mean time spent (min)

Mean time spent (min)

b

b

4 a

3 2 1 0

0 Test

Test

Control Control Control 1 2 3

Control Control Control 1 2 3

(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; ab (females), P=0.001.

6 b

5

b

b

4 3

a

2 1

Mean time spent (min)

Mean time spent (min)

6

0

b

5

b

b

4 3

a

2 1 0

Test

Control Control Control 1 2 3

Test

(a)

Control Control Control 1 2 3

(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; ab (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 Test stimulus olfactometer arm n χ2* P* i) White maize + 10% A. melegueta against controls T C 13.3 Males 2.50 5.06 12 2 0.004 Females 2.92 4.83 12 8.67 0.034 ii) White maize + 10% Z. officinale against controls Males 2.83 4.83 12 8.42 0.038 Females 2.33 4.17 12 8.20 0.042 iii) White maize + 10% P. guineense against controls Males 3.09 5.53 12 9.43 0.024 Females 2.92 4.58 12 6.48 0.091 Control arms contained clean filter paper discs T is the mean value of test arm C 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).

6 b

5

b

b

4 3

a

2 1

Mean time spent (min)

Mean time spent (min)

6

b

b

5

b

4 3

a

2 1 0

0 Test

Test

Control Control Control 1 2 3

(a)

Control Control Control 1 2 3

(b)

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; ab (females), P=0.035.

62

6

b

5

ab

ab

4 3

a

2 1

Mean time spent (min)

Mean time spent (min)

6

0

b b

5

b

4 3

a

2 1 0

Test

Control Control Control 1 2 3

Test

(a)

Control Control Control 1 2 3

(b)

5

5

4

4

Mean time spent (min)

Mean time spent (min)

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; ab (females), P=0.001.

3 2 1

3 2 1 0

0 Test

Test

Control Control Control 1 2 3

(a)

Control Control Control 1 2 3

(b)

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 combination with 10% non-host plant volatiles Mean no. visits in Test stimulus olfactometer arm n χ2* P* i) Winter wheat + 10% A. melegueta against controls T C Males 3.50 5.91 12 9.07 0.028 Females 2.67 4.53 12 8.59 0.035 ii) Winter wheat + 10% Z. officinale against controls Males 3.67 5.69 12 8.98 0.029 Females 2.75 4.67 12 8.41 0.038 iii) Winter wheat + 10% P. guineense against controls Males 3.67 5.53 12 8.87 0.031 Females 3.42 4.53 12 3.29 0.349 Control arms contained clean filter paper discs T is the mean value of test arm C 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 10 min duration when compared with control arms. Treatments Males Test Control 1 Control 2 Control 3 P-value Females Test Control 1 Control 2 Control 3 P-value

1

Dosage of A. melegueta (%) 10 33

2.31 ± 0.34a 2.49 ± 0.23a 2.37 ± 0.29a 2.28 ± 0.31a 0.964

1.00 ± 0.26a 2.93 ± 0.35b 3.17 ± 0.30b 2.50 ± 0.29b 0.001

0.70 ± 0.23a 2.83 ± 0.25b 3.36 ± 0.30b 2.74 ± 0.28b 0.001

1.82 ± 0.26a 2.80 ± 0.29a 2.44 ± 0.27a 2.41 ± 0.31a 0.112

1.18 ± 0.27a 2.83 ± 0.23b 2.99 ± 0.27b 2.55 ± 0.23b 0.001

0.69 ± 0.20a 4.04 ± 0.24b 3.30 ± 0.25b 2.73 ± 0.20b 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.

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Table 2.7b Mean number of entries made by Sitophilus zeamais in response to 2 g yellow maize + 3 dosage levels of Aframomum melegueta seed volatiles when compared to control arms in a 4-way olfactometer for 10 min bioassay period Mean no. visits in Dosage (test stimulus) olfactometer arm n χ2* P* i)Yellow maize + 1% A. melegueta T C Males 3.17 3.19 12 0.12 0.989 Females 3.33 3.78 12 0.59 0.899 ii) Yellow maize + 10% A. melegueta Males 2.50 4.47 12 9.31 0.025 11.8 Females 2.00 4.17 12 4 0.008 iii) Yellow maize + 33% A. melegueta Males 1.67 4.42 12 19.1 0.001 15.7 Females 1.92 4.44 12 1 0.001 Control arms contained clean filter paper discs T is the mean value of test arm C 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,

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P<0.001) suggesting that Z. officinale is more repellent at a higher concentrations (Table 2.8b). 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. Treatments Males Test Control 1 Control 2 Control 3 P-value Females Test Control 1 Control 2 Control 3 P-value

1

Dosage of Z. officinale (%) 10

33

3.61 ± 0.39a 1.88 ± 0.29b 2.09 ± 0.24b 2.08 ± 0.26b 0.003

1.33 ± 0.18a 2.89 ± 0.25b 2.52 ± 0.21b 2.96 ± 0.26b 0.001

1.01 ± 0.33a 2.89 ± 0.30b 2.66 ± 0.31b 2.92 ± 0.30b 0.001

3.26 ± 0.37a 1.90 ± 0.32a 2.28 ± 0.33a 2.02 ± 0.29a 0.068

1.25 ± 0.24a 2.52 ± 0.24b 2.96 ± 0.24b 2.84 ± 0.29b 0.001

0.78 ± 0.26a 2.60 ± 0.29b 3.25 ± 0.35b 2.98 ± 0.33b 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 Mean no. visits in Dosage (test stimulus) olfactometer arm n χ2* P* i) 2 g yellow maize + 1% Z. officinale T C Males 3.08 2.61 12 1.06 0.787 Females 4.17 2.97 12 4.04 0.257 ii) 2 g yellow maize + 10% Z. officinale Males 2.42 4.24 12 8.19 0.042 Females 2.58 4.53 12 8.85 0.031 iii) 2 g yellow maize + 33% Z. officinale 18.1 Males 1.67 4.39 12 8 0.001 21.4 Females 1.67 4.67 12 9 0.001 Control arms contained clean filter paper discs T is the mean value of test arm C is the mean value of the mean of three control arms 67

*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

(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 time spent 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 when compared with control arms. Treatments Males Test Control 1 Control 2 Control 3 P-value Females Test Control 1 Control 2 Control 3 P-value

1

Dosage of P. guineense (%) 10

33

1.98 ± 0.31a 2.52 ± 0.24a 2.28 ± 0.22a 2.85 ± 0.30a 0.143

1.46 ± 0.27a 2.61 ± 0.23b 2.88 ± 0.29b 2.67 ± 0.26b 0.001

1.46 ± 0.25a 2.83 ± 0.29b 2.65 ± 0.25b 2.57 ± 0.26b 0.001

2.04 ± 0.32a 2.53 ± 0.24a 2.57 ± 0.24a 2.51 ± 0.24a 0.376

1.25 ± 0.27a 2.99 ± 0.28b 2.74 ± 0.25b 2.69 ± 0.25b 0.001

1.29 ± 0.25a 2.69 ± 0.23b 3.09 ± 0.23b 2.55 ± 0.25b 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 Mean no. visits in Dosage (test stimulus) olfactometer arm n χ2* P* i) Yellow maize + 1% P. guineense T C Males 4.42 4.83 12 1.32 0.724 Females 3.92 4.25 12 0.40 0.940 ii) Yellow maize + 10% P. guineense Males 3.27 4.89 12 7.26 0.064 Females 2.58 4.14 12 5.91 0.116 iii) Yellow maize + 33% P. guineense Males 2.50 4.38 12 8.30 0.040 Females 2.55 4.56 12 8.71 0.033 Control arms contained clean filter paper discs T is the mean value of test arm 69

C 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

(iv) Dose response percent repellency (PR) The dose response mean percent repellency (%) showed that with A. melegueta, males (51.06%) and females (61.69%) were repelled at 1% (w/w) in the mean time spent in the test arm compared to the controls in olfactometer bioassays. At 10% (w/w), males (79.17%) and females (75.37%), and at 33% dosage males (85.65%) and females (87.11%) respectively were repelled from the test arm compared to the control arms (Figure 2.17a, b). For the number of visits, the mean percent repellency at 1% A. melegueta (w/w) was males (50.75%), females (54.25%), at 10% it was males (68.96%), females (71.01%), and at 33%, males (77.39%) and females (74.41%) (Figure 2.17c, d).

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Figure 2.17 Mean % repellency (PR) values for different dosages of A. melegueta volatile plus yellow maize grains against S. zeamais males (a) and females (b) in the mean time spent in the test arm; and males (c) and females (d) in the mean number of visits to the arm in a 4-way olfactometer bioassays. Bars = standard errors of the means.

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With Z. officinale, olfactometry bioassays revealed that the mean percent repellency at 1% (w/w) in the mean time spent in the test arm compared to the controls was males (25.68%) and females (31.65%). At 10% (w/w) dosage, the mean percent repellency for the males (72.52%) and females (73.95%) was observed, and with 33% (w/w) dose, males (78.68%) and females (83.56%) were repelled from the test arm compared to the control arms (Figure 2.18a, b). In the number of visits to the olfactometer arms, the mean percent repellency for males (40.01%) and females (36.55%) was observed at 1% Z. officinale. But at 10% (w/w), males (72.52%) and females (73.95%), and at 33% males (78.68%) and females (83.56%) were repelled from the test compared with the control arms (Figure 2.18c, d).

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Figure 2.18 Mean % repellency (PR) values for different dosages of Z. officinale volatile plus yellow maize grains against S. zeamais males (a) and females (b) in the mean time spent in the test arm; and males (c) and females (d) in the mean number of visits to the arm in a 4-way olfactometer bioassays. Bars = standard errors of the means.

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The mean percent repellency of 1% P. guineense against males (58.96%) and females (57.63%) was observed in the mean time spent by the weevil is the test arm compared to the control arms.

But at 10% (w/w), the mean % repellency against males

(69.56%) and females (74.32%) was achieved, and at 33% (w/w) males (69.36%) and females (73.18%) were repelled from the test arm (Figure 2.19a, b). For the number of visits, at 1% males (51.77%) and females (54.29%), at 10% males (66.97%) and females (66.49%), while at 33% males (68.64%) and females (67.47%) were repelled from the test arm compared to the control olfactometer arms (Figure 2.19c, d). Generally, olfactometry bioassays showed that the repellency of A. melegueta, Z. officinale and P. guineense volatiles against S. zeamais increased with dosage.

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Figure 2.19 Mean % repellency (PR) values for different dosages of P. guineense volatile plus yellow maize grains against S. zeamais males (a) and females (b) in the mean time spent in the test arm; and males (c) and females (d) in the mean number of visits to the arm in a 4-way olfactometer bioassays. Bars = standard errors of the means.

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2.4 DISCUSSION The results of the olfactometer experiments with individuals of S. zeamais showed that adult S. zeamais demonstrated clear orientation choices between the volatiles generated by host and non-host plants, and volatiles that had been mixed with both. Yellow maize, white maize and winter wheat kernels were found to be attractive to S. zeamais in single choice tests, and this behavioural response was independent of the sex of the weevil. Overall, the results of this study suggest that a variety of host plant complexes emit volatile blends that are broadly attractive to S. zeamais adults.

The fact that S. zeamais showed positive behavioural responses to the different host plant volatiles tested could suggest that this species may use the plant volatiles as cues during the search for food and oviposition sites. Under natural conditions this mechanism may occur when the first weevil preferably a male arrives at a food source and releases aggregation pheromone (4S,5R-sitophinone) which in combination with food odours contributes to aggregation of small colonies of con-specifics during the initial phase of weevil infestation (Walgenbach et al., 1987). This result is in agreement with Pike et al. (1994) who showed that S. zeamais was attracted to maize volatiles, and identified the main volatile compounds as hexanoic acid, nonanoic acid, nonanal, decanal, 2-phenylethanol, and vanillin which are common plant volatiles. These volatiles which are typically present at low concentrations responsible for food odour, and their sensory relevance is due to considerably lower odour thresholds (Grosch, 2001). For example, Kaškonienė et al (2008) reported that the composition of the volatile compounds of honey collected from various floral origins include nonanoic acid, hexanoic acid, nonanal and decanal. Also the volatile aroma compounds from different rice flavour types have been reported to include decanal

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and nonanal (Yang et al., 2008). Wakefield et al (2005) also reported the attraction of S. zeamais and S. oryzae to carob volatiles alone, and significantly greater attraction of the insects to fresh lures containing 4S,5R-sitophinone and carob volatiles in pitfall bioassays. Recently, Germinara et al (2008) reported that the congener, S. granarius adults have the ability to respond behaviourally to wide a range of cereal volatiles and that response may change as a function of concentration.

In the present study, the attraction of yellow maize to S. zeamais dropped when 1% A. melegueta and P. guineense were incorporated with yellow maize respectively. However, with 1% Z. officinale yellow was still attractive to the male but not female S. zeamais. Edde and Phillips (2006) working with Rhyzopertha dominica (Fab) another stored-product pest, using a dual-choice, still-air bioassay method, observed that both sexes of R. dominica were attracted to volatiles from wheat kernels, a plant species judged to be most suitable to the insect. S. zeamais has a broad host range including dry cassava tubers, sorghum and rice in addition to the host plants used for these studies. This could mean that the weevil would require specialized olfactory receptor neurons (ORN) for the recognition of volatile compounds associated with each host plant. In many insects, olfactory receptor neurons are found in two bilaterally symmetrical pairs of olfactory organs, the antennae and the maxillary palps which functions mainly as contact chemoreceptors. The surfaces of the olfactory organs are covered with sensory hairs (sensilla) which contain the ORN dendrites (Hallem et al., 2006). Despite considerable variations in the general morphology of olfactory organs across insect species, the structure of the olfactory sensillum is broadly similar and consists of a cuticular wall containing multiple pores through which odours can enter (Rospars, 1988; Shanbhag et al., 2000). Olfactory sensilla

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contain the dendrites of between one and five ORNs (Vermeulen and Rospars, 2004). The axons of the ORNs project to functional processing units called glomeruli in the antennal lobes of the brain (Hildebrand and Shepherd, 1997). In addition to ORNs, the olfactory

organs

of

several

insect

species

contain

smaller

numbers

of

mechanosensory, thermosensory, hygrosensory, and gustatory neurons (Rospars, 1988). Insect ORNs have been studied extensively by single-unit electrophysiology, which is an extracellular recording technique used to examine the responses of single ORNs to odours.

The high attractivity of yellow maize, white maize and winter wheat grains to S. zeamais in the present study provide a starting point in the understanding of the importance of volatile cues in host habitat location by the maize weevil. There are other examples in the literature that demonstrate the attraction of host plant volatiles to

field

insects

using

electrophysiology.

Using

gas

chromatographic-

electroantennographic detection analysis (GC-EAD), Shepherd et al (2008) reported the antennal responses of the western pine beetle; Dendroctonus brevicomis LeConte to 42, out of 64 stem volatile compounds of its primary host, Pinus ponderosa Dougl. et Laws (Pinaceae) and nine sympatric non-host angiosperms and conifers. Also, nonanal and decanal were reported among the volatile compounds identified from the shoots of riverbank grape, Vitis riparia that attracts the female grape berry moth, Paralobesia viteana which attacks the plant (Cha et al., 2008). Van den berg et al (2008) also showed that three antennally active components of sorghum, Sorghum bicolor (L. Moench) panicles namely; 2-phenylethanol, benzyl alcohol and linalool in specific ratios, played a significant role in the host attraction by the pollen beetle, Astylus atromaculatus Blanchard. Similarly, decanal and nonanal collected from S.

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bicolor and Z. mays by air entrainment have been reported to elicit a large response in the stemborer, Busseola fusca Fuller, a major pest of cereal crops in Africa (Birkett et al., 2006). In aphids, Powell et al (2006) reported that host plant selection occurs as a sequence of several successive behaviours. For instance, landing by flying aphids on plant has been described to involve phototactic response to plant-reflected wavelengths (Hardie, 1989), which is modified by plant volatiles (Nottingham and Hardie, 1993) detected by antennal olfactory sensilla (Hardie et al., 1994). Park et al (2000) using electroantennogram and linear-track olfactometer, demonstrated that the male bird cherry-oat aphid, Rhopalosiphum padi, utilises the sex pheromones (-)(4aS,7S,7aR)-nepetalactol and (+)-(4aS,7S,7aS)-nepetalactone, and benzaldehyde, a volatile which is released by the winter host plant, for mate and/or host-plant location in the autumn. Webster et al (2008) also reported that the winged Aphis fabae Scop., was attracted to volatiles from the air entrainment sample of the faba bean, Vicia faba in olfactometry behavioural and electrophysiological responses, and identified decanal and methyl salicylate among the 16 electrophysiologically active compounds.

In repellency olfactometry assays, adult S. zeamais that were given the choice between untreated controls containing clean filter paper discs and 2 g A. melegueta seeds, Z. officinale rhizome, or P. guineense seeds significantly preferred the control arms. This suggests that S. zeamais is able to detect these repellent plants through olfaction and avoid them when given the choice. This could explain, at least in part, how the application of these plants may protect grain from insect infestation in storage. It was observed that avoidance of these repellent odours by S. zeamais increases with dose increment because at 1%, none of these plant volatiles significantly repelled the weevil. This may suggest that S. zeamais, like many other

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insect species, can potentially tolerate low levels of harmful substances (Rajendran and Gunasekaran, 2002). However, at the higher doses of 10% and 33%, all the 3 plant products repelled S. zeamais during the behavioural evaluation in the mean time spent in the test arm compared to the control arms. The results indicate that volatiles of A. melegueta, Z. officinale, and P. guineense are repellent to S. zeamais at the right concentrations, and that the presence of these volatiles can inhibit attraction of the weevil to the host plant, stored maize. It is also possible that non-host plant volatiles caused a change to the maize and winter wheat seeds by adhering to the surfaces of the seeds so eliciting repellence, odour masking or other disruption that interfered with the attraction to the host plant grains. These repellent actions increase the potential practical value of these plants for grain protection against S. zeamais infestation. These results are in line with ethnobotanical use by resource-poor farmers in West Africa of plant powders and ashes to repel insects from stored products. It was observed that 10% (w/w) P. guineense was not as efficient in repelling the weevils compared to equal dosages of A. melegueta and Z. officinale in the mean number of visits especially when combined with yellow maize grains. Notably, both sexes of the weevil did not show any significant preference to the test or control arms when 10% P. guineense was tested in combination with white maize and winter wheat kernels. Considering the olfactometer results, it may be possible that the concentration of behaviourally active volatiles in A. melegueta and Z. officinale could be higher compared to P. guineense. However, the fact that insects’ response to particular plant volatiles can be markedly influenced by the concentration of that volatile is common in chemical ecology, as it has been reported for other weevil species (Chhabra et al., 1999; Belmain et al., 2005).

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The use of tropical plants to control stored-product pests through repellency, immobilization or deterrent activity has been studied elsewhere. For example, Fields et al. (2001) reported that wheat treated with Pisum sativum L. protein repelled C. ferrugineus and S. oryzae in multiple choice laboratory tests. Hassanali et al (1997) also reported that the bioactivity of materials derived from the leaves and succulent stems of Ocimum kenyense evoked high repellency against S. zeamais, moderate repellency against R. dominica and low repellency against S. cerealella in the laboratory. Belmain et al (2003) corroborated these findings when they reported that 7.5-10.0% (w/w) Tephrosia vogelli Hook repelled 87.5% S. zeamais adults, while T. vogelli at 2.5 % (w/w) and Lantana camara L. at 10% (w/w) repelled 65 and 62.5% of the insect respectively.

2.5 CONCLUSIONS The results reported here are of significance for the management of S. zeamais, because repellent compounds may be used to mask or alter odours emitted from stored maize to reduce the ability of the maize weevil to detect the source of food and oviposition sites. Identification and testing of substances responsible for the repellent effects of A. melegueta and Z. officinale reported here may yield further candidates for use in post-harvest crop protection especially at traditional African small-scale farmer level.

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CHAPTER 3: EXTRACTION AND IDENTIFICATION OF BIOLOGICALLY ACTIVE COMPONENTS FROM THE SEEDS OF A. melegueta AND RHIZOMES OF Z. officinale.

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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,

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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).

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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 5°00′ and 5°15′ North and longitude 8°04′ and 8°25′ 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

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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, 0.25µ m 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 oncolumn 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 Purity by Compound Source 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 >99 Citral (neral + geranial) Fluka >95 1,8-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.

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OLEORESIN PREPARATION

ESSENTIAL OIL PREPARATION

Methanolic Extraction

Diethyl ether extraction

Oleoresins (bioassayed)

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)

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.

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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)-2heptanol (2.78%), β-pinene (0.64%), linalool oxide (0.59%), (E)-4,8-dimethyl-1,3,7nonatriene (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%), γcadinene (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%), αterpineole (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%).

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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 melegueta Peak no. Compound % Peak area by GC 1 (S)-2-Heptanol 2.78 2 α-Pinene 0.19 3 β-Pinene 0.64 4 Myrcene 0.21 5 (S)-2-Heptyl acetate 16.22 6 (E)-Ocimene 23.22 7 Linalool oxide (furan) 0.59 8 (R)-Linalool 8.68 9 (E)-4,8-Dimethyl-1,3,7-nonatriene 0.32 10 m/z (unidentified) 1.18 11 β-Caryophyllene 19.17 12 Humulene 26.23 13 Germacrene-D 0.06 14 Bisabolene 0.07

DAU/Alligator 251006MAB01

X = impurities which accounted for 0.44% peak area by GC.

100 93

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.

DAU/Gingerro 261006MAB01 100 94

Table 3.3 Compounds identified from coupled GC-MS analysis of vacuum distilled essential oil of Zingiber officinale rhizome Peak no. Compound % Peak area by GC 1 Tricyclene 0.33 2 α-Pinene 6.39 3 Camphene 16.87 4 6-Methyl-5-hepten-2-one 0.05 5 Sabinene 0.84 6 β-Pinene 0.25 7 Myrcene 3.20 8 3-Carene 0.71 9 1,8-Cineole 22.63 10 Terpinolene 0.66 11 (R)-Linalool 0.83 12 Borneol 0.63 13 α-Terpineol 0.51 14 Neral 5.03 15 Geranial 11.25 16 Borneol acetate 0.15 17 Cyclosativene 0.20 18 Copaene 0.39 19 α-Curcumene 1.00 20 α-Muurolene 1.10 21 Zingiberene 10.63 22 (E,E)-α-farnesene 3.69 23 γ-Cadinene 3.21 24 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

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 38.26 2 (S)-2-Heptyl acetate A. melegueta 27.92 3 (R)-Linalool A. melegueta 33.81 4 1,8-Cineole Z. officinale 45.94 5 Neral Z. officinale 14.96 6 Geranial Z. officinale 34.72

96

O O

OH

OH

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

H

H

O

O

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®.

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3.4 DISCUSSION The main objective of this chapter was to isolate and identify the major components with repellent activity against S. zeamais. GC and GC-MS analyses of A. melegueta seed and Z. officinale rhizome essential oils, which were shown to be repellent, revealed the presence of a number of components, including monoterpenes, sesquiterpenes, oxygenated monoterpenes and alcohols. The repellent activity of the oils was accounted for by diethyl ether fractions that were isolated by liquid column chromatography. The volatile constituents of A. melegueta with repellent properties were (S)-2-heptyl acetate, (S)-2-heptanol and (R)-linalool, and from Z. officinale were 1,8-cineole, neral and geranial. There are few reports on the chemical composition of the essential oil or extracts from the seeds of A. melegueta. In Cameroon, Menut et al (1991) identified nine compounds from the essential oil composition of A. melegueta obtained by hydrodistillation with α-humulene and ß-caryophyllene (39.8%) and their epoxides (45.6%) as main constituents. In 1995, Escoubas et al isolated and identified four aryldecanones namely gingerdione, paradol, gingerol and shogaols, and eight minor compounds from n-hexane and methanolic seed extracts of A. melegueta obtained from south western Nigeria. Ajaiyeoba and Ekundayo (1999) isolated a total of 27 compounds including linalool from the essential oil of A. melegueta seeds from Nigeria, but with the quantitative preponderance of α-humulene and ß-caryophyllene making up 82.6% of the oil. Two other sesquiterpene hydrocarbons, germacrene-D and δ-cadinene, occurred in trace amounts (0.1%), while 3 oxides namely humulene oxide I, humulene oxide II and caryophyllene oxide were identified in relatively substantial amounts (9%) in the volatile oil. Lately, the compositions of absolute and supercritical CO2 extract of A. melegueta purchased from Côte d’Ivoire have also been analyzed by GC and GC-MS with 33 components, representing more than 98.3%

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of the absolute, 43 components representing more than 98.2% of the supercritical fluid extraction (SFE) products identified (Fernandez et al., 2006). The analysis revealed that absolute contained four oxygenated monoterpenes (0.2%), 11 sesquiterpene hydrocarbons (15.9%), six oxygenated sesquiterpenes (1.6%) and five phenolic alkenones (75.7%). For the SFE, eight monoterpene hydrocarbons (2.9%), five oxygenated monoterpenes (0.8%), 11 sesquiterpene hydrocarbons (12.2%), seven oxygenated sesquiterpenes and five phenolic alkenones (70.5%) were found. The result from this study is in agreement with Fernandez et al (2006) who identified heptan-2-ol from SFE, while 2-heptyl acetate and linalool were obtained from both absolute and SF extraction methods. The volatile constituents responsible for the characteristic pleasant smell are present in the essential oil, but the essential oil only contributes partially to the flavour impact. The pungency of the seeds has been reported to be due to non-volatile phenolic alkenones known as gingerols, shogaols and paradols with various biological activities (Lee and Surh, 1998; Tjendraputra et al., 2001; Chung et al., 2001). The oleoresin (when resins are associated with volatile oils) of A. melegueta obtained by solvent extraction is a combination of both the important characteristics such as aroma and pungency compounds in the same extract.

Several methods for the determination of aroma and pungent compounds in Z. officinale have been described, and the constituents of the rhizome have been examined for quality and quantity. Depending on the place of origin, the powdered rhizome contains 3-6% fatty oil, 9% protein, 60-70% carbohydrates, 3-8% raw fibre, up to 8% ash, 9-12% water, and 2-3% volatile oil (Langner et al., 1998). The volatile oil consists mainly of mono- and sesquiterpenes namely; camphene, ß-phellandrene, curcumene, cineole, geranyl acetate, terpineol, terpenes, borneol, geraniol, neral, α-

99

pinene,

1,8-cineole,

limonene,

linalool,

myrcene,

alpha-(-)zingiberene,

ß-

sesquiphellandrene, ß-bisabolene and (E)(E)-α-farnesene (Harvey, 1981; MacLeod and Pieris, 1984; Langner et al., 1998 ). The results from this study confirmed the report of Ekundayo et al (1988) who isolated 54 components including geranial, neral, 1,8-cineole, zingiberene, ß-sesquiphellandrene and ß-bisabolene from the essential oils of Nigerian Z. officinale rhizomes. Using solid-phase microextraction (SPME), combined with comprehensive two-dimensional (2D) gas chromatography (GC X GC), Shao et al (2003) reliably identified 36 compounds including neral, geranial, ßsesquiphellandrene,

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%), arcurcumene (9.09%), ß-sesquiphellandrene (8.24%), ß-phellandrene (7.97%), E,E-αfarnesene (5.52%), ß-bisabolene (5.40%), α-pinene (2.57%), geranial (2.08%), endoborneol (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-8shogaol

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

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main sesquiterpene hydrocarbon fractions. Z. officinale essential oil contain 29.30% monoterpenes with camphene (16.87%) as the major and sulcatone (6-methyl-5hepten-2-one)

as

minor

monoterpenes

respectively,

41.03%

oxygenated

monoterpenes, and 23.60% sesquiterpene hydrocarbons. The major oxygenated monoterpenes were 1,8-cineole (22.63%), geranial (11.25%) and neral (5.03%), and zingiberene (10.63%) the main sesquiterpenes hydrocarbon compound. There are major differences in chemical composition between samples of essential oils obtained from aromatic plants of the same species or genus, which may be due to genetic, environmental, developmental or other factors. The chemical composition of the oleoresins also depends on whether the plant product is fresh or dried, as well as the nature of solvents used for the extraction. This study shows that using analytical techniques such as liquid chromatography and GC-MS, it was possible to identify biologically active compounds that could eventually be used as repellents against S. zeamais in stored-product protection.

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

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4.1 INTRODUCTION Insect damage in stored food grains may amount to 10% - 40% in countries where modern storage technologies have not been introduced (Shaaya et al., 1997) or are inadequate. Among the stored-product Coleoptera, the maize weevil, Sitophilus zeamais is an important primary pest of stored maize in the tropics (Wakefield et al., 2005). It is an internal feeder and causes considerable loss to stored maize affecting the quantity and quality of the grains. In many storage systems, applications of contact chemical insecticides have been used for the control of this pest. More frequently, fumigation with appropriate chemical substances such as methyl bromide and aluminium phosphide are the most economical and convenient tools for managing the maize weevil because of their ease of penetration into the commodity while leaving minimal residues (Bond, 1984). But safety and environmental impact concerns about the continuous application of these chemical substances have prompted the search for more environmentally sound and novel methods for the control of storage pests. Globally, the management of stored product pests using plant substances has been the subject of much research (Isman, 2006), and there has been a growing interest in research concerning the possible use of plant extracts as alternatives to synthetic insecticides in stored-product protection (Obeng-Ofori and Reichmuth, 1997; Shaaya et al., 2003; Sahaf, et al., 2007). A large number of plant species used traditionally as medicines have been reported to possess bioactivities against several insect species (Ivbijaro and Agbaje, 1986; Singh and Upadhyay, 1993; Bekele et al., 1995). Among possible strategies aimed at reducing the use of synthetic insecticides and fumigants, natural repellents produced by edible plants represent a vital approach for ecochemical control of stored-product insect pests (Adler et al., 2000; Rozman et al., 2007). Plant essential oils may act as fumigants, contact

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insecticides, repellents, deterrents and antifeedants to storage insect species (Hassanali et al., 1997; Isman, 2000; 2006; Bekele and Hassanali, 2001; Huang et al., 2000; Stamopoulos, 1991; Shakarami et al., 2004; Rajendran and Sriranjini, 2008).

The chemical compositions of Aframomum melegueta and Zingiber officinale (both Zingiberaceae) essential oils have been reported (Ekundayo et al., 1988; Ajaiyeoba and Ekundayo, 1999; Fernandez et al., 2006; Ali et al., 2008). These studies show that the essential oils of these plants comprise mainly of monoterpenoid and sesquiterpenoid hydrocarbons some of which have been extracted from other plants and which have been reported to possess repellent properties against some storedproduct 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.

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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” chisquare 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).

3 Mean time spent (min)

Mean time spent (min)

3

2

1

2

1

0

0

Diethyl Control Control Control ether 1 2 3

Diethyl Control Control Control ether 1 2 3

(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.

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Table 4.1 Behavioural responses of Sitophilus zeamais to 10 µl redistilled diethyl ether and blank control arms in a four way olfactometer. Mean no. visits in Test stimulus olfactometer arm n χ2* P* 10 µl diethyl ether against blanks T C Males 4.67 4.83 12 0.99 0.8037 Females 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

4

4 b

b

b

b

3 Mean time spent (min)

Mean time spent (min)

3

b

2 a 1 0

2 a 1 0

A. D ie th y l D ie th y l m e le g u e ta e th e r ethe r

D ie t h y l e th e r

A. D ieth y l m e le g u e ta e th e r

(a)

D ie th y l e th e r

b

b

b

a

1

Mean time spent (min)

4

3 2

D ie th y l e th e r

(b)

4

Mean time spent (min)

b

b

3 2

b

b

a

1 0

0 Tes t

Tes t

Diethyl Diethy l Diethy l ether ether ether

(c)

Diethy l Diethy l Diethy l ether ether ether

(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

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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. Mean no. visits in Test stimulus olfactometer arm n χ2* P* i) 10 µl A. melegueta T C 14.5 0.00 Males 2.5 5.14 12 3 2 0.00 Females 2.67 5.19 12 13.2 4 ii) 10 µl A. melegueta + 2 g maize 0.05 Males 2.67 4.44 12 7.62 5 11.6 0.00 Females 2.5 4.81 12 8 9 Each control arm contained 10 µl diethyl ether loaded on clean filter paper disc T is the mean value of test arm C 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).

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4

4 b b

2 a 1

2 a 1

Z. D ie t h y l D ie t h y l D ie th y l o ffic in a le e t h e r e th e r ether

Z. D iethy l D ie th y l D ie th y l o ffic ina le e th e r e th er e th er

(a)

(b)

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Mean time spent (min)

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b

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a

1 0

Tes t

Diethy l Diethy l Diethy l ether ether ether

Tes t

(c)

Diethy l Diethyl Diethyl ether ether ether

(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.

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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. Mean no. visits in Test stimulus olfactometer arm n χ2* P* i) 10 µl Z. officinale T C Males 2.5 4.94 12 12.5 0.006 12.9 Females 2.5 4.97 12 9 0.005 ii) 10 µl Z. officinale + 2 g maize 12.9 Males 2.33 4.69 12 3 0.005 Females 2.83 4.75 12 8.48 0.037 Each control arm contained 10 µl diethyl ether loaded on clean filter paper disc T is the mean value of test arm C 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).

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4

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3 Mean time spent (min)

Mean time spent (min)

3 2 1

2 1 0

0

A fra m o m u mH e x a n e H e x a n e H e x a n e m e le g u e t a

A fra m o m u mH e x a n e H e x a n e H e x a n e m e le g u e t a

(a)

(b)

4

4

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a a

2 1

Mean time spent (min)

Mean time spent (min)

b 3

0

3 2 1 0

Zingiber Hex ane Hex ane Hex ane offic inale

Zingiber Hex ane Hex ane Hex ane offic inale

(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.

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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. Mean no. visits in Test stimulus olfactometer arm n χ2* P* i) A. melegueta hexane fraction T C Males 3.83 3.73 12 0.37 0.946 Females 2.75 2.78 12 0.38 0.944 ii) Z. officinale hexane fraction Males 4.33 4.58 12 2.93 0.403 Females 2.92 3.03 12 0.28 0.964 Each control arm contained 10 µl hexane loaded on clean filter paper disc T is the mean value of test arm C 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).

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Mean time spent (min)

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0 A fra m o m u mD ie t h y l m e le g u e t a e t h e r

D ie t h y l ether

A fra m o m u mD ie th y l m e le g u e ta e t h e r

D ie t h y l e th e r

(a)

4 b

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3

Mean time spent (min)

Mean time spent (min)

D ie t h y l e th e r

(b)

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2

D ie th y l e th e r

ab a

1 0

3 2

b

b

b

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1 0

Zingiber D iethy l offic inale ether

D iethy l ether

D iethy l ether

Zingiber Diethy l offic inale ether

(c)

Diethy l ether

Diethy l ether

(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.

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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. Mean no. visits to Test stimulus olfactometer arm n χ2* P* i) A. melegueta diethyl ether fraction T C 4.7 0.19 Males 2.83 4.22 12 5 1 5.5 0.13 Females 2.17 3.47 12 3 7 ii) Z. officinale diethyl ether fraction 5.6 0.12 Males 2.83 4.33 12 6 9 1.8 0.60 Females 3.5 4.42 12 5 4 Each control arm contained 10 µl diethyl ether loaded on clean filter paper disc T is the mean value of test arm C 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.

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4 Mean time spent (min)

Mean time spent (min)

4

3

2

1

0

3

2

1

0 Hexane Blank

Blank

Blank

Hexane Blank

(a)

Blank

Blank

(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 to 10 µl redistilled hexane and blank control arms in a four way olfactometer Mean no. visits in Test stimulus olfactometer arm n χ2* P* i) 10 µl Hexane T C Males 2.33 2.50 12 0.17 0.982 Females 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

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(R)-linalool in the ratio 1:6:3, and Z. officinale synthetic blend was prepared from 1,8cineole, 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).

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Test

Control Control Control 1 2 3

Test

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Control Control Control 1 2 3

(b)

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b

3

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2

b

ab

3

ab

a

1 0

Test

Control Control Control 1 2 3

Test

(c)

Control Control Control 1 2 3

(d)

Figure 4.7 Mean time spent in the arm out of 10 min by male S. zeamais in response to 10 µl Aframomum melegueta diethyl ether fraction synthetic blend tested individually (a) and in combination with 2 g maize grains (c), and mean time spent by females in response to 10 µl A. melegueta synthetic blend tested individually (b) and in combination with 2 g yellow maize grains (d) in a four way olfactometer. Bars = standard errors of the means, n = 12. Bars followed by the same letter are not significantly different from each other (P>0.05). a-d: a (males), P<0.001; b (females), P<0.001; c (males), P=0.002; d (females), P=0.022.

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Table 4.7 Responses of Sitophilus zeamais to volatiles from 10 µl Aframomum melegueta diethyl ether fraction synthetic blend and in combination with maize grains in a four way olfactometer Mean no. visits in olfactometer Test stimulus arm n χ2* P* i) 10 µl Aframomum melegueta synthetic blend T C 5.0 Males 1.58 2.67 12 3 0.169 8.9 Females 0.67 1.58 12 5 0.03 ii) 10 µl A. melegueta synthetic blend + 2 g maize 2.1 Males 2.08 2.89 12 9 0.534 4.3 Females 1.75 2.78 12 9 0.222 Each control arm contained 10 µl hexane loaded onto a clean filter paper disc T is the mean value of test arm C 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).

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Mean time spent (min)

Mean time spent (min)

4

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2 a 1 0

Test

Control Control Control 1 2 3

Test

Control Control Control 1 2 3

(a)

(b)

4 b

b

3

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Mean time spent (min)

Mean time spent (min)

4

2

b

0

b 3

ab

b

a 2 1 0

Test

Control Control Control 1 2 3

Test

(c)

Control Control Control 1 2 3

(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 Mean no. visits in olfactometer Test stimulus arm n χ2* P* i) 10 µl Zingiber officinale synthetic blend T C 1 9.1 Males 2.17 4.03 2 9 0.027 1 Females 2.17 3.72 2 7.6 0.055 ii) 10 µl Z. officinale synthetic blend + 2g maize 1 0.5 Males 2.25 2.61 2 5 0.908 1 Females 2.5 3.61 2 3.8 0.284 Each control arm contained 10 µl hexane loaded on clean filter paper disc T is the mean value of test arm C 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

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arm when tested alone. But both sexes failed to make any significant choice between 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).

3 Mean time spent (min)

Mean time spent (min)

3

2

1

0

2

1

0 Diethyl Blank ether

Blank

Blank

Diethyl Blank Blank Blank ether

(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 Mean no. visits in Test stimulus olfactometer arm n χ2* P* 10 µl Diethyl ether against blanks T C 1 0.97 Males 2.25 2.08 2 0.2 8 1 0.3 0.95 Females 2.33 2.56 2 3 4 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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b Mean time spent (min)

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2 a 1 0

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A. C o n tro l 1 C o n tro l 2 C o n t ro l 3 m e le g u e t a

A. C o n tro l 1C o n tro l 2C o n tro l 3 m e le g u e ta

(a)

(b)

4 ab

3

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b

0

ab

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ab

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a

1 0

Test

Control Control Control 1 2 3

Test

(c)

Control Control Control 1 2 3

(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 to volatiles from 10 µl Aframomum melegueta oleoresin tested alone and in combination with maize grains in a four way olfactometer. Mean no. visits in olfactometer Test stimulus arm n χ2* P* i) 10 µl Aframomum melegueta oleoresin T C Males 1.33 2.72 12 8.04 0.045 10.1 Females 1.17 2.56 12 5 0.017 ii) 10 µl A. melegueta oleoresin + 2 g maize Males 2.08 2.89 12 2.32 0.509 Females 1.5 2.28 12 2.8 0.424 Each control arm contained 10 µl diethyl ether loaded on clean filter paper disc T is the mean value of test arm C 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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Z. Control Control Control officinale 1 2 3

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0 Test

Test

Control Control Control 1 2 3

(c)

Control Control Control 1 2 3

(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 to volatiles from 10 µl Zingiber officinale oleoresin tested alone and in combination with maize grains in a four way olfactometer. Mean no. visits in olfactometer Test stimulus arm n χ2* P* i) 10 µl Zingiber officinale oleoresin T C 1 8.3 Males 2.25 4.08 2 9 0.039 1 7.5 Females 1.42 2.75 2 2 0.057 ii) 10 µl Z. officinale oleoresin + 2 g maize 1 Males 2.83 3.81 2 2.5 0.475 1 4.9 Females 1.67 2.81 2 2 0.178 Each control arm contained 10 µl diethyl ether loaded on clean filter paper disc T is the mean value of test arm C 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 127

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). 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 Stimulus Treatments presentation Males Females Control Hexane 2.36 ± 0.15a 2.34 ± 0.15a Blank 2.58 ± 0.23a 2.42 ± 0.15a Blank 2.27 ± 0.17a 2.48 ± 0.16a Blank 2.21 ± 0.21a 2.24 ± 0.21a (R)-linalool

Test Control 1 Control 2 Control 3

1.22 ± 0.22a 2.47 ± 0.20b 3.05 ± 0.21b 2.81 ± 0.24b

0.89 ± 0.28a 2.58 ± 0.26b 3.35 ± 0.30b 2.68 ± 0.23b

(S)-2-heptyl acetate

Test Control 1 Control 2 Control 3

1.81 ± 0.28a 2.65 ± 0.29b 2.62 ± 0.22b 2.46 ± 0.21ab

1.57 ± 0.27a 2.61 ± 0.25b 2.69 ± 0.26b 2.80 ± 0.25b

(S)-2-heptanol

Test Control 1 Control 2 Control 3

1.23 ± 0.27a 2.51 ± 0.19b 3.02 ± 0.25b 2.92 ± 0.24b

1.61 ± 0.24a 2.73 ± 0.23b 2.55 ± 0.19b 2.80 ± 0.28b

Citral

Test Control 1 Control 2 Control 3

1.18 ± 0.24a 2.75 ± 0.22b 2.92 ± 0.23b 2.75 ± 0.24b

1.84 ± 0.29a 2.38 ± 0.24b 2.65 ± 0.25b 2.71 ± 0.26b

1,8-Cineole

Test Control 1 Control 2 Control 3

2.09 ± 0.24a 2.42 ± 0.22a 2.57 ± 0.22a 2.48 ± 0.21a

1.89 ± 0.26a 2.33 ± 0.22ab 2.72 ± 0.22b 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)

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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). 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 Mean no. visits in Test stimulus olfactometer arm n χ2* P* i) Hexane T C Males 4.08 3.72 12 1.02 0.796 Females 4.33 4.42 12 0.02 0.999 ii) (R)-linalool Males 2.00 3.36 12 6.09 0.107 Females 1.58 3.00 12 10.1 0.018 iii) (S)-2-heptyl acetate Males 2.17 2.86 12 2.19 0.534 Females 2.08 2.44 12 0.66 0.883 iv) (S)-2-heptanol Males 3.00 2.67 12 3.98 0.264 Females 2.33 4.14 12 8.42 0.038 v) Citral Males 2.33 3.69 12 5.48 0.139 Females 2.75 3.42 12 1.38 0.710 vi) 1,8-Cineole Males 2.50 3.11 12 1.27 0.736 Females 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 discs T is the mean value of test arm C 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 129

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 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 Overall % Treatments Males Females repellency Control (Hexane) 0.00 ± 0.00 0.00 ± 0.00 0.00 A. melegueta vacuum distillate 77.86 ± 2.63 76.05 ± 2.96 76.96 A. melegueta vacuum distillate + maize 76.92 ± 1.24 76.55 ± 3.78 76.74 A. 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 Overall % Treatments Males Females 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 72.01 A. melegueta vacuum distillate + maize 62.16 ± 1.80 68.51 ± 4.28 65.34 A. 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

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vacuum distillate gave an overall 70.65% repellency, and Z. officinale vacuum 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 Overall % Treatments Males Females repellency Control (Hexane) 0.00 ± 0.00 0.00 ± 0.00 0.00 Z. officinale vacuum distillate 77.41 ± 3.29 81.63 ± 2.34 79.52 Z. officinale vacuum distillate + maize 71.67 ± 2.03 72.92 ± 3.28 72.29 Z. 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 Overall % Treatments Males Females 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 70.65 Z. officinale vacuum distillate + maize 65.65 ± 2.57 63.79 ± 3.81 64.72 Z. 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

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blends for both plant extracts but was significantly different compared to control arms. 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.

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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 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

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reported to elicit direct toxicity and fumigant activity against 3-day-old eggs, thirdinstar larvae and pupae of T. confusum (Stamopoulos et al., 2007). Obeng-Ofori et al (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)-βocimene (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%

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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, 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

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recognised important components of plant defence system against herbivores and pathogens, as well as shaping the diet of herbivores. Terpenes are the most widespread and important secondary plant compounds, and can exert toxic, deterrent, antifeedant and repellent effects on insect herbivores. They are the dominant components of many natural volatile blends and responsible for many of the characteristic smells of plant oils, resins, fruits and flowers (Paré and Tumlinson, 1999). Terpenoid chemistry may vary among plants due to many factors which may include environmental and genetic influences (Langenheim, 1994; Powell and Raffa, 1999; Wang and Lincoln, 2004).

4.5 CONCLUSIONS The results obtained in the present study are encouraging given the on-going search for environmentally safe and non-toxic natural products for the protection of stored grains globally. The results stressed the importance of evaluating plant essential oils or components in blends to elucidate their full potency in a given bioactivity. In the future, with proper formulation and production technology, these essential oils could be exploited for use against insect infestation of stored-products at the small scale farmer’s level especially in the developing world for a more sustainable food security. The effects of these essential oils are not particularly dangerous to human health and the environment (Isman, 2006) since the plants are edible and often incorporated in the diet as spices in soups, meat and stew.

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CHAPTER 5: FIELD

REPELLENT

ACTIVITY

AND

OVIPOSITION

DETERRENT

EFFECTS OF A. melegueta AND Z. officinale AGAINST S. zeamais IN STORED MAIZE.

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5.1 INTRODUCTION Maize or corn, Zea mays L. (Poaceae) is one of the most important cereal crops in the world (Ishaya et al., 2008). In Nigeria, agriculture is one of the most important sectors of the economy, because it contributes more than 30% of the total annual gross domestic products (GDP), employs about 70% of the labour force, accounts for over 70% of the non-oil exports and, perhaps provides well over 80% of the food needs of the country (Babatunde et al., 2007). Maize is an important crop widely grown across the different ecological zones of the country ranging from the rain forest belt in the south to the northern Guinea savannah as a major source for food, feed and raw material for agro-allied industries (Agboola and Fayemi, 1999).

Its annual

production is estimated at 5.4 million metric tonnes from about 3.4 million hectares of land (FAO, 2004). Nutritionally, maize contains 80% starch, 13% water, 10% protein, 4% oil, 2% sugar and 3% fibre. Yellow maize contains some vitamins notably vitamin A in addition (Purseglove, 1974). There is, however, an increasing concern about the sustainability of large-scale maize production in Nigeria, as a result of field to store insect pests’ infestation. After harvest, inadequate infrastructure and lack of economic means constrains smallholder farmers to store grain using traditional storage structures and procedures (Markham et al., 1994), such as cribs, baskets, jute bags, and earthen ware or in the open. Of all these methods, the open storage system allows air to flow freely through the grain, which enhances the drying process but also makes the store vulnerable to attack by insect pests (Holst, et al., 2000). One of the major pests of maize is the maize weevil, Sitophilus zeamais Motschulsky, an internal feeder of grains. S. zeamais is a very serious pest in Nigeria because environmental

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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 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

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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 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; ObengOfori 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 ethnobotanical 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.

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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 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 5°00′ and 5°40′ North and longitude 8°04′ and 8°62′ 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

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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 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.

Plate1. Map of Nigeria showing Cross River State where the experiment was conducted from November 2006 to February 2007.

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Plate 2. Map of Cross River State where the experiment was carried out.

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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.

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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.

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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.

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Plate 8. After the application of plant powders, the treatments were covered with dried banana leaves. December 02, 2006. 5.2.5 Data Analysis. Differences in the mean number of S. zeamais per 2 cobs sampled per treatment was determined by analysis of variance (ANOVA) using the statistical software Minitab 15.The germination percentage was calculated using simple percentage method.

5.3 LABORATORY OVIPOSITION DETERRENCE EXPERIMENTS 5.3.1 Materials and Methods Fifty grams of yellow maize were weighed out into 8 x 8.5 x 8.5 cm transparent plastic containers for each replicate. The dried seeds of A. melegueta and rhizomes of Z. officinale were ground into powders and applied by direct admixtures to the maize grains calculated on the weight of plant material/weight of grain (w/w) basis. Each plant powder was applied at three dosage rates of 1%, 5% and 10% in transparent plastic containers, while the controls received no plant powders. Fifteen pairs of 3 d

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old S. zeamais adults were introduced into each plastic container containing treatments for mating and oviposition for 6 days. The plastic containers had their covers drilled with holes to facilitate air circulation. They were then covered with nylon mesh and their perforated lids screwed in place to facilitate confinement of the weevils. Each treatment was replicated 4 times and laid out in a Completely Randomized Design (CRD) on the laboratory bench for 10 weeks. The experiments were conducted in a CTH room maintained at 25 ºC and 65% rh on a 12:12 L.D photoperiod. A mortality count was done every 24 h for 72 h after treatment by sieving out the contents into a clean white tray and counting the number of dead insects. Each time a count was done; dead individuals were discarded while live ones were returned to their respective treatments. After 6 days all live and dead weevils were removed and discarded and the seeds kept aside for F1 progeny emergence. After five weeks, the weevils emerging from each jar were counted to give a measure of productivity and sieved off subsequently every 2 days to prevent mating and subsequent oviposition by F1 as mating in S. zeamais does not occur before weevils are 3 d old (Walgenbach and Burkholder, 1987). The numbers of F1 progeny from each treatment were counted, weighed and recorded. Five weevils were randomly selected from each treatment and weighed on the first week of emergence (week 5 after

treatment)

using

Sartorius

weighing

balance.

The

oviposition

deterrence/inhibition experiment was terminated after 10 weeks, and germination tests were carried out. Thirty seeds were randomly selected from each replicate and soaked in water for about 30 min after which time the grains were removed and placed in labelled Petri dishes lined with filter paper which were covered and moistened daily with water. Germination was assessed after 4 days by calculating the number of seeds germinated out of the total of 30 in each Petri dish. Percentage oviposition deterrence

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of treatments to S. zeamais was also calculated using the formula: Mean No. of emerged adults in control – Mean observed adults in treatment)/Mean No. of emerged adults in control x 100.

5.3.2 Data analysis The experiment was laid out in Completely Randomized Block Design (CRBD) and data obtained analysed using analysis of variance (ANOVA) in MINITAB 15, and all treatment means were compared using Tukey’s Test.

5.4 RESULTS. 5.4.1 Repellent effects of A. melegueta and Z. officinale against S. zeamais in traditional storage granaries in Nigeria. The results of field trials in traditional granaries showed that 10% powders (w/w) of the 2 plant products (A. melegueta and Z. officinale) and a combination of the two (5% each) significantly repelled (p<0.001) S. zeamais from stored maize cobs when compared with the untreated control at 12 weeks post treatment. The prevalence of weevil associated with the combination of A. melegueta and Z. officinale was not significantly (p>0.05) different than either of A. melegueta or Z. officinale alone (Table 5.1) showing no synergistic or additive effects of the combination. Z. officinale alone appeared a poorer repellent than A. melegueta or the two together although not significantly different (p>0.05) from the other treatments.

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Table 5.1.Mean number of S. zeamais counted 12 weeks post treatment with A. melegueta and Z. officinale powders in traditional storage barns in Nigeria. Means in the same column followed by the same letter superscript are not significantly different at the 0.05 level as determined by Tukey’s test. N=4, a-b, P=0.01. Treatments

Mean 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 10th 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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Mean no. of weevils/ 2 cobs

A.melegueta Z.officinale A.melegueta + Z.officinale Control 7 6 5 4 3 2 1 0 0

1

2

3

4

5

6

7

8

9

10 11 12

Time (weeks)

Figure 1.Mean weekly population responses of S. zeamais to A. melegueta and Z. officinale powders in traditional storage facilities in Nigeria. Error bars represent standard errors of the means. n=4.

The percentage germination in the various treatments (Figure 5.2) was significantly higher (p< 0.01) in the treated than untreated cobs.

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+Z .o f

on tro l

A. m el

eg ue ta

C

fic in al e

al e cin Z. of fi

A. m el

eg ue ta

Mean (%) germination

100 90 80 70 60 50 40 30 20 10 0

Treatments

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

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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. Cumulative mean % mortality (± SE) of S. zeamais (h) Treatments 24 48 73 Control (Untreated) 0.83 ± 0.65d 6.67 ± 0.83c 10.83 ± 1.36c Z. officinale (1%) Z. officinale (5%) Z. officinale (10%)

1.67 ± 0.69d 9.17 ± 1.36ab 12.50 ± 1.12a

7.50 ± 1.36c 19.17 ± 1.31b 28.33 ± 1.04a

13.33 ± 1.34c 42.50 ± 2.32a 48.33 ± 1.26a

A.melegueta (1%) A.melegueta (5%) A.melegueta (10%)

0.00 ± 0.00d 4.17 ± 0.65c 7.50 ± 1.02b

2.49 ± 0.65d 9.17 ± 0.89c 18.33 ± 1.26b

8.34 ± 0.69c 23.33 ± 1.47b 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 Oviposition Treatments emergence at 5 weeks (mg) deterrence (%) Control (Untreated) 41.75 ± 1.06a 3.13 ± 0.23a 0.00 ± 0.00d Z. officinale (1%) Z. officinale (5%) Z. officinale (10%)

26.5 ± 1.09b 16.75 ± 1.07bc 6.25 ± 0.99c

3.09 ± 0.60a 3.23 ± 0.49a 3.33 ± 0.54a

35.58 ± 1.99c 59.35 ± 1.78b 84.22 ± 1.69a

A.melegueta (1%) A.melegueta (5%) A.melegueta (10%)

32.5 ± 1.58ab 23.25 ± 1.33b 14.25 ± 1.07bc

3.10 ± 0.49a 3.13 ± 0.51a 3.30 ± 0.53a

21.01 ± 2.35c 43.65 ± 2.02bc 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 10th week. It therefore meant that all viable eggs laid by the weevils had hatched by the 10th week post treatment.

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Control A. melegueta 5%

A. melegueta 1% A. melegueta 10%

Mean adult emergence

45 40 35 30 25 20 15 10 5 0 Week 5

Week 6

Week 7 Week 8 Time

Week 9

Week 10

Figure 5.3. Weekly S. zeamais F1 progeny emergence from maize grains treated with A. melegueta powders at 3 dosages. Bars= standard errors of the means, n=12.

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Control Z. officinale 5%

Z. officinale 1% Z. officinale 10%

45 Mean adult emergence

40 35 30 25 20 15 10 5 0 Week 5

Week 6

Week 7 Week 8 Time

Week 9

Week 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.

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% )

% )

(1 0

(5

ta

ta

ue m el eg

A.

m el eg

ue

ta A.

A.

m el eg

ue

al e

(1 0

(1

% )

% )

% ) of iic in Z.

in

al e

(5

% ) (1 of iic Z.

Z.

of iic

in

al e

C

on t

ro l

Mean (%) Germination

100 90 80 70 60 50 40 30 20 10 0

Treatments

Figure 5.5. Effect of A. melegueta and Z. officinale powders on germination (± SE) of maize grains.

5.5 DISCUSSION Results reported in this chapter show the toxicity, oviposition deterrent and repellent effect of the powders from two plant species against S. zeamais in southern Nigeria, and in laboratory studies. The powders from the seeds of A. melegueta and rhizomes of Z. officinale were effective in repelling the insect population from stored maize when compared with the control at 10 % (w/w) and at a combination of 5 % (w/w) of each plant powder treatments after 12 weeks in traditional storage environment in Nigeria. A combination of the two plants did not produce any significant synergistic or additive effect on their repellency against S. zeamais. It was observed that the mean number of visiting weevils to treated maize cobs fell sharply one week after the admixture with plant powders, while the number of insects in the untreated maize

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cobs continues to increase week by week. Maize cobs were protected from pest infestation up to 10 weeks in traditional storage granaries. Laboratory studies confirmed the results from field trials and showed that the powders of A. melegueta and Z. officinale are toxic and repellent to S. zeamais and can suppress oviposition as measured by progeny development of the weevils resulting in better protection of stored maize grains from pest infestation and damage. The mean number of progeny produced by S. zeamais in the untreated control was significantly higher than the one treated with 5% and 10% (w/w) concentration of plant powders. The efficacy of the powders was dose-dependent with higher doses providing greater protection with significantly fewer emergent adults. However, the treatments did not influence the weight of emergent adults. This finding is in agreement with Danho et al (2002) who reported that emergent S. zeamais adult weight was not affected by competition on different quantities of host grains.

Although the mode of action of these plant powders is not clearly understood, it was observed that the repellent and pungent odours from these plants caused the insects to climb to the walls of the containers soon after introduction thereby limiting adequate feeding and oviposition. Also the physical abrasion of the insect cuticle with the resultant loss of body haemolymph or partial blockage of the spiracles (Ogunwolu et al., 1998; Oparaeke and Kuhiep, 2006) may have contributed to mortalities in suffocation and death. The observation that high dosages of A. melegueta and Z. officinale powders caused significant adult mortality and a reduction in F1 progeny emergence could be due either to repellent, feeding or oviposition deterrence effects on the weevils or to a combination of all the three. This could also be due to plant material having toxic effects on the larvae hatching from eggs laid on grains resulting

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in reduced progeny emergence (Tapondjou et al., 2002, 2005; Akob and Ewete, 2007), however it was probably not likely that the active ingredients of the powders entered the seeds as after washing the maize seeds treated in this fashion tasted normal. Similar results have been reported elsewhere. For example, Bekele (2002) reported that seed powder of Milletia ferruginea (Hochest) Baker applied at 10% w/w to maize seeds was toxic to S. zeamais and caused significant reduction in F1 progeny production, and attributed the toxicity to rotenone. Powders and essential oil obtained from

dry

ground

leaves

of

wormseed,

Chenopodium

ambrosioides

(L.)

(Chenopodiaceae) at a dosage of 6.4% (w/w) have been reported to induce total mortality of S. granarius and S. zeamais two days after treatment, and inhibited F1 progeny production and adult emergence under laboratory conditions. The toxicity of C. ambrosioides was attributed to major constituents such as ascaridole, cymol and αterpinen (Tapondjou et al., 2002). Bekele et al (1995) also reported the repellent effect of dried ground leaves (25 g/250 g of maize seeds) and essential oil (0.3 mg/250 g of maize seeds) of Ocimum kilimandscharicum (Labiatae) against S. zeamais, R. dominica and S. cerealella resulting in lower weight loss and number of damaged maize seeds compared with untreated grains. Similarly, Silva et al (2005) reported that 2% concentration (w/w) powdered leaves of C. ambrosioides and boldo, Peumus boldus Mol. (Monimiaceae) exhibited 90.1% and 98.8% mortality of S. zeamais after 24 h exposure, and suppressed progeny production by 13% in Chile. While in Tanzania, 10% (w/w) leaf powders of eucalyptus, Eucalyptus macrorhyncha (F. Muell) (Myrtaceae), pawpaw, Carica papaya (L.) (Caricaceae), neem, Azadirachta indica (A. Juss) (Meliaceae) and lantana, Lantana camara (L.) (Verbenaceae) were toxic to S. zeamais and significantly reduced grain damage and weight loss (Mulungu et al., 2007). Under small-scale farmer conditions as is the case

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in this study, powder treatments may protect stored grains for some time (Bekele, 2002). It is quite obvious that phytochemicals have potential for use in modern stored product protection. As attractants or repellents, they may be used to modify insect behaviour. Repellent compounds could also be effective as control agents and some of these compounds may even be used as fumigants (Adler et al., 2000).

5.6 CONCLUSIONS The application of plant powders may minimize insecticide usage thereby reducing health hazards to applicators and the amount of toxic residues to the environment. Treatment of grains with repellents could also have important practical applications in the parts of the world where insecticides are expensive, in short supply or where these repellent plants are cheap and readily available. The results from this study indicate a possible scientific rationale for the traditional use of plant powders as grain protectants by resource poor farmers. The implication of this in practice is that A. melegueta and Z. officinale powders could constitute agents with sufficient broad spectrum of repellent activity for use as general purpose stored product insects repellent in developing countries.

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CHAPTER 6: GENERAL DISCUSSION

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The main aim of this study was the identification and use of semiochemicals for the control of the maize weevil, Sitophilus zeamais in Nigeria. The use of semiochemicals has stimulated much interest because they may be used to improve current monitoring as well as direct means of controlling stored-product insect pest species (Cox, 2004; Germinara et al., 2008). Laboratory olfactometry, oviposition deterrence and field experiments were designed to investigate the repellent properties of alligator pepper, Aframomum melegueta, ginger Zingiber officinale and the West African black pepper, Piper guineense against S. zeamais of stored maize. Gas chromatography (GC), coupled gas chromatography mass spectrometry (GC-MS), and liquid column chromatographic separation of A. melegueta and Z. officinale vacuum distilled essential oils were carried out to identify the chemical constituents that confer repellency to these plants. The aim was to investigate the possibility of using these non-host plants as sources of repellent signals for S. zeamais in a novel, environmentally sound, small-scale crop protection strategy in Africa.

6.1 BEHAVIOURAL RESPONSES TO HOST AND NON-HOST PLANT VOLATILES Insect host location frequently involves the detection of volatile chemical cues by olfactory receptors located on the antenna (Bruce et al., 2005). This implies that insect species are able to detect a suitable host while walking or in flight, and also that host selection can depend on a lack of repellency. Single choice olfactometer experiments were conducted with virgin male and female S. zeamais adults to study odour responses to their host and non-host plant volatiles. S. zeamais responded with positive anemotaxis to air plumes passed over 2 g white maize, yellow maize and winter wheat kernels, but negatively to air that passed over host plant plus 10% non-

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host plants (w/w), namely A. melegueta, Z. officinale and P. guineense respectively when compared to the control arms emitting clean air. Both males and females showed a general attraction to all three host plants tested, and were able to distinguish between host plants and non-host plants. Odours from a combination of host and nonhost plants, and non-host plants individually, repellent to the weevils in olfactometer bioassays. The repellency of non-host plant volatiles was dose-dependent. These results confirm similar reports that phytophagous insects employ volatiles from plant materials to locate suitable substrates for food, mating and aggregation (Visser, 1986). It has been shown that extracts from carob pods (Collins et al., 2004) and specific cereal volatiles attract the granary weevil S. granarius, a sibling species of S. zeamais (Collins et al., 2007; Germinara et al., 2008). S. zeamais adults oriented to a volatile blend emitted by grains of white maize, yellow maize and winter wheat kernels in olfactometry bioassays. The presence of phagostimulatory compounds have been considered crucial in the infestation process by this weevil (Kanaujia and Levinson, 1981). The use of host volatiles for host finding and colonisation is not limited to stored-product insects. Couty et al (2006) reported that the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae) the major insect pest of cruciferous crops such as cabbages, broccoli and cauliflower, uses volatile plant chemical cues to locate and to promote landing on their hosts, even in a complex mixed-crop environment in large cages. Amarawardana et al (2007) studied the odour-mediated effects of leek, Allium porum and chives, Allium schoenoprasum (Alliaceae), on the host searching behaviour of the peach-potato aphid, Myzus persicae Sulzer (Homoptera; Aphididae) in 4-way olfactometry experiments. They reported that odour of the host plant sweet pepper, Capsicum annum L. (Solanaceae), was significantly attractive, whereas odour of chives was significantly repellent. The combined odour

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of sweet pepper and chives was neither attractive nor repellent, but when sweet pepper plants were exposed to volatiles from chives for five days, their odour became repellent to M. persicae. An extract of A. porum plants was significantly repellent to aphids in the olfactometer, as were C. annum plants that were sprayed with this extract. Viewing this analogous report, it may be possible to mask host plant odours with non-host plants in order to disrupt host finding by insect pest species thereby protecting the desired stored product.

6.2 BIOACTIVITY OF A. melegueta AND Z. officinale ESSENTIAL OILS AND THEIR CONSTITUENTS AGAINST S. zeamais. The use of unattractive plant odours to repel insect pest species from stored commodities has resulted in some commercial pest control products in recent years (Isman, 2000; 2006). One of the most important sources of repellents is the essential oil extract from aromatic plants commonly used to flavour foods and in perfumery (Coppen, 1995). Plant essential oils or their constituents have been valued in crop protection due to their broad spectrum of biological activity. They consist of complex mixtures of mono and sesquiterpene hydrocarbons, aliphatics as well as aromatic compounds with a few major constituents (Rosell et al., 2008). In olfactometry experiments, vacuum distilled essential oils extracted from A. melegueta and Z. officinale were repellent to adult S. zeamais when tested individually and in combination with seeds of the host plant, Z. mays. GC-MS analysis of the vacuum distilled essential oil from A. melegueta and Z. officinale identified 13 and 24 compounds respectively. The repellency of the essential oils was accounted for by the Florisil® diethyl ether fractions. GC-MS analysis of the behaviourally active Florisil® diethyl ether fractions of A. melegueta identified (S)-2-heptanol, (S)-2-

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heptyl acetate and (R)-linalool as major components in a 1:6:3 ratio, and for Z. officinale vacuum distillates, 1,8-cineole, neral and geranial in a 5.48:1:2.13 ratio. To my knowledge, this is the first time (S)-2-heptyl acetate has been identified from A. melegueta seeds. Apart from (R)-linalool, the synthetic preparations of these compounds individually could not produce total protection of the maize grains, but the blend of each plant essential oil in their natural ratios gave significantly higher repellent activity against S. zeamais in laboratory studies than individual compounds. Both blends of essential oils produced percent repellency (PR) equivalent to a class IV repellent with between 60-80% repellency of the pest species. According to the percent repellency (PR) classes of Juliana and Su (1983) from 0 to V: class 0 (PR < 0.15), class I (PR = 0.1-20%), class II (PR = 20.1-40%), class III (PR = 40.1-60%), class IV (PR = 60.1-80%) and class V (PR = 80.1-100%). The implication of this result in practice is that the blend as whole rather than specific components could constitute a control agent with sufficient broad spectrum of bioactivity to be deployed in stored-product pest control. (R)-Linalool is a volatile oxygenated monoterpene compound found ubiquitously in the plant and even human odours (Logan, 2006), and different concentrations could result in either attractancy or repellency for a variety of insect species (Mauchline et al., 2008). Insects can determine ratios of volatile chemicals by comparing the stimulation of one type of receptor cell with that of another (Blight et al., 1995; Bruce et al., 2005). For example, using single cell recordings (SCR), Wadhams (1990) reported 166 responding olfactory cells on the antenna of the cabbage seed weevil, Ceutorhynchus assimilis Paykull, and most of these cells exhibited high specificity in their response profiles. Many insects use a specific blend and ratio of volatiles to find their host plant (Städler, 1992), a negative selection by the insect for specific odours associated with unsuitable host plants

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within the complex of odours plays a role in selection or avoidance of plant species (Dickens et al., 1992; Hayes et al., 1994; Poland et al., 1998). This avoidance is not always based on selection and learning of the insect species alone but can also involve an induced defence mechanism by plants after attack of the insect herbivore which makes them unattractive for the insect (Franceschi et al., 2005) and to attract natural enemies (Takabayashi and Dicke, 1996; van Tol et al., 2007). Specific ratios of all the behaviourally active compounds from Florisil® diethyl ether fractions of A. melegueta and Z. officinale may therefore be responsible for the repellent activity of their essential oils against the maize weevil. These results identified individual chemical constituents that were likely to be involved in the repellent activity of blends of A. melegueta and Z. officinale essential oils against S. zeamais in olfactometry bioassays.

Direct toxicity of essential oils to pest species appear to result from interaction with the insect nervous system, either by acetylcholinesterase inhibition or antagonism of the octopamine receptors (Rosell et al., 2008). The lack of octopamine receptors in vertebrates likely accounts for the profound mammalian selectivity of essential oils as insecticides and repellents because the octopaminergic system of insects represents a biorational target for insect pest management (Isman, 2000; Enan, 2001). Effects with the essential oil of A. melegueta and Z. officinale against S. zeamais such as repellency and feeding deterrence may be consistent with this mode of action. Applications of other essential oils such as citronella oil in stored-product protection, mosquito repellency and domestic pest control (cockroaches, ants and fleas), cinnamon oil in mite and urban pest control have been reported (Wong et al., 2005; Isman, 2006). This study presents a rational approach to investigating the chemical

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basis of repellent non-host plant volatiles, for assessing the behavioural effects of such repellent volatiles on stored-product pests such as S. zeamais in small-scale farmer conditions in Nigeria.

6.3 FIELD REPELLENT ACTIVITY AND LABORATORY OVIPOSITION DETERRENCE OF A. melegueta AND Z. officinale POWDERS AGAINST S. zeamais. The post-harvest system in Nigeria presents particular problems for pest control. The traditional storage facilities are varied and include sacks, baskets, earthenware pots, farmers’ houses, cribs, and sacks in small communal stores, wooden barns, and trader’s stores and in the open. Another problem is that the stored-product and its associated insect pests are constantly being moved through different stores as it goes through processing, marketing and distribution systems. Such post-harvest systems in which stored grain is being moved around with the commodity batches being split or combined are logistically problematic even for conventional pesticide application (Haines, 1991). However, research findings from several countries confirm that some plant powders, essential oils or their constituents not only repel insects, but also have contact and fumigant insecticidal actions against specific stored-product pests (Isman, 2000; Rajendran and Sriranjini, 2008). As a part of an effort aimed at the development of reduced-risk stored-product protection based on repellent plant products, 10% powders of A. melegueta and Z. officinale, and a combination of both (5% each) were evaluated for repellent activity against S. zeamais under traditional storage conditions in Obudu, southern Nigeria for 12 weeks. Results showed that maize cobs treated with powders of A. melegueta seeds and Z. officinale rhizomes were protected from insect attack for up to 10 weeks and gave significantly higher germination rates than

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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

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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 storedproduct protection is a common practice, and has more potential in subsistence and traditional farm storage conditions, in developing and under-developed countries (Weaver and Subramanyam, 2000; Nikpay, 2007). Under small-scale farmer conditions, protecting grains with indigenous repellent plants could lead to the development of a sustainable crop protection method.

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6.4 POTENTIAL FOR APPLICATION BY SMALL-SCALE FARMERS The ultimate goal of this study is the development of a push-pull strategy for the control of S. zeamais in small-scale traditional storage facilities in Africa. Push-pull or stimulo-deterrent diversionary strategies (S.D.D.S.) involve a combination of various behaviour-modifying stimuli to manipulate the prevalence and distribution of pest species and beneficial organisms for pest management. The principles of the push-pull strategy are to maximize pest control efficacy, efficiency, sustainability, and output, while minimizing negative environmental effects. The ‘push’ could be a repellent, antifeedant or an oviposition deterrent natural agent, and the ‘pull’ a kairomone, aggregation, sex and oviposition pheromones or a selective control agent (Pickett et al., 1997; Cook et al., 2007). There appears to have been no successful application of the push-pull strategy to protect stored grain from insect attack, although the literature is rich in reports of plant-derived repellents in stored-product protection (Obeng-Ofori and Reichmuth, 1997; Bekele et al., 1997; Isman, 2000; 2006; Huang et al., 2000; Nikpay, 2007). Findings from this study could be applied practically by small-scale farmers in Nigeria by direct administration of the seeds of A. melegueta and Z. officinale rhizomes powders with maize cobs for protection against S. zeamais. However, there may be a need to re-apply the repellent plant powders after 9-10 weeks to boost their efficacies. Framers could also be encouraged to expand the existing cultivation of A. melegueta and Z. officinale thereby boosting agriculture and more cash in their pockets. This study provides the underpinning science for use of the repellent plant materials in stored-product pest control, and provides chemical markers for quality assurance and control if the envisaged push-pull system breaks down.

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Monitoring is a vital component of all integrated pest management programmes. The foundation of any successful agricultural production programme is an effective pest monitoring system that supplies information on not only the number and type of pests present in the agro-ecosystem, but also detects changes in pest populations over time and locates foci of infestation and routes of entry (Campbell et al., 2002). The use of attractants in insect traps can lead to earlier detection of infestations, more accurate monitoring of pest population levels and development of proper control measures. Several trap designs specific for stored-product pests have been developed and are commercially available (Mullen and Dowdy, 2001). Since insects recognize suitable hosts by using key volatiles that are most often present in specific ratios (Bruce et al., 2005), host volatiles used in host location by Sitophilus species such as kibbled pods of the carob tree Ceratonia siliqua L. or their extracts (Wakefield et al., 2005), (E)-2nonenal 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

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(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

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of the synthetic plant compounds for use in applied research targeting semiochemical based stored-product pest management. Also, application of slow release formulations of a blend of attractive chemicals may be used to concentrate S. zeamais to traps from where they will be selectively destroyed.

The present study will contribute to the development of management tactics that rely on the exploitation of semiochemicals to manipulate oviposition behaviour of the maize weevils such as application of repellent blends of essential oils from A. melegueta and/or Z. officinale to the packaging materials of stored maize and mass trapping of visiting weevils with host volatiles deployed with sitophilure outside the granaries. Therefore, the use of a stimulo-deterrent diversionary strategy that takes advantage of naturally-occurring semiochemicals in traditional maize storage granaries in Africa appears to be feasible in the short term.

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