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INSECTICIDE RESISTANCE IN DIAMONDBACK MOTH, 
PLUTELLA XYLOSTELLA L. (LEPIDOPTERA: YPONOMEUHDAE) 
FROM SELECTED CABBAGE FARMS ASSOCIATED WITH 
PYRETHROID AND ORGANOPHOSPHATE USE IN SOUTHERN
GHANA
BY
JACINTER AT1ENO OTIENO ODHIAMBO (B. Sc. (Agric.) HONS, Egerton
A THESIS SUBMITTED TO THE AFRICAN REGIONAL POSTGRADUATE 
PROGRAMME IN INSECT SCIENCE (ARPPIS), UNIVERSITY OF GHANA, 
LEGON IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE 
AWARD OF MASTER OF PHILOSOPHY DEGREE IN ENTOMOLOGY
INSECT SCIENCE PROGRAMME*
UNIVERSITY OF GHANA
AUGUST 2005
*JOINT INTERFACULTY INTERNATIONAL PROGRAMME FOR THE TRAINING OF 
ENTOMOLOGISTS IN WEST AFRICA. COLLABORATING DEPARTMENTS: ZOOLOGY 
(FACULTY OF SCIENCE) & CROP SCIENCE (SCHOOL OF AGRICULTURE, COLLEGE 
OF AGRICULTURE AND CONSUMER SCIENCES).
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DECLARATION
I hereby declare that with the exception o f references to other people’s work which I have duly 
acknowledged all the experimental work described in this thesis was carried out by me and this 
thesis, either in whole or in part, has not been presented elsewhere for another degree.
Jacinter A. O. Odhiambo 
(Student)
t v
Rev. Dr. Winfred S. K. Gbewonyo 
(Supervisor)
Prof. Daniel Oheng-Ofori
(Supervisor)
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DEDICATION
To God, My Parents Mr. And Mrs. Charles Otieno, for offering me the best in life; my husband 
Jared, Daughter Alpha, for your unwavering thoughtfulness and sacrifice and the late Ben
Akoth who never lived to see this work.
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ACKNOWLEDGEMENTS
I wish to express my sincere gratitude to all the individuals who through their support 
contributed to the successful completion of this dissertation.
I reserve my sincere gratitude to my supervisors, Rev Dr. Gbewonyo W S. K o f 
Department Biochemistry and Prof Obeng-Ofori D. O of Faculty o f Agriculture. Their 
patience, expert guidance and treasured advice, concern, stimulating discussions and 
maximum co-operation contributed greatly to the final result of this work. Special thanks go 
Dr. Gbewonyo who went with me the extra mile and whose personal and spiritual guidance 
gave me the zeal to continue the rough road to success. I also thank Dr. Michael D. Wilson 
and Dr. Daniel A. Boakye, both of the Parasitology Unit of the Noguchi Memorial Institute 
for Medical Research (NMIMR) for their direct involvement in the molecular aspect o f this 
work I am also grateful to Dr. Owusu E O of Department of Zoology for his excellent 
expertise in various fields concerning insecticide resistance for making available his cages 
for rearing the insects and enzyme assay reagents which were very instrumental in successful 
completion of this project besides his accurate advice whenever needed.
I acknowledge the tremendous role played by Dr Charles Brown. His exemplary training in 
molecular biology, great contribution in identifying the specific primers and brilliant ideas 
throughout the laboratory work in addition to his personal support and guidance which were 
vital for the successful completion o f this research. My heartfelt appreciation goes to Mr. 
Richard Harry Asmah of Electron microscopy and Mr. Kusi Asamoah of pathology unit both 
of NMIMR, for their expertise and guidance through the zymogram studies.
tv
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I also express my gratitude to Dr Loehr of ICIPE Kenya for sending the reference laboratory 
cultured strains and Vincent Eziah for the useful literature in zymogram studies
I would like to acknowledge the tremendous help, encouragement and friendly support given 
me by lecturers and technical staff at Crop science; Mr. Tomgah, Mr Ankrah, Mr. Elvis 
Appiah, Ampah, Kurt, Otoo, Ben I am more indebted to Prof Obeng-Ofori the then head o f 
crop science department and Prof. Oduro for availing the pathology laboratory and the 
facilities therein for this research and MR  Cornelius and Mr. Asante for their assistance with 
data analysis. I also acknowledge the contribution and support of lecturers and technical staff 
of department of Biochemistry particularly; Mrs Doris Amanor, Mr. Sammy Feyi, Mr. 
Winfred-Peck, Emmanuel Anipa and Cosmos Oduro. Special thanks go to Dr Naa 
Adamafio the Head of the Biochemistry department for allowing this research to be 
undertaken in their laboratories. My appreciation also goes to the Parasitology Unit, 
NMIMR I am particularly indebted to D r Bosompem the head of the unit and for allowing 
me access to his laboratory and friends in parasitology including Naiki, Hellena , Richard, 
Dan Kwame, Mamuye, Isaei and Dr. Bismark Sarfo I am most grateful to all the cabbage 
growers in Accra-surburbs and Mampong-Akuapem who were very helpful during the 
execution of the survey.
I greatly appreciate the co-operation of Dr. Ochieng, Head of capacity building, ICIPE, 
Kenya and the administration of the African Regional Postgraduate Programme in Insect 
Science (ARPPIS). I acknowledge the essential role played by Prof. J.N Ayertey, 
coordinator, ARPPIS-West Africa in making this course a success. Special thanks to all my 
lecturers for helping me realize the potential in my work. My heartfelt thanks to all my 
classmates Olivia, Zakka, Abdulahi, Badi and Kahindi and the Kenyans Kipruto, Mbilinyi,
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Sospeter, Mary, John, Gachoka. I appreciate the companionship and the useful discussions 
we had.
I wish to register my utmost gratitude to Lydia Daddy and family, Dr. Emmanuel and 
Rosemary, Joseph, Michael, Sylvester, Omondi-Aman, Judy, Mike Osae, Wonder Awumey, 
all brethren of the S. D A Campus Fellowship and Prince Emmanuel fellowship, Aqua, Ben 
Kwansa, Emanuel Adom, Isaac, Hellena Osae. I thank you all for your unlingering support, 
prayers and encouragement.
Finally, I express my wannest obligation to the Otieno family for their prayers while I was 
away working on this thesis. To the Omollo family, especially for my mother in-law (“Nyar 
kuma chiegni”), my sister Everlyne for assuming the position of a mother to Alpha. Brothers 
Denis, John, Sunday, Ben, Reagan, Rasto amongst others, my cousin Charles Nyawuor I can 
never thank you enough for your love and attention throughout the time I was in Ghana. May 
God richly bless you. To my friends Rosemary Scott, Lisa Omondi, Ken Odira, Mrs. 
Obongo. I express my appreciation for all the encouragement. Above all, I am most grateful 
to the Almighty God for his provision.
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TABLE OF CONTENTS
TITLE PAGE..........................
DECLARATION....................
DEDICATION........................
ACKNOWLEDGEMENTS..
TABLE OF CONTENTS......
LIST OF ILLUSTRATIONS
LIST OF PLATES..................
LIST OF TABLES..................
LIST OF APPENDICES.......
ABSTRACT..............................
CHAPTER ONE............................................................................................................................1
GENERAL INTRODUCTION...................................................................................................1
1.1 Introduction..................................................................................   1
1.2 Justification........................................................................................................................... 4
1.3. OBJECTIVES....................................................................................................................6
1.3.1 Main objective.............................................................................................................. 6
1.3.2 Specific objectives........................................................................................................6
CHAPTER TWO.......................................................................................................................... 7
LITERATURE REVIEW........................................................................................................... 7
2.1 THE CABBAGE PLANT................................................................................................... 7
2.1.1 Origin, distribution and taxonomy............................................................................. 7
2.1.2 Agronomy......................................................................................................................8
2.1.3 Pests................................................................................................................................9
2.2 THE DIAMONDBACK MOTH ..................................................................................... 11
2.2.1 Description...................................................................................................................11
2.2.2. Origin and distribution o f DBM ........................................................................... 13
2.2.3 Biology and Ecology................................................................................................. 13
2.2.3.1 Life cycle............................................................................................................. 13
2.2.3.2 Host range and host specificity.........................................................................17
2.2.3.3 Effects of environmental factors on DBM......................................................18
2.2.3.4 Pest damage and economic importance...........................................................18
2.3 MANAGEMENT OF CABBAGE..................................................................................19
2.3.1 Small-scale farm ing...................................................................................................20
2.3.2 Large Scale Fanning........................................................................  20
2.4 CONTROL................................................................................................ZZZZZZZZZZ21
2.4.1 Cultural control...........................................................................................................21
2.4.2 Biological Control...................................................................................................... 21
2.4.3 Plant resistance...........................................................................................................22
2.4.4 Sex pheromone...........................................................................................................22
2.4.5 Insect-growth regulators........................................................................................... 22
2.4.6 Bacillus thuringiensis.............................................................................................. 23
2.4.6.3 Molecular studies of B.t. resistant gene........................................................24
2.4.7 Chemical control o f DBM .................................................................................  25
2.4.7.1 Chemical insecticide use patterns..................................................................  25
2.4.7.2 Effects of chemical control...............................................................................26
2.4.8 Pesticide Residues........................  oa
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2.4.8.1 Effectsof Insecticide Residues on Agriculture Systems.............................. 30
2.4.8.2 Pesticide Residue Legislation ...... ............................................................31
2.4.8 3 Violation of pesticide residue legislation....................................................32
2.4.8.4 Pesticide residues levels in Ghana..................................................................34
2.4.8.£ Residue level estimation................................................................................. 35
2.4.8.5.1 Methods for residue analysis...................................................................35
2 4 9 Integrated pest management (IPM ).......................................................................40
2 5 RESISTANCE DEVELOPMENT.................................................................................41
2.5.1 Mechanisms of insecticide resistance....................................................................45
2.5.1.1 Metabolic detoxification................................................................................. 4 5
2.5.1.2 Target site insensitivity....................................................................................45
2.5.1.3 Changes in behaviour as a resistance mechanism....................................... 47
2.5.1 4 Cross resistance and multiple resistance....................................................... 48
2.5.2 Resistance in Diamondback moth.......................................................................... 49
2.5.3 Management of resistance.......................................................................................53
CHAPTER THREE
MATERIALS AND METHODS_____________________________________________ 56
3.1 Chemicals, reagents, equipment and software.............................................................56
3.2 Study area.........................................................................................................................56
3.3 Questionnaire Survey...................................................................................................... 58
3.4 Insecticides.......................................................................................................................59
3.5 Culturing o f Diamondback m oth ...................................................................................59
3.5.1 Field Sample collection...........................................................................................60
3.5.2 Reference (Susceptible) strain ............................................................................... 61
3.5.3 Cultivation of insecticide free cabbage.................................................................61
3.6 Determination of level o f resistance............................................................................. 62
3.6.1 Dose-response bioassay...........................................................................................62
3 .6.2 Carboxyl esterase activity in field population of DBM.......................................67
3.6.2.1 Establishment of standard calibration curves............................................... 67
3.6.2.1.1 a-Naphthol.................................................................................................67
3.6.2.1.2. P-Naphthol..............................................................................................68
3.6.2.1.3 Calibration curve of bovine serum albumin (BSA)........................... 69
3.6.2.2 Enzyme preparation and assay........................................................................69
3.6.2.3 Protein determination...................................................................................... 70
3 .6.3 Polyacylamide gel electrophoretic analysis of esterases.....................................70
3.6.3.1 PAGE analysis o f DBM esterase isozymes................................................ 70
3.6.4 PCR amplification of B t. resistant gene...............................................................71
3.6.4. 1. DNA extraction..............................................................................................72
3 .6.4 2 PCR amplification............................................................................................73
3.6.4.5 Analysis of PCR products............................................................................... 73
3 .6.4 3 Polyacrylamide gel electrophoresis of PCR products................................. 74
3.7 Residue Level Estimation...............................................................................................75
3.7.1 Sampling................................................................................................................... 75
3.7.2 Cabbage samples......................................................................................................75
3 .7.3 Extraction, concentration and analysis of insecticide residues..........................76
3.7.4. Solid phase extraction.............................................................................................77
3 .7 .5 Bioassay of cabbage extracts................................................................... 77
3 .8 Data Analysis......................................................................................................  80
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CHAPTER FOUR-------------------------------  —..........................................................310 |
RESULTS
4.1 Insecticide use pattern survey.................. -.....................................................................
4.1.1 Agronomic practices......................   81
4.1.2 Pests and pest control.............................................................................................. 81
4.1.3 Insecticides and their use pattern........................................................................... 83
4.1. 4 Mode of insecticide application............................................................................ 86
4.1.5 Frequency o f insecticide application..................................................................... 87
4.2 Susceptibility of DBM to insecticides.......................................................................... 91
4.2.1 Pawa (Lambda-cyhalothrin)....................................................................................91
4.2.2 Cypercal (cypermethrin)...................................     91
4.2.3 Deltaplan (Deltamethrin).........................................................................................92
4.2. 4 Dursban (Chlopyrifos-methyl)..^...........................................   92
4.2.5 Dipel (Bacillus thuringiensis Var. Kurstaki)........................................................ 92
4.3. Cues for Cross and multiple-resistance .......       94
4.4 Carboxylesterase Activity in Field populations o f Diamondback M oth................. 95
4.4.1 a-naphthyl esterase...............................................................   .95
4.4.2 3-naphthyl esterase..................................................................................................95
4.5 Native Polyacrylamide Gel Electrophoresis (PAGE)...................   99
4.6 Molecular Identification o fB.t. resistance gene........................................................ 102
4.7 Polyacrylamide gel electrophoresis of PCR Products.............................................. 105
4.8 Residue Level estimation..............................................................................................108
4.8.1 Brine shrimp bioassay for the insecticide SPE standards fractions...............108
4.8 .2 Estimation of residue levels in cabbage samples............................................ 110
4.8.3 Brine shrimp mortality profile for solid phase extracted (SPE) fractions from 
cabbage samples.............................................................................................................. 1 10
4.8.4 Estimation of Maximum Residue Levels (MRL) o f insecticides in the cleaned 
cabbage extracts............................................................................................................... 112
CHAPTER FIVE_________  115
DISCUSSION____________________________________   ” ....115
CONCLUSIONS AND RECOMMMENDATION  127
REFERENCES_________________________ _____________ v .in
APPENDICES      * «
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Figure 1: 
Figure 2: 
Figure 3:
Figure 4: 
Figure 5 (A): 
Figure 5 (B): 
Figure 5(C): 
Figure 5(D): 
Figure 6:
Figure 7: 
Figure 9:
Figure 10: 
Figure 11: 
Figure 12: 
Figure 13: 
Figure 14: 
Figure 15:
Figure 16:
LIST OF ILLUSTRATIONS
Adult diamondback moth
Life of diamondback moth showing the four stages
The evolution of analytical methodology for organic toxicants in
environmental samples
Perceived pesticide treadmill
Hydrolysis of chlorpyrifos by carboxylesterase
Hydrolysis of cypermethrin by carboxylesterase
Hydrolysis o f lambda-cyhalothrin by carboxylesterase
Hydrolysis of deltamethrin by carboxylesterase
Duration under which the farms have been used for Brassicas 
production
Types o f irrigation equipment used in cabbage ferms
Comparison between the previous and currently used agrochemicals
in the cabbage farms
Interval between insecticide spraying and subsequent irrigation 
Frequency of insecticide spraying 
Pre-harvest interval observed by cabbage farmers 
Frequency of visits by the extension officers 
Sources of information on insecticide residues awareness 
Polyacrylamide gel electrophoresis of esterase isozymes o f DBM larvae 
showing the difference between fast- moving and slow moving esterase 
isozymes.
Polyacrilamide gel electrophoresis of DBM larvae showing higher
frequency of slow moving esterases in Mampong (gel A), than in a
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Table 1: 
Table 2: 
Table 3: 
Table 4: 
Table 5: 
Table 6:
Table 7: 
Table8:
Table 9:
Table 10: 
Table 11:
Insect pests associated with cabbage in Ghana (Source: Forsyth, 1966 
Fatal Pesticide poisoning in Ghana
Comparison of Records of Resistance to Pesticides in Arthropods 
Number o f respondents interviewed in each cabbage growing area 
Current insecticide use pattern in Accra suburbs and Mampong-Akuapem 
Previous insecticide use pattern in Accra suburbs and Mampong-Akuapem
Farmers’ Awareness about some agronomic practices and Insecticide 
residues.
The response o f Plutella xylostella (FI) generation o f field-collected 
Population to lethal concentrations o f active ingredients o f the selected 
insecticides.
Carboxylesterase activity o f DBM populations and insecticide used in 
cabbage farms in Mampong Akuapem and Accra suburbs
Lethal concentrations of insecticide standards
Fractionated Insecticides from cabbage samples and estimated insecticide 
residue content
LIST OF TABLES
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Figure 1: 
Figure 2: 
Figure 3:
Figure 4: 
Figure 5 (A): 
Figure 5 (B): 
Figure 5(C): 
Figure 5(D): 
Figure 6:
Figure 7: 
Figure 9:
Figure 10: 
Figure 11: 
Figure 12: 
Figure 13: 
Figure 14: 
Figure 15:
Figure 16:
LIST OF ILLUSTRATIONS
Adult diamondback moth
Life of diamondback moth showing the four stages
The evolution of analytical methodology for organic toxicants in
environmental samples
Perceived pesticide treadmill
Hydrolysis of chlorpyrifos by carboxylesterase
Hydrolysis of cypermethrin by carboxylesterase
Hydrolysis o f lambda-cyhalothrin by carboxylesterase
Hydrolysis of deltamethrin by carboxylesterase
Duration under which the forms have been used for Brassicas 
production
Types of irrigation equipment used in cabbage farms 
Comparison between the previous and currently used agrochemicals 
in the cabbage farms
Interval between insecticide spraying and subsequent irrigation 
Frequency of insecticide spraying 
Pre-harvest interval observed by cabbage farmers 
Frequency of visits by the extension officers 
Sources of information on insecticide residues awareness 
Polyacrylamide gel electrophoresis of esterase isozymes o f DBM larvae 
showing the difference between fast- moving and slow moving esterase 
isozymes.
Polyacrilamide gel electrophoresis of DBM larvae showing higher
frequency of slow moving esterases in Mampong (gel A), than in a
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Figure 17:
Figure 19:
Figure 20:
Figure 22:
typical Accra-suburb site (Airport) (gel B).
Ethidium bromide stained 0.8% agarose gel electrophoresis o f  genomic 
DNA isolated from the DBM.
Polyacrylamide gel electrophoresis o f PCR products obtained from 
amplification of DBM DNA with specific primers 
Polyacrylamide gel electrophoresis o f PCR products obtained from 
amplification of DBM DNA with specific primers showing the 
difference between susceptible and resistant DBM larvae.
Brine shrimp mortality profile o f solid phase extracted fractions from 
Cabbage samples
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Table 1: 
Table 2: 
Table 3: 
Table 4: 
Table 5: 
Table 6:
Table 7: 
Table8:
Table 9:
Table 10: 
Table 11:
Insect pests associated with cabbage in Ghana (Source: Forsyth, 1966 
Fatal Pesticide poisoning in Ghana
Comparison of Records of Resistance to Pesticides in Arthropods 
Number o f respondents interviewed in each cabbage growing area 
Current insecticide use pattern in Accra suburbs and Mampong-Akuapem 
Previous insecticide use pattern in Accra suburbs and Mampong-Akuapem
Farmers’ Awareness about some agronomic practices and Insecticide 
residues.
The response o f Plutella xylostella (FI) generation o f field-collected 
Population to lethal concentrations o f active ingredients o f  the selected 
insecticides.
Carboxylesterase activity o f DBM populations and insecticide used in 
cabbage farms in Mampong Akuapem and Accra suburbs
Lethal concentrations of insecticide standards
Fractionated Insecticides from cabbage samples and estimated insecticide 
residue content
LIST OF TABLES
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LIST OF PLATES
Plate 1:
Plate 2:
Plate 3.
Plate 4:
Plate 5:
Sample collection site at Dzorwulu. A cabbage farm destroyed by 
Diamondback moth.
A metal net cage (50cm3) with metal board floor. Inside is a potted cabbage 
plant about six weeks old used for oviposition and feeding o f the hatching 
larvae
Potted cabbage plant showing diamondback moth 3rd to 4th instars larvae.
Leaf disk dip (leaf residue) bioassay set up. Plastic petri dish with insecticide 
treated cabbage leaf disk and ten early fourth instar DBM larvae introduced into 
the petri dish.
Larvae immersion bioassay set up. Plastic cup “chalice” (about 2.74cm depth, 
bottom and top radii are 3.42cm and 2cm respectively).
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Appendix 1: 
Appendix II: 
Appendix III: 
Appendix IV: 
Appendix V: 
Appendix VI:
Standard solutions
LIST OF APPENDICES
Questionnaire survey
Bioassays
Enzyme assays
Residues analysis
Statistical analysis
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ABSTRACT
Over dependence on insecticides for the control of diamondback moth (DBM), Plutella 
xylostella (L ) has resulted in development of resistant strains and health hazards due to toxic 
residues in cabbage. The present study was undertaken to evaluate resistance in DBM and to 
assess the contribution of residues of insecticides used for DBM control on resistance 
development. A preliminary survey conducted in Accra and Mampong-Akuapem, revealed 
inappropriate agronomic practices as the main cause o f resistance development and health 
hazards due to insecticide residues on cabbage. Three pyrethroids viz lambda-cyhalothnn 
(pawa), cypermethrin (cypercal) and deltamethrin (deltaplan), an organophosphate- 
chlorpyrifos (dursban) and a biopesticide- Bacillus thuringiensis (B.t.) (dipel) were selected 
for the study. Wild DBM pupae were sampled from three sites in Accra (Dzorwulu, Airport 
and Madina) and a site in Mampong-Akuapem. These populations were used to establish a 
DBM colony, which was reared on potted insecticide-free cabbage in a screen house. Early 
4th instar larvae were used for leaf residue bioassays for B.t. and larvae immersion for 
organophosphate and pyrethroids. Compared to the recommended dosage the L C 9 5  of 
dursban were 106, 74, 193, 114 fold in DBM populations from Airport, Madina, and 
Dzorwulu and Mampong respectively. Similarly, for Pawa the LC95 to the recommended 
dosage were 103, 77, 100, 58 fold for Airport, Madina, Dzorwulu and Mampong 
respectively. In contrast, only 3-fold tolerance to dipel was recorded in Airport and Madina, 
2 fold in Dzorwulu and 4 fold in the Mampong DBM populations The study revealed that 
most of the field-observed resistance among the conventional insecticides might be 
attributed to cross and multiple resistance. There was however lack o f cues for cross­
resistance between the conventional insecticides and B.t. Molecular studies of B.L resistance 
using a PCR based method and further resolution using polyacrylamide gel electrophoresis 
(PAGE) showed the B. t. resistant geue 10 nave multiple bands, including the diagnostic band,
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as compared with bands from the susceptible strains. When the wild larvae were tested for
carboxylesterase activities using the naphthyl acetate-diazo blue coupling reaction,
significantly higher activities of a- and )3- naphthyl esterases were recorded in the Mampong
DBM population than the Accra samples. Compared to previous findings, the mean activity
of a-naphthyl esterase had doubled, while an eight-fold increase was registered for
p-naphthyl esterase. Although this result has enormous implications for cabbage farming,
there was no relationship between resistance levels obtained by bioassay and activities o f a-
and/or p -  esterases. The involvement of other metabolic detoxification enzymes or
resistance mechanisms is suggested. Nonetheless, polyacrylamide gel electrophoretic
zymogram study using a-naphthyl acetate revealed presence of bands, which are associated
with higher resistance in DBM  The Mampong population showed higher frequencies of
such bands than the Accra populations. Residues of chlopyrifos-methyL, pirimiphos-methyl
and pyrethroids (Cypermethrin, lambda-cyhalothrin and deltamethrin) were estimated on
cabbage samples using biotoxicity to brine shrimp nauplii after fractionation o f the
insecticide residues using solid phase extractor (SPE). The residue levels o f chlopyrifos were
found to be higher than the FAO/WHO recommended maximum residue level (MRL).
However, residues for pyrethroids could not be detected for most o f the study sites due to
low brine shrimp mortalities, except for Dzorwulu and Airport B. Compared to earlier
findings the residue levels recorded were not only above the WHO/FAO recommended
MRL but a 12-18 fold increase was also recorded in residue levels of pyrethroids. Results
from this study have shown that the insecticide residue problem for cabbage should be taken
seriously in Ghana. Farmers need to be educated on proper use and handling o f insecticides
in order not to compromise human health. The study suggests that a more integrated
approach using IPM principles, careful and selective use o f conventional insecticides
coupled with judicious use of B. t. could help reduce insect pests and the associated problems 
on cabbage.
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CHAPTER ONE 
GENERAL INTRODUCTION
1.1 Introduction
Cabbage and other Brassicas are a diverse group of crops of European origin 
(Purseglove, 1969). They are cultivated from the arctic to sub -  tropics, and at higher 
altitudes in the tropics as well as in the tropical lowlands on the West African coast (Hill, 
1983) certain species or varieties are more adapted to the tropics than others (Ooi, 1986).
In Ghana, the cultivation and consumption of cabbage has been on the increase especially 
in the urban areas where there is high demand for the vegetable. This high demand, 
which has led to vegetable growers resorting to monoculture and intensive cultivation has 
in turn altered the natural ecosystem in such a way that an insect such as Diamondback 
moth (DBM), once part of the background fauna, has become a major pest (Kumar, 
1986).
The diamondback moth (DBM) Plutella xylostella (Linnaeus) is an important pest of
Brassicae plants worldwide. It is implicated as notorious and cosmopolitan pest
(Brempong -Yeboah, 1992 and Talekar and Shelton, 1993). This insect is believed to be
the most universally distributed of all Lepidopterans. Out of the 191 countries
worldwide, DBM is listed to be present in 145 countries scattered over all possible
climatic and ecogeographical zones, implying that it has a high propensity to adapt to
variety of ecological habitats (Country Watch, 2002). It has been reported to attack both
cultivated as well as many wild Brassica plants (Harcourt, 1986). The host range of DBM
is limited to crucifers that contain mustard oils and their glucosides (Thorsteinson, 1953,
1955; Gupta & Thorsteinson 1960; Nayar & Thorsteinson, 1963; Hillyer & Thorsteinson, 
1971).
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In recent years, DBM has become the most destructive insect-pest of cruciferous plants 
throughout the world, and the annual cost of managing it is estimated to be $1 billion 
(Talekar, 1992). The pest is able to attack the crop from the nursery stage causing about 
52 % loss in marketable yield in cabbage by rendering the vegetable unattractive, 
obnoxious, and therefore unmarketable.
There are various DBM control approaches such as cultural, biological, host-plant 
resistance, sex pheromone and chemical methods among others. Among all the control 
methods, chemical crop protection is the most effective and convincing, optimizing crop 
yields and improving quality of farm produce (GTZ, 1979).
In recent times, protected cabbage production, mainly by use of insecticides has been 
stepped up considerably worldwide. Brempong-Yeboah (1992) observed that cabbage 
growers in Accra plains used unnecessarily large quantities of insecticides, and in various 
concoctions against DBM. Some of the insecticides documented to have been used in 
Ghana include; Permethrin, Deltamethrin, Decis, Biobit & Dipel (.Bacillus thuringiensis), 
and Dursban (chlorpyrifos). Others are Karate (Lamdacyhalothrin), Perfeckthion 
(Dimethoate), Ripcord and Actellic (Pirimiphos-methyl) [Mawuenyegah, 1994],
Large scale and indiscriminate use of insecticides for the control of this pest, necessitated
by the ever increasing demand for quality food and better public health has resulted in a
number of problems (Joia et al., 2004) such as insecticide resistance and health hazards
due to toxic residues that may persist in or on food after their application in amounts
above prescribed maximum residue limit (MRL) [Oudejans, 1991]. The latter problem
becomes more acute if the xenobiotics are used close to harvest as well as during transit
and in vegetable yards (Kumari et al., 2002). The residues also have hazardous effects on
both the ecosystems and the environment in general including the atmosphere, soil and
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water bodies (Carson, 1962). The toxic effects are more apparent in vegetables since they 
are mostly consumed fresh.
Insecticide resistance in DBM has occurred in many parts of the world since Ankersmit 
(1953) first reported its resistance to DDT in Indonesia. Resistance has been reported in 
Hawaii (Tabashnik et al., 1987), Japan and Australia (Kao et a l, 1989). However 
insecticide resistance may not be limited to these areas only (Kao et al., 1989). 
Diamondback moth is notorious for resistance to a wide range of insecticides (Syed et 
al., 1989; Syed, 1992). These cover all major groups of insecticides, chlorinated 
hydrocarbons, organophosphorus insecticides, carbamates, synthetic pyrethroids (Sun et 
al., 1986; Lin, 1988) and even some bacterial pesticide, that is, Bacillus thuringiensis 
(B.t.) based products (Tabashnik et al., 1990; Hama, 1992; Kfir 1997; Wright et al. 
1997), as well as insect growth regulators (Yuxian, 2001).
As hazards of conventional, broad-acting pesticides are documented, researchers look for 
pesticides that are toxic only to the target pest, have low toxicity to non-target organisms 
including beneficial insects (Edelson et al., 1993; Liu, 1999; Liu and Sparks, 1999), and 
fewer environmental hazards. One of these pesticides is the soil bacterium Bacillus 
thuringiensis Berliner which has demonstrated its potential as effective and 
environmentally safe alternative to synthetic chemical insecticides for lepidopterous 
pests (Wilding, 1986) thus fulfilling the requisites of a microbiological control agent 
against agricultural pests and vectors of diseases (Aronson, 1994; Kumar et al., 1996). 
However the use of genetically engineered crops to produce the B.t. toxin may greatly 
expand its use, and speed up the development of higher level of resistance.
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Lepidopteran larvae being the world’s most damaging crop pest are, the primary target of 
B.t. producing transgenic plants (McGaughey and Whalon, 1992). With many of such 
plants grown yearly, other insects are likely to develop resistance quickly unless effective 
countermeasures are designed and implemented (Mellon and Rissler, 1998). This calls 
for a better understanding of genetic basis of resistance to develop such countermeasures.
Resistance mechanisms in DBM proposed for synthetic chemicals include decreased 
penetration (Noppun et al., 1987), enhanced detoxification by esterases (Maa and 
Chuang. 1983), Glutathione-S-transferases and reduced sensitivity of 
acetylcholinesterase (Wu, 1983; Hama, 1987). However, resistance to pyrethroids can be 
attributed to an inherited or induced mixed-function oxidase complex (Hama, 1987). 
Esterases in general have been noted to play significant roles in resistance to insecticides 
particularly organophosphates (Owusu et al., 1996)
1.2 Justification
The repeated and indiscriminate use of pesticides in crop protection has created problems 
such as human health hazard due to toxic residues that persist in/on food after their 
application and environmental pollution among others. Although consumer awareness 
and legislation on pesticide residues in food are not that high in Ghana, pesticide 
management specialists are very concerned about the risks posed by pesticide residues in 
food to the Ghanaian public. This concern, specifically in respect to cabbage 
wholesomeness, is due to suspected indiscriminate use of insecticides on the crop.
Despite extensive use of pyrethroids in agriculture, comparable studies on DBM
resistance to chemical pesticides are lacking (Beeman and Schmidt, 1982). This is
because when new and more potent insecticides replace those that have become less
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effective, laboratory tests to confirm the existence of resistance are not always 
performed. The increasing trend of insecticide resistance in insect pest of Ghanaian 
vegetables has led to scientists calling for the establishment of a national pesticide 
resistance-monitoring network (Owusu, 1997). Therefore, susceptibility study and 
identification of the biochemical and genetic mechanisms of resitance development by 
DBM and their correlation to insecticide residue levels in cabbage crop is a necessary 
first step to provide baseline data for the success of any insecticide resistance-monitoring 
programme. This information can be used for the establishment of a more effective 
resistance management scheme.
Apart from resistance development, the success of conventional pesticides is also 
threatened by increasing awareness of their toxicity to natural enemies (Johnson and 
Tabashnik, 1991) and their harmful effects to the environment. Microbial insecticides are 
a promising alternative. The most widely used microbial insecticide, B.t., is highly toxic 
to certain pests, yet it has little or no adverse effect on most non-target organisms, 
including humans (Wilcox et al., 1986). Resistance of DBM to conventional insecticides 
has been documented in Ghana (Brempong-Yeboah, 1992). Kaiwa (2000) also reported 
high activity of carboxylesterase as an indicator of insecticide resistance in populations 
of DBM in Ghana, yet B.t. is considered to be highly effective against DBM that are 
resistant to conventional insecticides (Sun et al., 1986). At the same time, evidence of 
B.t. control failures has been documented in Florida (Shelton & Wyman 1992), 
Philippines (Kirsch & Schmutterer, 1988) and Malaysia (Syed et al., 1990). Therefore 
there was the need to establish a baseline data for susceptibility status of DBM 
population in Accra suburbs (Ghana) to B.t. alongside the conventional insecticides 
should DBM show increased resistance to the conventional insecticides, since an
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insecticide use pattern survey conducted in 1997 proved B.t. to be the most highly used 
insecticide by the cabbage growers in Accra suburbs (Ninsin, 1997).
1.3. OBJECTIVES
1.3.1 Main objective
The main objective of this study was to determine the level of DBM resistance to some 
commonly used insecticides, evaluate some of the resistance mechanisms and correlate 
these with insecticide residue levels in cabbage.
1.3.2 Specific objectives
To achieve the main objective the following specific objectives were considered.
i) To conduct a preliminary survey to determine the insecticide use patterns on 
cabbage in Accra-suburbs and Mampong-Akuapem.
ii) To determine and compare the level of insecticide resistance in P. xylostella 
to formulations of Lambda-cyhalothrin, Deltamethrin, Cypermethrin, 
Chloipyrifos and Bacillus thuringiensis using bioassay.
iii) To determine the levels of non-specific carboxylesterases as a measure of 
resistance levels in DBM.
iv) To use polyacrylamide gel electrophoresis to establish qualitative differences 
in carboxylesterases isozymes in DBM.
v) To evaluate B.t. resistance mechanism of DBM using polymerase chain 
reaction (PCR) -based method.
vi) To determine levels and types of insecticide residues using Brine shrimp 
bioassay and compare the residue levels detected on cabbage to the 
FAO/WHO MRL.
vii)To correlate the level of resistance in DBM with residue levels in cabbage.
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CHAPTER TWO 
LITERATURE REVIEW
2.1 THE CABBAGE PLANT
Cabbage, Brassica oleracea var. capitata L. (Brassiceae) is an important global 
vegetable (Rice et a l, 1993). It is a biennial herb with a short thickened stem surrounded 
by a series of fleshy overlapping expanded leaves, which form a compact head; the 
edible part of the plant (Purseglove, 1969). The older leaves surround the younger, 
smaller, tender ones and the miniature stem. The head shape may be pointed or round 
and leaf colour and shape are variable.
2.1.1 Origin, distribution and taxonomy
The crop is of very ancient cultivation and has been grown in Europe since 2500 BC. It
was introduced into England by the Romans and is now grown throughout the world
(Purseglove, 1969). However, CPC (2001a) asserted that the wild cabbage (B. oleraceae
L. var. oleraceae) is indigenous to the Mediterranean region, South-West Europe and
Southern England UK, where it grows on sea cliffs. It was brought into cultivation about
5000 years ago and gave rise to numerous cultivated forms, varying widely in vegetative
morphology. Cabbages are cultivated from the arctic to the subtropics and at high
altitudes in the tropics as well as in the tropical lowlands on the West African coast (Hill,
1983). Cabbage (Brassica Oleracea var. Capitata) belongs to the family Brassiceae
(=Cruciferae) and the order Brassica (=capparidates). Members of the family cmciferae
occur in temperate and tropical climates and represent a diverse, widespread and
important plant group that include cabbage, broccoli, cauliflower, collards, rapeseed and
Chinese cabbage, the most important vegetable crop grown in China (Li, 1981).
The Brassica is an agriculturally diverse group of high value crops (Tsunoda, 1980).
Members of this plant group are cultivated for various edible parts, such as roots of
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radishes and turnips, stems of kohlrabi, leaves of cabbage and other leafy brassicas. The 
seeds of mustard and rape are consumed as fresh, cooked, or processed vegetables. 
Crucifers are grown in tropical and temperate climates and in a variety of cropping 
systems from back yard gardens to larger-scale fully mechanized farms. The cultivated 
Brassica group is generally considered to belong to one species Brassica oleraceae, 
including white, red, and savoy cabbage, cauliflower, broccoli, Brussels sprout, kohlrabi 
and different kinds of kale (CPC, 2001a). Although information on distribution or 
introduction of the crop to Ghana is not well documented, it is believed that the British 
introduced it into this country and the crop has been grown on small scale since 1940 
(Sinnadurai, 1992).
2.1.2 Agronomy
Cabbages like other brassicas are grown from the seed. Sowing is done in nurseries in the 
field. The land should be well drained and fertile. It should be well cultivated to achieve 
fine tilth for planting. Proper weed control is important for optimum yield. The crop 
responds well to organic manure and inorganic fertilizers, particularly nitrogen (CPC, 
2001a).
Although cabbage production in Ghana has been on a steady increase over the last 
decade, there are certain constraints to attaining optimum yield and profit (Brempong- 
Yeboah, 1992). These constrains include shortage of land, clean water, marketing 
problems and insect pest infestation, which happens to be a global problem (Miyata et 
a l, 1986; Cartwright et a l, 1987; Hill and Waller, 1994). Insect pest infestation has 
arisen from monoculture and intensive cabbage production in peri-urban areas. 
Monoculture has created suitable conditions favourable for specialized insect species to 
flourish and become notorious pests (Kumar, 1986).
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The insect pest problem has been augmented by the cultivation of other varieties of 
Brassica oleracaea such as Chinese cabbage and cauliflower on adjacent plots to 
cabbage farms, since as a group the varieties of B. oleraceae tend to have a similar insect 
pest spectrum (Hill and Waller, 1994). This practice of cultivating related crops together, 
according to Way (1976), bridges gaps in the host plant sequence of insect pests resulting 
in upsurges in pest infestation.
2.1.3 Pests
Like other brassicas, cabbage has a wide spectrum of pests, Hill (1983), listed 14 major 
pests of brassicas, of which Plutella xylostella is one, and a range of minor pests. The 
crop protection compendium (CPC, 2001a) listed 57 major pests and 28 minor ones 
including pathogens for brassicas and also listed DBM as one of the major pests of 
brassicas.
Hill and Waller (1994) mentioned the cabbage aphid, Brevicoryne brassicae (L.) 
(Homoptera; Aphididae) and leaf-eating caterpillars, which include the DBM, as the 
insect pests that cause the most damage to the Brassica spp. in the tropics. Forsyth 
(1966) recorded several insects on the cabbage plant (Table 1.) His findings have been 
confirmed by other workers (PPRSD, 2000).
The cabbage aphid transmits turnip mosaic virus and several other viruses specific to the 
cruciferous crops (Hill and Waller, 1994). The leaf-eating caterpillar also disfigures the 
cabbage plant and may completely defoliate it.
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Table 1. Insect pests associated with cabbage in Ghana (Source: Forsyth, 1966)
Name Order and Family
Allogista serricome (Kibe) Coleoptera: Alleculidae
Lagria villosa (F.) Coleoptera: Lagriidae
Melanagromyza Iambi (Hend) Diptera: Agromyzidae
Diacrisia investigatorum (Karosh) Lepidoptera: Arctiidae
Plusia signata (F.) Lepidoptera: Noctuidae
Amauris psyttalea (Plotz.) Lepidoptera: Nymphalidae
Spodoptera littoralis (F.) Lepidoptera: Noctuidae
Appias epaphia (Cram) Lepidoptera: Pieridae
Crocidolomia binotalis (Zeu) Lepidoptera: Pyralidae
Hellula undalis (F.) Lepidoptera: Pyralidae
Hymenia recutyalis (F.) Lepidoptera: Pyralidae
Gymnognylus lucen (Wik.) Orthoptera: Gryllidae
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Thus, these insects acting in concert can devastate many cabbage plants over a very short 
time if growers do not institute control measures (Kaiwa, 2000). Diamondback moth has 
become the key pest of cabbage in Ghana (Brempong-Yeboah, 1992)
2.2 THE DIAMONDBACK MOTH
2.2.1 Description
The diamondback moth, Plutella xylostella Linnaeus (Lepidoptera: Yponomeutidae), is a 
small moth of about 1/3 inch (6mm) long, grey or brownish in colour, with pronounced 
antennae and a wingspan of about % of an inch. It is recognized by the three pale or 
yellow triangular markings along the inner margin of each of the forewings (Sorensen, 
1996). This is more pronounced in the males. When at rest, the closed /folded wings 
present an image of light coloured diamond shapes along the mid-dorsal line of the wings 
where they meet, which gives the moth its common name (Fig. 1). When viewed from 
the side, the tips of the wings can be seen to turn upward slightly (Talekar and Shelton, 
1993; Sorensen, 1996; Hutchison et al. 2004). The eggs are small measuring about 0.44 x 
0.26 mm and are spherical, yellowish to white (Sorensen, 1996). The larvae vary in 
coloration from light brown at hatching through pale to dark green when fully grown. 
The larval body is wider in the middle than both ends and slightly tapering at both ends 
with prolegs on the last segment forming a distinct V-shape at the rear end (Suterra, 
2004). The body is covered with tiny, erect black hairs. The larva wiggles rapidly when 
disturbed, and spins down from the plant on a strand of silk and drop over the edge. It 
climbs back on the leaf once the danger is gone (Talekar and Shelton, 1993). This 
behaviour is believed to be a parasitoid evasion response but can result in contamination 
of leaf surfaces with pathogens if they exist in the soil reservoir (Capinera, 2001) this 
behaviour distinguish them from other cabbage-infesting species.
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Pronounced antenna
Three pale o r yellow 
triangular markings 
presents an image o f 
diamond shapes.
Fig 1. Adult diamondback moth (Credit: James Castner, University o f  Florida)
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Pupation takes place in a net-like silken cocoon about 9-12 mm long. The new pupae 
appear greenish and are normally found on the underside of the leaves along the ridges of 
the leaf veins (Hill, 1983) or in soil debris (Hutchison et al., 2004).
2.2.2. Origin and distribution of DBM
The diamondback moth is probably of Mediterranean region (European) origin, but is 
now found throughout the North Americas, southern portion of South America and in 
Europe, India, Southeast Asia, Australia, and New Zealand (Hardy, 1938). It is reported 
to be highly dispersive (Capinera, 2001). Although DBM had been considered to have an 
European origin, (Hill, 1983; Talekar et al., 1990; Talekar and Shelton, 1993), on the 
basis of the large complex and sexual forms of parasitoids and host plants of DBM found 
in South Africa, a recent report by Kfir (1998) suggested that DBM may have originated 
from South Africa and dispersed to Europe (Palaearctic Region) and not vice versa. 
However, other reports indicate that the parasitoid complex of DBM is even more diverse 
in Eastern Europe and so that may be the origin (Talekar and Shelton, 1993). 
Furthermore, DBM feeds exclusively on Brassicas, which are of Mediterranean origin; 
there should be no doubt that its origin is Europe and not South Africa. Li (1981) 
asserted that, DBM has a very high propensity to adapt to a wide variety of ecological 
habitats.
2.2.3 Biology and Ecology
2.2.3.1 Life cycle
The DBM goes through four separate and distinct stages of its life cycle; egg, larva, 
pupa, and adult (Fig. 2).
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Eggs
Adult
Larva
• • v - .  ‘
Pupa
Fig. 2 Life cycle o f diamondback moth showing the four stages (Credit: James Castner, 
University o f Florida)
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Eggs: After mating, the female deposits small (0.44x 0.26mm), eggs singly or in small 
groups of 2-8 eggs in a depression on the surface of foliage or occasionally on the stem 
and leaf-petiole (Capinera, 2001). Females lay between 250 -300 eggs, but an average is 
about 150 eggs (Capinera, 2001).
Oviposition is influenced by secondary chemicals, temperature, trichomes, and waxes on 
leaf surface (Uematsu and Sakanoshita, 1989; Pivnick, 1990). In addition, lack of light 
during normal daylight stimulates oviposition, but light during night hours does not 
completely inhibit it (Sorenson, 1996). Incubation lasts 5 to 6 days averagely ranging 
from 3 days at high temperatures (Ooi and Keldesman, 1979) to 8 days at low 
temperature (Harcourt, 1957). Just before hatching, they darken and young larvae can be 
seen coiled beneath the chorion or egg -shell (Harcourt, 1957).
Larvae: After emergence, neonate larvae crawl to the underside of the leaf and then 
mine the spongy mesophyll tissue thereby forming a gallery (Hill, 1983; Ooi, 1986) 
whereas older larvae feed by chewing the lower leaf surface resulting in irregular 
patches. Larvae usually consume all tissue except the wax layer on the upper surface and 
the veins thus creating the characteristic “windowing”, (Bhalla and Dubey, 1986; Salinas, 
1986; Lu et al., 1988; Samthoy et al., 1989). The DBM has four larval instars. The heads 
of the first and second larval stages are black in colour and distinct from the green to 
brown coloured heads of the 3rd and 4th instars (Mau and Kessing, 2004). Overall length 
of each instar rarely exceeds 1.7, 3.5, 7.0 and 11.2 mm respectively (Capinera, 2001). 
Average duration of the larval instars is about 4.5 (3-7), 4 (2-8), and 5 (2-10) days 
respectively (Capinera, 2001). The fully grown fourth instar larvae pupate within a 
woven silk cocoon fasten^ tCL-tlje veins on the under surfaces of leaves (Hill, 1983)
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Pupae: The fully-grown larva constructs an open network cocoon where it feeds and 
spends a two-day period of quiescence marking the pre-pupal stage. The pre-pupa sheds 
its larval skin, which remains attached to the caudal end of the green pupa, which is 
encased in a delicate, netlike cocoon on the under surfaces of leaves or in other protected 
areas on the plant or in soil debris (Hutchison et al., 2004; Mau and kessing, 2004). The 
pupal period varies from 4 to 15 days, (averaging at about 8.5 days) depending on 
temperature (Chelliah and Srinivasan, 1986; Hoy, 1988; Capinera, 2001), with optimum 
development temperature being at 27.5°C and the threshold temperature at 9.8°C 
(Yamada and Kawasaki, 1983). The yellowish pupa is 7 to 9 mm in length (Capinera, 
2001). Adult moths emerge primarily between 1:00 and 4:00 pm with a peak at 2:00 pm 
(Sakanoishita and Yanagita, 1972; Pivnick et al., 1990)
Adult: Most adults emerge during the first 8 hours of photo phase (Pivnick et al, 1990). 
They feed on water droplets / dew or nectar from wild flowers and have a 2-3 weeks life 
span. Adult males live from 3-58 days averaging 12.1 days and females live from 7-47 
days, an average of 16.2 days (Capinera, 2001; Mau and kessing (2004). The moth is 
nocturnal, active at dusk and continues so into the night (Harcourt, 1954). Mating occurs 
at dusk of the same day the adults emerge. Female moths start laying eggs soon after 
mating and the oviposition period lasts between 4-10 days. The majority of the eggs are 
laid before midnight with peak oviposition occurring between 7- 8 pm. of the first night 
of oviposition (AVRDC, 1987 and Pivnick et al., 1990). Photoperiod, temperature, age, 
larval foods significantly affect adult survival, oviposition rates and lifetime fecundity 
(Harcourt, 1957; Sivapragasam and Heong, 1984). The moths are weak fliers, usually 
flying within 2m off the ground, and not flying long distances (Capinera, 2001).
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2.2.3.2 Host range and host specificity
The diamondback moth feeds only on members of the family Cruciferea. Virtually all 
Cruferous vegetable crops are eaten, including cabbage (Brassica oleracea var. capitata), 
cauliflower (B. oleracea var. botrytis), broccoli (B. oleracea var. italica), radish 
(Raphanus sativus), turnip (B. rapa pekinesis), brussels sprouts (B. oleracea var.
gemmifera) Chinese cabbage (B. rapa cv. gr. pekinensis), kohlrabi (B. oleracea var.
gongylodes), mustard (B . juncea), rapeseed (B. napus), collard (B. oleracea var.
acephala) kale (B. oleracea) and watercress (Nasturtium officinale). Not all are equally
preferred, however, and ovipositing moths relative to cabbage will usually choose collard 
(Talekar and Shelton, 1993). In addition, DBM feeds on numerous cruciferous plants that 
are considered to be weeds. It maintains itself in the absence of more favoured cultivated 
hosts. The following crucifers have been reported to sustain feeding and reproduction of 
DBM: Arabis glabra, Armoracia lapathifolia, Barbarea stricta, Barbarea vulgaris and 
Basela alba. Others include Beta vulgaris, Brassica caullorapha, Galinsoga ciliate, G. 
parviflora, Sinapis alba, Brassica napobnrassica etc. Apart from both cultivated and 
wild plants of family Cruciferae, several ornamentals, such as wallflower, candytuff, 
stocks, and alyssum are host plants of DBM (Mau and Kessing, 2004). Alternate weed 
hosts are especially important to maintain DBM population in temperature countries in 
spring (early in the season) before cruciferous crops are planted (Louda, 1986). The host 
range of DBM is limited to crucifers that contain mustard oils and their glucosides 
(Nayar and Thorsteinson, 1963; Hillyer & Thorsteinson, 1971), recently a group of 
ICIPE scientists have reported growth, development and survival of DBM on snow peas 
(Loehr et al, 2002).
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2.2.3.3 Effects of environmental factors on DBM
Various environmental factors influence the biology and therefore abundance and 
occurrence of the DBM. For example rainfall is specifically important in the tropics. 
Although the pest breeds all year round in the tropics, it is reported to be most abundant 
during cool or warm and dry seasons (Talekar and Lee, 1985; Talekar and Shelton, 
1993). The weather during the egg laying period influences DBM abundance; cool, 
cloudy weather reduces moth flight activity. Heavy rain can disrupt mating and egg- 
laying. If inclement weather persists, many female moths die before egg- laying is 
completed (Moller, 1988). Heavy rains appear to be detrimental to infestation (Talekar 
and Lee, 1985) and are reported to be a major mortality factor for this pest since young 
larvae are easily dislodged from plants by rain and can drown on the soil surface or in 
water trapped on the plants, or are washed off the leaves (Harcourt, 1963; Capinera, 
2001). During rainy weather and high humidity more than half of the first three larval 
stages may perish by drowning (Waterhouse, 1987). Harcourt (1957) reported an average 
mortality of 56%. Hence it is not surprising that cruciferous crops with overhead sprinkle 
irrigation tend to have fewer DBM larvae than drip or furrow- irrigated crops. Best 
results were obtained with daily evening application of sprinkle irrigation (Capinera, 
2001).
2.2.3.4 Pest damage and economic importance
Plutella xylostella causes considerable damage to leaves, stems, growing point’s 
inflorescence and fruits/pods (CPC, 2001b). Larval feeding mainly causes the damage. 
Initial damage results in small incomplete holes caused by young larvae and larger 
complete holes caused by mature larvae (Hurchison et al., 2004). The entire plant may 
become riddled with holes under moderate to heavy populations. It attacks the growing 
tips of young plants and is particularly damaging to seedlings, since it can arrest
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development and disrupt head formation causing headless plants or multiple undersized 
heads (Suterra, 2004). Sometimes, it causes formation of deformed heads and also 
encourages soft rots in cabbage, broccoli, and cauliflower. In case of severe infestation 
the entire plant could be lost (Capinera, 2001). Injury to leaves is not usually serious, 
except when the wrapper or cap leaves of cabbage are perforated /injured, down grading 
the quality and thus the value of the harvested crop (Suterra, 2004).
The pest causes serious economic losses. Even though proper economic impact o f the 
pest is difficult to asses especially in Africa, since it occurs in diverse areas in large and 
small scale farms, and there seems to be less reliable data on the value of crucifers and 
losses incurred due to DBM. Besides, accurate data on the cost of control is rare due to 
the large volumes of insecticides used to control it, their variable costs, variable number 
of applications and efficacy (Capinera, 2001). DBM has developed resistance to most 
insecticides (Talekar and Shelton, 1993). The high pest status of DBM can be attributed 
to several factors mainly: high fecundity and reproductive potential; this results in rapid 
turnover of generation (Talekar and Shelton, 1993). This is further enhanced by the 
pest’s ability to migrate over long distances, absence of effective natural enemy, and its 
ability to establish faster in newly planted crucifers than its natural enemy complex (Lim, 
1986). Effective control by natural enemy complex is further weakened by over 
dependence on chemical pesticides and high ability of DBM to develop resistance to all 
forms of pesticides used against it (Talekar et al. 1990).
2.3 MANAGEMENT OF CABBAGE
Cabbage is grown both on large scale and small-scale farming and in both cases DBM 
has been the key pest and the main constraint in cabbage production.
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2.3.1 Small-scale farming
In developing countries of the tropics and sub-tropics, production of crucifers is 
characterised by small-scale farms and intensive use of land, labour and pesticides. For 
fresh vegetable production, for the large cities residents, farms are located on the 
outskirts of city centres or cleared areas in the highlands accessible from the cities. 
Production of healthy looking, damage free vegetables for the wealthy city dwellers is 
important consideration in cultivation, especially plant protection. The mainstay of 
control is frequent use of insecticides (Talekar and Shelton, 1993). This sole reliance on 
insecticide for control facilitates the rapid build up of resistance. Nevertheless the first 
report on DBM resistance to an insecticide in 1953 came from one intensive production 
area in the tropics, Indonesia (Ankersmit, 1953; Johnson, 1953) decades before resistance 
in warm areas of United States (Magaro and Eldelson, 1990; Shelton and Wynam, 1992). 
To overcome resistance, fanners often increase doses of insecticides, sometimes every 
two days. These high levels of use have caused the DBM to become resistant to 
practically all insecticides in many areas. Additionally, high insecticide use, has led to 
excessive residue on produce. Moreover because pesticide residue monitoring is absent 
or not enforced, insecticide contaminated crucifers often pass easily through marketing 
channels.
2.3.2 Large Scale Farming
In developed countries, crucifers’ production is characterized by large-scale fanning, 
which includes the reduction of labour, the increase of management and capital, and the 
consolidation of land into larger holdings. Large-scale farming is common in North 
America and Europe, and is becoming increasingly common in Mexico and Central 
America. However crop protection decisions tend to be similar over relatively large
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areas. The primary control method of DBM here involves insecticides applied by air or 
ground rigs (Hoy et al., 1983, Cartwright et al., 1987, Beck, 1992).
2.4 CONTROL
Several approaches have been used to control DBM infestation in brassicas. The most 
popular control approaches include:
2.4.1 Cultural control
Prior to introduction of synthetic insecticides in the late 1940s, DBM were not reported 
as major pests on crucifers because they were relatively well managed by the natural 
enemies and cultural methods. Due to the failure of insecticides to control the DBM, 
interest is growing in the use of cultural practices in commercial crucifer production. 
Some of the classical control measures that have been tried with some success are 
intercropping, use of sprinkler irrigation trap cropping, crop rotation and clean 
cultivation (Talekar and Shelton, 1993).
2.4.2 Biological Control
The use of living organisms, such as parasitoids has also been exploited in controlling
DBM. Numerous parasitoids attack all stages of DBM. Although over 90 parasitoids
species attack DBM, only about 60 of them appear to be important (Godwin, 1979).
Among these, 6 species attack the eggs, 38 attack larvae and 13 attack pupae (Lim,
1986). Egg parasitoids belonging to genera Trichogramma and Trichogrammatoidea
require mass release to be effective. The most predominant and effective larval
parastioids include Diadegma insulare (Cresson) Hymenoptera: Ichneumonidae (Suterra,
2004) and Diadromus subtilicornis (Gravenhorst) (Hymenoptera: Ichneumonidae, and
Cotesia (Apanteles) plutellae, which attacks the three larval stages (Mau and kessing,
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2004). This approach has considerable promise, although widespread, indiscriminate use 
of insecticides has frustrated recent efforts, and delayed the establishment of parasitoids, 
and their beneficial effect (Talekar & Shelton, 1993). Along side parasitoids, fungi and 
granulosis virus sometimes occur in high-density in DBM larval populations (Capinera, 
2001).
2.4.3 Plant resistance
Several studies have surveyed existing germplasm for resistance to Lepidoptera, 
including DBM, in crucifers. Crucifers differ in their susceptibility to attack by DBM. 
Mustard, turnip, and Kohlrabi are among the more resistant crucifers (Eigenbrode et al., 
1990). The most notable resistance came from germplasm in United States North Eastern 
Plant Introduction Station (Eigenbrode et al., 1990)).
2.4.4 Sex pheromone
The use of DBM sex pheromone has also been exploited, particularly when used in 
combination with the augmentation or conservation of natural enemies (CPC, 2001b).
2.4.5 Insect-growth regulators
Insect-growth regulators and pathogens offer a promising control measure as alternatives 
to broad-spectrum insecticides, which often disrupt the control exerted by natural 
enemies (Kobayashi et al., 1992).
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2.4.6 Bacillus thuringiensis
Biologicals like B. thuringiensis have since their introduction provided fairly good 
control. The use of B.t. is considered important since it favours survival of parasitoids. 
B. thuringiensis contains crystal proteins which are toxic only to certain insects, but are 
harmless to non-target organisms including people, wildlife, and most beneficial insects 
(Schnepf et al., 1998). Genes encoding B.t. toxins have been incorporated into and 
expressed by crop plants, thus providing environmentally benign control of insect pests 
(Schnepf et al., 1998).
2.4.6.1 Mode of Action
When conditions are unfavourable for bacterial growth Bt. forms a spore (the dormant 
stage of the bacterial life cycle) and it also creates the toxic protein crystal. After the 
insect ingests B.t. during feeding, the crystal is dissolved in the alkaline gut of the insect. 
The insect's digestive enzymes then break down the crystal structure and activate the 
insecticidal component, called delta-endotoxin, which binds to the cells lining the midgut 
membrane and create pores in the membrane, upsetting the ionic balance in the gut. The 
insect soon stops feeding and starves to death (Swadener, 2004).
2.4.6.2 Resistance to Bacillus thuringiensis
Bacillus thuringiensis offered tremendous hope for the control of DBM in the first two 
decades of its introduction. Due to its complex mode of action, and lack of documented 
cases of resistance from field population (Devriendt and Martouret 1976; Krieg and 
Langebruch, 1981), scientists presumed that DBM was unlikely to develop resistance to 
B.t. under field conditions (Talekar and Griggs, 1986). However, eight insect species 
including the Indian meal moth which was the first insect to develop resistance to B.t. in 
the laboratory ((McGaughey, 1990; McGaughey and Whalon, 1992) and others include
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gypsy moth (Rossiter et al., 1990). Moreover DBM and tobacco budworm, have 
exhibited multiple resistance to B.t. strains (Ellis, 1991; Gould, 1992). Evolution of 
resistance has therefore become the most serious threat to the continued efficacy of B.t. 
toxins. Although DBM is the only insect with resistance to B.t. in open-field populations 
(Tabashnik, 1994), however the wide spread use of B.t. toxin-producing transgenic 
plants, may hasten evolution of resistance by other pests (McGaughey and Whalon, 
1992; Mellon and Rissler 1998). Since genetic analysis indicated that resistance to B.t. 
was autosomal, recessive and controlled primarily by one or few loci, a better 
understanding of the genetic basis of resistance is essential for developing good 
countermeasures to reduce further resistance to this important biopesticide.
2.4.6.3 Molecular studies of B.t. resistant gene
Extended Polymerase Chain Reaction (PCR) methodology has recently been exploited to 
rapidly identify the B.t. resistant gene. It requires minute amounts of DNA and allows 
quick, simultaneous screening of many samples for faster identification and classification 
(Kumar et al, 1996).
Principles of Polymerases Chain Reaction (PCR)
The PCR is an in vitro method used for the enzymatic synthesis of speS^^DNA  
sequences, using two oligonucleotide primers which hybridize to opposite strands and 
flank the region of interest in the DNA (Saiki et al, 1985). The reaction uses the DNA 
polymerase enzyme and its many repetitive series of cycles results in accumulation of 
large amounts of the target DNA. The utilization of a heat stable polymerase isolated 
from the thermophilic bacteria Thermus aquaticus commonly denoted as Taq polymerase 
has transformed PCR into a simple, robust reaction, which is now automated using a 
programmable thermal cycling device (Saiki et a l 1985). The reaction mixture contains
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the DNA template, oligonucleotide primers, Taq polymerase, deoxynucleotides, and 
reaction buffer and the amplification reaction is done by cycling the temperature withm 
the reaction tube (Saiki et al., 1988). Precisely, the PCR reaction involves three main 
steps of repeated cycles: heat denaturation to separate the two strands of DNA, primer 
annealing to their complementary sequence at lower temperature and extension of the 
new strands determined from the 5' — 3' end by DNA polymerases, with the base 
sequence of the new strands determined by the DNA template. The reaction mixture is 
repeatedly heated and cooled until the desired amount of DNA template is amplified.
2.4.7 Chemical control of DBM
The use of chemicals has become the mainstay of DBM control.
2.4.7.1 Chemical insecticide use patterns
Because DBM larvae feed on cruciferous vegetables, which are highly valued, effective 
crop protection is necessary. This is because the production of healthy-looking damage- 
free vegetable for the relatively wealthy city dwellers is an important consideration for 
better economic gain from crucifer production (Talekar and Shelton, 1993). Historically, 
the mainstay of control has been the use of synthetic insecticides. Since farmers reason 
that so long as it is profitable, and no better alternative is available, the spraying of 
pesticides is a good investment (Hardy, 1995). Chemical control is effective, rapid in 
curative action, adaptable to most situations, flexible in meeting changing agronomic and 
ecological conditions and economical (Metcalf, 1975). There are majority of pest 
outbreaks for which chemical control remains the only method of choice (Hill, 1983). 
Protection of crucifer crops from damage often requires application of insecticide to plant 
foliage, sometimes as frequently as twice per week (Capinera, 2001). Farmers tend to 
increase their use of pesticides, despite their rising costs, in Senegal; farmers increased
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application frequency from 15 days to every 3 days in cabbage and this can be attributed 
to increased pest infestations levels; repeated ineffective pesticide treatments. This made 
farmers to suspect fraudulent use of poor quality insecticides and as a consequence 
anxiety to minimize damage and increase yields increased (Williamson et al., 2003).
The general use patterns of insecticides vary widely over geographic locations and 
decades. The driving forces behind these changing patterns are the development of new, 
more effective insecticides and the lost usefulness of older insecticides because of 
resistance. Factors that influence the development of resistance in DBM include high 
fecundity and reproduction potential, rapid generation turnover, long growing season, 
extensive acreage of crucifers, and frequent insecticide application (Yamada and 
Koshihara, 1978; Magaro and Edelson, 1990). DBM has a long history of becoming 
resistant to every insecticide used extensively against them. Ankersmit (1953) noted 
development of resistance to DDT in Lembang, Indonesia. In the Philippines, Barroga 
(1974) reported development of resistance by DBM, by confirming failures with EPN 
and Mevinphos.
2.4.7.2 Effects of chemical control
Indiscriminate or misuse of insecticides has hazardous effects on both the ecosystem and 
the environment in general. Carson (1962) highlighted the hazards and environmental 
consequences associated with the use of pesticides. Many researches and publications 
have since been carried out to support the concerns raised by this publication (Metcalf, 
1980). Hazards caused by the prolonged use of pesticides may affect human health, 
directly and indirectly through residues in food and other biotic systems.
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2.4.7.2.1 Effects on human health
Insecticides are chemicals designed to kill insects but most of the widely used 
insecticides are nerve poisons and general biocides with acute toxicity on a weight basis 
approaching equivalence between mammals and insects (Metcalf, 1980). Therefore, the 
insecticides are not only toxic to insects but also to humans (Oudejans, 1991).
Insecticide toxicity to humans can either be acute or chronic. Acute toxicity is an 
immediate poisonous effect of a single dose of a toxicant involving few target organs or 
systems (Hassal, 1990). Acute poisoning occurs when people come in direct contact 
with insecticides. This phenomenon in Africa is known as “the new developing world 
disease” (Anonymous, 1989). Most of the banned or heavily restricted pesticides 
produced in the developed world are sent to developing countries where the illiterate 
farmers hardly have knowledge of the associated hazards with the use of such chemicals 
(Atteh, 1987). From United States of America alone, out of 210,000 metric tons of 
pesticides produced, 53,000 are either banned or not recommended for use in the U.S.A. 
However, these chemicals find their way into markets of developing countries, which 
may have dire consequences on the users (Weir and Schapiro, 1981). Even though only 
20% of the total world pesticide consumption is in the developing countries, where 
majority of users are illiterate, about half of the poisoning cases and nearly three-quarters 
of the deaths are estimated to occur in the developing countries (Oudejans, 1991). FAO 
compiled a list on pesticide poisoning cases in Ghana as shown it Table 2.
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Table 2. Fatal Pesticide poisoning in Ghana (Souce, FAO, 1989)
Year No. of reported Remarks 
cases
1986 4 All staff of plant protection and Regulatory services
1987 9 All volunteers, one died at Navrongo in the Upper East
Region (Armyworm control)
1988 6 All farmers one died in the Volta Region (Armyworm
control)
1989 4 Two staff of Plant protection and Regulatory services the
others were farmers
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These accidents happened because the users had inadequate knowledge on the proper 
handling and use of pesticides and improper disposal of containers after use, or because 
the containers were used to store water or food. Acute poisoning may also result from 
the infection of food exposed to high concentrations of highly toxic chemicals or 
containing residues of such chemicals. Many deaths have occurred due to pesticides 
poisoning. The International Organization of Consumer Union puts the figure for 1986 
at 375,000 human poisoning cases in developing countries of which 10,000 died 
(Oudejans, 1991). At Kadjebi in the Volta Region of Ghana, five members of a 
household died after eating okra that was sprayed with an insecticide (Atsu, 1996), and as 
recent as July 2004, several people were admitted to hospital at Tarkwa after eating 
cabbage sprayed with excessive amounts of organophosphates (GNA, 2004). However 
reliable statistics on the true extent of human morbidity and mortality due to insecticide 
use is difficult to obtain, since most of these poisoning cases are not reported and are 
rarely subjected to laboratory verification (Davies, 1977).
Chronic toxicity on the other hand is associated with repeated and prolonged exposure to 
non-lethal doses of potentially harmful chemical (Yang, 1987). Known responses include 
lung cancer, brain damage and necrosis of the liver or kidneys. Others may cause 
chromosomal mutation or damage the foetus during pregnancy (tetratogenic effect) and 
inrmuno suppressive effects (Hassal, 1990; Mathews, 1994).
2.4.8 Pesticide Residues
Pesticide residues in food are remnants of a pesticide or its metabolites that can be found
in or on a crop after it has been used for pest control purposes. The small concentration
of these toxic residues that persist after application may have serious biological
consequences (Kumar, 1986). They may cause damage to the liver, heart and kidneys
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(Jackai, 1995). They may also be neurotoxic, teratogenic, mutagenic or carcinogenic. 
To evaluate chronic toxicity and hazard of pesticides to humans, an array of long term 
toxicity studies and hazard evaluation are carried out on smaller mammals like rats, 
rabbits or dogs. Extension Toxicology Network (EXTONET) [1993, 1995] compiled 
properties of chronic toxicity of some insecticides as follows:
Dimethoate: - it is a possible human tetratogen. It is also a mutagen and carcinogen. It 
causes organ toxicity. The testicles of male rats exposed to dimethoate decreased in size. 
The rats also developed chronic kidney problems.
Deltamethrin: suspected chronic exposure effects on man include choreogthretosis,
hypertension, abortion and shock.
Cypermethrin: is a possible human carcinogen. Long-term exposure that may cause 
pathological changes in the cortex of the thymus, liver, adrenal glands, lungs and skin 
observed in rabbits repeatedly fed on cypermethrin.
Bacillus thuringiensis: Suspected exposure to B.t. has caused respiratory, eye, and skin 
irritation, and one comeal ulcer after direct contact with its formulation. Also allergies to 
the “inert" (secret) ingredients. People with compromised immune systems may be 
particularly susceptible.
2.4.8.1 Effects of Insecticide Residues on Agriculture Systems
Insecticides disrupt the natural equilibrium between insects-pests, their parasites and 
predators. Natural enemies are said to be more susceptible to insecticides than the insect 
pests (Jackai, 1995). The resultant effect is the resurgence of the pest or new pest species 
whose populations were regulated by natural enemies. For example DBM was long 
considered a relatively insignificant pest before 1940s, but with the introduction of 
broad-spectrum synthetic insecticides, which destroyed the natural enemies, by 1970s it
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was reported as a major pest and by 1980s become a more serious pest of crucifers due to 
insecticide resistance and presently the most troublesome since it has resisted virtually all 
insecticides available in the modem markets (Talekar and Shelton, 1993; Capinera, 
2001).
Statistics of harm from pesticides show that each year 25 million people, from the 
Southern hemisphere, are poisoned through occupational exposure to pesticides; out of 
which 220, 000 die. Pesticides have also been linked to cancer: like higher concentrations 
of DDT have been found in the breast milk of mothers in Central America (Instant Essay, 
2004).
2.4.8.2 Pesticide Residue Legislation
Consumer awareness and concern about perceived risks that potential residues of 
pesticides may pose on human health is challenging the agro-industry worldwide 
especially the fruit and vegetable industry, to minimize pesticide residues in food. In 
order to protect the health of consumers, many countries have set maximum permissible 
levels for residues of particular pesticides in food. For purposes of trading foodstuffs on 
the world market, most developing countries rely on international standards, established 
jointly by (FAO) and (WHO) through Codex Alimentarius Commission, which have 
since 1961 set maximum residue limits (MRL) for pesticides in foods (Gbewonyo, 2004). 
They have also been concerned about the “maximum acceptable daily intake” (ADI) 
value, which is expressed in milligrams per kilogram body weight, and is defined as “that 
daily intake which during an entire life-time appear to be without appreciable risk on the 
basis of all known facts at the time” (FAO/WHO, 1993). In most developed countries, 
and some developing countries, strict legal controls have been enacted which lay down 
reasonable safe levels of pesticide residues in food. For instance, in 1979, the Federal
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Republic of Germany pesticide residues legislation covered more than 200 different 
ingredients (GTZ, 1979).
While in Denmark, the Danish National Food Institute continuously controls pesticide 
residues in Food by continuously analysing various food samples on the market (Hewleg, 
1991). The Indian Institute of Horticultural Research also published recommended 
pesticides, concentrations, terminal residues, and waiting periods for specific crops. 
While in USA, the first country to enact pesticide residue laws (GTZ, 1979) to ensure the 
safety of food supply by regulating pesticide residues through the cooperative effort of 
the United States Environmental Protection Agency (EPA) and the Food and Drug 
Administration (FDA) (Gal and Mathews, 1992).
Maximum Residue Limits (MRL) and ADI set by FAO/WHO and specific pesticide 
residue legislation of any country are based on Good Agricultural Practice (GAP). GAP 
emphasizes the official recommended usage of pesticides under practical conditions at 
any stage of production, storage, transportation, distribution, and processing of food, 
agricultural commodities and animal feed. It accounts for variations in different regions 
concerning minimum quantities necessary to achieve adequate control, yet leaving 
smallest residue, which is toxicologically acceptable (Oudejans, 1991). Therefore 
official recommended usage of pesticides need to comply with the procedures, including 
formulation, dosage rates, frequency of application and pre-harvest intervals (PHI) 
approved by the national authorities (Oudejans, 1991).
2.4.8.3 Violation of pesticide residue legislation
Cases of pesticide residues violation have been reported in both developing and 
developed countries alike. Elderkin et al. (1995) reported that; there are 2.5 billion
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pounds of pesticides being applied to agricultural products each year in the United States. 
This is ten times more than was applied 40 years ago. Moreover each year there are
10,000 pesticide related poisonings. For example, on July 4th 1985, over 300 Californians 
became sick after eating watermelons treated with the pesticide TENIK. Besides 44% of 
the foods (carrots, tomatoes and lettuce) tested in supermarkets were found to have some 
traces of pesticide residue on them (Elderkin et al., 1995).
The developing countries are the worst hit by this catastrophe. In Benin; cotton 
production is the largest consumer of insecticides, utilizing about 80 % of imported 
chemicals followed by cowpea and garden vegetable production. Among the most 
commonly used insecticides is endosulfan, which during 1999/2000 growing season, 
contributed to the food poisoning deaths of approximately 70 Benin citizens. 
Consequently there was increased demand for chemical free vegetables, which were 
grown mainly by using neem, and papaya leaf extracts (Vodouhe, 2001).
In India, Kumari et al. (2002) concluded that 100% of the vegetables tested were 
contaminated. Out of 60 samples, 92% were contaminated with organochlorines, 80% 
with organophosphates, 41% with pyrethroids and 30% with carbamates. About 23% 
showed residues of organophosphate insecticides above the respective MRL values. In 
Mauritius, Choy and Seeneevassen (2004) sampled 115 vegetables and fruits and 
extracted pyrethroids residues. They recorded cypermethrin in 73% of tomatoes, 37% of 
the watercress and 31% of the beans, whereas deltamethrin was found in 53% of 
tomatoes and 19% of watercress samples analyzed.
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2.4.S.4 Pesticide residues levels in Ghana
Mawunyega (1994) carried out a study on residue levels of chlorpynfos sprayed on 
cabbage fields in Legon. Brine Shrimp lethality test was used after observing four weeks 
of post harvest interval. The residue detected was 0.8mg/kg, which was lower than 
1 .Omg/kg recommended by Codex Alimentarius Commission.
Osafo and Frempong (1998) analyzed water and fish samples from three rivers that flow 
through areas of intensive vegetable farming from 1993 to 1995. In 1993, only low level 
of Lindane was recorded, however in 1995 significant levels of Lindane and endusulfan 
residues were recorded. Higher levels of residues were recorded in fish than in water, 
indicating accumulation of pesticides in fish. The highest quantities were found in River
Analysis of street vended food in Accra, Ghana, carried out in 1999-2000 revealed some 
levels of contamination by heavy metals, pesticides, micro-organisms and mycotoxins. 
Organophosphate chlorpyrifos was detected in six out of eight samples of waakye (rice 
and beans) and one out of eight samples offufu (cassava and plantain dough).
Botchway (2000) analysed pesticide residues in exportable cocoa beans from selected 
growing districts and two shipping ports. Analysis by Gas liquid chromatography 
showed detectable levels of lindane residues but the levels were about 10% of the MRL 
of 1.0 jj.g/g permitted by Codex Alimentarius Commission. Aboagye (2002) used Gas 
Chromatography to analyse residues of chlorpyrifos, and fungal inhibition to determine 
levels of carbendazim in exportable pineapples in Ghana. The range of chlorpyrifos 
detected was 0.005 ± 4.8x 10'3 to 0.02±lxl0'2|ng/g. This was below European Union 
MRL and therefore was no threat to the pineapple industry in Ghana.
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Gbewonyo (2004) reported studies on residue levels in a number of agricultural products. 
It was found that residues detected on cabbages sprayed with Lambda-cyhalothrin 
(karate) did not exceed FAO/WHO levels, the levels of Deltaphos 262 EC (250g/l 
triazophos and 12g/l deltamethrin) used then (1996) by farmers, were above 
recommended MRL. It was concluded that 33% of cabbage samples assayed within 
Accra-Tema Metropolitan area of Ghana showed residue levels, which were likely to be 
2-3 fold higher than the FAO/WHO MRL levels. It is evident from these examples that 
the extent of pesticide residue violation even in countries with residue legislations is 
high. This reaffirms the need to minimize pesticide residues in agricultural produce, 
especially vegetables. To minimize the pesticide residues in vegetables, pest control 
must balance economic pesticide management (Sances et al., 1993) considering the 
requirement for multiple insecticide application in vegetable production. This level of 
decision making certainly requires greater information not only from the field with 
respect to pest density, location and potential to increase (Sances et al., 1993), but also 
on insecticide use patterns (FAO/WHO, 1994) and chemical behaviour of insecticide 
once they are applied in the environment. Other variables such as surfactant, formulation 
type, micro and macro environmental factors and individual chemical properties of the 
pesticide in question, may also have profound effect on degradation and hence the 
amount of residues in the market place (Sances et al., 1993).
2.4.8.5 Residue level estimation
The following method has been adopted in residue estimation.
2.4.8.5.1 Methods for residue analysis
Residues are present in very small quantities in heterogeneous compounds including 
biological materials. The process of residue analysis consists of the following steps:
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i) Sampling: This procedure aims at obtaining a final sample representative of the lot in 
order to determine is average pesticide residue content. It is based on the nature of the 
crop and the history of the crop (NRI, 1994).
ii) Extraction: This is done in a solvent to remove the residues from other components 
of the sample matrix. Insecticides are soluble in both polar and non polar solvents e.g. 
methanol is used for extraction of fatty substances, while polar water miscible solvents 
like acetronitrile, methanol or acetone are recommended for non-fatty samples and those 
with high to medium moisture contents (Yeboah, 2001). Hexane has been exclusively 
utilised for extraction of chlorinated hydrocarbon and organophosphorus insecticides 
(Matsumura, 1985).
iii) Clean up: Clean up removes extraneous materials that are co-extracted from the
analytical sample. Methods involved in general clean up include liquid-liquid
partitioning, adsorbent column chromatography, gel permeation chromatography and
solid phase extraction (SPE) (Olson, 1988: Hetzel, 2000; Yeboah, 2001). The basic
principle of SPE (accumulator or concentrator) is based on other clean up techniques
such as adsorbent column chromatography, which is based on the interaction between a
chemical dissolved in a solvent and an adsorptive surface. Where the clean up is either
achieved by the extractives or the pesticides being adsorbed onto the absorbent i.e. the
solvent passes through the column, dissolving and removing residues (eluting) leaving
extractives attached to the absorbent or the co-extractives pass through the column and
pesticides are eluted with appropriate solvent systems. Using the latter principle
therefore, the SPE packing materials or cartridges retain the pesticides when the extract is
passed through without retaining co-extractives and then eluted with appropriate solvent
system. Conversely SPE can retain the co-extractives and allow the pesticides to pass
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through. The column materials commonly used are florisil, alumina, silica gel, 
magnesium oxide and carbon (Yeboah, 2001).
iv) Sample concentration: This is aimed at reducing the volume of the solvent carrying 
the insecticide residues without losing residues, thus reducing the residues to detectable 
level (Hetzel, 2000). This is done by using either nitrogen gas evaporator or rotary 
evaporator.
v) Identification and quantification
After providing the residue containing extract with or without cleanup, the steps for 
resolution, detection, measurement, quantification, and confirmation are performed 
sometimes, after derivatization. Advances in the technologies available in these areas 
have been responsible for the improvements in both selectivity and detection limits over 
the past half century.
vi) Evolution of detection methods
In 1940s and 1950s, gravimetric and bioassay were the mainstay in trace analysis with 
detection level of about 1 ppm (Seiber, 1982). By 1950s to 1960s colometric and 
spectrophotomeric methods responded to whole classes of compounds rather than 
individual chemical species but with improved detection limits in magnitude, e.g. the 
Schechter Haller method for DDT. In 1950s paper and thin layer chromatography added 
the analysts’ ability to resolve individual chemicals in a fairly complex mixture. These 
qualitative techniques were best suited for screening for presence of specific pesticides. 
Quantitatively they could also compare spot intensities visually. Since 1960s to the 
present, gas and high-performance liquid chromatography (HPLC) has nearly eliminated 
PC and TLC (Beckman, et al, 1963).
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Trend in cost
G
&
a
< • rt
C
0
*0
>1
0.1
2  o .o i  
0
g 0.001
'8 <0,0001 
2
&
<
Bioassay, gravimetry
wmm
Colorimeiry, spectrophotometry
__T Paper, thin-layer chromatography
Gas and HP liquid chromatography
GC* and HPLC-mass spectrometry ^
IS
ettf*
TJ
C
B
h
JM  1950 i960 1970 If
Year (approxiraale)
1990 2000
Fig. 3 The evolution o f analytical methodology for organic toxicants in environmental 
samples (Seiber, 1982).
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Detection limits of low ppb and even ppt are attainable, especially with clean samples of 
water, air, and soil and biological matrices. The use of mass spectrophotometry in 
connection with GC and HPLC has increased their detectability and selectivity although 
the cost has limited their use. Lastly, immunoassay, which has the potential of cutting 
into chromatography-based bastion of pesticide residue methodology, is currently under 
development.
vii) Bioassay method
The methods of analysis employed include Biological (Bioassay) and Chemical 
(analytical). Test animals are selected on the basis of high pesticide sensitivities and the 
ease with which large numbers of them can be reared (Matsumura, 1985). Bioassay has 
been used to determine pesticide residue levels (Sarode and Rattan, 1981). 
Ramasubbaiah and Rattan (1978) indicated that there is a high degree of coefficient of 
correlation (agreement) between bioassay and calorimetric chemical assay. Brine 
Shrimp, Artemia salina leach, a test organism is commonly used in the bioassay to detect 
and estimate levels of the insecticide residues (Grosch, 1967). This is because it is found 
to be sensitive to a broad range of compounds at concentrations of 0.01 ppm in about 45 
minutes to two hours (McLaughlin, 1991). Although bioassay is rapid, inexpensive, and 
convenient, it lacks the ability to distinguish metabolites, impurities and alteration 
products of pesticides from the parent pesticidal compound. Hence Gas chromatography 
(G C) has been offenly used to determine the quantity and confirm the identity o f the 
residue detected. GC can be fitted with Electron capture detector (ECD), Flame Ionising 
Detector (FID) or nitrogen Phosphorus detector (NPD) depending on the type of 
compounds being analysed (GTZ, 1979). The methods used for analysis can be multiple 
residue method (MRM), which can determine various residues of different pesticide;
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while selective (MRM) is used on small numbers of chemically related pesticides and 
Single residue method (SRM) determines one pesticide (FDA. 1994).
2.4.9 Integrated pest management (IPM)
Sole reliance on insecticides is not an effective or sustainable practice for the 
management of DBM in the long run. DBM is often a secondary pest, becoming a 
problem when insecticide use for primary pests destroys its natural enemies and selects 
for resistance in the population (Endersby, 2004). The probable way forward in 
controlling DBM is therefore to develop IPM programmes given that even chemicals, 
which have been the mainstay of its control, can no longer hold its population. A number 
of successful IPM programmes have been developed for DBM in large-scale brassica 
cultivation areas; one of the most successful is in the Bajio region in Mexico where about
15,000 ha of brassicas are grown annually. This programme was initiated in 1987 after a 
control failure of DBM despite an average of nine applications of synthetic insecticides. 
The programme relies on scouting thresholds, crucifer free periods and the judicious use 
of B. thuringiensis, and has resulted in over 50% reduction in the use of insecticide 
sprays (Talekar and Shelton, 1993). Similar success has been achieved in Jamaica (Ivey 
and Johnson, 1998), Singapore (Ng et al., 1997), and the Philippines (Rejesus et al, 
1996). At Weija-Accra, Ghana, farmers abandoned cabbage production due to crop loss 
due to DBM infestation. But using agronomic skill acquired through training in Framers 
field schools coupled with the use of Neem extracts, they resumed cabbage production on 
a highly profitable way and environmentally sound manner (Youdeowei, 1999).
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2.5 RESISTANCE DEVELOPMENT
Diamondback moth has developed resistance to most of the commonly used insecticides. 
Long before environmental concern over negative effects of the use of organochlorine 
insecticides developed, entomologists were already encountering a major problem in 
insect pest control. The organochlorine insecticides, which initially had been so 
effective, were performing erratically or in some cases failing. It became apparent that 
these insects had developed resistance to the organochlorine (Soderlund and Bloomquist, 
1990).
Sawicki (1987) defined resistance as a genetic change in response to selection pressure 
by toxicants that may impair control in the field, while Wegorek et al. (2002) defined 
resistance as the naturally-occurring, inheritable adjustment in ability of an individual in 
a population to survive a plant protection product treatment that would normally give 
effective control. However, different views exist based on individual’s focus/interests on 
resistance, for example; a biologist would view resistance in terms of “X-fold increase in 
lethal doses or percent survival at discriminating dose”, while a geneticist would view it 
as “a change in gene frequencies,” and a biochemist would view it as “a modification at 
the pesticide’s target site”, while to an industrialist, it is when the target pest’s response 
to a commercial product development to a level where the product no longer performs as 
intended (Thompson, 2004), This circle between resistance development and insecticides 
spraying is presented in figure 4. Resistance is therefore exhibited as progressive 
inability of a given treatment to control a pest population (O’Brien, 1967). As a result, 
the insecticide is applied more frequently and in large quantities to compensate for the 
decline in effectiveness. Consequently, further resistance is developed within the insect 
population until the usefulness of the insecticide is greatly reduced and may totally loose
effectiveness in that particular population in the long run.
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Spray New Chemistry
Insects Become 
More Resistant
Fig. 4. Perceived pesticide treadmill (Thompson, 2004)
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The resistant genes are usually present at very low frequencies in normal populations and 
resistant strains are derived from the initial population by the selective mortality o f the 
more susceptible genotype growing during the application of an insecticide (Crow, 1957; 
O’Brien, 1967; Sawicki, 1979). Resistance development becomes rapid when selection 
pressure exerted by insecticide is widespread and continuous (Brown, 1964) and occurs 
after the insect has been exposed to chemicals for several generations. Graves et al. 
(1967) reported that it took 25 generations of intensive selection for the boll weevil 
Anthonomus grandis to develop resistance to organochlorine compounds. DDT was used 
for 15 years before Heliothis virescens developed resistance to it (Kumar, 1984). Brown 
(1977) has stated that development of resistance to the organochlorines (DDT and 
Cyclodienes) took about 10 years that for cyclodienes developed somewhat faster than 
the DDT group. It was further stated that development of resistance to organophosphates 
came to fruition 10 years after cyclodienes resistance. However, the resistance, to 
carbamates takes as much time as the organophosphate resistance but developed a little 
faster when it builds on a base of OP resistance (Kumar, 1984). The more persistent the 
poison is and the more rapid the life cycle of the insect or acarine and the greater the risk 
that the development of resistance will be rapid (Hassal, 1990). Table 3 is an indicator of 
broad trend or probably wider-estimate of the extent of resistance since many cases have 
either not been investigated or remain unreported (Georghiou and Mellon, 1983; 
Goerghiou and Lagunes-Tejeda, 1991).
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Table 3. Comparison of Records of Resistance to Pesticides in Arthropods (Source, 
Georghiou and Legunes-Tejeda, 1991)
Pesticide group 1970 1980 1989 Percentage
1971-80
Increases
1981-89
No. of species with resistance 
cases
224 428 504 91.1 17.8
DDT 98 229 23 133.7 14.8
Cyclodienes 140 269 291 92.1 8.2
Organophosphorus 54 85 260 270.4 30.0
Carbamates 3 48 85 1600.0 66.7
Pyrethroids 3 22 48 633.3 118.2
Fumigants 3 17 12 466.7
Other 12 41 40 241.7 -
Total for all pesticides groups 313 829 999 164.9 20.5
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2.5.1 Mechanisms of insecticide resistance
Insecticide resistance is a dynamic multi-dimensional phenomenon, which is affected by 
biochemical, physiological, genetic and ecological nature of the insect (Brattsten et al., 
1985). Considerable information on the biochemistry and genetics of resistance is 
discussed by (Busvine, 1971; Matsumura, 1985; Oppenoorth, 1985; Georghiou, 1986). 
They identified resistance mechanism to include cuticular penetration of toxicants, 
enhanced metabolism by cytochrome P450 dependent monoxygenase, hydrolases or 
glutathione- S- transferases, and reduced sensitivity of mutant acetylcholinesterases to 
organophosphates and carbamates and of other neuronal targets to pyrethroids, DDT and 
its analogues and chlorinated cyclodienes (Oppenoorth, 1985). Resistance mechanisms in 
DBM proposed for synthetic chemicals include, decreased penetration (Noppun et al. 
1987), enhanced detoxification by esterases (Maa and Chuang, 1983) and Glutathione-S- 
transferases, and reduced sensitivity of acetylcholinesterase (Wu, 1983; Hama, 1987). 
However, resistance to pyrethroids can be attributed to an inherited or induced mixed- 
function oxidase complex. Esterases in general have been noted to play significant roles 
in resistance to insecticides particularly organophosphates (Owusu et al., 1996).
2.5.1.1 Metabolic detoxification: This refers to the process by which non specific 
enzymes break down toxic, lipophilic compounds into less toxic usually more soluble 
compounds for excretion thereby greatly decreasing their biochemical activity or toxicity 
(Wilkinson, 1983; Mallet, 1989). Biochemical reactions leading to detoxification include 
oxidation, hydrolysis, dealkylation and dehydro chlorination caused by several enzyme 
systems including mono-oxygenases (mixed function oxidases, microsomal oxidases and 
cytochrome P450 dependent oxidases), hydrolases (esterases) and transferases 
(glutathione-S-transferase) and DDT dehydrochlorinase.
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Mixed-Function Oxygenases (MFO): Mixed function oxidases are located in the 
microsomal portions of several tissues particularly the Malpighian tubule. Microsomal 
enzymes have evolved as protective mechanisms against naturally occurring toxicants 
such as nicotine, rotenone and natural pyrethrins (Yamamoto et al., 1969; Elliot et al., 
1972).
Glutathione-S-transferases: Play a key role in the resistance of organophosphate 
compounds (Tanaka et al., 1981). Many researchers have demonstrated glutathione 
dependent degradation of parathion and diazinon in resistant houseflies (Lewis and 
Sawicki, 1971). Mechanisms of physiological resistance to toxic chemicals include 
reduced penetration, sequestration and excretion. Delayed penetration provides more 
time for detoxification of the incoming dose (Brattsen et al., 1985).
Alternative biochemical pathway; This mechanism provides an alternative pathway to 
the one blocked by the insecticide. The blocked site is by-passed by the use of the 
alternative pathway. Altered acetylcholinesterase gives rise to resistance to certain 
organophosphates in cattle tick, in the mosquito Aedes albimanus, and the two-spotted 
spider mite. The Indian meal moth became resistant to B.t. by using altered binding sites 
on the gut wall, likewise, the California red scale, Aonidiella aurantii, became resistant 
due to alternative pathway to the terminal stage of cellular respiration and was no longer 
dependent upon enzyme systems disabled by cyanide.
2.5.1.2 Target site insensitivity
When compared with the case of metabolic resistance mechanisms, a modified target site 
causes relatively few types of resistance. Some arthropods develop resistance by altering 
the properties of the target site of action of a given insecticide. This mechanism has been
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demonstrated in Spotted spider mite, Tetranychus urticae (Smissaert, 1964), leafhopper, 
Nephotettix cincticeps (Iwata and Hama, 1972), and in numerous resistant insect species 
with cross-resistance to cyclodienes (Hama, 1987). The best example of altered target as 
a cause of resistance to insecticides is that of acetylcholinesterase with reduced 
sensitivity to organophosphates and carbamates. A single gene has been shown to be 
responsible for the difference in acetylcholinesterase sensitivity and resistance caused by 
it in many arthropods (Oppenoorth, 1985). The esterase responsible for resistance 
development in the green peach aphid Myzus persicae has high binding affinity but low 
catalytic reactivity and hence functions as a storage protein for carbamates, 
organophosphates and pyrethroids. A major factor knockdown resistance (kdr) located on 
chromosome 111 in houseflies which confer resistance to DDT and pyrethroids through 
target insensitivity mechanism has been demonstrated (Sawicki, 1973).
2.5.1.3 Changes in behaviour as a resistance mechanism
Resistance mechanism can strictly be behavioural e.g. in Mazoa valley of southern 
Zimbabwe, Muirhead-Thomson (1960) found that Anopheles mosquitoes originally were 
abundant both outdoor and in human dwellings. The endophagic (indoor) mosquitoes 
eventually disappeared from both treated and untreated dwellings after eight years of 
BHC treatments in human dwellings and this condition persisted even after insecticidal 
treatment were discontinued. This suggests that during the period of insecticide 
treatment, an exophagic strain evolved that lived and fed outdoors.
In some cases the mechanism of resistance may represent an enhanced ability of the pest 
to detect a toxicant and initiate a response to avoid it. This phenomenon has been studied 
by Kirkpatrick and Schoof (1958) for houseflies and for mosquitoes (Gerold and 
Laarman, 1964).
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2.5.1.4 Cross resistance and multiple resistance
An insect population is considered resistant if its response to an insecticide in detection 
tests drops significantly below its normal response (Georghiou & Mellon 1983). 
Resistance develops mostly due to a genetic mutation that enables the insect to 
counteract or circumvent the activity of an insecticide.
Cross-resistance denotes the resistance of a strain of insects to chemicals other than 
those they were selected against. This is possible if the compounds are closely related 
and have the same or a similar mode of action. As a result one compound causes 
selection for detoxification mechanism common to members of a group of chemical and 
the population becomes resistant to several closely related chemicals, in absence of 
selection pressure against each (French et al., 1992).
Multiple-resistance on the other hand, is caused by the presence of separate 
detoxification mechanisms for unrelated insecticides, selected for independently (French 
et al., 1992). Thus a single strain becomes resistant to several different compounds, 
using different mechanisms. This may be the result from intensive (indiscriminate) use of 
different chemical groups without following resistance management principles 
(Oppenoorth & Welling 1976). Multiple resistance often results from the simultaneous or 
consecutive use of several insecticides. However, it is sometimes difficult to discriminate 
between cross-resistance and multiple resistance, since genetic linkage may result in 
different cross resistances, particularly during selection in the laboratory (Oppenoorth & 
Welling 1976).
These two phenomena have been greatly exploited by many insect species. By 1984 there 
were 1797 cases of resistance in arthropods to insecticides, and by 1991, resistance to at
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least one insecticide had been recorded for 504 species (Georghiou, 1986; Georghiou and 
Lagunes-Tejada, 1991). So far as at 1989, insect species resistant to the greatest number 
of insecticides is the green peach aphid, Myzus persicae (Sulzer) (Homoptera: Aphidae), 
which has a documented resistance to 71 synthetic insecticides (Georghiou and Lagunes- 
Tejada, 1991), followed by DBM resistant to 51 compounds and Colorado potato beetle, 
Leptinotarsa decem-lineata (Say) (Coleoptera: Chrysomelidae), resistant to 37 
compounds (Georghiou and Lagunes-Tejada, 1991). Other species that have developed 
resistance to most insecticides used against them include the cotton leaf worm, 
Spodoptera littoralis, in Egypt; the cattle tick, Boophilus microplus, in Australia; the 
housefly Musca domestica; and many species of Anopheles mosquitoes worldwide 
(Forgash 1984; Georghiou, 1986). Colorado potato beetle and DBM have developed 
resistance to all synthetic insecticides used against them including the biopesticide B.t. 
(TalekaT and Shelton, 1993). While the biochemical resistance mechanism in M. persicae 
is based on increased levels of esterase-4 (Scott, 1990).
2.5.2 Resistance in Diamondback moth
Insecticide resistance in DBM has occurred in many parts of the world since Ankersmit 
(1953) first reported DDT resistance in Indonesia. More recentiy, resistance has been 
reported in Hawaii, Japan and Australia. However, the occurrence of insecticide 
resistance in this insect pest may not be limited to these areas (Kao et al., 1989). The 
resistance spectrum of DBM covers all groups of insecticides; that is chlorinated 
hydrocarbons, organophosphorus insecticides, carbamates, pyrethroids (Sun et al., 1986; 
Lin, 1988) and even B.t. (Kao et al., 1989).
Resistance mechanisms in DBM proposed for synthetic chemicals include, decreased 
penetration Noppun et al. (1987), enhanced detoxification by esterases (Maa and
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Chuang, 1983). Glutathione-S-transferases and reduced sensitivity of 
acetylcholinesterase (Wu, 1983; Hama, 1987). However, resistance to pyrethroids can be 
attributed to an inherited or induced mixed-function oxidase complex. Esterases in 
general have been noted to play significant roles in resistance to insecticides particularly 
organophosphates (Owusu et al., 1996). Esterases are important in degrading 
organophosphates. The hydrolysis of organophosphates and pyrethroids by 
carboxylesterases, including site of action on some pyrethroids are shown in Fig 5 (A-D). 
The larvae of mosquito, Culex tarsali was found to have higher carboxyl esterase activity 
than susceptible strains (Matsumura and Brown, 1961). The DBM populations collected 
around Accra in Ghana, showed varying levels of Carboxylesterases activity, and hence 
insecticide resistance levels (Hama and Hosoda, 1988). The majority of individuals 
collected were classified as moderately resistance and may be low as compared with the 
populations in places like China, (Zhu, et al. 1996), New Zealand (Cameron et al., 1997), 
Japan (Hama, 1990) or South Africa (Sereda et al., 1997), however, there is evidence of 
probable increasing use of insecticides on cabbage indicating that insecticide resistance 
in DBM will become a limiting factor in the commercial cultivation of cabbage in the 
near future and therefore, there is need to urgently address the problem before it gets out 
of control (Kaiwa, 2000)
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Cl
1 ^  ^  —  ch,-c~0-p-0 - v  >ci h2o
ii \ = /
O Cl
Chlorpyrifos
carboxylesterase 
 ►
CH3-C-OH + HO-P-O 
II
o
Fig. 5 (A) Hydrolysis of chlorpyrifos by carboxylesterase (ref)
CLC=CH
H,0 carboxylesterase
C-OH + O^
o
i  h
Fig. 5 (B) Hydrolysis of cypermethrin by carboxylesterase
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Site of cleavage
Fig. 5 (Q  Hydrolysis o f lambdacyhalothrin by carboxylesterase
Fig. 5(D) Hydrolysis o f deltamethrin by carboxylesterase
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2.5.3 Management of resistance
The evolution of resistance according to Georghiou (1983) is determined by genetic, 
biological and operational factors. Some of the operational factors that promote 
resistance development include; prolonged exposure to insecticide, lack of refuges, large 
geographical area and selection before mating, while the biological factors comprises of 
little migration between population, monophagous species, short generation time, large 
numbers of offspring / generation, mobile species and increasing potential for exposure. 
Though control failure is attributed mainly to resistance, there are other factors that may 
lead to control failure. This includes poor chemical application methods and 
environmental condition, which may be due to old and obsolete application equipment, 
which provides poor coverage. Hot and dry conditions are known to be conducive to 
outbreaks of DBM and, this was probably the key reason for DBM outbreak in broccoli 
in California in 1997 (Shelton and Zhao, 2004).
For effective management of DBM, there is need to delay resistance development, this
can be achieved by starting resistance management program before resistance is detected
and strategically applying insecticides to vulnerable developmental stages and under
environmental conditions that render the insects susceptible and the insecticide effective.
Secondly, the choice of insecticides to be used may also decelerate resistance
development; for example choosing insecticides with short persistence or residual effect
while avoiding formulations that release their active ingredients slowly since these leads
to prolonged exposure to insecticides which, is a prerequisites to resistance development.
Consequently, insecticides should be used intermittently at the lowest possible effective
rates/dosages and on the other hand; higher doses strategy is needed to kill the rare
heterozygous population developing resistant. Besides, insecticides, which help conserve
natural enemies, should be preferred like the B.t. ’s, spinosad, emamectin benzoate and
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indoxacard which will be friendly to natural enemies than pyrethroid, carbamate and 
organophosphate insecticides.
Since IPM is the most effective and promising method for controlling DBM, some of the 
IPM principles need to be incorporated to delay resistance development, this is based on 
using other control methods like cultural practices, host plant resistance etc. whenever 
possible, coupled with judicious use of insecticides like restricting insecticides to local 
instead of area-wide treatments to include spot treatment. Other cultural practices like, 
avoiding continuous cultivation of crucifers by having a host-free period and 
preservation of untreated refuges can help delay resistance. It is also important to monitor 
susceptibility to all insecticides on a regular basis in representative fields in each region. 
This is achievable through regular field scouting and treatment according to threshold 
levels.
Even with the judicious use of insecticides in IPM, care should be taken to avoid mixing 
insecticides for use against DBM. Since mixtures usually cannot delay the onset of 
resistance but often accelerate and complicate it. Mixtures are also undesirable due to 
cost, environmental risk, and residues or ineffective (differential persistence of 
components of the mixtures), despite their popularity with pesticide manufacturers and 
distributors, mixtures and high doses have many negatives to be practical in pest 
management (Mallet, 1989).
However attempts have been made to manage DBM by using multiple attacks that aims
at achieving control through the action of independent acting forces by the use of
insecticides in mixtures and using unrelated insecticides in rotations or in combination
with other chemical and non-chemical measures and with synergists, which counteract
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the insect defence mechanisms (Georghiou, 1983). This requires that the member 
chemicals are reciprocally unaffected by cross-resistance. Insecticides remain the most 
reliable and effective means of controlling pests, but should be used carefully, selectively 
and only when alternative methods do not exist or are uneconomical. So far, minimizing 
use is the only strategy really proven to work satisfactorily (Mallet, 1989).
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CHAPTER THREE 
MATERIALS AND METHODS
3.1 Chemicals, reagents, equipment and software
The sources and or manufacturers of the chemicals and reagents, and equipment used are 
listed in Appendix I. The various buffers and solutions used were prepared as described 
in Appendix I.
3.2 Study area
The study was conducted in three localities within Accra suburbs and Mampong- 
Akuapem. The global positioning system (GPS) device was used to determine the 
specific geographical coordinates of the sampling sites. Dzorwulu (05° 37.05N, 
00°11.70W), Airport (05° 35.72N, 00° 10.89W), Redco-Madina (05° 40. 36N, 00° 
10.24W) and Mampong-Akuapem (05° 24.74N, 00°36.25W). These sites were chosen 
because of the differences in agronomic practices of the farmers, rainfall and insecticide 
use patterns. Accra suburb is located within the greater Accra Region in Southern Ghana. 
It is a coastal savannah ecological zone, characterised by dry climatic conditions with 
two peak rainy seasons from April to June and September to October. The annual rainfall 
ranges between 740 and 890 mm per annum and a temperature range of 26°C and 30°C. 
The relative humidity is 65-75 % (Dickson and Benneh, 1988). While Mampong- 
Akuapem records the highest mean monthly temperature of 30°C between March and 
April and the lowest of 26°C in August. Relative humidity is 65-75% throughout the year 
and the vegetation consists mainly of grass with isolated patches of shrub and sparse 
trees. The major rainfall season falls in June -July and followed by a long dry season 
(Yawson et al., 2004). A sampling site in Accra suburbs is shown in plate 1
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Plate 1. Sample collection site at Dzorwulu. A cabbage farm destroyed by Diamondback 
moth.
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3.3 Questionnaire Survey
A preliminary survey to determine insecticide use patterns on cabbage was carried out at 
the beginning of the work. A questionnaire (Appendix II) was prepared to obtain 
information needed for quantification (Horton, 1995) on insecticide usage, type of 
insecticides used, frequency of use, mode of application, history of the farms and the 
prevailing agronomic practices was administered to the fanners. The questionnaire was 
structured to suit the farmers understanding, and they responded to the questions in the 
presence of an administrator who helped to translate the questions into the local language 
in some cases, or to further simplify the questions. The data obtained was analysed with a 
view to making a decision on the appropriateness of the site for sample collection and the 
most commonly used insecticides to be selected for the study. To guide on planning and 
conducting of the formal survey, a reconnaissance survey (Rhoades, 1995) was done to 
obtain basic information on the location of cabbage farms, insect pests of cabbage, and 
other agronomic practices that may affect insecticide residues and insecticide resistance.
Locating cabbage farms for the survey was difficult and growers in perennial cabbage 
growing farms were approached for information on where to find more growers. Besides, 
extension officers, pesticide sellers, grocers and friends were also approached for further 
information on the same.
The choice of farmers from a particular cabbage growing area depended on the number 
of growing areas identified from the informal survey and the number of growers in a 
particular area. In each growing area, a random representative number of growers (at 
least 3) were selected and spoken to (Table 4).
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Table 4 . Number of respondents interviewed in each cabbage growing area
GROWING AREA NUMBER OF RESPONDENTS
Airport-West-Accra suburbs 4
Airport-(CSIR) -Accra suburbs 4
Dzorwulu-Ablemkpe- Accra suburbs 4
Adabraka -Accra suburbs 4
Atomic area- Madina Accra suburbs 3
Redco-Madina -Accra suburbs 6
Mampong-Akuapem 5
3.4 Insecticides
The insecticide formulations used were dependent on the results of the preliminary 
survey. The commercial formulations of test insecticides were purchased from the local 
chemical sale points namely Dizengoff, Agrimat House and Aglow-Accra. The products 
were diluted with water to obtain a range of test concentrations. The three most 
commonly used pyrethroids (Pawa 2.5 EC, Deltaplan 12.5 EC and Cypercal 250 EC), 
one organophosphate (Dursban 4E EC) and a B.t. formulation (Dipel 4E DF) were 
selected.
3.5 Culturing of Diamondback moth
For obtaining reliable and reproducible information from the physiological and 
toxicological experiments of any insect, it is desirable to have uninterrupted supply of 
physiologically homogeneous individuals. Therefore, development of laboratory rearing 
techniques is necessary (Sing, 1977). Mass rearing aims at producing the maximum 
number of insects within a short time using the most economical conditions such as 
minimum labor and space. Therefore the rearing materials and equipment should be kept
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as simple and inexpensive as possible but they must be nutritionally and behaviorally 
optimal for the insect.
3.5.1 Field Sample collection
Wild fourth instar larvae and pupae were collected from infested plants in various 
cabbage growing areas of the selected sites from September to December 2004. A 
composite of pupae collected from 2-4 fields within a radius of 2-3 km served as the 
sample for each location as recommended by (Vastrad et al., 2004). Samples were 
collected into plastic Petri dishes using fine camel hairbrush. The Petri dishes were lined 
with moistened tissue paper to avoid overheating during collection and transportation. 
The larvae were sent to Noguchi Memorial Institute of Medical Research (NMIMR) of 
University of Ghana and stored at a temperature of -80°C to prevent loss of the esterase 
activity (Zhu and Brindley, 1990) and later used for enzyme assay. The Pupae collected 
from the fields were used to establish a DBM colony at the Sinna’s Garden of Crop 
Science department of University of Ghana in rearing cages (30 cm3) as shown in Plate 2. 
The temperature during the period of study was 28+1 °C with relative humidity of 65­
70% and a photoperiod of 12h: 12h (L: D). The method of rearing was modified from 
Liu and Sun (1984) as adopted by Osae (2002), in which the adult moths were sustained 
on cotton wool impregnated with 50% (v/v) honey solution and potted-cabbage seedlings 
were provided as oviposition substrate. Hatched larvae were fed on pesticide-free potted 
cabbages grown in a screen house. Oviposition was synchronized in order to attain same 
age of insects at anytime. This was done by putting every potted cabbage into the 
oviposition cage for 24h to allow a maximum of 24h difference in age. Early fourth 
instar larvae of uniform size and weight from preferably FI and F2 generations were 
tested for susceptibility to the range of insecticides used for the bioassays.
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3.5.2 Reference (Susceptible) strain
Samples of the reference strain were provided by International Centre of Insect Ecology 
and Physiology (I.C.I.P.E.) Kenya. The samples were preserved in absolute ethanol. The 
origin of the colony is in Coast Province, Wundanyi District, Chawia (03° 28 39.6S and 
038° 20 54.7E) and Werugah (03° 21 48S, 038° 19.43E).
3.5.3 Cultivation of insecticide free cabbage
Soil samples collected from a virgin land were sterilized using hot water treatment and 
left to cool in conjunction with the use of natural sun’s heat on soil mass covered by 
black polythene sheet for 2 weeks. The samples were put into a tray and used for nursing 
the seedlings. Cabbage (var Oxylus) seeds were thinly sown in a nursery tray and shaded 
immediately after sowing. The shade was removed after one week when germination 
commenced and the seedlings were watered twice a day. After the seedlings were 
established, thinning was done to maintain one plant per spot. The sterilized soil was 
well mixed with poultry and farmyard manure and then put into 15cm diameter plastic 
pots. Four weeks after sowing, the seedlings were pricked out and transplanted into the 
pots. Agronomic practices like watering, weeding and mulching were carried out where 
necessary. Insect pests were controlled by non-chemical methods like handpicking and 
crushing. Six weeks after transplanting, the seedlings had 10-12 leaves and were used to 
rear DBM larvae. The cabbage was ready for harvesting 60 days after transplanting. 
Some heads were sampled and harvested at the root junction. The cabbage heads were 
sliced longitudinally and 50 grams of each sample were weighed into labeled beakers and 
then transferred into conical flasks containing 25ml hexane solution. The flasks were 
stored in a refrigerator at 4°C and the cabbage used as controls in the residue level 
estimations.
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3.6 Determination of level of resistance
Various methodologies were employed to determine insecticide resistance of DBM to the 
selected insecticides as described below;
3.6.1 Dose-response bioassay
Resistance can be verified through a combination of laboratory bioassay and field 
performance. Laboratory bioassay is when a set of test insects are subjected to different 
doses of the insecticides, and then after a specific period of time the insects are judged to 
be dead / moribund or alive. This is underpinned by the principle of toxicology, which 
asserts that there is a relationship between a toxic reaction (the response) and the amount 
of poison received (the dose). It operates between two extremes; a low dose below, 
which no response can be measured and a maximum response which when reached, any 
further increases in the dose, will not result in any increased effect. From the assay, a 
dose-mortality line can be determined and the LC50 value calculated. The LC50 value is 
the dose required to kill 50% of individuals within a population. Bioassay has therefore 
become the principal method used by entomologists to determine and compare the 
toxicity of insecticides to insect pests. This is possible by using the dose mortality 
standard measures like 50% and 95% mortality points which can be used to compare 
different populations (Cochran, 2004).
3.6.1.1 Dose-response bioassay for Organophosphates and Pyrethroids
Since organophosphate and pyrethroids act by contact, the larvae immersion bioassay 
protocol was adopted from Zhao et al. (1994) with slight modification. Plastic cups 
chalices (about 2.74 cm depth) were improvised by having both ends open (see Plate 4). 
The wide bottom end (radius 3.42 cm) was fitted with a netting material and the top end 
(radius 2cm) left open. Different concentrations of the insecticides below and above the 
critical levels (chlorpyrifos: 3g/L, cypermethrin: 0.1 g/L, deltamethrin: 0.0375g/L and
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lambda-cyhalothrin: 0.083 g/L) were prepared in Petri dishes. Ten early 4th instar larvae 
were introduced into the container and immersed into serially diluted test solution in a 
Petri dish for 2 seconds. Excess solution was drained off with tissue paper. Cabbage 
leaves were provided for food and para film was used to seal the top open end. Mortality 
data was taken every 12h for three days. Any larvae that did not respond to pencil-tip 
prodding were considered dead.
3.6.1. 2. Dose-response bioassay to Dipel
A Bacillus thuringiensis var. kurstaki) insecticide formulation Dipel was used in the 
bioassay to establish the susceptibility status of DBM., using leaf residue bioassay 
shown in Plate 3 as described by Tabashnik et al., (1987; 1990), Ninsin (2004) with 
slight modifications. One week after eggs were laid on cabbage plants, larvae were used 
for bioassays. Different concentrations of Bacillus thuringiensis var. kusrtaki formulation 
below and above the recommended rates (0.206 g/L) were serially diluted in distilled 
water assuming a spray volume of 200 litres/ha (Marina and Gatehouse, 2001), into 5-10 
concentrations. Larvae of uniform age were selected from the reared DBM population 
and exposed to cabbage leaf discs (5 cm diameter) which were previously immersed in 
test solutions for 5 second and air-dried for one hour at room temperature (25°C) in 
standard size Petri dishes. Once the leaf discs had dried they were placed individually in 
clean Petri dishes (6 cm diameter), lined with a slightly moistened tissue paper and 10 
early-fourth instars larvae were transferred to each dish.
For each concentration, four replicates of 40 larvae were tested, for each toxin and strain. 
Untreated cabbage leaves were dipped in distilled water as a control and 10 larvae 
introduced likewise. The Petri dishes were cleaned daily to avoid moisture build up and 
the larvae were provided with freshly treated leaf materia] for each bioassay every
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second day until they pupated. After exposure, mortality was assessed after every 12 h at 
25°C for 3 days. Larvae that did not show coordinated movement or did not respond to 
pencil-tip prodding were considered to be dead (Sun and Johnson, 1960). Whenever the 
control mortality exceeded 20 per cent the data was rejected d and fresh batch of larvae 
were used for the treatment. Resistant insect groups were preserved separately in 70% 
alcohol for further investigation to detect the presence of resistance genes as 
recommended by (Vastrad et al., 2004). To help predict field performance using 
laboratory data, discriminating concentration method for resistance monitoring was used 
for comparison of concentration mortality tests (Zhao et al., 1994). This enabled the 
population to be classified as either resistant/tolerant or susceptible to a single dose 
(Shelton and Zhao, 2004).
When a resistant strain was established, cross and multiple resistance was evaluated to 
determine which insecticides could be used to manage the resistant DBM populations. 
The protocol was modified from Liu et al. (2001) and Ninsin (2004). Larvae that 
survived during the bioassay were again reared to adults and their FI generation used to 
test for cross-Tesistance with other insecticides, which were not used for resistance 
selection on the parents. The protocol described under section 3.6.1.1 was used to test the 
larvae using the critical dosages and mortality data was recorded every 12h for three 
days.
The data for each replicate were pooled and analyzed by probit analysis (Finney, 1971), 
using MINITAB software programme to determine lethal concentration (LC) values at 
50% and 95% levels at 95% confidence intervals (Cl). When LC values were compared, 
they were judged to be significantly different when the corresponding 95% Cl values did 
not overlap. The slope of the regression line was also recorded.
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Plate 2. A metal net cage (50cm3) with metal board floor. Inside is a potted cabbage 
plant about six weeks old used for oviposition and further feeding o f  the hatching larvae.
DBM larvae
Plate 3. Potted cabbage plant showing diamondback moth 3rd to 4th instars larvae.
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Plate 4. Leaf disk dip (leaf residue) bioassay set up. Plastic petri dish with insecticide 
treated cabbage leaf disk (about 5cm diameter) and ten early fourth instars DBM larvae 
introduced into the petri dish.
Plate 5. Larvae immersion bioassay set up. Plastic cup “holy communion cup” (about 
2.74cm depth, bottom and top radii are 3.42cm and 2cm respectively). With net-like 
material at the bottom and an open top end.
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3.6.2 Carboxylesterase activity in field population of DBM
The approach is based on the activity of carboxylesterase, which has been confirmed, to 
be positively linked to resistance in DBM (Doichuanngam and Thornhill, 1989) and 
involves hydrolysis of naphthyl acetate by carboxylesterase. Naphthyl acetate is a general 
substrate for a variety of hydrolases and is commonly used for determining 
carboxylesterase activity (Brown and Brogdon, 1987).
Quantitative hydrolytic activity of esterase enzymes was determined with two model 
substrates, ct- and j8- naphthyl acetate at predetermined optimum substrate concentration 
(30 mM), temperature (40°C) and pH value (7.2) (Kaiwa, 2000). The specific esterase 
activity was quantified by the amount of naphthol produced and special staining solution 
added to allow colour development to enable spectrophometric readings to be taken at 
specific wavelengths.
DBM specimens were assayed for carboxylesterase activity by naphthyl acetate- diazo 
blue coupling reaction (Owusu, 1992). The individual larvae were homogenised in a 
buffer and carboxylesterase assay was performed based on the original method 
developed for housefly esterase (Van Asperen, 1962) and adopted for cotton aphid 
carboxylesterase (Owusu et al., 1996). A standard curve was plotted as reference.
3.6.2.1 Establishment of standard calibration curves
3.6.2.1.1 o-Naphthol
A 0.2M solution of a-Naphthol (MW 144.2) in 10ml of absolute ethanol was prepared. 
This solution was serially diluted 1 in 10 to give a concentration of 0.002M. This 
concentration was further serially diluted 1 in 2 to obtain the concentrations lOxlO^M, 
5x10"^, 2.5x10'4M, 1.25xl0^M, 6.25x10"5M in test tubes. The different concentrations 
were then used to establish the calibration curve. Into each of the five test tubes
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containing 2.8 ml of 0.07M phosphate buffer (pH 7.2), was added separately 0.1 ml of 
the prepared a-naphthol solution. A test tube containing ethanol (0.1ml) was also set up 
as a blank. Sodium dodecyl sulphate-fast blue B salt (SDS-FBS) solution (0.5ml) was 
added to each test tube and the mixture incubated for 15 minutes for colour development. 
The absorbances were measured using a Shimadzu Double -beam spectrophotometer at 
600nm against the blank that had oi-naphthol being replaced by absolute ethanol. The 
absorbance values were converted to micromoles of c l-  Naphthol produced by reference 
to the standard curve. The calibration curve was obtained from the results using linear 
regression in MINITAB statistical software programme (See Appendix IV).
3.6.2.1.2. p-Naphthol
A 0.5M solution of /3-Naphthol (MW 144.2) in 10ml of absolute ethanol was prepared. 
This solution was serially diluted 1 in 10 to give a concentration of 0.005M. This 
concentration was further serially diluted 1 in 2 to obtain the concentrations 25x10"4, 
12.5x10^, 6.25x10^, 3.125x10"* in test tubes. The different concentrations were then 
used to establish the calibration curve. Into each of the five test tubes containing 2.8 ml 
of 0.07M-phosphate buffer (pH 7.2), was added separately 0.1 ml of the prepared /3- 
naphthol solution. A test tube containing ethanol (0.1ml) was also set up as a blank. 
SDS-FBS solution (0.5ml) was added to each test tube and the mixture incubated for 15 
minutes for colour development. The absorbances were measured at 450nm against the 
blank that had j3-naphthol being replaced by absolute ethanol. The absorbances of the 
sample measurements were converted to micromoles of /3-Naphthol produced by 
reference to the standard curve. The calibration curve was obtained using linear 
regression in MINITAB statistical software programme (See Appendix IV).
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3.6.2.1.3 Calibration curve of bovine serum albumin (BSA)
Bovine serum albumin (0.1 g) was dissolved in 100 ml distilled water to obtain 0.1 /o 
stock solution. This was first diluted 1 in 10 after which 1 in 2 serial dilutions were made 
to obtain concentrations ranging between 10 x 10"2 g/L -  0.625 x 10 2 g/L which were 
used to establish the calibration curve. A volume of 0.1 ml BSA solution was transferred 
into test tubes and 2 ml of reagent B [50 ml of Reagent A (10 g of sodium carbonate and 
2 g sodium hydroxide in 500 ml distilled water) to 0.5 ml each of 1 % Q 1SO4 and 1 % 
sodium tartrate] was added and the mixture was allowed to stand for 20 minutes for 
colour development. Readings were taken on a spectrophotometer at 750 nm against a 
control that lacked BSA solution.
3.6.2.2 Enzyme preparation and assay
Individual larvae of DBM were separately homogenized using sterilized plastic pestle in 
a 1.5 Eppendorf tube-containing 0.3ml of potassium phosphate buffer (pH 7.2). The 
resultant homogenate was centrifuged at 4000 rpm in an Eppendorf centrifuge 5415C for 
2 min at 1 min interval and was then used as enzyme source for carboxylesterase assay.
A reaction mixture consisted of incubating 0.1 ml of the homogenate with 0.1 ml of 30 
mM CMiaphthyl acetate, in absolute ethanol for 10 min. at 40 °C in 2.8 ml of phosphate 
buffer (pH 7.2) in a shaking water bath. After incubation, 0.5 ml of (SDS-FBS) was 
added for colour development. This mixture was incubated for 15 min and read at 600 
nm with a spectrophotometer against a control that lacked en2yme. The homogenate 
(0.1ml) was also incubated for 10 min. at 40°C with /3-naphthyl acetate in absolute 
ethanol as substrate. The mixture was incubated with SDS-FBS for 15 minutes to allow 
colour development after which it was read at 450 nm on the spectrophotometer against a 
control that lacked the enzyme. For DBM larvae with higher activity above the
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measmable range, the homogenate was appropriately diluted 1 in 10 with phosphate 
buffer before assay.
3.6.2.3 Protein determination
Protein contents of all enzyme preparations used were determined by the method of 
Lowry et al. (1951) as adapted by Owusu et al. (1994) and Kaiwa (2000) with slight 
modifications. Aliquots (O.ml portions) of the homogenized enzyme solution were 
transferred into test tubes and 2 ml Reagent B (see 3.5.2.1.3) was added to it. This 
mixture was allowed to stand for about 30 minutes. The dilute phenol reagent (0.25 ml) 
was added and the reaction mix was incubated for further 20min to allow for a blue 
colour development. Readings were taken on a spectrophotometer at 750nm against a 
control that lacked an enzyme source.
3.6.3 Polyacylamide gel electrophoretic analysis of esterases
Resistance mechanism for organophosphate and pyrethroid insecticides in DBM was 
reported to involve the combined effect of increased oxidative and hydrolytic enzymes 
(Siegfried et al., 1990; Siegfried and Scott, 1991). Even though the hydrolytic enzymes 
have been quantified and documented, polyacrylamide gel electrophoresis can be used to 
further characterize the esterase enzyme based on the band sizes, electrophoretic 
nobilities and staining intensity of different isozymes.
3.6.3.1 PAGE analysis of DBM esterase isozymes
Each insect was homogenized in 33ml of sample buffer using sterilized plastic pestle in a 
1.5ml Eppendorf tube to break the cells and centrifuged at 4000 rpm in an Eppendorf 
centrifuge 5415C for 15-20 minutes and supernatant used for electrophoresis.
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Non-denaturing discontinuous (2.5%; 7%) polyacrylamide gel electrophoresis (PAGE) 
was performed in tris-glycine buffer (pH 8.3) with slab plate ATTO CORPORATION, 
Japan. Fifteen millilitres of supernatant was loaded along with 50g sucrose in running 
buffer at pH 8.9, into wells of the polymerized stacking gel. The electrophoresis was 
conducted at a constant voltage of 150V supplied by an electrophoresis power supply 
pack ATTO AE-3121 (ATTO- Corporation, Japan) until the tracking dye had moved to 
the interface between the stacking gel and separating gel.
Staining of the gel for protein bands corresponding to esterase activity was done in a 
plastic tray containing staining solution [(100ml of 0.2 M phosphate buffer, pH 6) 
containing 2ml a-naphthyl acetate (30mM) and 0.2g fast blue BB salt], at room 
temperature in the dark for 45 minutes. After the protein bands appeared, the gel was 
transferred from the staining solution into the fixing solution (7% acetic acid) overnight 
in a slow mechanical shaker to destain the background. The destained gel was then 
visualized under an ordinary light illuminator and stored in distilled water to prevent it 
from drying up. While still soaked in distilled water, the destained gel was transferred 
onto chromatographic filter papers 3mm thick (Tokyo Roshi Kaishi, Ltd, Tokyo, Japan) 
and overlaid with a cellophane membrane previously softened by immersion in distilled 
water then left to dry over a flat hard board for 3 days.
3.6.4 PCR amplification of B.t resistant gene
The DBM genomic DNA was extracted and Polymerase Chain Reaction (PCR) was used 
to detect B.t. resistant gene.
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3.6.4. 1. DNA extraction
The PCR method was slightly modified from Heckel et al. (1999) for amplification of Bt 
resistance gene in DBM. The amplification process utilised two specific primers c39- 
45lp 1 (5'-CCG TGC TGA GCA TTG GAC AGT GAG-3') and c39-451p2 (5'-TTA 
ACT ATA TTT GTT GGT GAC GAT AAG GTG-3'). The primers anneal on the rDNA 
of both susceptible and resistance strains of DBM, the amplified products are expected to 
be 325 bp.
DNA was extracted from the adult DBM using the Bender buffer method modified from 
Collins et al. (1987). Each adult was homogenized using sterilized plastic pestle in a 
1.5ml eppendorf tube containing 200|i.l Bender buffer (preheated at 65°C) followed by 
incubation at 65°C for 30 minutes. Samrated phenol+chloroform buffer (125fil) was 
added to the homogenate, vortexed briefly and spun at 14000 rpm for 10 minutes. The 
supernatant was transferred into a fresh tube, and 250pi of chloroform was added. This 
mixture was vortexed briefly and spun at 14000 rpm in the Eppendorf centrifuge 5415C 
for 10 minutes. The supernatant was then transferred into a fresh tube and 250|il of pre­
chilled absolute ethanol and lOfxl of potassium acetate 8 M added, and mixed well by 
tube inversion followed by incubation at -40°C for 1 hour. This was followed by final 
centrifugation at 10,000 rpm for 10 minutes to pellet the DNA after which the 
supernatant was discarded. The pellet was rinsed with 200|il of 70% ethanol the tube 
swirled gently and the DNA re-pelleted by centrifugation at 10,000 rpm in the Eppendorf 
centrifuge 5415C for 5 minutes. The supernatant was discarded and the tube opened and 
inverted over a paper towel to evaporate to dryness before being re-dissolved in 25^1 TE 
+ RNAse (5 fig/ml) or sddH20  and incubated in ice for about an hour followed by storage 
at -20°C till needed for PCR.
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3.6.4.4 Polyacrylamide gel electrophoresis of PCR products
Polyacrylamide gel electrophoresis was used to resolve the bands whose sizes were close 
and beyond the resolution of the agarose gel electrophoresis of the susceptible and the 
resistant DBM strains. The composition of the 7% polyacrylamide gel preparation used 
and solution preparation are outlined in Appendix II. A vertical electrophoresis gel tank 
(BIORAD, USA) was used. Before loading the samples the wells were flushed with 
sdc!H20  to remove excess urea. Fifteen microliter of the PCR products of each reaction 
was mixed with 10 fxl of bromophenol blue dye and loaded into each well and the gel run 
at 13mA and 100 volts for 7 hours. Thereafter, the glass plates were separated and the gel 
carefully transferred into a plastic tray containing 5% ethidium bromide for 5-10 minutes 
to stain. The gel was then photographed as described under section 3.6.4.4
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3.7.4. Solid phase extraction
One millilitre aliquot of cabbage extract was pipetted into a vial and the solvent 
completely dried (evaporated) under nitrogen gas and then redissolved in 0.5 ml of 
hexane. The SPE was equilibrated by allowing 2 ml methanol to run through the tube till 
the solvent front was about 1 mm above the column packing. After equilibration, the 
sample taken up in hexane (0.5 ml) was applied. Methanol, ethyl acetate, and hexane 
were used in that order to elute the insecticide residues in order of polarity. The eluent 
was collected in 2ml fractions for each solvent into pre-weighed vials. The eluted 
fractions were dried under nitrogen gas and the vials reweighed to obtain weights of 
extracted residues. The residues were re-dissolved in appropriate volumes of eluted 
solvents to give sample concentrations of lOmg/ml. Portions of the samples (250(xl) 
were used for brine shrimp toxicity bioassay and mortality data was recorded after 24 
hours.
3.7.5 Bioassay of cabbage extracts
A modified methodology outlined by Meyer et al. (1982) was adopted.
3.7.5.1 Hatching of Brine shrimps
The brine shrimps eggs used for the bioassay were obtained from Brine Shrimp Direct, 
California, USA. To hatch the eggs, a saline solution of concentration 25 g/L was 
prepared by dissolving sea salt in distilled water. This was filtered through Whatman 
number 1 filter paper and used to fill a small-perforated dividing tank (about % full). A 
spatula was used to transfer the brine shrimp eggs to the covered half of the tank, the 
other half of the tank being open, allows shrimps to move towards light after hatching.
also subjected to solid phase extraction (SPE) in Alltech Prevail Cig solid phase
extractor.
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3.7 Residue Level Estimation
Residues are present in very small quantities as heterogeneous compounds, including 
biological materials like plants. For the successful analysis of residues in cabbage head 
samples, several steps were followed, these included sampling to get a representative of 
the whole lot of material for average quantification of residues and extraction, in a 
solvent to remove the residues from other components of the sample matrix. The extract 
was cleaned up to remove extraneous materials that were co-extracted from the analytical 
sample. The eluates were then concentrated to reduce the volume of the solvent 
containing the insecticide residues and the residues were finally quantified and identified 
using brine shrimp Artemia salina Leach, as a test organism (Bioassay).
3.7.1 Sampling
Insecticide residue level was determined using mature and marketable cabbage heads 
from the cabbage farms in the selected study sites as well as on the insecticide free 
cabbage heads grown in a screen house, which served as control.
3.7.2 Cabbage samples
Guided by the survey, particular farms were selected for collection of cabbage samples. 
The sampling was based on the following facts
i. The farms with the highest or lowest pest infestation, observed from the level of 
destruction on the leaves.
ii. The most commonly used organophosphate and synthetic pyrethroid insecticides.
iii. The farmers’ agronomic practices like the mode of watering or concentration of 
the insecticides applied.
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3.7.2.1 Sampling for cabbage
After selecting the farms, sampling was only done when the farmers were harvesting 
their cabbage for sale. This precaution was necessary to ensure that cabbage heads 
selected were ready for sale and for consumption. To randomize the cabbage head 
sampling, the field was mapped on a piece of paper, and numbers given to rows of plant 
and each plant in each row. The numbers of the rows were balloted for before picking 
ballots for plants in each row and the particular heads harvested were kept in polythene 
bags. The cabbage heads were sliced longitudinally into cone shapes and 50 grams of 
each sample was weighed into labelled beakers, and then transferred into a conical flask 
and 25ml hexane was added, and then was stored at 4°C for residue extraction.
3.7.3 Extraction, concentration and analysis of insecticide residues
Extracting mixture consisting of 55 ml hexane and 20 ml butanone (ethyl-methyl ketone) 
was used for extraction. Thirty millilitre of hexane was used for extraction after which 
the sample was blended for 4 minutes, at a minute interval, and the extract decanted into 
a 250ml flask. Further portions of the extracting solvent (35 ml and 40ml) were added 
and the homogenate blended, the extracts obtained were decanted into a flask. The 
homogenate was centrifuged in a bench top centrifuge at 3000 rpm (rotor radius, 11.5 
cm) for 5 minutes at room temperature. The pooled extracts were concentrated using the 
Rotary Vacuum Evaporator to about 2 ml.
The concentrate was transferred into a 10 ml vial. The rotary vacuum flask was then 
rinsed twice, each time with 2 ml of hexane, and added to the concentrated extracts in the 
vial. The samples were dried under nitrogen gas and re-dissolved in 5 ml hexane. The 
vials were wrapped in aluminium foil and stored in refrigerator. The crude extract was
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3.7.4. Solid phase extraction
One millilitre aliquot of cabbage extract was pipetted into a vial and the solvent 
completely dried (evaporated) under nitrogen gas and then redissolved in 0.5 ml of 
hexane. The SPE was equilibrated by allowing 2 ml methanol to run through the tube till 
the solvent front was about 1 mm above the column packing. After equilibration, the 
sample taken up in hexane (0.5 ml) was applied. Methanol, ethyl acetate, and hexane 
were used in that order to elute the insecticide residues in order of polarity. The eluent 
was collected in 2ml fractions for each solvent into pre-weighed vials. The eluted 
fractions were dried under nitrogen gas and the vials reweighed to obtain weights of 
extracted residues. The residues were re-dissolved in appropriate volumes of eluted 
solvents to give sample concentrations of lOmg/ml. Portions of the samples (250^1) 
were used for brine shrimp toxicity bioassay and mortality data was recorded after 24 
hours.
3.7.5 Bioassay of cabbage extracts
A modified methodology outlined by Meyer et al. (1982) was adopted.
3.7.5.1 Hatching of Brine shrimps
The brine shrimps eggs used for the bioassay were obtained from Brine Shrimp Direct, 
California, USA. To hatch the eggs, a saline solution of concentration 25 g/L was 
prepared by dissolving sea salt in distilled water. This was filtered through Whatman 
number 1 filter paper and used to fill a small-perforated dividing tank (about % full). A 
spatula was used to transfer the brine shrimp eggs to the covered half of the tank, the 
other half of the tank being open, allows shrimps to move towards light after hatching.
also subjected to solid phase extraction (SPE) in Alltech Prevail Ci8 solid phase
extractor.
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Hatching tank was left under fluorescent light at 22°C for 48 hrs before nauplii were used 
for bioassay.
3.7.5.2 Calibration curves for brine shrimp toxicity to the standard insecticide
The standard curves for the insecticide standards; chlorpyrifos-methyl, pirimiphos- 
methyl, deltamethrin, cypermethrin, lambda-cyhalothrin and permethrin were established 
to be used in estimation of the insecticide concentrations in the cabbage heads. A 
preliminary bioassay was done to determine the concentration that gives 10% and 90% 
mortality. Based on this, 100^1 of each of the standard solutions of chlorpyrifos-methyl, 
deltamethrin, cypermethrin and permethrin were serially diluted (1 in ten dilution) to 
give two stocks of concentrations lOfig/ml and 1 ng/ml using these solutions as stock, 
0.5ng/ml, 5ng/ml, 50ng/ml, 500ng/ml concentrations of each of the standards were 
transferred into a 10ml vials, dried under nitrogen gas and used for the bioassay. 
However 200 |il of each of standard solutions of lambda-cyhalothrin and pirimiphos- 
methyl were used, to make a stock solution of 20|ig/ml and (1 in 10 dilution) 2jag/ml. 
Using these two stock solutions, lng/ml, lOng/ml, lOOng/ml and lOOOng/ml 
concentrations of these insecticides were used for the bioassay. The insecticide standards 
were dissolved in hexane. The dried sample in each vial was taken up in acetone (40p.l) 
and sea-water (5ml) was added for the brine shrimp bioassay. Four replicates were 
bioassayed for each insecticide standard. Mortality data was recorded for each insecticide 
standard dilution and the control. The data was analysed using probit package in 
MINITAB 12 windows software to determine LC5o and L C 9 5 , and the curves were 
obtained for all the standard insecticides.
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3.7.5.3. Bioassay to determine appropriate solvent that elutes the insecticide 
standards
Preliminary bioassay was conducted using known volumes of the insecticide standards to 
determine the solvents, which eluted the insecticides from the Cig SPE tubes. Different 
solvents were selected in order of their polarity: methanol, ethyl acetate and hexane were 
used. The SPE tubes were preconditioned using 2ml of methanol, after which lOOjxl of 
each of the insecticide standards (pirimiphos-methyl, chlorpyriphos, deltamethrin, 
cypermethrin and lambda-cyhalothrin) was passed through the SPE. Two ml of each of 
the extracting solvents from methanol, ethyl acetate and hexane were added to the tube. 
The eluents were collected into 10ml vials and dried under nitrogen gas. Forty microlitres 
of acetone was added to re-dissolve the content in the vials. A volume of 2ml sea salt (25 
g/L) was added to each vial after which 10 brine shrimps were added into each vial and 
made up to 5 ml mark with sea water. The set up was kept under fluorescent light and 
mortality count taken after 24 hr and another set up without the extract served as a 
control.
The Abbots formula (Abbot, 1925) was used to correct for deaths in the control samples.
CM = (% T - %C1 xlOO 
(100 -%C)
Where, CM = corrected mortality
%T = percent test effect mortality 
%C = percent control mortality
The corrected percentage mortalities were used to estimate the concentration of 
insecticide residues in cabbage, from the linear regression equation of dosage- mortality 
curves of the standard insecticides.
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3.8 Data Analysis
SPSS version 10 (SPSS Inc. USA) analytical package was used for analyzing the survey 
data, all mortality data was corrected for natural mortality using Abbott’s formula 
(Abbott, 1925) and Probit package in MINITAB 12 windows software was used to 
determine LC 50  and LC 9 5  values (Finney, 1971). Analysis of variance was used to test for 
significance in the activity of carboxylesterase enzymes and MRL values of the residues 
and Least significant Difference (LSD) used to separate means in Genstat 5 Release 3.2 
Copyright 1995, Lawes Agricultural Trust (Rothamsted Experimental Station) statistical 
package.
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CHAPTER FOUR
RESULTS
4.1 Insecticide use pattern survey
4.1.1 Agronomic practices
The survey covered a total o f 30 cabbage farms in Accra suburbs and Mampong- 
Akuapem. It revealed that all the cabbage growers visited were small-scale farmers since 
14 (47%) of the farms cultivated were less than Vi hectare (ha); 7 (23%) o f the farms 
were about a hectare; 7 (23%) were 1-2 ha. and only 2 (7%) of the farms were more than 
2 ha in size. Most o f them were perennial cabbage farmers. Since 11 (36%) o f the 
farmers had been involved in cabbage production for a minimum period o f 5-10 years. 
Five (17%) o f the farms have been cultivated continuously for over 20 years (Fig.6). The 
main equipment mostly used on the farms was watering can. This was fitted to the 
shower type nozzle by only 3 (9%) of the farmers, while the rest fitted flat metal plate. 
Farmers at Mampong were using bucket and cup and only 3 (10%) o f the farmers used 
water hose (Fig. 7).
4.1.2 Pests and pest control
The survey showed that 28 out o f 30 of the farms surveyed had a problem with insect 
pest infestation Twenty-two o f the farmers reported that DBM was the most serious and 
destructive pest o f cabbage in the farms (Fig. 8). With regard to period o f prevalence, 
DBM was mostly found in dry season; 23 (77%) of the farmers confirmed this, while 6 
(20%) said the pest was present all year round. Chemical control was the most 
predominant method used by 29 (97%) of farmers to control pests while a few practiced 
IPM, which include cultural approach like crop rotation and frequent watering.
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> 20 Years
5-10 Years 
30%
Fig. 6 Duration under which the farms have been used for Brassicas production
80 -
70 -
60
S' 50 - 
c «
g. 40 
£
J  3 0 ­
20 
10 
0
Sprinkler Watering can Water hose Bucket and cup
Irrigation Equipment
Fig. 7 Types o f irrigation equipment used in cabbage farms
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6.7
Combination
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4.1.3 Insecticides and their use pattern
The survey sought information on current and previously used insecticides. Currently the 
most widely used insecticides to control cabbage pests in the study areas are as shown in 
Table 5 and the most popular insecticides on cabbage farms previously are shown in 
Table 6. When all the insecticides were grouped into their various classes, the results 
showed that apart from growth regulators, the use o f organophosphates was on the rise 
(Fig.9). On the contrary, pyrethroids and the biopesticides: (B.t. formulations; Biobit and 
Dipel) usage has declined over the years. Twenty (66.67%) of the farmers attributed the 
decline in usage o f these insecticides to ineffectiveness against the target pests especially 
Dipel, Biobit and cymbush while 8 (26.67%) respondents said the chemicals were out of 
stock, compelling them to look for alternatives even though the previous ones such as 
(karate and biobit) had not failed. Some o f the fanners stopped because the chemicals 
were too expensive.
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Table 5. Current insecticide use pattern in Accra suburbs and Mampong Akuapem
Agrochemicals Class Total (%) usage
Rimon Growth regulator 10 (32.39%)
Regent Growth regulator 6(19.72%)
Dursban Organophosphate 3 (9.86%)
Dize DDVP Organophosphate 2 (7.04%)
Cyperdim super Pyrethroid+organophosphate 2 (5.63%)
Dipel Biopesticide 1 (4.23%)
Pawa Pyrethroid 1 (2.82%)
Amektin Growth regulator 1 (2.82%)
Others (karate1, Cypercal2, 
Deltpaz3, Deltaplan4, Decis5, 
Actelic6, Cyperphos7, D3368 
and Polytrine9, Biobit10
l-5=Pyrethroids 
6-9=Mixtures (Sps+Ops) 
10=Biopesticide
4 [12.68%]
Table. 6. Previous insecticide use pattern in Accra suburbs and Mampong-Akuapem
Agrochemicals Class Total (%) usage
Dipel Biopesticide 5 (18.18%),
Karate Pyrethroid (SP) 5 (18.18%),
Biobit Biopesticide 4 (12.73%),
Regent Growth regulator (IGR) 3 (10.91%),
Cymbush Pyrethroid (SP) 2 (7.27%),
Polytrine Mixture (Op + SP) 2 (5.45%),
Cydimsuper Mixture (Op + SP) 2 (5.45%),
Actelic Mixture (Op + SP) 2 (5.45%),
Orthine Pyrethroid (SP) 2 (5.45%),
Dursban Organophosphate (OP) 1 (3.64%),
Others (Cyperphos1, 
Rimon2, Neem3 and 
Thionex4
l=Mixture, 2-1GR 
3=Biopesticide, 4= Op
2 [7 27%]
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% 
Fr
eq
ue
nc
y
80 -I 73.3
70 -
60 ■
50 Pi
40 ■ lltl
30 • sli
20 (jfgfl
10 • 
0 8
Diamondback moth
33
Aphids
16.7
3.3 3.3
Diamondback moth Diamondback moth Diamondback moth, 
& Aphids & Cabbage looper Aphids & Mole
Pest
Fig. 8 Various pests o f Brassicas in the cabbage farms
crickets
Growth Regulators Organophosphates Pyrethroids Biopesticides
Categories of Agrochemicals
Previous ■  Current
Mixtures
Fig. 9 Comparison between the previous and currently used agrochemicals in the 
cabbage farms
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On insecticide use, spraying in 26 out of 30 farms started at seedling stage and these 
insecticides were either sprayed alternatively by 19 (63 .33%) of the farmers or applied as 
cocktail mixtures by 11 (36.67%) o f the farmers The various combinations o f 
insecticides used by growers were as follows:
i Regent and Rimon [growth regulators]
ii Dize DDV [organophosphate], Rimon [growth regulators] and Cydunsuper
[pyrethroid + organophosphate]
iii Regent [growth regulators] and Dursban [organophophate]
iv Cydim super [pyrethroid + organophosphate] and Dursban 
[organophosphate],
v Rimon [growth regulator] and Deltapaz [pyrethroid]
vi Rimon, Regent [growth regulators] and Dize DDVP [organophosphate]
4.1.4 Mode of insecticide application
The growers were able to give the various volumes of insecticides used per sprayer. 
Majority used the lid o f chemical containers as standard measure o f dosages and for 
Dipel, in Mampong, farmers used matchbox. In addition to this, dosages in the farms 
were difficult to estimate because the application equipment was not calibrated before 
use. The main mode o f application was manually operated knapsack sprayer. This was 
attached to different nozzles Fourteen (46.43%) used yellow polyjet, 12 (39.29%) 
blue/black, 3 (10.71%) green, and 1(3.57%) used red polyjet. It was also observed that 18 
(60%) of the growers did not wear any protective clothes during spraying while 8 
(26 67%) were partly protected by using gloves and facemasks and only 4 (13.33%) wore 
protective clothes.
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4.1.5 Frequency of insecticide application
Apart from the use of various modes o f application, the interval between insecticide 
spraying and irrigation varied greatly among the respondents. Ten (33%) of the 
respondents watered within the same day o f insecticide spraying while majority, 16 out 
of 30 gave 1-3 days interval and the longest was by 1 (3%) farmer who used more than 5 
days interval (Fig. 10). The growers also sprayed the insecticides frequently and at short 
intervals, having 21 (70%) o f them spraying at a frequency less than a week (Fig. 11).
This high frequency of spraying was reflected by 14 (46%) respondents who observed 
pre-harvesting interval (PHI) as short as less than a week (Fig. 12). Although some 
growers used biopesticides like Biobit and Neem towards harvesting time with short PHI, 
others claimed they obeyed manufacturers’ instructions concerning every group o f 
chemicals used. Yet a few farmers during heavy pest infestation could spray the cabbages 
and sell them immediately.
The improper use o f insecticides reflected above was because the common source o f 
professional advice on use and handling o f insecticides were fellow farmers and pesticide 
dealers, except for 11 (36%) who received advice from Agricultural extension agents of 
this number 3 (10%) were visited within 1-2 weeks, while 3 (10%) received their visits 
well above two years span and some could not tell the frequency of the visits due to their 
irregularity or they were not visited at all (Fig. 13).
With regards to insecticide residue awareness 19 (63.33%) had no idea about it (Table 7). 
Of those who were aware, the majority (82%) heard about the perceived risks caused by 
improper use of insecticides through the mass media (radio, news paper, television) as 
the main source of information (Fig. 14).
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> 5 days 
4-5 days 3%
Same day 
33%
Fig. 10 Interval between insecticide spraying and subsequent irrigation
> 2 w eeks
3%
4-7 days
37%
Fig. 11 Frequency o f insecticide spraying
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% 
Fr
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y
2-3 w eeks 
37%
Manufacturers
recommendations
7%
3-4 w eeks 
10%
< 1 week 
46%
Fig. 12 Pre-harvest interval observed by cabbage farmers
1-2 weeks 1-3 months 5-12 months Once a year >2 years None
Period
Fig. 13 Frequency o f visits by the extension officers
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Table 7. Farmers’ Awareness about some agronomic practices and
insecticide residues.
Practice/problems Yes No
Pest problem 93.3 6.7
Nursery treatment 86.7 13.3
Keeping o f farm records 16.7 83.3
IPM training 16.7 83.3
IPM application 13.3 86.7
Residue awareness 36.7 63.3
Fellow farmers
82%
Fig. 14 Sources o f information on insecticide residues awareness
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4.2 Susceptibility of DBM to insecticides
Diamondback moth was found to be tolerant to all the insecticides assayed. Due to lack 
of reference strain, the recommended rate expected to give 95 % mortality was used as 
point o f reference. This was compared to the L C 9 5  to get the number of fold tolerance. 
Dose response mortality curves were used to determine the L C 5 0  and L C 9 5  values. The 
L C 9 5  values were found to be much higher than the recommended dosage for all the 
insecticides assayed, which included Dursban (chlorpyrifos-methyl), Deltaplan 
(deltamethrin), Pawa (lambda-cyhalothrin), Cypercal (cypermethrin) and Dipel (B.t. var 
kurstaki). However, there were also wide variations in the response o f  the insect 
populations from different sites (Tables. 8)
4.2.1 Pawa (Lambda-cyhalothrin)
The L C 9 5  estimated for Pawa for the Airport population was 8527.3mg/L which when 
compared with the recommended dosage of 83mg/L gave 102 7 fold tolerance. The 
Dzorwulu population o f DBM followed closely with 100.3 fold tolerance and the least 
tolerant population was from Mampong which recorded 57.9 fold tolerance (Table 8). 
Pawa was the most highly tolerated pyrethroid in all the sites and overall it was second to 
dursban in all the study sites with respect to resistance.
4.2.2 Cypercal (cypermethrin)
Airport DBM population showed the highest level o f tolerance to Cypercal, recording an 
LC 9 5  of 8565.2 mg/L which when compared to the recommended dosage o f lOOmg/L 
gave 85.7 fold tolerance. Other Accra suburb sites recorded nearly similar values o f fold 
tolerance. Mampong however recorded the lowest which was 54 6 fold tolerance
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4.2.3 Deltaplan (Deltamethrin)
This was shown to be the most effective o f all the pyrethroids assayed. Among the 
conventional insecticides it had the lowest LC95 value for all the sites However it was 
least effective in Dzorwulu where DBM population recorded an LC95 value o f 
2988.4mg/L against a critical dosage of 37.5mg/L giving 79.7 fold tolerance.
4.2.4 Dursban (Chlopyrifos-methyl)
The bioassay revealed that Dursban was the least effective o f all the insecticides assayed. 
It generally recorded the highest L C 9 5  value in all the sites and except for Madina and also 
recorded the highest fold tolerance. Specifically, Dzorwulu population o f DBM proved to 
be the most tolerant to Dursban with L C 9 5  of 579438 Img/L against recommended dosage 
of 3000mg/L giving a 193.2 fold tolerance. The Madina DBM population was 
comparatively less tolerant
4.2.5 Dipel {Bacillus thuringiensis Var. Kurstaki)
In contrast to the conventional insecticides, DBM was found to show some significant 
level of susceptibility to Dipel in all the sites. The highest recorded LC95 for B.t. 892.7mg/L 
was recorded in Mampong as against a critical dosage o f 206 mg/L, representing 4.3-fold 
tolerance. On the other hand, most o f the Accra suburb populations were more susceptible 
giving about 2.8 fold in Airport with the Dzorwulu DBM population showing the least 
tolerance In general, while Dzorwulu population showed the highest level of tolerance to 
the organophophates and pyrethroids tested, it showed the highest level o f susceptibility to 
B.t.
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The slopes o f the curves indicated the potency of the insecticides Mortality curves with 
high slope values were more potent in controlling DBM, since low insecticide 
concentrations gave high larval mortality for example dipel (B .t). The most potent o f the 
pyrethroids was deltaplan and this was shown with relatively higher slope values compared 
to the other pyrethroids. While for dursban the least potent o f the insecticides recorded the 
lowest slope values meaning higher concentrations only resulted in low larval mortality
4.3. Cues for Cross and multiple-resistance
The DBM larvae were shown to indicate development o f cross-resistance to the commonly 
used pyrethroids and multiple resistance to the pyrethroids and organophosphates. These 
phenomena were tested across some selected sites The DBM population that tolerated 
Pawa could be fairly controlled by dursban in Madina, where 42 (52%) mortality was 
recorded. Similar populations were tolerant to dursban in Mampong and Airport where 
only 21 (26%) and 24 (30%) out of 80 mortalities were recorded for both sites respectively. 
The DBM population tolerant to pawa when exposed to deltaplan at Airport recorded only 
21 (30%) mortality. However, Deltaplan tolerant DBM population at the same site gave 
mortality of 8 (10%) for dursban. Conversely, when selection was done using Dursban and 
the population exposed to Pawa, mortalities ranging between 12 to32 (15-40%) were 
recorded in all the study sites. There was 90% mortality recorded in the DBM population 
selected for resistance by all the synthetic conventional insecticides bioassayed when 
exposed to B.t.
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4.4 Carboxylesterase Activity in Field populations of Diamondback 
Moth
For each o f the seven sub-study sites, 25 samples o f DBM larvae were assayed for the 
two enzymes a- and p-carboxylesterases, giving a total o f 350 larvae samples. The study 
sites were Mampong-Akuapem, Madina A (near Redco Junction), Madina B (Redco 
Area), Airport A (Airport West), Airport B (Opaibea Area near CSIR), Dzorwulu A 
(Around Power Station) and Dzorwulu B (Dzorwulu East near Kotobabi)
4.4.1 a-naphthyl esterase
The highest level of activity for a-naphthyl esterase was recorded in Mampong (Table 9). 
The Mampong DBM population was significantly different from all the other sites (P < 
05) However the lowest activity was recorded in Madina B, which was significantly 
different from Madina A and Dzorwulu A. The enzyme activity in Madina A and 
Dzorwulu A were however not significantly different
4.4.2 f3-naphthy] esterase
B-naphthyl esterase activity for Mampong DBM population was also very high while 
very low activities were recorded for Accra sub-sites. Comparatively, higher activities 
were recorded in Madina and Airport A. However, statistical analysis proved that the 
activity of the enzyme was only significantly different between Mampong population and 
the rest of the sites, with the other six sites in Accra suburbs showing no significant 
difference in activity.
Analysis of variance showed that a- and p- naphthyl esterase activities were highly 
significant at both (0.05 and 0.01) probabilities (Appendix IV).
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The frequency profiles of activity for the two enzymes for the various sites were shown 
to vary greatly (Appendix IV). In Mampong the modal frequency for a-naphthyl esterase 
activity was 1 Opmol/min/mg protein and p-naphthyl esterase 50mol/min/mg protein. 
While for the Accra surburb sites modal frequencies for both esterases varied greatly 
even within the same population. Specifically, for Dzorwulu DBM population a- 
naphthyl esterase activity was mostly recorded at 4pmol/min/mg in site A and 1 
pmol/min/mg protein in site B. For P-naphthyl esterase the modal frequency was 5 and -2 
pmol/min/mg protein in sites A and B respectively. While Airport populations had most 
frequencies o f activity between 1 and 2 ^mol/min/m activity for a- naphthyl esterase and 
P-naphthyl esterase had most frequencies at 15 and as low as -4  to Opmol/min/mg 
protein activity in Airport A and B respectively. Likewise Madina sites had the most 
frequencies for a-naphthyl esterase at 4 and 1 (jmol/min/mg protein activity recorded in 
site A and B respectively and for P-naphthyl esterase the modal frequencies were -5  to 0 
and 5 (jmol/min/mg activity for DBM populations in Madina A and B respectively 
(Appendix IV).
The study revealed that carboxylesterase had higher affinity for p-naphthyl acetate than 
for a-naphthyl acetate. This was indicated by the mean values o f the two enzymes. In 
contrast, for DBM population in Accra surburbs, a-naphthyl acetate proved to be a better 
substrate for reaction with a mean of 1.77 pmol/min/mg protein and a range o f 2.589­
0.915 (imol/min/mg protein, while P-naphthyl acetate recorded a mean o f 0 72 
jjniol/min/mg with arrange o f 2.925 and -0.798 pmol/min/mg protein.
Mampong DBM population showed the widest disparity in activity for both enzymes but 
the Accra DBM populations for example recorded no significant difference for p- 
naphthyl esterase. This implied that though all the insects quantitatively showed some
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carboxylesterase activity however there might be qualitative differences within insects 
even from the same population in one study site.
Concerning the insecticides used in the cabbage farms, lambda-cyhalothrin formulations 
(karate and pawa) were found in all the study sites except in Madina A and Mampong 
(Table 9). The bioassay showed DBM population in Mampong to be comparatively the 
most susceptible to Pawa (Table 8). Bacillus thuringiensis formulations (biobit and dipel) 
and dursban was used in all the farms except Madina B and Dzorwulu A  However, 
comparing with the susceptibility studies, Dzorwulu DBM samples were the least 
susceptible to dursban at the same time the most susceptible to dipel while the Madina 
populations were the most susceptible to dursban. Comparing these results to 
carboxylesterase activity, Madina B showed the least activity o f  a-naphthyl esterases 
followed by Dzorwulu A. However, the Madina populations comparatively exhibited 
relatively higher activity among the Accra surburb DBM samples for (3-naphthyl 
esterases. While cypermethrin formulations were used in all the other sites except in 
Dzorwulu and Airport B. Contrastingly, DBM population in Airport was the most 
resistant to cypermethrin. In addition deltamethrin, which was used in most o f the farms 
except Dzorwulu and Mampong, was least effective in DBM population in Dzorwulu.
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Table 9. Carboxylesterase activity of DBM populations and insecticide used in cabbage 
farms in Mampong Akuapem and Accra suburbs
Population/
Site
Insecticides used Carboxylesterases activity Mean 
(+ S.E) (jumol/min/mg protein
a-NA p-NA
Mampong-
Akuapem
■ i ----------H-------------
Dipel , dursban , 
cypermethrin4, orthine4,
9.55+1.253 49.3+10.46 *
Madina A Dize-DDVP2, dursban2, Decis4, 
orthine4, polytrine5, biobit1, 
actelic2,
2.18±0.4b 1.14+1.03°
Madina B Dize-DDVP2, decis4, 
cydimsuper5, karate4, actelic2, 
polytrine5, thionex2
0.92+0.14° 2.92+0.95°
Airport A Amektin , dursban , dize-ddvp , 
deltapaz4, actelic5,biobit1, 
dipel1, karate4,
1.05+0.25°° 1.26±0.8B
Airport B Dize-DDVP2, pawa4, polytrine5, 
karate4, dipel1, deltaplan4, 
cydimsuper5, 
biobit, dursban2
1.55+0.39bc 0.25+0.65 D
Dzorwulu A karate4 2.59±0.25b 0.45±0.8b
Dzorwulu B Amektin3, karate4, dursban"1, 
dipel1, biobit1, neem extract1
1.49±0.23“° -0.8+0.5115
*Regent3 and rimon3 were common to all the study sites
*The same letter within a column denotes no significance difference at 95% confidence
level (0.05) probability level
*1 Biopesticides
*2 Organophosphates
*3 Growth regulators
*4 Pyrethroids
*5 Insecticide mixtures
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4.5 Native Polyacrylamide Gel Electrophoresis (PAGE)
The results obtained from 120 DBM larvae collected from the four study sites revealed 
various distinct banding patterns in the field populations of DBM The variants were 
classified based on the number of bands, staining intensity, and electrophoretic mobility. 
Between two to seven bands were detected in the zymograms. These bands were 
scattered throughout two zones o f the gel: a fast moving upper zone and a slightly slower 
moving middle zone (Fig. 15). All the isozymes migrated to the cathode end of the gel. 
The fast moving heavily stained zone, composed of one narrow and one wide band, was 
detected in all the zymograms examined. The middle zone-bands however varied in 
number, width, and staining intensity, with some faint bands in front o f the main body o f 
the middle zone, being present in most of the zymograms.
Diamondback moth esterases were shown to be highly heterogeneous in nature. This was 
shown through polymorphism of isozyme patterns and general variations. Thus more 
than one zymogram pattern was observed for DBM larvae even within the same 
population (Fig. 15). The electrophoregram revealed higher frequency o f slow moving 
esterases, which confer more resistance in Mampong samples (Fig 16) than for the Accra 
suburb sites which had majority of the zymograms with the fast moving esterases which 
are associated with less resistance. Besides, the zymogram patterns for the Accra 
populations were relatively similar, compared with the Mampong population, which 
reflected a wide range of zymogam patterns. Among the Accra populations Madina 
samples had relatively higher frequency of the slow moving esterases and the site with 
the least frequency of this type of esterase was Dzorwulu.
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Fig. 15 Polyacrylamide gel electrophoresis o f  esterase isozymes o f DBM larvae showing 
the difference between fast- moving (heavily stained) and slow moving (lightly stained) 
esterase isozymes. And the various zymogram patterns o f the esterase isozymes within 
the same DBM population (Mampong).
Lanes 1-6 = carboxylesterase isozymes, S = Slow moving esterases, F =  Fast- moving 
esterases, Arrow = direction o f gel movement
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4.6 Molecular Identification of B.L resistance gene
Bender buffer protocol was successfully used to extract DNA from a total o f 140 samples 
of DBM. The genomic DNA was found to run together with 23.13kb in ethidium 
bromide stained 0.8% agarose ge! electrophoresis (Fig. 17). PCR amplification was 
successfid for 65 out o f the total 140 (46%) that were processed. The 65 consisted o f 24 
samples from Madina, 14 from Dzorwulu, 17 DBM samples from Mampong and 10 
samples were from Airport. In addition to this, 14 laboratory reared reference/ 
susceptible DBM strains from ICIPE, Kenya were also analyzed for B.t. resistance gene.
Those that were PCR positive 41 out o f  65 (61%) were characterized by the expected 325 
bp amplified DNA product (Fig. 18). This 325bp band was shown by all the samples 
collected from Airport. In addition to this Mampong, Dzorwulu and Madina DBM  
populations had 26 out o f 65 (40%) showing 760bp fragment (Fig. 19). Some samples 
from Madina and Mampong showed an extra 1100 bp band size. Most notably, were 
double bands o f sizes 325 bp and 776 bp sizes amplified in four samples from Madina 
and a triple band o f 325 bp, 776 bp and 1100 bp shown by two samples from Madina and 
Mampong. The susceptible strains also possessed a band size similar to the diagnostic 
325bp DNA fragment. Since the size difference between the PCR products o f the 
resistant and the susceptible gene differ by only 20bp, this cannot be easily resolved by 
agarose gel, but PAGE hence the latter was used.
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Fig. 17 Ethidium bromide stained 0.8% agrose gel electrophoresis o f genomic DNA  
isolated from the DBM larvae Lanes 1-6 and molecular weight marker (Lane M). 
Population
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Fig. 18 Ethidium bromide stained 2% agarose gel elecrophoresis o f  PCR products 
obtained after amplification o f DBM genomic DNA with specific primers (c39-451pl 
and c39-45 lp2) for the B.t. resistant gene.
Lane M=molecular weight marker, Lane 1- 4 = 325 bp (expected gene band), Lanes 5 
and 6 = 776 band size
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4.7 Polyacrylamide gel electrophoresis of PCR Products
Polyacrylamide gel electrophoresis of PCR products of DBM larvae revealed presence o f 
double and sometimes triple bands among the resistant populations, which were observed 
in all the study sites. The DNA fragments amplified were 325 bp, 776 bp and 1100 bp 
band sizes (Fig. 19). Seven out 65 PCR products showed 325 bp and 1100 bp single bands 
from DBM samples from Airport and Mampong respectively. While 26 samples from 
Madina exhibited the 776 bp fragment. The double bands of sizes 325 bp and 776 bp 
were amplified in 13 DBM samples from Mampong, Madina and Dzorwulu. Nine out o f 
65 samples showed the triple bands of sizes 325 bp, 776 bp and 1100 bp. PAGE results 
also showed that all the susceptible strains o f DBM produced single bands which were 
slightly above 20 bp the expected 325 bp bands size o f the resistance strains, however the 
resistant strains had multiple bands (Fig. 20).
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1 2 3  4 5 M 6 7 8 9 10
Fig. 19 Polyacrylamide gel electrophoresis o f PCR products obtained from amplification 
of DBM DNA with specific primers (c39-45 lp l  and c39-45 lp2).
Lane 2 = single band 325 bp, Lanes 1, 3, 5 and 9 = Single band 776bp, Lane 10 = Single 
band 1100 bp, Lanes 4 and 7 = Double band sizes 325 bp and 776 bp), Lanes 6 and 8 — 
Triple band sizes 325 bp, 776 bp and 1100 bp, Lane M  = 100 bp molecular weight 
marker.
1100 bp 
776 bp
325 bp
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1 2 3 4 M 5 6 7  8
345 bp
1100 bp
440 bn 
325 bp
Fig. 20 Polyacrylamide gel electrophoresis o f PCR products obtained from amplification 
of DBM DNA with specific primers (c39-451pl and c39-451p2) showing the difference 
between B .t  susceptible and resistant DBM larvae.
Lanes 1-4 ^susceptible strains, M = 100 bp ladder and Lanes 5-8 resistance strains o f 
DBM
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4.8 Residue Level estimation
4.8.1 Brine shrimp bioassay for the insecticide SPE standards fractions
Mortality data recorded following bioassay of SPE fractions obtained by elution o f  the 
standard insecticides (permethrin, cypermethrin, deltamethrin, lambda-cyhalothrin, 
chlorpyrifos-methyl and pirimiphos-methyl) using methanol, ethyl acetate and hexane are 
shown in (Fig. 21). It was observed that pyrethroids were mostly eluted in hexane 
fractions which gave nearly 100% mortality, except for lambda-cyhalothrin which gave 
only 60% mortality. On the other hand, chlorpyrifos was eluted in methanol giving 
100% brine shrimp mortality, while pirimiphos-methyl was eluted in ethyl- acetate which 
also gave 100% brine shrimp mortality. Thus, cypermethrin gave no mortality in ethyl 
acetate fraction, 10% and 100% of the brine shrimps died in methanol and hexane 
respectively. Furthermore, deltamethrin and permethrin gave about 40% mortality for 
ethyl acetate fractions but 100% mortality was recorded for hexane fractions. 
Chlorpyrifos only gave 100% mortality in methanol fraction while pirimiphos also gave 
100% mortality in ethyl acetate, with no mortality being observed in the other fractions. 
Dose-response mortality curves o f the insecticide standards were obtained from probit 
analysis and their L C 5 0  and L C 9 5  values and the slopes o f the curves were recorded as 
shown in Table 10. The potency o f pyrethroids based on LC50 was as follows: 
cypermethrin > permethrin>deltamethrin> lambda-cyhalothrin (Table 10). For 
organophosphates, chlorpyrifos was three times as potent as pirimiphos-methyl
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120
Permethrin Cypermethrin Lambda- Deltamethrin Pirimiphos- Chtorpyrifos-
cyhalothrin methyl methyl
Fractions of insecticide standards
0 Methanol ■  Ethyl acetate □  Hexane |
Fig. 21 Brine shrimp mortality profile o f SPE fractions o f insecticide standards
Table 10. Lethal concentrations of insecticide standards
Insecticides LCso (mg litre'1) 
(95% CI)*a
LC<js (mg litre1) 
(95% Cl)
Slope
Deltamethrin 374.0(286.9-524.7) 970.4(749.1-1411.9) 0.032
Cypermetherin 179.7(108.1-293.1) 634.0 (459.4-1053.5) 0.019
Pirimiphos-mehyl 684.1(483.9-1269.0) 1420.8(982.3-2805.8) 0.015
Lambda- 561.3(422.9- 64.5) 1413.6(116.8- 1960.3) 0.019
cyhalothrin
Chlorpyrifos- 222.0(177.1- 284.7) 489.2(402.0-628.1) 0.067
methyl
Permethrin 234.7(171.3-327.9) 766.3(596.9-1089.8) 0.037
*a Cl- is the confidence interval
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4.8.2 Estimation of residue levels in cabbage samples
The differential elution of the two organophosphate standards and pyrethroids from SPE 
(Fig. 21) with methanol, ethyl acetate and hexane were used as basis for fractionation o f 
these insecticides from extracts o f the residues obtained from cabbage. To estimate 
insecticide residue content in the different fractions, regression equations for standard 
insecticide concentration against brine shrimp probit mortality calibration curves were 
used (Appendix IV). Calibration curve o f chlorpyrifos-methyl was used for methanol 
fractions, while pirimiphos-methyl and cypermethrin were used for ethyl acetate fraction 
and hexane fractions respectively.
4.8.3 Brine shrimp mortality profile for solid phase extracted (SPE) fractions 
from cabbage sample
A total of 60 brine shrimp nauplii were used for the bioassay o f each of the fractions 
eluted from SPE. Fractions obtained for each of the 7 sub-sites were bioassayed for 
activity.
Fig. (22) Shows biological activity recorded for fractions obtained from all samples 
using the three selected solvents in the order: methanol, ethyl acetate and hexane 
Much higher mortality was generally recorded for methanol fractions compared with the 
hexane and ethyl acetate fractions. Between 42-93% mortality was recorded for methanol 
fractions The ethyl acetate and hexane fractions gave relatively higher mortality in the 
Dzorwulu samples than for Airport and Madina. Level o f mortality in ethyl acetate and 
hexane were lower for Mampong samples. Mortalities in Airport A samples were 
relatively low.
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4.8.4 Estimation of Maximum Residue Levels (MRL) of insecticides in the 
cleaned cabbage extracts.
The results of the MRL values of the cleaned cabbage extracts are shown in Table 11. 
Chlorpyrifos was eluted in methanol fraction, pirimiphos-methyl and pyrethroids in ethyl 
acetate and hexane respectively. The results showed very high values for chlorpyrifos- 
methyl in all the study sites. The highest value however was recorded in Mampong 
(124.1 mg/kg), likewise Airport B and the Madina samples recorded 114 mg/kg and
111.5 mg/kg respectively with the least MRL value being given by Airport A (55mg/kg) 
However, Least Significant Difference (LSD) at 95% confidence level showed no 
significant difference in MRL means for the study sites (Appendix VT)
For pirimiphos-methyl, cabbage samples from Dzorwulu sites though not significantly 
different recorded the highest values (14 6 mg/kg and 10.9 mg/kg) But MRL value for 
Dzorwulu A was significantly different from all the sites. Moderate MRL values were 
recorded for the Madina and Airport cabbage samples and the least value was recorded in 
Mampong.
Estimation o f brine shrimp lethal activity o f hexane fraction eluents was difficult due to 
the low mortalities recorded hence the pyrethroid concentration in Mampong, Madina A 
and B, Airport A and the control cabbage samples could not be determined from the 
calibration curve. The concentrations of pyrethroids determined in the three sites 
Dzorwulu B and A, Airport B were between 1.2 and 1.8 mg/kg respectively The three 
were however not significantly different at 0.05 probability.
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Cabbage samples from Dzrowulu were found to have high concentrations o f both 
pyrethroids and pirimiphos-methyl, while Airport samples had comparatively high 
residue o f all the three insecticides. Mampong cabbage samples recorded the highest 
concentration of chlorpyrifos but the least in pirimiphos-methyl and the pyrethroids 
levels in the samples were too low to be detected therefore could not be determined.
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Table 11 Fractionated Insecticides from cabbage samples and estimated insecticide
resiaue content _____________
Total
cleaned Total Active % Residual MRL
Insecticide residue % residues Active activity (mg/kg) of
Site/Sample fractions (g) Mortality (mg) x 103 residues (kg)
cabbage
Mampong Chlorpyrifos 318 93.3 488.5 0.020 124144.1 124.1
Pirimiphos 2.7 11.7 95.7 0.004 2086.3 2 1
Pyrethroids 1.7 13.3 ND* ND* ND* ND*
Madina A Chlorpyrifos 31.6 85 441 0.018 111524.5 111.5
Pirimiphos 4.4 22.5 180.3 0.007 6400.7 6.4
Pyrethroids 2 7 2.5 ND* ND* ND* ND*
Madina B Chlorpyrifos 31.6 85 441 0.018 111524.5 111.5
Pirimiphos 2.6 35 277.9 0.011 5835 9 5.8
Pyrethroids 5.8 10 ND* ND* ND* ND*
Dzrowulu A Chlorpyrifos 31.5 76.7 393.5 0.016 99022.7 990
Pirimiphos 3.0 76.7 603.3 0.024 14640.1 14.6
Pyrethroids 2.1 35 74.1 0.003 1266.1 1.3
Dzrowulu B Chlorpyrifos 31.1 60 298.5 0.012 74373.7 74.4
Pirimiphos 4.3 40 316.9 0.013 10922.5 10 9
Pyrethroids 2.2 38.3 101.4 0.004 1804.9 1.8
Airport A Chlorpyrifos 30.9 46.7 222.5 0.009 54994.9 55.0
Pirimiphos 3.4 15 121.7 0.005 3334.6 3.3
Pyrethroids 1.4 21.7 ND* ND* ND* ND*
Airport B Chlorpyrifos 31.6 86 7 450.5 0.018 114038.7 114.0
Pirimiphos 3.4 31.7 251.9 0.010 6818.1 6.8
Pyrethroids 2.1 35 74.1 0.003 1235 1.2
Control*0 Chlorpyrifos 30 0 5 ND* ND* ND* ND*
Pirimiphos 1.6 2.5 ND* ND* ND* ND*
Pyrethroids 1.2 0 ND* ND* ND* NTD*
*c Control- Insecticide free cabbage samples grown in the screen house.
*a Active residue- the estimated concentration (jig/ml) of toxic residues per solvent using the calibration 
curves of the insecticide standards
*b % active residues- This is the fraction of the active residue in relation to the total residues extracted from 
the cabbage heads.
N/B Active residues for mortalities below 26% of pyrethroid fractions could not be estimated from the 
calibration curves of the insecticide standards
ND* (Not detected) Are fractions that could not be estimated (detected) from the calibration curve
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Table 11. Fractionated Insecticides from cabbage samples and estimated insecticide
residue content
Site/Sample
Insecticide
fractions
Total
cleaned
residue
(g)
%
Mortality
Total Active 
residues 
(mg) x 10'3
%
Active
residues
Residual
activity
(kg)
MRL 
(mg/kg) of 
cabbage
Mampong Chlorpyrifos 31 8 93.3 488.5 0.020 124144.1 124.1
Pirimiphos 2.7 11.7 95.7 0.004 2086.3 2.1
Pyrethroids 1.7 13.3 ND* ND* ND* ND*
Madina A Chlorpyrifos 31.6 85 441 0.018 111524.5 111.5
Pirimiphos 4 4 22.5 180.3 0.007 6400.7 6.4
Pyrethroids 2.7 2.5 ND* ND* ND* ND*
Madina B Chlorpyrifos 31.6 85 441 0.018 111524.5 111.5
Pirimiphos 2.6 35 277.9 0.011 5835.9 5.8
Pyrethroids 5.8 10 ND* ND* ND* ND*
Dzrowulu A Chlorpyrifos 31.5 76.7 393.5 0.016 99022.7 99.0
Pirimiphos 3.0 76.7 603.3 0.024 14640.1 14.6
Pyrethroids 2.1 35 74.1 0.003 1266.1 1.3
Dzrowulu B Chlorpyrifos 31.1 60 298.5 0.012 74373.7 74.4
Pirimiphos 4.3 40 316 9 0.013 10922.5 10.9
Pyrethroids 2.2 38.3 101.4 0.004 1804.9 1.8
Airport A Chlorpyrifos 30.9 46.7 222.5 0.009 54994.9 55.0
Pirimiphos 3.4 15 121.7 0.005 3334.6 3.3
Pyrethroids 1.4 21.7 ND* ND* ND* ND*
Airport B Chlorpyrifos 31.6 86.7 450.5 0.018 114038.7 114.0
Pirimiphos 3.4 31.7 251.9 0.010 6818.1 6.8
Pyrethroids 2.1 35 74.1 0.003 1235 1.2
Control*0 Chlorpyrifos 30.0 5 ND* ND* ND* ND*
Pinmiphos 1.6 2.5 ND* ND* ND* ND*
Pyrethroids 1.2 0 ND* ND* ND* ND*
*c Control- Insecticide free cabbage samples grown in the screen house.
*a Active residue- the estimated concentration (ng/ml) of toxic residues per solvent using the calibration 
curves of the insecticide standards
*b % active residues-This is the fraction of the active residue in relation to the total residues extracted from 
the cabbage heads.
N/B Active residues for mortalities below 26% of pyrethroid fractions could not be estimated from the 
calibration curves of the insecticide standards
ND* (Not detected) Are fractions that could not be estimated (detected) from the calibration curve
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Table 11. Fractionated Insecticides from cabbage samples and estimated insecticide
residue conl ent
Site/Sample
Insecticide
fractions
Total
cleaned
residue
(g)
%
Mortality
Total Active 
residues 
(mg) x 10 3
%
Active
residues
Residua]
activity
(kg)
MRL 
(mg/kg) of 
cabbage
Mampong Chlorpyrifos 31.8 93.3 488.5 0.020 124144.1 124.1
Pirimiphos 2.7 11.7 95.7 0.004 2086.3 2.1
Pyrethroids 1.7 13,3 ND* ND* ND* ND*
Madina A Chlorpyrifos 31.6 85 441 0.018 111524.5 111.5
Pirimiphos 4.4 22.5 180.3 0.007 6400.7 6.4
Pyrethroids 2.7 2.5 ND* ND* ND* ND*
Madina B Chlorpyrifos 31.6 85 441 0.018 111524.5 111.5
Pirimiphos 2.6 35 277.9 0.011 5835.9 5.8
Pyrethroids 5.8 10 ND* ND* ND* ND*
Dzrowulu A Chlorpyrifos 31.5 76.7 393.5 0.016 99022.7 99.0
Pirimiphos 3.0 76.7 603.3 0.024 14640.1 14.6
Pyrethroids 2.1 35 74.1 0.003 1266.1 1.3
Dzrowulu B Chlorpyrifos 31.1 60 298.5 0.012 74373.7 74.4
Pirimiphos 4.3 40 316.9 0.013 10922.5 10.9
Pyrethroids 2.2 38.3 101.4 0.004 1804.9 1.8
Airport A Chlorpyrifos 30.9 46.7 222.5 0.009 54994.9 55.0
Pirimiphos 3.4 15 121.7 0.005 3334.6 3.3
Pyrethroids 1.4 21.7 ND* ND* ND* ND*
Airport B Chlorpyrifos 31.6 86.7 450.5 0.018 114038.7 114.0
Pirimiphos 3.4 31.7 251.9 0.010 6818.1 6.8
Pyrethroids 2.1 35 74.1 0.003 1235 1.2
Control*' Chlorpyrifos 30.0 5 ND* ND* ND* ND*
Pirimiphos 1.6 2.5 ND* ND* ND* ND*
Pyrethroids 1.2 0 ND* ND* ND* ND*
*c Control- Insecticide free cabbage samples grown in the screen house.
*a Active residue- the estimated concentration (jig/ml) of toxic residues per solvent using the calibration 
curves of the insecticide standards
*b % active residues- This is the fraction of the active residue in relation to the total residues extracted from 
the cabbage heads.
N/B Active residues for mortalities below 26% of pyrethroid fractions could not be estimated from the 
calibration curves of the insecticide standards
ND* (Not detected) Are fractions that could not be estimated (detected) from the calibration curve
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Table 11 Fractionated Insecticides from cabbage samples and estimated insecticide
residue conl pnt
Site/Sample
Insecticide
fractions
Total
cleaned
residue
(g)
%
Mortality
T otal Active 
residues 
(mg) x 10 3
%
Active
residues
Residua]
activity
(kg)
MRL 
(mg/kg) of 
cabbage
Mampong Chlorpyrifos 31.8 93.3 488.5 0.020 124144.1 124.1
Pirimiphos 2.7 11.7 95.7 0.004 2086.3 2.1
Pyrethroids 1.7 13.3 ND* ND* ND* ND*
Madina A Chlorpyrifos 316 85 441 0.018 111524.5 111.5
Pirimiphos 4.4 22.5 180.3 0.007 6400.7 6.4
Pyrethroids 2.7 2.5 ND* ND* ND* ND*
Madina B Chlorpyrifos 31.6 85 441 0.018 111524.5 111.5
Pirimiphos 2.6 35 277.9 0.011 5835.9 5.8
Pyrethroids 5.8 10 ND* ND* ND* ND*
Dzrowulu A Chlorpyrifos 31.5 76.7 393.5 0.016 99022.7 99.0
Pirimiphos 3.0 76.7 603.3 0.024 14640.1 14.6
Pyrethroids 2.1 35 74.1 0.003 1266.1 13
Dzrowulu B Chlorpyrifos 31.1 60 298.5 0.012 74373.7 74.4
Pirimiphos 4.3 40 316.9 0.013 10922.5 10.9
Pyrethroids 2.2 38.3 101.4 0.004 1804.9 1.8
Airport A Chlorpyrifos 30.9 46.7 222.5 0.009 54994.9 55.0
Pirimiphos 3.4 15 121.7 0 005 3334.6 3.3
Pyrethroids 14 21.7 ND* ND* ND* ND*
Airport B Chlorpyrifos 31.6 86.7 450.5 0.018 114038 7 114.0
Pirimiphos 3.4 31.7 251.9 0.010 6818.1 6.8
Pyrethroids 2.1 35 74.1 0 003 1235 1.2
Control*0 Chlorpyrifos 30.0 5 ND* ND* ND* ND*
Pirimiphos 1.6 2.5 ND* ND* ND* ND*
Pyrethroids 1.2 0 ND* ND* ND* ND*
*c Control- Insecticide free cabbage samples grown in the screen house.
*a Active residue- the estimated concentration (jig/ml) of toxic residues per solvent using the calibration 
curves of the insecticide standards
*b % active residues- This is the fraction of the active residue in relation to the total residues extracted from 
the cabbage heads
N/B Active residues for mortalities below 26% of pyrethroid fractions could not be estimated from the 
calibration curves of the insecticide standards
ND* (Not detected) Are fractions that could not be estimated (detected) from the calibration curve
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Table 11. Fractionated Insecticides from cabbage samples and estimated insecticide
residue conl ent
Site/Sample
Insecticide
fractions
Total
cleaned
residue
(g)
%
Mortality
T otal Active 
residues 
(mg) x 10 3
%
Active
residues
Residual
activity
(kg)
MRL 
(mg/kg) of 
cabbage
Mampong Chlorpyrifos 31.8 93.3 488.5 0.020 124144.1 124.1
Pirimiphos 2.7 11.7 95 7 0.004 2086.3 2.1
Pyrethroids 1.7 13.3 ND* ND* ND* ND*
Madina A Chlorpyrifos 31.6 85 441 0.018 111524.5 111.5
Pirimiphos 44 22.5 180.3 0.007 6400.7 6.4
Pyrethroids 2.7 2.5 ND* ND* ND* ND*
Madina B Chlorpyrifos 31.6 85 441 0.018 111524.5 111.5
Pirimiphos 2.6 35 277.9 0.011 5835.9 5.8
Pyrethroids 5.8 10 ND* ND* ND* ND*
Dzrowulu A Chlorpyrifos 31.5 76.7 393.5 0.016 99022 7 99.0
Pirimiphos 3.0 76.7 603.3 0.024 14640.1 14.6
Pyrethroids 2.1 35 74.1 0.003 1266.1 1.3
Dzrowulu B Chlorpyrifos 31.1 60 298.5 0.012 74373.7 74.4
Pirimiphos 4.3 40 316 9 0.013 10922.5 10.9
Pyrethroids 2.2 38.3 101.4 0.004 1804.9 1.8
Airport A Chlorpyrifos 30.9 46.7 222.5 0.009 54994.9 55.0
Pirimiphos 3.4 15 121.7 0.005 3334.6 3.3
Pyrethroids 1.4 21.7 ND* ND* ND* ND*
Airport B Chlorpyrifos 316 86 7 450.5 0.018 114038.7 114.0
Pirimiphos 3.4 31.7 251.9 0.010 6818.1 6.8
Pyrethroids 2.1 35 74.1 0.003 1235 1.2
Control*0 Chlorpyrifos 30 0 5 ND* ND* ND* ND*
Pirimiphos 1.6 2.5 ND* ND* ND* ND*
Pyrethroids 1.2 0 ND* ND* ND* ND*
*c Control- Insecticide free cabbage samples grown in the screen house.
*a Active residue- the estimated concentration (ng/ml) of toxic residues per solvent using the calibration 
curves of the insecticide standards
*b % active residues- This is the fraction of the active residue in relation to the total residues extracted from 
the cabbage heads.
N/B Active residues for mortalities below 26% of pyrethroid fractions could not be estimated from the 
calibration curves of the insecticide standards
ND* (Not detected) Are fractions that could not be estimated (detected) from the calibration curve
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CHAPTER FIVE 
DISCUSSION
The questionnaire survey showed that insect pests were a major constraint to cabbage 
production in the selected sites. DBM was considered a key pest of cabbage in Ghana 
causing damage in 73% of the farms surveyed. DBM has been reported as a key pest of 
cabbage and other crucifers, in many other parts of the world (Miyata et a l, 1986; 
Cartwright et al., 1987; Hill and Waller, 1994). The increasing pest infestation could be 
attributed to improper agronomic practices in the farms as was revealed by the present 
study. Most farmers in Accra suburbs applied water to the entire crop during irrigation, 
this washed off the insecticides applied especially those with contact action such as the 
pyrethroids and organophosphates, which are the most commonly, used insecticides 
against DBM. The duration between insecticide spraying and irrigation therefore was 
vital depending on the method used. Most farmers in Mampong justified watering the 
same day with spraying with insecticides since their bucket and cup method of watering 
was done underneath, hence did not wash off the chemicals to the extent done by the 
method used by their counterparts in Accra-suburbs.
Monoculture enhanced pest infestation on cabbage by lepidopterous insects and aphids 
since it creates conducive environment for particular insect pest to flourish (Kumar, 
1986). Creating a host free period was difficult due to lack of coordination of activities 
amongst the growers with the Tesult that growers cultivate cabbage at different times of 
the year. Consequently, growers were unable to synchronise time for insecticide spraying 
schedules, resulting in unsprayed cabbage plants serving as refuge at a time when some 
fanners would have sprayed their crops.
Since Brassicas (cabbage, cauliflower and Chinese cabbage) have similar pest spectrum
(Hill and Waller, 1994), all year round cultivation of these varieties of Brassica in
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adjacent plots to cabbage bridged the gap in host plant sequence of DBM (Miyata et al., 
1986) resulting in upsurges in its infestation (Way, 1976). In addition, DBM can survive 
on host residues throughout the off seasons. This was a major source of re-infestation in 
the farms since most farmers either left the residue on the farms or threw them by the 
side of the farms. These practices made food available for the pest, and created a 
favourable condition for their survival, development and multiplication even during off 
seasons, leading to increased pest infestation and damage.
On pest control, growers relied exclusively on the use of insecticides since they believed 
they gave faster results once applied and most of them used the insecticides as 
prophylactic measure. The high rate and dosages of application lead to development of 
resistance in the pest. Consequently, the survey showed a decline in the use of 
pyrethroids and the biopesticide B. thuringiensis (Fig.9), which were the most widely 
used against pests of cabbage (Ninsin, 1997). This was attributed to their ineffectiveness 
against DBM. Thompson (2004) has proposed a perceived treadmill that clearly 
illustrates the circle between resistance development and insecticide spraying. The result 
of the present study further supports Thompson’s assertion. This is because the use of 
organophosphates had increased, with a worse consequence due to their persistence in 
the environment, and this could enhance development of resistance due to prolonged 
exposure to the pest and also cause health ha2ards due to insecticide residue 
accumulation in edible materials.
Cabbage farmers were using pesticides labelled in French (cyperphos, Polytrine, D336
EC and Thionex). These could be a major source of hazards to consumers since the
relevant information on the products label concerning the safe and effective use of these
insecticides were written in French. Furthermore, these products were not registered
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contrary to the pesticide Control and Management Act, 528 of Ghana. Davis (1997) 
reported that about one third of chemical products on the shelves in a shop in Kumasi 
were labelled in French. In addition, products that were recommended for use in cotton, 
coffee and cocoa were sold for use on all crops.
Farmers also reported the use of insecticide mixtures. This practice defied the principles 
of insecticide management. Metcalf (1980) recommended that the use of such mixtures 
should be avoided since it results in the simultaneous development of pest resistance 
because each compound seems to develop the residual inheritance of the supporting 
genome for resistance in the other (Metcalf, 1980. This gives rise to control failure for 
such a notorious pest as DBM, which is capable of quickly developing resistance to all 
insecticides, exposed to it (Miyata et al., 1986). Growers were observed to use sub- 
lethal-doses of the insecticides and this also increases the development of resistance in 
the pest.
With regard to personal protection against contamination, it was found out that the 
application of insecticides by the cabbage growers was not carried out properly. Only 
four (13.33%) wore protective clothes during spraying, claiming that the equipment were 
expensive and uncomfortable. Thus these growers were predisposed to the risk of 
insecticide poisoning, “the New Developing World Disease” (Anonymous, 1989). Most 
of their knapsack sprayers often leaked and it was common for the sprayers to have skin 
rashes, headaches, and dizziness for a few days after insecticide application (Davis, 
1997).
Farmers observed short Pre-harvest intervals. Indeed during heavy infestation, farmers
sprayed and sold their produces the same day (Fig. 12). This exposed consumers to great
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health risk since the cabbages were sold directly to consumers without passing through a 
marketing quality control board. Fanners also turned to pesticide sellers and fellow 
farmers for professional advice concerning identification of new pests and their control. 
The former recommended their new products that needed field trials for the new pest 
while the latter recommended the insecticides they had tried on their farms and were 
successful, although their practices in terms of rate of application and safety measures 
were not up to standard.
Although the increase in the use of OPs and the high residue levels of chlorpyrifos was a 
great health hazard since farmers were revealed to observe Pre-harvest interval as short 
as one day, yet OPs, require 21 days (PHI). The use of biopesticide should therefore be 
encouraged towards maturation of cabbage since its PHI is zero days (Shelton and Zhao, 
2004) and it has less health hazards. More extension should be encouraged in conjunction 
with the Farmers Field Schools, which were reported to have reduced indiscriminate use 
of insecticides (Kaiwa, 2000).
Concerning pesticide residue awareness 19 out of 30 (63%) of the respondents were 
ignorant about perceived risks caused by improper use of insecticides. They attributed 
pesticide-poisoning incidences e.g. (Tarkwa and Sunyani) to lack of proper training on 
insecticide use since the farmers were reported to have used insecticides meant for cocoa 
on cabbage farms.
In order to ascertain the effect of the indiscriminate use of insecticide on DBM, a
susceptibility study was carried out. The study showed that DBM was highly resistant to
all the synthetic insecticides assayed. However, resistance to B.t. was very low (Table 8).
Shelton and Zhao (2004) reported that resistance to CrylA toxins of B.t. in DBM tends
to be unstable, and one generation without selection can make the population susceptible
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but resistance can quickly return when populations are challenged. Tabashnik et al.
(1991) also reported that B.t. resistance in DBM declines in absence of selection. Hama
(1992) also documented that DBM resistance to B.t. decreased within few generations. 
Conversely, McGaughey & Beeman (1988) reported that Plodia interpunctella resistance 
to B.t. did not decline even after two generations without selection. This apparent 
unstable resistance was demonstrated by the survey findings, which indicated that most 
fanners in Accra suburbs had reduced their use of B.t. due to low efficacy in controlling 
DBM. As a result the DBM populations in Accra were almost susceptible to B.t. whereas 
the Mampong population showed some level of resistance since B.t. is presently widely 
used by farmers in the area.
On the other hand the synthetic insecticides assayed (pyrethroids and organophosphate) 
have been reported to have more stable resistance (Shelton and Zhao, 2004). This was 
substantiated from the study, which recorded very high resistance to insecticides assayed 
even in areas where those particular formulations were not currently in use. For example, 
Dursban recorded very high level of tolerance (193 fold) in Dzorwulu, although only a 
few farmers were currently using it. Similarly, DBM population in Airport showed the 
highest tolerance to cypermethrin although currently cydimsuper, a mixture of 
cypermethrin (36g/L) and dimethoate (400g/L) was in common use in the area and not 
cypermethrin alone. This suggested not only the stability of resistance to conventional 
insecticides but also that multiple resistance and cross-resistance might be the major 
contributors to low efficacy of most conventional insecticides to DBM.
The study suggested the presence of cross-resistance among the pyrethroids assayed and
multiple-resistance between the pyrethroids and organophosphates. In contrast, all the
DBM resistant strains to the conventional insecticides were susceptible to B.t. This
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suggests that B.t. resistance results from selection by B.t., but not cross-resistance to 
other insecticides. This is not surprising because the mode of action for B.t. differs from 
that of the conventional insecticides. The 5 .t.’s 8 endotoxin lyses cells in the midgut 
membrane while the pyrethroids and organophosphates, targets cholinesterases the high 
voltage sodium ion channel (Horway et al., 1986). DBM populations showed cues of 
multiple resistance to both Pawa (lambda-cyhalothrin a pyrethroid) and Dursban 
(chlorpyrifos an organophosphate) in Mampong and Dzorwulu and therefore the two 
should not be used in rotation. The low tolerance recorded for Deltaplan (deltamethrin a 
pyrethroid) in Madina suggest that it may be judiciously used to control DBM population 
that were tolerant Pawa however this was not the case for Airport area where Deltaplan 
only recorded 30% mortality on a similar DBM population. This observation suggests 
similar mechanisms underlying DBM resistance to the assayed groups of insecticides. 
This confirmed the findings by other workers that DBM resistance covers all major 
groups of insecticides (Sun et al., 1986 and Lin, 1988). As a result insects resistant to 
pyrethroids may develop resistance to organophosphates even without exposure to the 
latter group of insecticides. It was therefore not advisable to use pyrethroids with dursban 
in the management of DBM. The use of pyrethroids and organophosphates in rotation 
with dipel could be a better option, as this will reduce the selection pressure of the 
insecticides and preserve susceptible genes in DBM population. These results confirm 
the findings of Sun et al. (1986) that Bt is highly effective against DBM populations that 
were resistant to conventional insecticides.
The different levels of non-specific carboxylesterase activities observed in the DBM
(Table 9) from different study sites indicate some level of resistance (Doichuanngam and
Thornhill, 1989). The high carboxylesterase activity observed in DBM was a
consequence of a long period of exposure to organophosphates and pyrethroids, which
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was prerequisite for resistance development. Mampong recorded very high activity of the 
two enzymes followed by Madina and Dzorwulu population. The grand mean recorded in 
this study for a  and p naphthyl esterases were 3.05/miol/min/mg and 9.1 /imol/min/mg 
respectively. Comparing these findings with the work of Kaiwa (2000), which recorded 
means of 1.5jUmol/min/mg and 1,2jttmol/min/mg for a  p-naphthyl esterase activities 
respectively; it was observed that the mean activity of a-naphthyl esterase had doubled 
while an eight-fold increase was registered for p-naphthyl esterase. However, in general, 
higher a—naphthyl esterase activity was recorded for Accra suburb sites. This confirmed 
the findings of Kaiwa (2000) on carboxylesterase activity in DBM in Accra suburbs. 
Similar findings were also reported by Villania et al. (1983) on Culexpipiens mosquitoes 
in selected locations around Accra by Poku (1999).
The results of the carboxyleterase activity did not reflect the findings of the insecticide 
use pattern survey, which showed high use of both groups (OP and SPs) of insecticides 
in the Accra suburbs than in Mampong (Table 9). Likewise this was contrary to the 
bioassay results, which recorded high resistance to all the conventional insecticides 
assayed (Table 8). This may suggest that apart from carboxylesterases, other enzymes 
such as oxidases and Glutathion-S-transferases may be involved in DBM resistance as 
reported elsewhere (Maa et al., 1997). Carboxylesterase enzymes were also shown to 
have wide variations in activity even from same site especially in Mampong. This may 
suggest development of resistance at different times under selection by different 
insecticides.
An attempt was made to characterise the esterases to identify the isozymes present. 
Based on electrophoretic mobility and staining intensity, two main groups of esterase
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bands were identified. These bands were scattered throughout two zones of the gel 
(Fig. 15) a fast moving-heavily stained upper zone and a slow moving-light stained lower 
zone. Based on the preliminary characterization of esterases of DBM (Appendix IV) 
reported by Maa et al. (1990); Maa and Liao (2000) high level of resistance is more 
likely to be associated with slow moving esterases, which are more important than fast 
moving esterases in resistance monitoring. However, without genetic data it was difficult 
to attribute each isozyme to a given locus. The esterases isozymes combined in different 
patterns and intensities to give varying levels of resistance, although negative correlation 
between the banding patterns and resistance had been reported (Maa et al., 1997). These 
workers argued that DBM had developed multiple resistance mechanisms, and its 
resistance to OP and SPs did not only depend on hydrolysis. In addition, enzyme 
activities of soluble esterases of different cultures were found not to correlate with 
frequency of slow moving esterases. However, the work of Owusu et al. (1995), on 
aphids emphasized that, band intensities rather than numbers were likely to be the 
principal factors determining the quantity of resistant-linked enzymes produced by an 
individual insect.
DBM population in Mampong-Akuapem showed higher frequency of slow moving
esterases than the Accra surburb sites (Fig. 16), which had majority of the zymograms
with the fast moving esterases, associated with low resistance. This confirms the findings
of the enzyme assays, for which the Mampong population had higher a -  and
(B-carboxylesterase activity than the Accra sites. Besides the zymogram patterns for the
Accra populations were nearly similar, while the Mampong population reflected a wide
range of zymogam patterns (Fig. 15). This pattern was also shown by the results for
enzyme assay, which depicted wide deviations in activity, indicative of wide qualitative
differences within the same population. Comparatively, Madina DBM population had
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higher frequency of slow moving esterases while Dzorwulu was shown to record the 
least frequency. This confirmed the results of the carboxylesterases assay, which showed 
Dzorwulu population to be nearly susceptible for both enzyme substrates. The exhibition 
of different forms of electrophoretic banding patterns by resistant DBM strains and the 
presence of esterase heterogeneity suggest that resistance might have developed over 
different periods of time under varying degrees of pesticide selection. This supports the 
view that resistance development has become rapid when selection pressure exerted by 
insecticide is widespread and continuous (Brown, 1964). The heterogeinity shown in the 
characterization of carboxylesterase as well as the variation in gradient of the dose- 
mortality curves suggests the possibility of having different DBM species particularly in 
Mampong and some taxonomic work need to be done to elucidate this fact in Ghana 
Polimerase chain reaction (PCR) was used to detect B.t. resistant gene. This molecular 
identification of B.t. resistant gene of DBM showed the expected 325 bp PCR fragment 
in both resistant and susceptible strains, implying that B.t. resistance was not as a result 
of deletion of the entire genome but of change in the genome (Heckel et al., 1999). This 
finding supports the leading hypothesis that B.t. resistance was conferred by altered B.t. 
toxin binding sites in the larval midgut epithelium (Ferre et al., 1991). The amplification 
of the additional 776 bp fragment could be because the primers may not be as specific as 
they were reported to be. Moreover the primers could be amplifying the section that 
represents the sequence-tagged site (STS) for the chromosome containing B.t. R-l, which 
corresponds to the B.t. resistance gene in DBM where the primers were cloned (Heckel 
et al., 1999). The extra 1110 bp fragment however could also be a product of further 
mutation of B.t. resistant gene in DBM or a change in the target site in DBM, therefore 
sequencing the new areas amplified would have shown whether or not these mutations 
are relevant to B.t. resistance.
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The first insect reported to have developed resistance to B.t. in open field was DBM 
(Mcgaughey and Whalon, 1992) and Indian meal moth in laboratory experiments 
(Mcgaughey, 1990). Others that have shown resistance in laboratory set up are tobacco 
budworm and Colorado beetle. DBM and tobacco budworm have even shown multiple 
resistance to other B.t. strains (Gould, 1992).
The varying levels of resistance recorded for DBM from the various sites may have 
arisen from continued exposure of these DBM populations to conventional insecticides. 
The present work showed stability of resistance to organophosphates and pyrethroids, 
which these farmers have been using. Brine shrimp toxicity bioassay directed SPE 
fractionation of extracted residues from cabbage was used to establish the identity and 
levels of organophosphates and pyrethroids the extracts. The organophosphates were 
eluted in the polar solvents, while the pyrethroids were generally eluted in hexane, anon 
polar solvent Yeboah (2001) has found that methanol extracted a lot fatty substances and 
extraneous materials, which may explain the high extractable levels of total residue in 
methanol fraction (Table 11). While Matsumura (1985) exclusively used hexane for 
extraction of organochlorines and organophosphate insecticides, this study proved 
otherwise. On the bother hand, the low residue levels obtained for hexane fractions may 
be due to hexane/ petroleum ether selective extraction of insecticides from plant 
materials found by the work of Gbewonyo (1991). Ethyl acetate with medium polarity 
may also demonstrate some levels of selective extraction, hence the lower level of ethyl 
acetate extracted residues.
In the Mampong cabbage samples, methanol fraction gave significantly high
chlorpyrifos residues and consequently MRL, but the lowest levels of pirimiphos-methyl
ethyl acetate fraction and pyrethroid hexane fraction residues. The survey showed that
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the farmers were currently using chlorpyrifos (dursban) (Table 9) and DBM also in the 
bioassay recorded the highest (106 fold) resistance for chlopyrifos (dursban) at the same 
site (Table 8). In addition to these carboxylesterase activity for both enzymes was highest 
in Mampong. Additionally, the esterase isozymes recorded the highest frequencies of 
slow-moving esterases associated with high resistance for Mampong samples (Fig. 16) 
justifying the high chlopyrifos residues detected.
Unlike Mampong, the Accra suburb sites gave much higher yield for all the three 
insecticides. This was substantiated by the insecticide use pattern survey, which indicated 
the use of most of the insecticides chosen for the residue estimation. Notably Dzorwulu 
A had significantly higher amounts of pirimiphos-methyl and low to undetectable levels 
of pyrethroid residues, in some sites due to low brine shrimp mortalities. The 
susceptibility studies showed that DBM population in these sites was highly resistant to 
all the conventional insecticides assayed (Table 8). Similarly, Airport samples generally 
showed all round presence of the three insecticides including high levels of pyrethroids. 
This concurred with the bioassay results, which showed Airport DBM population to 
record the highest resistance to cypermethrin and lambda cyhalothrin. This may be an 
indication of long-term use of these insecticides in the sites, and therefore prolonged 
exposure of the insect to them, which is prerequisite for resistance development (Brown, 
1964). However, these high pyrethroids residues and resistance was not reflected in 
carboxylesterase activity and esterase zymogram pattern, studies suggesting that 
carboxylesterase was not solely responsible for the resistance observed in these DBM 
populations.
The generally low residual activity and MRL for pyrethroids could partly be because
pyrethroids are photosensitive and are therefore less persistent in the environment as
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compared to the OPs. However comparing these findings with the work of Ninsin (1997) 
on lambda-cyhalothrin there was 18 fold increase in MRL values at Dzorwulu B and 13 
and 12 fold increase in Dzorwulu A and Airport B respectively over results obtained by 
Ninsin (1997). This was 6 to 9 fold above the FAO/ WHO MRL of 0.2mg/kg. The 
residue levels of pirimiphos-methyl ranged between 2.1mg/kg and 14.6mg/kg, which 
gave 2- 15, fold, increase above the recommended MRL lmg/kg. The insecticide use 
pattern survey also showed an increase in the use of organophosphates and a reduction 
in the use of pyrethroids (Fig. 9). This was confirmed by the highest resistance recorded 
for dursban (chlorpyrifos-methyl) when bioassayed, and an equally high chlorpyrifos 
level detected in all the sites. The highest chlorpyrifos residue was recorded in Mampong 
124. lmg/kg and the lowest 55mg/kg in Airport A samples (Table 11). This was 55 to 124 
fold above the recommended MRL of lmg/kg recommended by Codex Alimentarius 
Commission.
Works reported by many scientists in Ghana have shown concern for the use of 
chlorpyrifos. Mawunyega (1994) and Aboagye (2002) analysed residues on cabbage and 
on exportable pineapples respectively. Chlorpyrifos was also detected in 6 out of 8 
samples of street vended waakye (rice and beans) and 1 out o f 8 samples offiifu (cassava 
and plantain dough that were analysed in 1999-2000. However, residue levels recorded 
were lower than the level permitted by Codex Alimentarius Commission
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CONCLUSIONS AND RECOMMMENDATION
Poor agronomic and insecticides application practices by cabbage farmers have resulted 
in high increase in DBM resistance to all the conventional insecticides. As a consequence 
of the indiscriminate use of these conventional insecticides to control DBM, increased 
residue levels on cabbage heads has become a great source of health hazards to cabbage 
consumers. However, low resistance was recorded for B.t. leaving it as the only possible 
alternative for controlling DBM in the study sites.
A substantial increase in carboxylesterase activity compared with earlier studies together 
with esterase zymogram pattern studies further confirmed that DBM population have 
developed resistance to these pyrethroids and organophosphates assayed. The low 
carboxyleterase activity recorded suggests that apart from carboxylesterase, other 
enzymes could be involved in DBM resistance. Given that DBM has developed 
resistance to different classes of insecticides and most of the field observed resistance are 
due to multiple resistance activities of other enzymes such as oxidases and glutathione 
transferase should be determined since they have also been reported to be involved in 
DBM resistance. Use of enzyme inhibitors in the electrophoretic studies of the esterases, 
could help identify the particular esterase isozyme (acetylcholinesterase or 
carboxylesterase) that confers resistance to this group of insecticides.
The expression of B.t. resistance genes and the possibility of further mutations should 
serve as appoint of caution to scientists who are using transgenic plant campaign as the 
ultimate solution to insect pest problems. Rather, more integrated approach using IPM 
principles, including the use of parasitoids coupled with judicious use of the 
environmentally friendly biopesticides could be more effective in the management of 
DBM and other insect pests. This should be strengthened by using the Farmers Field
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schools which was shown to be successful in controlling DBM at Weija cabbage farms in 
Accra especially when Neem extracts were incorporated in insect pest control.
Furthermore the use of spreader stickers could help increase efficacy of the insecticides 
with contact action this is because of the wax on cabbage leaves. The same has led to low 
efficacy of B.t. in controlling DBM, which stays on the underside of the cabbage leaves. 
Care should therefore be taken to ensure proper coverage during spraying and proper 
timing of application since B.t. has a field life of 2-3 days and would be most effective if 
applied soon after eggs of DBM emerge for larvae to feed when it is active (Mau and 
Kessing, 2004).
As a consequence of DBM resistance to conventional insecticides and further increase in 
their rate and frequency of application very high levels of insecticide residue were shown 
in Brine shrimp toxicity test. Chlopyrifos levels were far higher than the recommended 
FAO/WHO established MRL levels likewise pyrethroids levels compared to the works of 
earlier scientists and recommended MRL were also higher. The results of the present 
study have shown that the insecticide residue problem for cabbage should be taken 
seriously in Ghana. Urgent steps must be taken to help fanners control insect pests of 
cabbage without compromising human health. To achieve this, insecticide companies and 
researchers should educate farmers on proper use and handling of insecticides. In 
addition, the Environmental Protection agency of Ghana should prevent the importation 
of insecticides not registered for use in Ghana and the National Agricultural Extension 
Service should be strengthened since the extension service’s education of cabbage 
farmers was found to be seriously inadequate.
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For effective management of DBM, there is need to delay resistance development. This 
can be achieved by starting resistance management before resistance is detected and 
strategically applying insecticides at the most vulnerable developmental stages o f the 
insect. The use of insecticides with short persistence is preferred to avoid prolonged 
exposure to insecticides, which is a prerequisite to resistance development. These 
insecticides should also be used intermittently at the lowest possible effective dosages/ 
rates. Alongside, IPM principles like cultural practices, host plant resistance should also 
be incorporated to delay resistance development. It is also important to monitor 
susceptibility of DBM and other major pests to all the insecticides on a regular basis in 
representative fields in each region. Finally, insecticides remain the most reliable and 
effective means of controlling pests but should be used carefully and selectively and only 
when alternative methods do not exist, or are not economical, so far, minimiying their 
use is the only strategy really proven to work satisfactorily.
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APPENDICES 
APPENDIX 1: STANDARD SOLUTIONS 
Preparation of standard solutions and buffers
All chemicals used were of the highest grade commercially available,
a) Solutions for Enzyme assays
a. 0.558 g a. naphthyl acetate (MW 186.21 g/mol) was dissolved in 100 ml absolute ethanol 
to make a solution with a concentration o f 30 mM.
b. 0.558 g (3 naphthyl acetate (MW 186.21 g/mol) was dissolved in 100 ml absolute ethanol 
to make a solution with a concentration o f 30 mM. As above.
c. 4.5135 g Sodium dodecyl sulphate (SDS) (MW 260.32 g/mol) was dissolved in 100ml- 
distilled water to give 5% solution. Another 0.0498g (0.05) o f fast blue B salt (MW 317.2 
g/mol) was dissolved in 10ml o f distilled water to make 1% fast blue B salt solution. 
These two solutions were mixed in the ratio o f 5:2 (mixture o f 2 parts o f a 1% solution o f 
azoic diazo blue B salt and 5 parts o f a 5% solution o f SDS by volume and stored in the 
dark one hour before the assay.
d. 11.933 g Na2 H P04 12H2 O (MW 358.14 g/mol) and 4.533 g KH2 P 0 4. (MW 136.09 
g/mol) were each dissolved in 500ml-distilled water. These were mixed in a ratio o f  6:4 
(Na2 HPO4: KH2 PO4) by volume to make (0.07 M) phosphate buffer (pH 7.2).
Protein assay
a. Reagent A was prepared by dissolving 10 g o f sodium carbonate (MW 105.99 g/mol) and 
2 g sodium hydroxide (MW 40.0 g/mol) in 500 ml distilled water to give 2% sodium 
carbonate. This was replaced at the end o f every month.
b. 5.595 g CUSO4 (MW 249.68) were dissolved in 500 ml distilled water in a beaker to 
obtain 1 % CuS04 solution.
c. 5.925 g of sodium tartrate (MW 230.08 g/mol) were dissolved in 500ml o f distilled 
water in a beaker to obtain 1 % sodium tartrate solution.
d. Reagent B was prepared by adding 50ml o f Reagent A to 0.5ml each o f 1% CuS04 and 
1% sodium tartrate, to give a ratio of 50:1. Mixing was done just before the start o f assay.
e. Phenol (folin-Ciocalteau) Reagent: water (1:1) by volume was prepared just before the 
assay started because o f the instability of the mixture.
NB/ All stocks solutions were stored at 5°C in a refrigerator with exception o f SDS and
Phenol reagent,
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b) Standard Solutions Used In Molecular Studies
Solution for DNA extraction 
Bender buffer (pH 8.0)
0.1 M NaCl, 0.2 M sucrose, 0.1 M Tris-HCL pH 7.5, 0.05 M EDTA pH 9.1, 0.5% SDS stored at
4°C
0.5 M EDTA (pH 8.0)
186.1 g/1 in 800ml of sddw, pH adjusted with NaHO pellets and stored at room temperature. 
Ethidium bromide, EtB(10 mg/ml)
lg of EtBr was completely dissolved in 100ml ddw and stored in the dark at room temperature. 
KAc(5M K 8M Acetate)
60 ml of 5M KAc and 11.5 ml glacial acetate acid in 28.5 ml distilled water autoclaved and
stored at 4cC
RNase - lOmg/ml in water. Sterilised by filtration and stored at -20° C.
TE (pH 8.0) - lOmM Tris-HCL, 1 mM EDTA (pH 8.0),stored at room temperature.
TE+RNase
(5pg/ml) 5 pi of RNase (lOmg/ml) solution, 995 pi o f TE (pH 8.0). Stored at -20° C
Solution for Electrophoresis 
Agarose Gels
10x TAE buffer, 242 g Tris-base, 57.1 ml glacial acetic acid, 100 ml 0.5 M EDTA. pH adjusted to 
7.7 (with glacial acetic acid) and the volume made to 1 litre with sddw
Urea Polyacrylamide Gels
10 X  TBE electrophoresis buffer
108g/L tris base, 55g/L Boric acid, 9.3g/L Na2 EDTA and distilled water added to make up the 
volume to 1 litre and store at room temperature on the bench. It was Dilute to IX  working 
solution for electrophoresis.
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40 % Acrylamide
38g acrylamide (MW 71.08), 2g bis-acrylamide (MW 154.2) in 100 sddH20 . This was filtered 
and stored in the dark at 4 °C. The required constituents needed to prepare the 7% 
polyacrylamide; plug and running (separating) is given in the following table.
Ingredients 5 % Polyacrylamide gel solution
Water (ml) 30.00
Urea (g) 8.25
40 % acrylamide (ml) 11.66
10 X TBE buffer (ml) 5.50
10 pi ammonium persulphate (APS) and 5 [J.1 o f TEMED were added to 2.4 ml o f the gel to plug 
the plates to avoid leakage. 60 jxl o f APS and 30 pi o f TEMED were added to the rest o f the 
solution to make the separating gel.
The following table gives the required quantities o f ingredients needed to prepare stacking gel 
for the above running gel and plug.
Ingredients 4.5 % Polyacrylamide gel solution
Water (ml) 31.00
Urea (g) 8.25
30 % acrylamide / 0.8 Bis-acrylamide(ml) 7.50
10 X TBE buffer (ml) 5.50
3.5 ml of this solution were taken per gel, 30 jLtl o f  APS and 10 pi TEMED were added and used 
as stacking gel. The gel was stained in EtBr.
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ii. Gel loading buffers
5x Orange G
20% (w/v) Ficoll, 25 Mm EDTA, 2.5mM EDTA. 2.5% (w/v) orange G. Stored at 4°C
6 X  Bromophenol blue
0.25 % bromophenol blue was added to 40 % sucrose in water and stored at 4°C. Bromophenol 
blue xylene cyanol; 1 volume o f bromophenol blue xylene cyanol and 4 volumes o f cyanide.
DNA molecular weight size markers
The following DNA molecular weight markers were used at various stages o f the study: those 
that were obtained from the manufacturers like lOObp ladder marker was diluted according to 
their recommendations.
i. hind HI molecular weight maker
Digestion of y/DNA with Hind III generates 8 fragments o f the following lengths: 
23130 bp, 9416 bp, 6682 bp, 4361bp, 2322 bp, 2027 bp, 564 bp, 125 bp.
ii. 100 bp ladder molecular weight marker:
The first band size is 100 bp, the subsequent ones measure 200, 300...... 1000 bp.
A reaction mixture of 4 samples was set 
SDH20
Buffer ( MgCl2) (NH, )2S04 Tris-HC) 
dNTP mix 1.6
16
16
0.5
m
20|il
64.8
8
Cpl
CP2
Taq
DNA template
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c) Protein (Esterases) Analysis By Polyacrylamide Gel Electrophoresis (PAGE)
The electrophoresis and PCR standard solutions were prepared using sterilised double distilled 
water (sddw). Where applicable, the solutions were autoclaved at 121 lb/sq in. for 15 minute in 
an Eyela Autoclave (Rikikkaki, Tokyo).
0.558 g a  naphthyl acetate was dissolved in 100ml absolute ethanol to make a solution with a 
concentration o f 30mM.
Tris-HCL buffer (pH 8.9)
54.513g o f Tris (121.14 MW) was dissolved in 300ml of distilled water to make 1.5M and pH 
adjusted using HC1.
Tris-HCl buffer (pH 6.7)
16.67ml of the above solution was made up to 50ml with distilled water to make a concentration 
of 0.05M.
0.1% Bromophenol Blue
O.lg of 1% aq bromophenol was dissolved in lOmls o f  distilled water and 1/10 dilution taken. 
Homogenising Buffer
5ml of Tris-HCL buffer (pH 8.9) was added to 50g sucrose and 0.4 ml o f 1% aq bromophenol 
blue, this was made up to 100ml solution.
Electrophoresis Buffer of (pH 8.3)
3.0285g of Tris (121.14 MW) was used to make 0.05M and 14.2633g o f Glycine(75.07 MW) 
was used to make 0.38M , the two were dissolved in 500ml o f distilled water.
10% Ammonium persulfate (APS)
O.lg was dissolved in 1ml of distilled water
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7% Separating Gel
40% Acrylamide/Bisacrylamide (19:1) 
1.5M Tris-HCL pH 8.9 (4x)
10% APS in (IH2O 
TEMED 
Distilled water
5.0ml
0.5ml
0.01ml
10.9ml
20ml
3.5ml
2.5% Stacking Gel
40% Acrylamide/Bisacrylamide (19:1) 
1.5M Tris-HCL pH 8.9 (4x)
10% APS in dH20  
TEMED 
Distilled water
0.625ml 
2.5ml
0.1ml
0.005ml
6.8ml 
10 ml
Phosphate Buffer (pH 6)
13.8g of Sodium phosphate (138 MW) was dissolved in 5000ml of distilled water to make a 
solution of concentration 0.2M.
Staining Solution
0.1 g of fast blue salt was added to 50ml of 0.2M phosphate buffer (pH6)/gel. And 1ml, o f 30mM 
a naphthyl acetate also added and kept in the dark.
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APPENDIX II: SURVEY
Preliminary survey to determine insecticide use patterns in the major cabbage growing
areas in Southern Ghana
Questionnaire for Cabbage Producers
1. Survey Agent................................................. 2. Date.......................
3. Farmer Name................................................... 4. Site........................
5. What is the size o f your farm per season?
1 = < 1/2 ha
2 = 1 ha
3 = 1-2 ha
4 = >2 ha
6. How long has your field been under brassica cultivation..................................................
7. What irrigation equipment do you use?
1 = sprinkler
2 = watering can
3 = water hose
8. If watering can, what type o f nozzle is attached..............................................................
9. Are insect pests a problem on your farm Yes [ ]No [ ]
10. Which is the most serious pest on your farm?
1 = diamondback moth
2 = cabbage looper
3 = aphids
4 = cabbage worm
5 = all the above
6 = other (specify)
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1 1 . Which season is it most serious
1 = dry season
2 = wet season
12. How do you control these pests?
1 = spraying insecticides
2 = cultural methods
3 = botanicals
4 = biological control
5 = IPM
6 = Other (specify)
13. If by use o f chemicals, which type o f insecticide do you use
currently?.......................................................................................................................
14. Which others have you ever used........................................................................
15. Why did you stop using them?............................................................................
16. Do you treat your cabbage nursery before transplanting Yes [ ] No [ ]
17. How do you use the various insecticides?
1. Alternatively (one after the other)
2. as mixture
18. State the precise dosage applied by you of the insecticides you
use..........................................................................................................................
19. How many cabbage plants do you spray with your stated concentration o f 
insecticides?............................................................................................................
20. What insecticide application equipment do you use?
l=Knapsack sprayer (hand operated)
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2= other (specify).................................................................
21. Give reasons for your choice of equipment........................................................................
22. If you use a knapsack sprayer (hand operated), what nozzle type do you use?
l.Po ly je tred  2.Polyjet yellow 3.Polyjet green 4 .Polyjet blue 5.Cone
23. Where do you buy your pesticides?.......................................................................................
24. Do you wear protective clothing during insecticide application? Yes [ ] No [ ]
25. What is the interval (hours/days/weeks) between insecticide application and 
watering?..........................................................................................................................
26. How many times do you treat your cabbage field before harvest.................................
27. How long do you wait after last insecticide application before you harvest..................
28. Where do you get professional advice on proper insecticide use and handling?
1 = From Agric Extension Officers
2 =Fellow farmers
3 = Pesticide sales points 
4=other (specify)
29. How often do you receive such advice from extension officers?......................................
30. Do you keep farm records on your insecticide use patterns? Yes [ ] No [ ]
31. Have you had any training in EPM Yes [ ] No [ ]
32. Do you apply the methods you learnt from the EPM training Yes [ ] No [ ]
33. What percentage of your income is derived from the sale o f cabbage.............................
34. Do you have any knowledge on the problems associated with insecticide residues in foods?
1- Yes [ ] 2. No [ ]
35. If yes, please elaborate......................................................................................................
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Results of Determination o f a- -Naphthol Calibration Curve
Concentration x 10'7 (M) Absorbance (600nm)
0.20 1.433
0.10 0.673
0.05 0.331
0.025 0.146
0.0125 0.064
0.00625 0.027
Table 7: Results of Determination of (3 -Naphthol Calibration Curve
Concentration (M) Absorbance (450nm)
0.5 x 10" 0.379
0.25 x 10" 0.289
0.125 x 10* 0.148
0.625 x 10° 0.086
0.3125x10'" 0.042
Table 8: Results of Determination of Calibration Curve For 0.1% BSA
Concentrationx IO'"1 (mg/mL) Absorbance (750nm)
10 0.612
5 0.594
2.5 0.493
1.25 0.315
0.625 0.187
’J A 12
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Zymgram patterns of 5 strains of DBM populations and zymogram patterns showing the fast-and 
slow-moving esterase isozymes (Maa and Liao, 2000)
172
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Ab
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Ca l i b r a t i on  cu r ve  for  Be ta  N a p h t o l
Y = 5 .01E -02  + 0.7 15957X 
R -Sq = 0 3.1 %
Conc/M
C a l i b r a t i o n  c u r v e  for  A l p h a  N a p h t o l
Y = -3 .1E -02  + 7 .26289X  
R-Sq = 99.9  %
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Ca l i b r a t i o n  C u r v e  f o r  0.1 % B o v i n e  S e r u m  A l b um i n
Z = Log ten(X )
Y = 0 .273159  + 0 .51224 1Z 
R -Sq = 98.5  %
Conc/M
173
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APPENDIX V: RESIDUES ANALYSIS
Site Solvents
Residues 
wet( Dg/ml) Total mort. % mort.
Active*3 
resd. Dg)
% Active*b. 
residues
Mampong Methanol 127066.6667 56 93.3333 0.4885 0.0004
Ethyl acetate 10900 7 11.6667 0.0957 0.0009
Hexane 6733.33 8 13.3333 -0.1032 -0.0015
Madina A Methanol 47150 51 85 0.4410 0.0009
Ethyl acetate 17750 14 22.5 0.1803 0.0010
Hexane 10950 2 2.5 -0.1918 -0.0018
Madina B Methanol 58350 51 85 0.4410 0.0008
Ethyl acetate 10500 21 35 0.2779 0.0026
Hexane 3650 6 10 -0.1304 -0.0036
Dzorwulu
A Methanol 89666.66667 46 76.6667 0.3935 0.0004
Ethyl acetate 12133.33333 46 76.6667 0.6033 0.0050
Hexane 4800 21 35 0.0741 0.0015
Dzorwulu
B Methanol 405033.3333 36 60 0.2985 7E-05
Ethyl acetate 17233.33333 24 40 0.3169 0.0018
Hexane 8900 23 38.3333 0.1014 0.0011
Airport A Methanol 54200 28 46.6667 0.2225 0.0004
Ethyl acetate 13700 9 15 0.1217 0.0009
Hexane 5433.333333 13 21.6667 -0.0350 -0.0006
Airport B Methanol 108233.3333 52 86.6667 0.4505 0.0004
Ethyl acetate 13533.33333 19 31.6667 0.2519 0.0019
Hexane 8333.333333 21 35 0.0741 0.0009
Control*0 Methanol 120100 2 5 -0.0151 -1.3E-05
Ethyl acetate 6400 1 2.5 0.0241 0.0004
Hexane 4800 0 0 -0.2123 -0.0044
*c Control- Insecticide free cabbage samples grown in the screen house.
N/B Active residues for mortalities below 26% could not be estimated from the calibration curves o f the insecticide 
standards
N/B A total o f 60 Brine shrimp nauplii were used for each fractions of the sample extract while a total o f  40 brine
shrimps wee used for each of the control fractions
N/B A volume o f 250^1 of the cabbage extracts was used.
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Calibration curve for Cypermethrin
Concentration(ng/ml)
Calibration curve for chlorpyrifos
Concentration(ng/ml)
Calibration curve for Pirimiphos-methyl
0  100  2 0 0  3 0 0  4 0 0  5 0 0
Concentration(ng/ml)
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APPENDIX VI: STATISTICAL ANALYSIS
Analysis of variance
Variate: Alpha-naphthy esterase
Source of variation d.f. s.s. m.s. v.r. F pr.
Lo ca t i on  6 1784.856 297.476 40.33 <.001
Residual 168 1239.049 7.375
Total 174 3023.905
F tab at 5 (2 .16 )  0.01 ( 2 .92 )
Analysis of variance 
Variate:Beta naphthyl esterase
Source of variation d.f. s.s. m.s. v.r. F pr.
Location 6 73191.1 12198.5 30.14 < -001
Residual 168 68002.9 404.8
Total 174 141194.0
F tab at 5 (2 .16 )  0 .01 (2 .92)
Analysis of variance
Variate: Chlorpyrifos-methyl
Source of variation d.f. s.s.
Location 7 42623.
Residual 13 96212.
Total 20 138835.
m. s .
6089 . 
7401.
v.r. F pr. 
0.82 0.586
Analysis of variance 
Variate: Pirimifos-methyl
Source of variation d.f. s.s.
Location 7 350.82
Residual 13 716.44
Total 20 1067.26
m.s. 
50 . 12 
55 . 11
v.r. F pr. 
0.91 0.529
Analysis of variance
Variate: Pyrethroids
Source of variation
Location
Residual
Total
f .
7
13
20
s . s. 
112 .302 
103.701 
216.003
m.s. 
16.043 
7 . 977
v.r. F pr.
2.01 0.131
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