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QL”G355591 ')7' , Hiiiiiiilli3 0 6 9 2  1 0 0 0  4 0 9 3  8
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STUDIES ON THE CHEMORECEPTION PROFILE OF BULINUS  
TRUNCATUS {AVDOUIN) AS AN AID TO THE DEVELOPMENT OF 
CONTROLLED RELEASE MOLLUSCICIDES.
BY
JOHN KOFI DOMEH 
B. Sc (Hons), (Legon)
• . ‘ "  “  i
A THESIS SUBMITTED TO THE UNIVERSITY OF GHANA 
IN PARTIAL FULFILM ENT OF THE REQUIREM ENTS FOR 
THE AWARD OF M ASTER OF PHILOSOPHY IN ZOOLOGY 
(APPLIED PARASITOLOGY).
DEPARTM ENT OF ZOOLOGY 
UNIVERSITY OF GHANA 
LEGON. GHANA.
FEBRUARY, 1998.
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Table of Contents:
Acknowledgement
Dedication
Declaration
List of Figures IV
List of Plates v
List of Tables 1/1.
Abstract ™
CHAPTER 1: INTRODUCTION 1
1.1 Epidemiology and geographical distribution of Schistosomiasis 1
1.2 Water and Human Life “
1.3 Schistosomiasis in Ghana 3
1.4 Historical account of Schistosomiasis 6  
1 5 Life cycle of Parasite 7 
1 6  Location of adult worms 7
1.7 Eggs, Miracidia and Sporocysts 8  
1 .8 Cercaria 9
1.9 Morbidity of infection 9
1.10 Schistosomiasis control 10
1.10.1 Chemotherapy 10 
1 10 .2 Modification of Human behavior 11
1.10.3 Snail Control 12
1.10.3.1 Manipulation of the Physical environment 13
1.10.3.2 Manipulation of the Biological environment 13
1.10.3.3 Manipulation of the Chemical environment 15 
1.11 Scope of present study 16. 
CHAPTER2: Chemoreception response of Bxdimts truncatus to some unprocessed
bioactive plant materials 18
2.1 INTRODUCTION 18
2.2 MATERIALS AND METHODS 20
2.2.1 The snail 20
2.2.2 Preteatment of snails 20
2.2.3 Preparation of materials 24
2.2.4 The Olfactometers 24
2.2.5 The bioassay 28
2.2.6 Scoring snail positions 30
2.3 RESULTS 31
2.3.1 Attractant Effects 3 ]
2.3.2 Arrestant Effects 3 7
2.3.3 Repellent Effects 3 7  
2 4  DISCUSSION 4 3
2.4 .1 Discriminatory Response 4 3
2.4.2 Response to Fruit materials 4 4
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CHAPTER 3: Chemoreception response o f  Bulinus truncatus to some processed 
bioactive plant materials
3.1 INTRODUCTION
3.2 MATERIALS AND METHODS
3.2.1 Processing o f  materials
3 .2.2 Choice of materials for processing
3.2.3 Processing procedures
3.2.4 The bioassay
3.3 RESULTS
3.3.1 Attractant Effects
3.3.2 Arrestant Effects
3.3.3 Repellent Effects
3 .3 .4 Response to fermented materials 
3 4 DISCUSSION
3.4.1 Age-specific response
3 .4 .2 Effect of fermented materials 
CHAPTER 4: Relative effectiveness of unprocessed and processed bioactive materials 
attractants, arrestants and repellents
4.1 Effect of processed leaves
4.2 Effect of processed stems tubers
4.3 Effect of processed roots tubers
4.4 Processed repellents
4.5 Comparing processed and unprocessed materials
4.6 Effectiveness of processing techniques
4.6.1 Sun-drying and fermentation
4.6.2 Oven-drying and fermentation
4.6.3 Boiling and fermentation
4.6.4 Boiling and oven-drying
4.7 Best processing technique
4.8 Suggestions for further work
CHAPTER 5: Effectiveness of attractants and arrestants under Simulated Natural 
Conditions (SNC)
5.1 INTRODUCTION
5.1.1 Criteria for choice of materials tested under SNC
5.2 MATERIALS AND METHODS
5.2.1 Snail breeding
5.2.2 Simulated Natural Environmental Conditions (SNC)
5 .2.3 Trap design
5.2.4 Test materials
5.2.5 The SNC experiment
5.3 RESULTS
5 .3 .1 Number of snails caught
5.3.2 Unprocessed materials under SNC test
5.3.3 Processed materials
5.3.4 Bioactive fruits
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5.3.5 Trapping efficiency of bioactive materials
5.4 DISCUSSION
5.4.1 Concordance with bioassay tests
5 .4.2 Conversion of bioactive ingredients
5.4.3 Fermented materials and fruits
CHAPTER 6 : GENERAL DISCUSSION
6 .1 Choice of materials for bioassay tests
6.2 Bioassay experiments
6.2.1 Unprocessed materials
6.2.2 Processed materials
6.3 Evaluation of efficiency of bioactive materials under Simulated Natural 
environmental Conditions
6.3.1 Choice of bioactive materials
6  .3 . 2  Choice of trapping devices 
CONCLUSION
Appendix 1 
Appendix 2 
Appendix 3 
Appendix 4 
Appendix 5 
Appendix 6  
Appendix 7 
Appendix 8 
References
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ACKNO WLEDGEM ENT.
I would like to acknowledge the Danish Bilharziasis Laboratory (DBL) for providing generous grant 
for this research work. I also want to thank sincerely Shell Ghana Limited for their assistance, that 
came in the form of University of Ghana Graduate Fellowship, covering the entire MPhil. Program 
of study
I am very grateful to my supervisor; Dr. John Kpikpi, whose tolerance, resourcefulness and wonderful 
supervision skills contributed immensely to produce this work
My sincere thanks also go to the Head and entire staff of Department o f Zoology, University of 
Ghana, for use of facilities. I am particularly grateful to the following for personal assistance I 
received in the course of the research work: Mr Joseph Ofosu-Barko, Mr. Boni, Mr. Simon Mensah 
(of blessed memory), Mr. R. Adjoh, Mr. Assiedu and Mr Daniel Kyei-BafFour.
I am also thankful to the Herbarium staff of the Department of Botany, especially Mr. Abbiw and Mr. 
Amponsah, for assisting me to identify some of the test materials used I also thank the chief 
technician, Mr. Sekou,for providing me with weighing devices for the snails.
I wish to give special thanks to my colleague, Mrs. Juliana Adjei-Bieni, for her wonderful 
encouragement and many useful suggestions she offered. Special thanks also go to the following for 
their encouragement : Johnson, Emmanuel, David, Joe, Nana, Nana Yaa, Kofi and others.
I am very grateful to my assistant, Mr. Prince Avonyo, and also the driver, Ernest, for readily offering 
any help required of them during the research period. May God bless everyone o f you for your time 
spent on me.
My final thanks go to my friends and roommates; Mr. Addo Nyamekye, Mr. Sam Boateng, Mr. 
Ahenkorah and Mr. Mike Dade for encouraging me especially during the thesis write up period.
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DEDICATION:
WITH ALL HUMILITY TO
THE LORD JESUS CHRIST
AND TO
ALL FAMILY MEMBERS WHOSE ASSISTANCE HAS BROUGHT ME THIS FAR
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DECLARATION:
This thesis is original work produced by the author alone. Where information from other works ha: 
been used, references to those sources have been duly cited.
This work in its original form has never been submitted to this or any University for the award o f a 
degree, or any other qualification.
(Author)
(Supervisor)
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C H A P T E R  O NE.
1 GENERAL INTRODUCTION.
1.1 Epidemiology and geographical distribution of Schistosomiasis:
Schistosomiasis is the second most prevalent parasitic infection apart from malaria. Unlike the 
latter, whose prevalence tends to fluctuate with the rains in many areas of high endemicity for the 
year, schistosome infection prevalence may be usually high throughout the year in areas where the 
disease is endemic. Available WHO(1991) document indicates that the disease afflicts more than 
200million people while 500-600million are considered to be at risk of infection in 76 countries. 
Furthermore, mortality rate is estimated to be in excess of 200,000 per year and 20million people are 
known to suffer from various forms of clinical morbidity and disability due to the infection each year 
(TDR, 1995; Hodasi, 1995).
Essentially, schistosomiasis is a tropical disease . It affects people of all countries in Africa, except 
Lesotho, especially those south of the Sahara where the disease is endemic. In South America it is 
endemic in Brazil, Surinam and Venezuela. In the middle-east it occurs in Yemen, Saudi Arabia, 
Israel and UAR. Some countries in the Caribbean such as St Lucia, Puerto Rico and Antigua and 
also the West Indies, parts of China and India have considerable numbers of schistosome infections 
(Rollinson and Southgate, 1987; Correa and Paraense, 1973). Out of the 76 countries affected by the 
disease more than 50 occur in Africa and it is estimated that 90% of all cases of schistosomiasis occur 
in Africa. In addition, every 3 out of 4 children may be infected (TDR, 12th Program report, 1995). 
One of the most important causes of the increase in prevalence of the disease in most areas has been 
the rapid increases in the development of freshwater resources . These developments sought to
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provide sufficient freshwater for agriculture, hydroelectric power and also for domestic use. 
Unfortunately, the health implications of these developments were not given any serious attention 
before such programs were undertaken.
1.2 WATER AND HUMAN LIFE
Water is essential to man, animals and plants and without water life on earth would not exist. 
From the very beginning of human civilization people have settled close to water sources. Indeed 
where people live some water is normally available for drinking, for domestic use and possibly for 
watering animals (Hofke, 1983).
Mayer (1987) remarked that all forms of life on our planet depend on water. Water is the most 
precious resource on earth. Although there are microorganisms which can do without oxygen, even 
the most primitive unicellular life forms cannot live without water. Since it is ubiquitous, the 
presence of water, like that of air, is taken for granted (Ihenkoronye and Ngoddy, 1985).
In general,71% of the earth’s surface is covered with water, of which only 1.9% is freshwater; less 
than a quarter of that occurs in the form of groundwater, lakes or springs (WHO, 1994) .
Due to the unique importance of water to human life the rapidly increasing world population calls 
for development of traditional water resources for increased output in agriculture , industry and also 
for domestic supplies. Creation of dams, ponds, and irrigation systems to boost up agriculture and 
to supply hydroelectric power and also freshwater for domestic use has resulted in rapid increases in 
prevalences of water-related diseases including schistosomiasis in most of the affected areas. 
However, in most of these areas no serious thought is given to the health implications o f such 
projects. Even where scientific research gives indications of the health consequences that are bound
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to emanate from such projects no preventive measures are taken in most cases before these projects 
are carried out. This, in many cases, is due to lack of adequate financial resources as in the case of 
Ghana and most African countries.
13 SCHISTOSOMIASIS IN GHANA
Available records indicate that both urinary and intestinal schistosomiasis have been in Ghana since 
the colonial days. Earliest reports of the disease occured in the Annual Report of the Colony of the 
Gold Coast in 1895. All reported cases of the disease by then, which were derived from hospital 
records, were only on urinary schistosomiasis . According to Odei (1961) the earliest reported case 
of intestinal schistosomiasis was in 1920, when Macfie reported the presence of lateral-spined eggs 
of schistosoma in the urine of a male lunatic.
Field work done by many workers since then showed that the disease was present in all the regions 
of the country with varying levels of prevalence. Edington (195 7, as cited by Odei 1964) found that 
90% of children in an area in Western Ashanti had the infection whereas in nearby villages there was 
little or no infection McCullough (1965) also reported similar variations of infection rates in his 
work within southern Ghana. He found that more than 75% of children examined at Pokoasi and 
Maiyara were infected whereas the infection was less than 30% at neighbouring areas like Agbogba, 
Ashongman, etc. Such variations in infection, the workers noted , was largely due to factors which 
govern the frequency of exposure of the inhabitants to infection. In addition, it was noted that 
urinary schistosomiasis was more widely distributed than intestinal schistosomiasis. This was 
attributed, in part, to the reluctance of the inhabitants to submit stool samples for examination. A 
more important reason was the relative distribution of the respective intermediate host snails for the
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two schistosome parasites responsible for each form of the disease. Bulinus globosus, the then 
known intermediate host snail for Schistosoma haematobium responsible for urinary schistosomiasis 
was more widely distributed whereas Biomphalaria pfeifferi, the intermediate host for Schistosoma 
mansoni was more restricted in distribution, mainly around Tarkwa and in the Northern sector of the 
country (Odei, 1965).
One point worthy of note is that before 1964 (ie the pre Volta lake period) prevalence of both urinary 
and intestinal schistosomiasis was very low and especially along the Volta river the estimated 
prevalence was as low as 0.5% ( Chinery, 1990). According to Hodasi (1995) the disease was 
virtually absent from the area during this period. Major foci then were in the Upper and Northern 
regions and also in large areas of Brong Ahafo, Ashanti, Eastern, Western and Central regions. Two 
years after construction of the Volta lake, both forms of the disease had spread rapidly to all parts of 
the country such that by 1967 infection rate was 37% in some villages along the Volta lake and 
reaching 100% by 1968 (Chinery, 1990).
At the moment, both urinary and intestinal schistosomiasis are endemic in the country but urinary 
schistosomiasis is more prevalent and has a more widespread distribution. The reasons for this 
include the following :
1. Creation of the lake gave rise to extensive aquatic vegetation due to nutrients from decaying 
plants from catchment area. These detached and transported the entrapped snails and their eggs to 
other parts of the lake downstream where they became established.
2. More extensive distribution of the two main intermediate hosts of S. haematobium; B. truncatus 
rohlfsi which is limited virtually to the savanna zones and B. globosus which has both forest and 
savanna distribution ( McCullough, 1965).
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3. Localized distribution of Biomphalaria pfeifferi, the intermediate host of S. mansoni.
4. The fact that urinary schistosomiasis depends more on water than the intestinal type as direct 
contamination of water by urine is a more common occurrence than ingestion of food and water 
contaminated with eggs of intestinal worms.
Although the Volta lake is the largest artificial water body worldwide other smaller dams created 
all over the country also contributed significantly towards the increase in prevalence rate of 
schistosomiasis in the country. A WHO(1993) document indicated that between 1958 and 1960, 
104 small dams were built in the northern parts of the country to provide enough freshwater for both 
domestic and agricultural purposes especially during the dry seasons. Consequently, prevalence rate 
of urinary schistosomiasis within 38 areas of these dams surveyed increased from 17% before the 
dams were constructed to 51% with some riparian communities recording 1 0 0 % prevalence. 
Another epidemiological study carried out in settlement areas along the Weija lake near Accra also 
indicate prevalence rates between 34.2% and 89.4% of urinary schistosomiasis (Zuta, 1994).
It can be said that, until recently, there has been a lack of serious effort at controlling the spread of 
the disease in the country. One reason for this is the insiduous nature of schistosomiasis in general. 
Unlike other parasitic infections such as malaria in which victims usually exhibit symptoms within 
a few days after infection, symptoms of schistosomiasis usually appear after two to three months 
depending on the state of immune responsiveness of the victim At the moment, some efforts are 
being made towards the control of schistosomiasis along the lower Volta basin by the Volta River 
Authority by means of health education and chemotherapy. It is hoped that this would be extended 
to other parts of the country where the disease is endemic. If this is not done the desired effect of 
this control measure along the Volta cannot be achieved as human migrations could easily
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re-introduce the disease to the Volta basin again.
1 4 HISTORICAL ACCOUNT OF SCHISTOSOMIASIS.
Available records indicate that the recorded history of schistosomiasis dates back to the 16th 
century . The Ebers papyrus discovered at that time contained what was thought to be a reference 
to its treatment or prevention (Macpherson and Craig, 1991).
Both Schistosoma haematobium and Schistosoma mansoni are believed to originate in Africa, 
possibly in Central Africa, and S. Mansoni was spread around the world during the slave trade 
especially to South America (Wright, 1966,as cited by Macpherson and Craig, 1991). The first 
evidence of the earliest records of the disease in Africa was provided by Ruffer in 1910 when he 
found characteristic eggs of S. haematobium in mummies dating back to 1250-1000 B C. During 
a post-mortem examination of a patient in Cairo, Egypt, in 1851 the German pathologist, Theodore 
Bilharz, recovered the first schistosome worms and he later linked it to the haematuria in Egyptians 
discharging terminally spined eggs in their urine. In 1902 Manson described the presence of laterally 
spined eggs in human faeces in the West Indies and suggested a possibility of there being more than 
one species of schistosomes. This view was supported by other workers and in 1907 Sambon 
named the worms with laterally spined eggs as Schistosoma mansoni. Other species which do not 
occur in Africa were discovered ; Katsura recovered adult S. japonicum  from the portal sysem o f a 
cat in 1904. Leiperinl915
finally demonstrated the existence of S. haematobium and S. mansoni as two distinct species which 
have morphologically different adult worms and eggs, different in distribution in the definitive hosts 
and a dependence on snails of different genera as intermediate hosts ( Abdel- Wahab, 1979)
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1 5 LIFE CYCLE OF PARASITE
Schistosomiasis is caused by the digenic trematode (commonly called blood flukes) of the genus 
Schistosoma. These flukes belong to the Phylum Platyhelminthes and Family Schistosomatidae. 
At the moment, nineteen species of the genus are known to cause the disease in mammals and birds 
but only five of them are significantly responsible for schistosomiasis in man. These include 
Schistosoma haematobium and Schistosoma mansoni, which have man as the principal definitive 
host, and also S. japonicum, S. intercalatum and S. mekongi which are zoonotic in nature infecting 
mainly herbivorous mammals (sheep, water buffalo, etc.)
1.6 LOCATION OF ADULT WORMS IN DEFINITIVE HOSTS:
The adult female schistosome is permanently held in the gynaecophoric canal of the male and the 
paired worms inhabit various anatomical locations of the definitive hosts depending on which species 
of the worms are involved Adults of S. haematobium are found in the vesical plexus and 
sometimes in the portal veins and its mesenteric tributaries. Adults of S. mcnisoni are commonly 
found in the inferior mesenteric veins and its tributaries . Copulation and oviposition occur at these 
locations.
1.7 EGGS, MIRACIDIA AND SPOROCYSTS:
The eggs, which are oval in shape and non-operculate are characterised by either lateral (those of
S.mansoni) or terminal (S.haematobium) spines. They secrete enzymes which enable them to move 
into the lumen of the intestine or the urinary bladder by passing through the venules and tissues
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Approximately half of the eggs produced are voided ‘normally’. The other half go astray and end 
up in ectopic sites where they embryonate in six days and live for another 15 days During this 
period they secrete antigenic materials which are toxic to the surrounding tissues This invokes 
immune reactions which lead to granuloma formation. The unshed (unexcreted) eggs are eventually 
destroyed by macrophages through phagocytosis over a period of months 
Under favourable conditions of temperature, light and osmotic pressure the eggs deposited in the 
external environment, on reaching a freshwater body , hatch into free-swimming ciliated larvae, the 
miracidia. These seek and infect by penetration the appropriate snail intermediate host a few hours 
after hatching to proceed with development Lytic secretions as well as muscular action are 
thought to be involved in the mechanism of penetration which usually occurs along the tentacles, 
mantle collar or the head-foot regions of the snail. There is development into a mother sporocyst 
which later gives rise to numerous daughter sporocysts. These migrate to the hepatopancreas of the 
snail and continue development there for several weeks to form cercariae which are later shed by the 
snail into the water.
18 CERCARIA.
This larval stage of the parasite is infective to the definitive hosts including man. When shed from 
the snail host into the water it remains infective for approximately 20 hours . Infection is by direct 
penetration of the unbroken skin and mucus membrane of the host. During the penetration process, 
the cercaria loses the tail, external layer and penetration glands and transforms into a schistosomulum 
which is known to be capable of surviving only in salt water (Warren, 1973) . The schistosomula 
move through the tissues of the portal vein into the lymph and blood vessels and eventually reach
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the lungs where they remain for several days. They then migrate to the liver via the blood stream 
or directly through the tissues In the liver, they develop into adult male and female worms, move 
back to either the blood vessels of the bladder or the mesenteric veins of the intestines, depending on 
the type of worm involved They then mate and start a new cycle (Warren, 1973).
1.9 MORBIDITY OF INFECTION
Morbidity of schistosomiasis infection depends on the intensity of the infection. Only a small 
proportion of the victims have high intensity of infection (Sturrock et al., 1987). The majority o f the 
victims without urgent need for clinical attention remain untreated and act as reservoir hosts of the 
disease and thus maintain the transmission cycle.
Generally, schistosomiasis morbidity is due to the massive egg output by the adult worms of both S. 
haematobium and S. mansoni (Cheever,1968, as cited by Kpikpi, 1990). Only a few of these eggs 
are expelled from the victim. The rest cause various organomegalies in their victims, the most 
common ones being splenomegaly and hepatomegaly (Friis and Byskov, 1987). Other notable 
effects include ureteric strictures, irregularities of the contour of the bladder, hydronephrosis as well 
as carcinoma of the urinary bladder (Honey and Gelfand, 1960). Most often, some of the eggs may 
be transported to the brain and spinal cord forming various granulomas which may need surgical 
operation to remove (Cosnnet and Van Dellen, 1986).
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1 10 SC H IST O SO M IA SIS C O N T R O L .
Considering the increasing incidence of schistosomiasis the world over it is apparent that 
measures which have been put in place to check the spread of the disease have not been quite 
successful. At the moment, the emphasis is on reduction of transmission to levels at which the more 
serious morbidity associated with the advanced stages of the disease are unlikely to occur 
(WHO, 1983a). The various control methods in use at the moment are discussed below.
1.10.1 CHEMOTHERAPY.
Over the years, schistosomiasis control has relied on chemotherapy as the method of choice The 
expectation of this method is that by using drugs, in the correct dose, the adult worm load of the 
victim would be reduced and thereby reducing egg output and hence the disease transmission rate. 
Among the several drugs that have been tried the most outstanding ones include thioxanthone, 
antimony compounds (Astiban) (Gothe et al.,1965), Niridazole and Hycanthone. In recent times the 
drugs have included Metrifonate which is an organophosphorous cholinesterase, Oxamniquine and 
Praziquantel (WHO, 1993). Most of these drugs, when taken in the correct doses, are able to destroy 
all adult worms within the victim. Chemotherapy is thus thought to achieve rapid success in 
schistosomiasis control.
However, the rapid re-infection that follows drug administration tends to obliterate the perceived 
successes. It has been reported that in order to contain transmission, drug administration needs to 
be carried out annually or biennually (Sturrock et al., 1987, Polderman and Mashande, 1981, Bensted- 
Smith et al ,1987,Tingley et al., 1988). It has been argued that schistosomiasis transmission would
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be broken if drugs were used to kill the adult parasites. However, the high cost, logistics and 
difficulty in getting the full coorperation of all the people makes chemotherapy a less feasible option 
in schistosomiasis transmission control. It should be seen as a way of offering some relief to victims 
with serious clinical complications due to high intensity of infection (Kpikpi, 1990).
1.10.2 MODIFICATION OF HUMAN BEHAVIOUR
Many aspects of human behavior patterns tend to encourage the transmission of schistosomiasis. 
These include ignorance of the transmission patterns of the disease due to lack of appropriate 
education . A number of surveys conducted in areas where the disease is endemic indicates that 
there is lack of basic knowledge of the disease (Agudogo, unpublished, Stephenson et al., 1986). 
Beliefs and customary practices also contribute to increased schistosomiasis transmission. Due to 
lack of knowledge the disease is considered a natural phenomenon associated with some traditional 
practices. For example, haematuria in males among the Gongola people in Nigeria is taken to signify 
the coming of age of the victim (Akogun, 1991).
Some occupations such as agriculture and also recreational activities that draw humans to water 
bodies also promote schistosomiasis transmission Furthermore, efforts to increase water supplies 
and provide irrigation facilities have encouraged the spread of the disease 
Education of the people in areas where the disease is endemic is normally by means of speech and 
visual aids to desist from practices that promote and bring them in close contact with infected waters 
and those which result in more snails becoming infected. Coupled with this is the provision of 
alternative sources of water for domestic use and toilet facilities for hygienic disposal of human 
excreta and urine
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Unfortunately, poor implementation and the lack of adequate logistics have so far resulted in very 
little being achieved by this control method (WHO/UNDP, 1979; el Kholy et al., 1989). In addition, 
the difficulty of getting people to change from certain normal practices, such as fishing, that requires 
constant contact with water and the absence of toilet facilities in canoes have all been contributory 
factors against modification of human behavior (Jordan and Webbe,1982).
1.10.3 SNAIL CONTROL.
The intermediate host occupies an important part of the life cycle of the Schistosome parasites. Their 
control is of vital importance towards the successful control of schistosomiasis. Some features of the 
snails, however, tend to make their control quite difficult. These include their hermaphroditic nature 
that enables only a few of them to repopulate new water bodies, their ability to survive out of water 
over a relatively long period (Olivier, 1955; Chu et al.,1967a) and their ability to survive long periods 
of aestivation in sediments, detritus or vegetation (Thomas and Tait,1984).
Notwithstanding these factors, there have been various strategies for manipulating the biological, 
chemical and physical environments of the snails with a view to controlling their population 
dynamics. These strategies are discussed below .
1 Manipulation o f  the physical environm ent:
This can be done by measures aimed at increasing the mean velocity of moving water in channels 
beyond certain thresholds. Such measures include stream canalisation, canal relocation with deep 
burial of snails, removal of vegetation, earth filling, improved agricultural practices and proper 
drainage in irrigation schemes All these methods have been observed to increase water velocity and
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thereby decreasing snail populations in them especially when these methods are combined with 
biological control (Jordan, 1975; Jobin & Michelson,1969, Madsen and Christensen, 1992; Ayeh- 
Kumi,1996 ; Hicklin, 1988,Thomas and Compston, 1980, Jobin et al.,1984).
2. Manipulating the Biological Environm ent:
This involves measures directed at using the biotic components of the environment to control the 
population of the snails . This may be done by
a) Using competitor snails:
This involves competitive interactions between host and non-host snails. There have been a lot of 
evidence indicating the elimination of host snails from ponds upon the introduction of competitor 
species . Barbosa (1973) reported the exclusion of Biomphalaria glabrata from a pond when 
Marisa conuarieties were introduced. Also, Helisoma duryi has been shown to compete 
successfully with Bulinus and Biomphalaria under laboratory conditions (Frandsen and Madsen, 
1979; Madsen, 1982, 1983). Other intra molluscan competition tested and proved to be successful 
recently involved Melanoides tuberculata against Bulinus trimcatus (Ayeh-Kumi, 1996). These 
competitor snails have been found to also adversely affect miracidial penetration into the snail host 
by acting as a miracidial sponge (Frandsen, 1987). Most of this work is, however, at the experimental 
stage at the moment.
Much work still needs to be done on this aspect of biological control of the schistosome host snails 
before large scale field application can be feasible .
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b) Using Microbial Pathogens, Parasites, Parasitiods and Predators :
Parasites and predators are biotic agents that could be used to control population of host snails. 
Predators such as sciomyzid flies, malacophagous fishes such as Geophagus brasiliensis, 
Serranochromis sp ., THapia melanopluera , some prawns, as well as aquatic birds have long been 
considered as good biological control agents for schistosome host snails (McCullough, 1981, 
Slootweg, 1989; Weinzettl and Jurberg, 1990). Recently it has also been found in Kenya, that the 
American crayfish, Procambarus clarkii, is capable of preying on Bulinus africanus in large 
permanent pools of water and thereby reducing schistosomiasis transmission for at least two years 
(Mkoji, 1997). Micropathogens such as Bacillus thuringiensis as well as viruses have been used 
in biological control of host snails (Osman and Mohammed, 1991).
As with the use of competitor snails, most of this work is still at the laboratory stage and a lot more 
work is needed to be done before field testing can be feasible.
3 Manipulating the Chemical Environment:
This involves the use of molluscicides in the control of the host snails. Although there are a large 
number of plants with molluscicidal activity such as Phytolacca dodecandra, Ambrosia maritima 
Balanites aegytiaca and Anacardium sp.(Webbe and Lambert, 1983, Appleton, 1985) synthetic 
varieties such as Bayluscide are most preferred at the moment. This is due to their availability and 
the rapid results they give in bringing down the snail population. Also apart from ‘Endod’ which 
has been given a few field trials, studies on most of these plant molluscicides are still in the laboratory 
stage of research and development.
Despite their quick action, molluscicide use gives rise to some problems which can best be considered
14
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as more serious than the snail problem. Notable among these are their adverse effects on non target 
organisms due to their broad-spectrum activity and the high cost of the chemicals in the areas where 
they are needed (Okunji and Iwu,1988).
Owing to these problems it has been suggested that cheaper, species-specific molluscicides should 
be developed which could selectively kill snail hosts without much effect on non-target organisms 
(WHO, 1977). To enhance the efficiency of such molluscicides the use of a controlled release system 
has been proposed (Cardarelli, 1977). Thomas and Assefa (1979) suggested that such slow release 
molluscicides incorporated into specific snail attractants, arrestants and phagostimulants in the form 
of ingestible and digestible capsules would result in the release of the toxic component in the digestive 
system of the host snails only after ingestion . This would ensure a higher selective destruction of 
target snails without adversely affecting non-ta.rget organisms.
Since then, much research effort has been directed towards the search for specific attractants, 
arrestants and phagostimulants for schistosome host snails. Although some successes have been 
achieved in the identification drive (Thomas and Assefa 1978; Thomas et al., 1980,1983, 1985, 1986, 
1989; Thomas, 1982, 1986, 1989, Kpikpi, 1990, 1991; Kpikpi and Thomas, 1992, 1993 ;), all the 
materials found have been pure synthetic chemicals which are more expensive than the synthetic 
molluscicides in current use. Furthermore, such molecules may possess only one active factor for 
attracting and arresting the snail hosts and may therefore not be ideal materials.
To this end, Kpikpi (1990) suggested that it should be possible to obtain naturally occuring bioactive 
materials that could be used as attractants, arrestants and phagostimulants for use in the development 
of controlled release formulations The potency of such materials is thought to be due to the 
possesssion of complex chemical factors. Refinement or processing of some of these materials may
15
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remove any repellent factors and thereby increasing their attractant, arrestant and phagostimulant 
properties .
1.11 SC O PE  O F TH E P R E SE N T  STUDY:
The present study had three major objectives .
First, to identify naturally occurring bioactive materials which act as attractants and arrestant for B. 
truncatus snails under laboratory conditions using diffusion olfactometers The findings for this are 
reported in chapter 2  .
Secondly, to explore the possibility of improving upon the bioactive effects of naturally occurring 
materials by subjecting them to various processing regimes . This is reported in chapter 3 .
In chapter 4, a comparison between the effectiveness of processed and unprocessed naturally occuring 
bioactive materials used in chapters 2 and 3 is made .
Thirdly, to determine the applicability of the bioactive materials for field conditions by investigating 
their effects under simulated natural environmental conditions. This is reported in chapter 5 .
16
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C H A P T E R  T W O .
CHEMORECEPTION RESPONSE OF BULINUS TRUNCATUS 
TO SOME UNPROCESSED BIOACTIVE PLANT MATERIALS.
2.1 INTRODUCTION :
The control of the intermediate host snail of schistosome parasites forms a major aspect of any 
integrated program towards control of schistosomiasis. Currently, the method of choice for snail 
control is the use of synthetic molluscicides due to the rapid kills they tend to produce. The rising 
costs of these chemicals and their ecologically unacceptable effects are, however, major drawbacks 
that require a search for more cost-effective and ecologically acceptable alternatives.
Owing to these problems associated with synthetic molluscicides, the Scientific Working Group of 
the World Health Organization in 1993 recommended the development of cheaper, environmentally 
compatible and more species-specific molluscicides for control of schistosome host snails WHO 
(1977). In response to these recommendations, Thomas and Assefa (1978) suggested that known 
snail toxicants incorporated into specific attractants, arrestants and phagostimulants could be used 
to selectively remove the schistosome host snails from their natural environments.
Since then some work has been done towards the identification of the species-specific attractants, 
arrestants and phagostimulants of the main intermediate hosts of the schistosome parasites, Bulinus 
and Biomphalaria species (Daldorph and Thomas, 1988; Kpikpi, 1990; Kpikpi and Thomas, 1992, 
1993; Thomas and Assefa, 1979; Thomas, 1986, 1989; Thomas et al, 1985a,b) by means of a
17
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bioassay technique developed by Thomas and Assefa (1979).
Most of the substances so far identified as attractants, arrestants and phagostimulants have been 
pure synthetic chemicals that are not easily available in the areas where schistosomiasis is endemic. 
Furthermore, the acquisition of these chemicals may even be more expensive than the molluscicides 
in current use.
In response to a proposal by Kpikpi (1990) that it should be possible to obtain naturally occurring 
bioactive substances which could serve as more potent attractants, arrestants and phagostimulants, 
Kpikpi et al (1995) assayed 26 naturally occurring substances of plant origin using a modified form 
of the diffusion olfactometer, of these 14 (ie 53.85%) were in their raw unprocessed forms. Among 
the 5 bioactive materials identified as strong attractants and arrestants, 3 (ie 60%) were unprocessed 
This work indicated the need for expansion of the search for even more potent, naturally occurring 
bioactive materials with multiple chemical factors that can be used as attractants, arrestants and 
phagostimulants for selective removal of Schistosome intermediate host snails.
In line with these findings, 35 naturally occurring substances in their crude unprocessed forms were 
obtained from common plants found in schistosomiasis endemic areas in Ghana By means of similar 
bioassay technique, these materials were tested for their bioactive effects on both adult and juvenile 
Bulinus truncatus, the intermediate host of Schistosoma haematobium, the causative agent of urinary 
schistosomiasis in Africa.
The results of this study and its possible implications for future snail control are discussed.
18
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2.2 M ATERIALS AND METHODS:
2.2.1 The Snails:
Healthy Bulinus trimcatus snails were selected from laboratory stocks (plate 2.2.1) 
originally obtained from the Weija lake, a Schistosomiasis endemic area near Accra(plate 2.2.2). 
These were fed on fresh lettuce leaves (Latuca sativa) over a period of four months. Eggs laid by 
these snails during the period were separated and allowed to hatch. The freshly hatched snails were 
fed on fresh lettuce leaves till they attained the desired age for the experiments. Two groups o f snails 
were selected for the assay; the juveniles and the adults. The juveniles were obtained by allowing 
freshly hatched snails to grow up to 4-6 weeks after hatching. The adults were at least 10 weeks
2.2.2 Pre treatment o f Snails:
For the bioassay, 25 snails from each age group were carefully selected on the basis of health 
(mainly those observed to be feeding normally) and weight. Weight of the juveniles was 20- 60mg 
while that of the adults was 400mg. The snails were weighed just before each assay. Those whose 
weights fell outside the range were discarded. Individual juvenile snails used for the bioassay were 
placed in a separate 150ml plastic cylindrical container with 100ml o f tap water. The adults were 
similarly placed in a 250ml container with 150ml of tap water. This was to minimise variations in 
behavior of the snails (plate 2.2.3). They were kept under a temperature of 26±1°C and 
photoperiod of 1 2  hours light and 1 2  hours darkness and were allowed to acclimatize for 3  days
19
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PLATE 2.2.1
LABORATORY SETUP FOR BREEDING „F BULINUS TRUNCATUS
20
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PLATE 2 . 2 . 2
SITE WHERE SNAILS WERE COLLECTED FOR BREEDING.
21
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PLATE 2 . 2 . 3
EXPERIMENTAL SET UP FOR PRETREATMENT OF SNAILS 
BEFORE BIOASSAY.
22
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The snails were handled with plastic spoons due to their fragile nature.
Each adult snail was fed daily with a disc of lettuce leaf of diameter 2.0cm while each juvenile snail 
was given a leaf of diameter 1 1cm The water medium was changed every other day. In order to 
reduce response differences among the snails that might be due to their individual differences in 
physiology, each of the selected snails was deprived of food 20-24 hours prior to the bioassay.
2.2.3 Preparation of Materials:
Thirty-five fresh, unprocessed materials obtained from 26 different common plant species were 
used for the experiment (Table 1). These were obtained 3 days prior to the test in order to use them 
in the fresh state. To ensure their cleanliness each sample was washed with tap water and kept in 
the refrigerator. Ripe fruits used were those that have naturally ripened on the plant.
2.2.4 The Olfactometers:
Diffusion or gradient olfactometers were used for the test. These were similar to those used 
by Kpikpi et al (1995). Each olfactometer consisted of a central chamber measuring
7.8 *1.9 x 2.0 cm joined at each end to a cylindrical end chamber measuring 2.4cm in diameter and 
2.0cm in depth. A smaller version of the olfactometer used for the assay response of the juvenile 
snails measured 3.9 * 0.9 x 1.0cm with an end chamber of 1.2cm diameter and 1.0cm depth.
23
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24
PLATE 2 . 2 . 4
ADULT BULINUS TRUNCATUS FEEDING ON LETTUCE 
LEAVES.
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Ti aaboliee  1i  Pi llaanmt ss   aain'ud the various parts used as Test Materials.
Plant Name Part(s) Usee
Fruits Leaves Stems Roots
Anacardium occidentalis *
Annona muricala *,(*)
Azadirachta indica (*) *
Borassus aethiopium *
Calotropis procera *
C.arica papaya *
Chrysophyllum albicum *
Citrulus vulgaris *
Citrus sinensis
Citrus aurantifolia *
Dialium m inensis *
Duranta plumieri *
Eleais suinenesis *
Ipomea batatas *
Latuca sativa *
Maneifera indica * f*')
Manihot esculentus * *
Musa paradisiaca 1
Parlaa clappertoniana *
Phoenix dactvlifera *
Saccharum officinarum *
Svnsepalum dulcilicum *
25
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Plant Name Part(s) Used
Fruits Leaves Stem Roots
Theobroma cacao *
Thevetia neriifolia (*)
Xanthosoma maffafa * *
Zingiber officinale *
Note : (*) denotes unripe form o f fruits used. 
@ denotes peels o f fruits used.
26
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One perspex block of 2 0  olfactometers was used (plate 2.25). This made it possible to replicate 
each test material twenty times. Since the snails have been shown to respond to gravity (Lever and 
Geuze, 1965), care was taken to ensure that the olfactometers were level. Prior to the experiment 
each olfactometer was thoroughly cleaned with tap water and kept at the laboratory temperature of 
26± 1 degree Celsius.
2.2.5 The Bioassay :
The test materials were cut into smaller sizes such as can fit into the olfactometer chambers. 
The solid test materials such as fruits were cut into small discs of sizes between 7.5X 1.5x0.7 and 
6 .8 x1 .0 x0 .4mm using a knife.
The test material was placed at one chamber of the olfactometer while a polystyrene material, 
which served as the control, was in the opposite chamber. For the non solid test materials which 
were mainly in powdered form, a small quantity of the sample was placed into the chamber of the 
olfactometer and covered with a disc of No. 1 Whatman filter paper or cotton wool. In order to 
counteract any directional bias due to gradients, the test material and the control were alternated in 
successive olfactometers. 25ml and 5ml of tap water were pipetted into each larger and smaller 
olfactometer respectively and one appropriate aged snail was placed into the centre of each 
olfactometer Sminutes later. The time lapse was to allow for the establishment of a diffusion gradient 
between the test and control chambers.
27
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PLATE 2 . 2 . 5 .
DIFFUSION OLFACTOMETERS FOR BIOASSAY TESTS
28
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2.2.6 Scoring Snail Positions:
The position of each assay snail in the olfactometer relative to the test material was recorded at 2 ' / 2 
minutes interval on a specially designed chart for a period of 30minutes. They were scored positive 
if found on the test side +, or on test disc (+), and negative if found on the control side or on 
control discs ( - )  .
Difference between the + scores and -  scores was calculated for each olfactometer and the mean 
difference computed for the 20 replicates to give the attractant index for the test material. Similarly, 
the mean difference between (+) and ( - )  was calculated to give the arrestant index for the material. 
The student t-test ( Bailey, 1981) was used to determine the level of significance of the attractant and 
arrestant effects recorded.
29
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2.3 R E SU L TS:
2.3 1 ATTRACTANT EFFECTS:
The present results show that 15 (ie 42.86%) of the unprocessed materials tested emerged 
as statistically significant attractants to adult Bulinus truncatus snails at either p<0.05, 0.01 or 0.001 
significant levels (Table 2.3.1) These include mango > water melon > fresh sugar cane > ripe date 
palm > fresh pawpaw leaves > locust beans > fresh cocoyam leaves > ripe cashew, fresh cassava root 
tuber, lettuce leaves > sweet potato stem >ripe miraculin > sweet potato + lettuce leaves > sodom 
apple > showers of gold . With response index ranging between 6.7 - 8 .8 , the five most potent 
attractants for the adult B. truncatus include ripe mango > water melon > sugar cane > date palm > 
fresh pawpaw leaves (Fig. 2.3.1).
Only 6  (ie 17.14%) of the materials showed significant attractant effects on the juvenile snails at 
either p< 0.05, 0.01 or 0.001 . These include fresh sugarcane > sweet potato stem tuber > locust 
beans >fresh lettuce leaves >showers of gold > ripe cashew . Of these, fresh sugarcane > sweet 
potato > locust beans emerged as the 3 strongest attractants with response indices of 10.0, 6.3, and
4.6 respectively.
Since all 6  significant attractants for the juvenile snails are a subset of the attractants for the adult 
snails, it means that 6  (ie 17.14%) of the 35 unprocessed materials assayed proved to be statistically 
significant attractants to both adult and juvenile B.truncatus at either p< 0.05, 0.01 or 0.001 
significant levels.
30
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T able 2.3.1
MATERIALS.
TEST SCIENTIFIC A D IJ  L T S J U V E N I L E S .
MATERIAL NAME
MEAN t -value MEAN t-value
INDEX (p< 0.05) INDEX (p<0.05)
FRUITS:
Ripe cashew Anacardium 5.2 4.7720* 3.6 5.9142*
occidentalis
Mango: Mangifera indica
ripe 8 . 8 9.9173* - 1 . 6 -0.8619
unripe -2 . 0 -1.1608 -4.9 -2.3390
Water melon Citrulus vulgaris 8.7 15.3412* 0.7 0.3251
Ripe lime Citrus aurantifolia -3.9 -2.3442 -7.9 -4.2846**
Ripe miraculin Synsepalum dulcilicum 4.8 4.1223* 3.0 1.9407
Orange : Citrus sinensis
ripe 2.4 1.6250 0.4 0.1819
peels 1 .1 0.5165 3.0 1.9407
Ripe date palm Phoenix dactylifera 7.7 8.6917* 1.9 0.8444
Long banana peels Musa paradisiaca 3.0 2.2712 0 . 6 0.9112
Ripe cocoa : Theobroma cacao
pod 2 . 8 2.0881 0 . 2 0.1966
beans 5.0 4.1943* 1.5 1.5607
Ripe fan palm Borassus aethiopium 0 . 6 0.5011 1.1 0.9280
Ripe milk bush Thevetia nerifolia 2.9 1.9462 0 . 1 0.0594
Ripe royal palm Eleais guinensis 2.4 1.2972 2 . 8 1.3028
Showers of gold Duranta p/umieri 3.8 2.5470* 4.0 4.1558*
Sodom apple Calotropis procera 3.9 2.4590* -2 . 6 -2.2739
31
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TEST SCIENTIFIC A D U I , T S ITT V E N I L E S
MATERIAL NAME MEAN t-value MEAN t-value
INDEX (p<0.05) INDEX (p<0.05)
Unripe neem Azadirachta indica -4.9 -2.6252** -5.0 -2.4837**
Locust beans Par/aa 6 . 2 5.3880* 6 . 0 6.5381*
clavvertoniana
Sweet potato lpomea batatas
+ + -1.5 -0.7069 1 .1 0.5806
locust beans Par/aa
clappertoniana
Velvet tamarind Dialium guinensis 0.5 0.2425 -4.0 -2.5379
Star apple Chrysophyl/unt -5.2 -3.1628** -8 . 2 -4.6628**
albicum
Sour sop : Annona muricata
ripe 2 . 1 1 . 2 2 1 1 0 . 2 0.1297
unripe -2 . 1 -1.5411 1.3 0.9038
LEAVES:
Cocoyam Xanthosoma maffafa 5.6 3.9268* 1.4 0.7510
Lettuce Latuca sativa 5.2 3.4296* 4.6 3.4544*
Neem Azadirachta indica 2.3 1.3197 0 . 0 0 . 0 0 0 0
Pawpaw Carica papaya 6.7 5.9476* 3.5 1.4935
Cassava Manihot esciilenta 2 . 2 1.1953 2.3 1.8150
STEMS: 
Cocoyam tuber Xanthosoma maffafa 0.4 0.3043 0.4 0.2670
Ginger Zingiber officinale -7.2 -8.5868** -9.3 -10.5000**
32
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TEST SCIENTIFIC ADUL"rs JUVEN][LES
MATERIAL NAME MEAN t-value MEAN t-value
INDEX (p<0.05) INDEX (p<0.05)
Sugar cane Saccharum 8.5 13.1477* 10 20.7802*
ojficinarum
Sweet potato Ipomea batatas 5.0 5.5624* 63 6.9000*
Sweet potato Ipomea batatas 
+ + 4.4 3.5501 0.9 0.8112
lettuce leaves Latuca sativa
ROOTS: 
Cassava tubers Manihot esculenta 5.2 5.6387* -2.9 -2.1371
Note: * denotes significant attractants at 0.05 confidence limit 
** denotes significant repellents at 0.05 confidence limit.
33
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ATTRACTANT INDEX
00 O) N> K) <J) 00
Anacardium occidentalis = □
ripe Mangifera indica
I
unripe Mangifera indica 
Citrulus vulgaris 
Citrus aurantifolia j- 
Synsepalum dulcilicum 
Citrus sinensis
Citrus sinensis peels
m
C/>
fresh Xanthosoma 
maffafa leaf
s
^  fresh Xanthosoma stem
m
3
^  Phoenix dactilifera
f
Musa paradisiaca peels t r
Theobroma cacao pod 
Theobroma cacao bean 
Borassus aethiopium 
fresh Thevetia nerifolia 
Eleais guinensis 
Duranta plumieri
c□_
< >
□
CQ.
CO
C</r>T
34
Fig. 2.3.1 ATTRACTANT RESPONSES OF BULINUS TRUNCATUS TO SOME 
UNPROCESSED BIOACTIVE PLANT MATERIALS.
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ATTRACTANT INDEX.
O O o 6 ) i . K ) O K ) ^ C D O O O
Calotropis procera 
Chrysophylium albicum 
Saccharum officinale
fresh Carica papaya 
leaf
fresh Manihot leaf
fresh Manihot root
fresh Azadirachta leaf
unripe Azadirachta
fresh Latuca sativa
fresh Ipomea stem
Parlaa clappertoniana
Ipomea stem + Latuca 
sativa
Dialium guinensis
Ipomea stem + P. 
clappertoniana =r
ripe Anona 
unripe Anona 
fresh Zingiber
J ____ 1__
□ □
35
ATTRACTANT RESPONSES OF BULINUS TRUNCATUS TO SOME 
UNPROCESSED BIOACTIVE PLANT MATERIALS.
TEST MATERIAL.
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2.3 2 ARRESTANT EFFECTS:
Only 5 (ie 14.29%) of the unprocessed materials tested emerged as statistically significant 
arrestants to both adult and juvenile snails at either p< 0.05, 0.01 or 0.001 significant levels 
(Table 2.3.2). These include sugarcane, lettuce leaves, showers of gold, sweet potato and cashew. 
In addition, 8  other materials also proved statistically significant as arrestants to the adult conspecifics 
alone while 2 were similarly significant only to the juveniles. These were ripe mango > water melon
> fresh pawpaw leaves > date palm > fresh cocoyam leaves > fresh lettuce leaves > sweet potato + 
lettuce leaves > ripe cocoa beans > fresh cassava root tuber
In the case of the juveniles, ripe miraculin and fresh cassava leaves with respective response 
indices of 3.6 and 2.5 were significant arrestants at p<0.05 (Fig. 2.3.2).
2.3.3 REPELLENT EFFECTS:
The results also show that 3 materials emerged as statistically significant repellents to B. 
truncatus of both ages at p<0.05, 0.01 and 0.001 confidence limits. These were fresh ginger stem
> star apple > unripe neem fruit In addition, lime fruits, ripe cocoa pod and fruits of sodom apple 
were significant repellents to the juvenile conspecifics alone at p<0.05,0.01 and 0 . 0 0 1  significant 
levels.
36
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Table 2  3 2  ARRESTANT RESPONSES TO UNPROCESSED M ATERIALS
A D L I L T S . J U V E N I L E  S.
TEST SCIENTIFIC MEAN t - value MEAN t - value 
MATERIAL NAME INDEX (p<0.05) INDEX (p<0.05)
FRU ITS:
Ripe cashew Anacardium 2.4 3.1722* 1.9 3.3838*
occidentalis
Mango : Mangijera indca
ripe 7.3 8.5104* 0.9 0.9873
unripe 0.3 0.6882 -1.9 -2.2549
Water melon Citrulus vulgaris 6 . 8 9.6372* 2 . 0 1.5744
Ripe lime Citrus aurantifolia -0.5 - 1 . 0 0 0 0 -2 . 8 -3.2432**
Ripe miraculin Synsepalum dulcilicum 1 . 6 2 . 0 1 0 1 3.6 2.4956*
Orange: Citrus sinensis
ripe 0.4 0.5674 -0.3 -0.4483
peels -0.4 -0.4163 2 . 2 1.9530
Ripe date palm Phoenix dactylifera 5.0 5.6844* 1 . 8 1.7832
Long banana peels Musa paradisiaca 0 . 6 0.9112 0 . 2 0.1482
Ripe cocoa: Theobroma cacao
pod 0 . 2 0.1966 -4.9 -4.7840**
Beans 3.2 3.3672* 0 . 6 0.9524
Ripe fan palm Borassus aethiopium 2.5 1.9493 0.4 0.4963
Ripe milk bush Thevetia nerifolia 2.4 1.5800 1 . 8 1.5340
Ripe royal palm Eleais guinensis 2.4 1.2972 2.5 1.9493
Showers of gold Duranta plumieri 3.4 4.5888* 3.2 5.1069*
Sodom apple Calotropis procera 3.6 2.2272 -2.3 -2.6706**
37
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TEST SCIENTIFIC A D U L T S Ti l  V E N I L E S .
MATERIAL NAME MEAN t -value MEAN t- value 
INDEX (p<0.05) INDEX (p<0.05)
Unripe neem Azadirachta indica -3.2 - -3.6 -3.0869**
Locust beans Parlaa clappertoniana 3.9516* * 2 . 2 0.7021
Ipomea batatas 0 . 2 0.2912
Sweet potato +
+ Parlaa clappertoniana 1 . 6 1.4220
locust beans Dialium guinensis -1.7 -1.9450
Chrysophyllum
Velvet tamarind albicum -0.5 -1.2538
Star apple Annona muricata 0 . 2 1.2952 - 1 . 0 -1.4001
0 . 1 0.4382
Sour sop : 
ripe 1.9 2.3818*
unripe 2 . 1 1.7653 0 . 8 0.6000
Xanthosoma maffafa -0.7 -0.8812
LEAVES: Manihot esculenta 
Cocoyam Latuca sativa 1.1 0.6408
Cassava Azadirachta indica 4.5 2.5 3.5668*
Lettuce Carica papaya 4.3700* 2.4 2.8710*
Neem 2 . 0 1.7900 0 . 0 0 . 0 0 0 0
Pawpaw Xanthosoma maffafa 4.6 1.7 1.1759
38
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TEST SCIENTIFIC A D U L T S J U V E N I L E S
MATERIAL NAME MEAN t-value MEAN t-value
INDEX (p<0.05) INDEX (p<0.05)
STEMS : 
Cocoyam tuber Xanthosoma maffafa -0.3 -0.2855 1 . 2 1.1982
Ginger Zingiber officinale -4.7 -7.4261** -2 . 2 -2.9794
Sugar cane Saccharum officinarum 5.0 6.3444* 6.7 9.1393*
Sweet potato Ipomea batatas 2.9 3.9612* 2.7 3.7037*
Sweet potato Ipomea batatas
+ + 3.5 3.9294* 1.3 1.4163
lettuce leaves Latuca sativa
ROOTS : 
Cassava tubers Manihot esculenta 3.2 4.0097* 0 . 1 0.1275
Note : * denotes significant arrestants at 0.05 confidence limit 
** denotes significant repellents at 0.05 confidence lim it.
39
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ARRESTANT INDEX.
CD N) O  K> CD oo
Anacardium occidentals (Q
ro
ripe Mangifera indica w
k>
unripe Mangifera indica >
73
citrulus vulgaris 5C-  m»
citrus aurantifolia 2  -• 5O  z>
synsepalum dulcilicum o  Hm ,,
Ui 73
citrus sinensis c/) mcP m co
o  ■?
Citrus sinensis peels 
m ro
c/> c/>
fresh xanthosoma leaf O
> mo CO
raw xanthosoma stem 
<5m 
 O-n 
Phoenix dactylifera , ,  DO
T J  C
Musa paradisiaca peels 
M
Theobroma cacao pod 
s
Theobroma cacao bean >  Sd)
m
Borassus aethiopium 3J i
J>
r~ 2
fresh Thevetia neriifolia U)
§
Eleais guinensis 
Duranta plumieri
(Si
O
S□ □ st > m
40
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ARRESTANT INDEX.
& ro o  m CD OD
Calotropis procera
>
I 73
Chrysophyllum albicum r= ) 71m
V) 
Saccharum officinale c >H  z z
fresh Carica papaya leaf 13 H
7J _
fresh Manihot leaf 1 O £O m
m Sg 
fresh Manihot root iinn  O3 
rmj i™n 
fresh Azadirachta leaf m
m OD W
HC/) unripe Azadirachta < 
o O
>o T1
> fresh Latuca sativa H  ODH <  C
ZmJ m fresh Ipomea stem
S -o ^
Parlaa clappertoniana
Ipomea stem+ P. 
clappertoniana
> i 
Dialium guinensis J Hm 2
Ipomea stem+ Latuca 2
sativa > Ii-
w
ripe Anona 
in
unripe Anona O
3
fresh Zingiber stem m
□ o
41
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2.4 DISCUSSION:
2.4 i Discriminatory responses:
The results indicate that both adult and juvenile Buhnus truncatus exhibit significant 
levels o f discriminatory responses towards the materials tested Thus whereas the adults 
responded significantly towards 51.43% o f the materials juvenile responses were significant 
towards only 28 .57 % o f these materials.
This selective chemoreception response is not unique to Buliniid snails alone but has long been 
observed and documented among other freshwater Planorbid snails and some invertebrates as well 
as vertebrates (Thomas and Assefa, 1978; Thomas et al, 1980a,b; Thomas, 1982; Kpikpi and Thomas, 
1992,1993). Using pure chemical, these workers have demonstrated that the adult and juvenile 
Bulinus rohlfsi and the South American schistosome intermediate host snail, Biomphalaria 
glabrata, are discriminatory to different extents in their chemoreception responses. Furthermore, 
the juveniles of both species of snails were found to exhibit a much wider response pattern than the 
adults.
The pattern o f chemoreception niches o f  Bulinus truncatus as shown by the results o f  this 
present work seems to be in contrast to those found by earlier workers in that the adults rather than 
the juvenile exhibited a wider chemoreception niche . A number o f factors may be responsible for 
this observation.
First, unlike in previous works in which single chemical species were used as test materials, the 
materials assayed in the present work are crude forms (unprocessed) o f naturally
42
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occurring plant materials. These are thought to consist o f an array o f  complex chemical factors 
(Kpikpi, 1990) some o f which may be stimulatory, inhibitory or repelling to these snails to  different 
extents. The summation o f these different chemical factors determines the direction o f  response o f 
the snails towards the test material in question.
Secondly, the narrower chemoreception niche showed by the juvenile snails o f  B. truncatus 
could be attributed to their ages. A juvenile bulinid snail such as B. truncatus, unlike the 
adult, has a relatively immature olfactory sense to be able to significantly respond towards 
materials with such complex chemical factors. Similar observations were made by Kpikpi et al 
(1995) on the chemoreception niche o f B. rohlfsi on some forms o f natural bioactive materials with 
similar complex chemical factors.
2.4 2 RESPONSE TO FRUIT MATERIALS:
One of the important features o f the chemoreception niche o f  adult and juvenile B. truncatus 
as shown by the present results is found in their responses towards the fruit materials assayed 
(Figs 2.4.1 and 2.4.2). Whereas 9 (ie 39.13 %) o f the 23 materials emerged as significant attractants 
and arrestants to the adults only 3(ie 13.04 %) proved to be significant attractants and arrestants to 
the juvenile snails. Also, 4 (iel 7.39 %) o f these fruits were found to be statistically significant 
repellents to adults while 7(ie 30.43 %) were similarly repellent to the juveniles .
All the fruit materials that emerged as statistically significant attractants and arrestants to  these 
snails are in their ripened states. In addition, with the exception o f  the date palm fruits these ripe 
fruits are very succulent and juicy (eg ripe mango, water melon). In contrast, those fruits that
43
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emerged as significant repellents were unripened and generally hard in texture.
It is known that all green unripe fruits contain protopectins and large stores o f  carboxylic acids. 
During ripening, the protopectins are converted to pectins which are complex polysaccharides similar 
to starches. A large proportion o f the acids are also converted to sugars and other flavours 
The ripe fruits thus contain far greater amounts o f  sugars and pectins than the unripe fruits.
The observed preference for ripe fruits by these snails could therefore be due to the presence o f  the 
large amounts o f sugars and other polysaccharides in them. These observations resemble those 
described by Kpikpi (1991) for sugar chemoreception niche o f B. rohlfsi and B. globosus, although 
in that case the materials used were pure chemicals. He found that 29.4 % and 23.5% o f various 
polysaccharides and oligosaccharides assayed by means o f the diffusion olfactometer were 
statistically significant as attractants or arrestants to  B. rohlfsi and B. globosus respectively. 
Thomas et al (1985a) also found that 27.8% o f various carboxylic acids were statistically significant 
as repellents to Biomphalaria glabrata It is possible that the unripe fruits acted as repellents 
because of the high concentrations o f carboxylic acids they contain which may therefore give rise to 
high H+ concentrations. As pulmonate snails are generally known to be intolerant to waters o f  low 
ph values (Boycott, 193 6 ) the probable cause o f  the repellent effects o f  the unripe fruits may be the 
presence o f the H+ concentrations in them.
44
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ATTRACTANTINDEX
o o c n - k r o o M - t ^ c n o o o (Qro
ripe Anacardium 
occidentalis
>
ripe Mangifera indica H
73
>
unripe Mangifera indica O
Citrulus vulgaris & z  mn   
Da ~n 
Citrus aurantifolia C  rn
6 3
PP Synsepalum dulcilicum 
C/> S ?
>  ripe Citrus sinensis
Hm  i  |  O rn
2
^  Citrus sinensis peels ^  °
d  o
oo 3
unripe Azadirachta indica
ro
Parlaa clappertoniana o
>o
Dialium guinensis H
<
m
-n
ripe Annona muricata 73
c
H
unripe Annona muricata C/i
□ □
45
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ATTRACTANT INDEX
o o o c r ) i . r o o i v ) ^ c n c D
H
Chrysophyllum albicum j 70
>
O
H
Phoenix dactylifera >
m
Musa paradisiaca
mo  
Theobroma cacao pod 00 H  
S  O
H 2  co Theobroma cacao beans
m C  O  co oo s
~h m 
Borassus eathiopium 2 o
2  s  
Eleais gumensis
Duranta plumieri o  
>o
H
Calotropis procera <
m
n
fresh Thevetia nenifolia c
H
CO
sun-dried thevetia 
neriifolia o
a □
46
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ARRESTANTINDEX
-K rb O  N3 ^  O) 00
University of Ghana                              http://ugspace.ug.edu.gh
ARRESTANT INDEX
Chrysophyllum albicus
Phoenix dactylifera
Musa paradisiaca peels
Theobroma cacao pod
Theobroma cacao beans
Borassus eathiopium
Eleais guinensis
Duranta plumieri
Calotropis procera
fresh Thevetia neriifolia
sun-dried Thevetia 
neriifolia
□ □
c_ >
< CLc
a to
t(oV
48
ARRESTANT EFFECT OF SOME BIOACTIVE FRUITS ON BULINUS
TRUNCATUS.
TEST MATERIAL
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C H A P T E R  3.
CHEMORECEPTION RESPONSE OF BULINUS 
TRUNCATUS TO  SOME PROCESSED BIOACTIVE 
MATERIALS.
3.1 INTRODUCTION:
The search for potent, naturally occurring bioactive substances to serve as attractants,
arrestants or phagostimulants for incorporation with known snail toxicants is an important step 
towards the development o f  a more cost-effective environmentally friendly molluscicide for 
schistosomiasis control
Since Markowski(1955) demonstrated the possibility o f using baits such as decaying palm leaves 
for the selective removal o f snails under field conditions in Africa, it has been shown that plant and 
animal materials in their early stages o f decomposition are generally attractive to  pulmonate snails 
(Pimentel and White, 1959; Sterry et al, 1983; Thomas et al, 1985b; Thomas and Tait, 1984). These 
materials are known to release various oligosaccharides which form important components o f 
dissolved organic matter (DOM) on which various aquatic organisms such as bacteria 
(Lodge,1985), freshwater molluscs such as Pisidium sp.(Eftbrd and Tsumura.1973), bulinid snails 
(Kpikpi and Thomas, 1992) rely for cues and nutrition.
These observations point to the fact that there exist some chemical factors in these decomposing 
natural materials that could be utilised as strong attractants, arrestants and
49
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phagostimulants This view was demonstrated when Ndifon (1979) observed that the Prosobranch 
molluscs, Lanistes lybicus tend to congregate around a soaked cassava tuber which is known to 
contain high levels o f  starch.
Earlier work by Kpikpi et al (1995) in which 12 o f  the 26 naturally occurring materials assayed 
were processed by boiling, only 2 of them emerged as significant attractants to the B. truncatus snails. 
This work suggested the need for other methods o f processing natural plant materials for the 
bioassay towards the identification of bioactive natural materials for use in snail control.
Accordingly, naturally occurring materials from 7 different common plants were processed by 
either fermentation, sun-drying or oven-drying methods to obtain 19 different test materials.
Using the bioassay technique, these were tested for their bioactive effects separately on the adults 
and juveniles o f B. truncatus. The results and the implications for development o f cheaper and target 
specific molluscicides for snail control are discussed.
50
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3.2 M A T E R IA L S  A N D  M ET H O D S :
3.2.1 PROCESSING OF MATERIALS :
Oven-drying, sun-drying and fermentation were used to process the materials for the bioassay. 
The objective of this processing is to alter the form and texture o f the materials to find out if a higher 
chemoresponse could be elicited from these snails under the experimental conditions. In all the 
processing procedures yielded 19 different materials for the bioassay.
3 2 2 CHOICE OF MATERIALS FOR PROCESSING:
Certain factors were considered in selecting the materials for processing in the present experiments. 
These include the chemoreception effect of the unprocessed form of the materials on the snails as well 
as availability and cost o f the materials,
a) Chemoreception effect:
Some of the materials selected for processing were those that elicited significant chemoresponses 
from either the adult or juvenile snails in their unprocessed forms. This was to  investigate the 
possibility o f obtaining higher response indices from their processed forms 
Similarly those unprocessed materials that proved to be significant repellents were also selected for 
processing . It was expected that the processed forms could be much stronger repellents.
Based on this criterion leaves o f pawpaw (Carica papaya), cocoyam (Xanthosoma maffafa) and 
neem (Azadirachta indica) as well as stem o f ginger (Zingiber officinale) were selected for 
processing .
51
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b) Availability and cost:
Although some o f the materials selected for processing were weakly stimulating in their 
unprocessed forms their abundance and cheaper cost qualify them for use in the present test. It was 
envisaged that in case their processed forms prove to be significant attractants or arrestants (or both) 
it would be much easier and cheaper to obtain them for use in snail control programs.
Based on these criteria, materials selected for processing include milk bush fruits (Thevetia 
nerifolia), cassava root tuber and leaves (Manihot esculentus), cocoyam stem tuber (Xanthosoma 
maffafa) and sweet potato stem tuber (Ipomea batatas).
3.2.3 PROCESSING PROCEDURES:
a) SUN-DRYING:
Materials processed under this method include leaves o f cocoyam, pawpaw, and fruits o f  milk 
bush. Fresh samples o f  each material were placed in a clean open tray and left on a table under 
sunlight for a period o f 7 hours (8 am to 3pm) each sunny day for 3 successive days.
After the period the leaves were observed to be changing colour from green to brown and softer 
whereas the fruits were turning biack and softer. These changes were indications o f possible onset 
of decaying due to the action of atmospheric microbes.
b) OVEN-DRYING:
Stems o f ginger, root tubers o f sweet potato and leaves o f  cassava were processed under this 
method. Samples of each material were placed in an oven maintained at a temperature o f  70°C for 
48 hours.
52
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c) FERMENTATION:
Materials processed under this method include root tubers o f cassava, sweet potato, stem tubers 
of cocoyam and leaves o f pawpaw, cocoyam and cassava.
Fermentation procedure:
Fresh sample o f each material was cut into smaller discs o f size 1 Ox 1.2 x 1.0cm suitable for the 
bioassay of the juvenile snails. Larger forms o f size 1 .5 x 1 4 x 1 ,2cm were also cut for the adult snail 
bioassay. Each group o f materials was placed in a clean 300ml plastic container, covered completely 
with 250ml of tap water and closed with a tight fitting lid
The set up was left under laboratory conditions and allowed to ferment at a temperature o f 
29± 1°C . For one day fermentation, the samples were allowed to remain in the water for 24 hours 
whereas for 3 days fermentation they remained in the water for 72 hours In the case o f  7 days 
fermentation, 168 hours o f fermentation was allowed before the bioassay.
In the case of the leaves, each sample was placed in a clean plastic container and closed with a tight- 
fitting lid. No water was added in this case This was to slow down the rate o f decomposition o f  the
leaves.
3.3.4 THE BIOASSAY.
Diffusion or gradient olfactometers used were same as described in section 2.2.4 o f chapter 2.
By means of a plastic spoon each fermented material was transfered into the olfactometer chamber 
while an inert material made of polystyrene was placed in the opposite chamber. 25ml and 5ml o f tap 
water was added to each large and smaller olfactometer respectively as discussed in section 2 .2 . 5
53
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The position o f each snail was noted at intervals o f  21/, minutes for 30 minutes.
The scores were computed after the period to generate the attractant and arrestant indices for each 
processed material used as described in section 2 .2 .6 .
54
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3.3 R E S  U L T S.
3.3.1 An rad an! effects:
The results show that 4 (21 05%) o f the 19 processed materials proved to be statistically 
significant attractants to both adults and juvenile B. truncatus snails at either p< 0.05,0.01 or 0.001. 
These include one-day fermented sweet potato >3 days fermented sweet potato > lday  fermented 
cassava root tuber > 3 days fermented cocoyam stem (Table 3.3.1, Fig. 3.3.1) In addition, oven-dried 
sweet potato stem, sun-dried milk bush fruits, sun-dried cocoyam leaves and 1 day fermented 
cocoyam stem are also significant attractants to  only the adult snails. For the juveniles,7 days 
fermented sweet potato, 3 days fermented cassava, 7days fermented cassava, 3days fermented 
cocoyam leaves and 7 days fermented cocoyam stem tuber also proved to be significant attractants. 
The most potent processed bioactive attractants as found from this experiment for both the adults 
and juvenile snails were respectively 1 day fermented sweet potato root tuber and 7 days fermented 
cocoyam stem tuber.
3.3.2 Arrestant effects:
Unlike the attractant responses, there were no overlaps in the arrestant response patterns o f 
the adult and juvenile snails as shown by the present results (Table 3.32, Fig 3.32). Whereas 
9(ie 47 4%) of the 19 processed materials emerged as statistically significant arrestants to the 
juveniles, only 3 ( ie 15. 8 % ) showed similar significant effects on the adults at either p< 0.05, 0.01 
or 0.001 confidence limits. For the adults, oven-dried sweet potato root, sun-dried
55
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Table 3.3.1
ATTRACTANT RESPONSE OF liUUN US TRUNCATUS JO  SOME 
PROCESSED BIOACTIVE MATERIALS_____________________
TEST MATERIAL A D U L T S I II V E m i  i: s
SCIENTIFIC NAME MEAN t-value MEAN t-value
INDEX (p<0.05) INDEX (p<0.05)
FRUITS:
Sun-dried milk bush Thevetia nerifolia 3.1 2.4042* 2.3 1.6326
LEAVES
Pawpaw, 
sun-dried Carica papaya
3 days fermented -0 . 6 -0.3639 -1.7 -0.9892
3.1 2.1750 2 .1 1.5052
Cassava; Manihot esculetita
oven-dried -2.4 -2.2592 0 . 0 0 . 0 0 0 0
3 days fermented 3.5 3.3201 4.3 4.3442*
Cocoyam; Xanthosoma maffafa
sun-dried 3.8 3.4124* 0 .1 0.0601
3 davs fermented 1 .6 1 8973 3.6 3.4112*
Neem, sun-dried Azadirachta indica -3.2 -2.4596** -2 . 0 -1.0603
STEMS
cocoyam, Xanthosoma maffafa
lday fermented 3.8 2.3068* 2 . 8 1.7742
3 days fermented 3.4 2.3421* 8 .2 9.8534*
7 days fermented 2 . 0 1.3942 9.7 5.1805*
Oven-dried ginger Zingiber officinale -4.2 -3 3008** -2 .1 -0.9617
Sweet potato; Ipomea batatas
oven- dried 2 .8 3.1236* 1.3 0.6729
1 day fermented 5.0 3.7268* 6.5 3.8573*
3 days fermented 3.9 3.5455* 5.1 6.0750*
7 days fermented 2.4 1.7983 5.7 4.5894*
ROOT
cassava Manihoi escnlenta
1 day fermented 4.6 3.2172* 4.1 3.2966*
3 days fermented - 1.8 -1.3878 5.3 3.2505*
7 days fermented 0.7 0.4017 8.5 9.9195*
Note: * -  Significant attractants ** = Significant repellents.
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ATTRACTANT INDEX
C D - k - N O O h O - t ' . O O O O
i_____ i_____ i-------- 1-------- 1-------- 1-------- 1-------- !-------- |
sun-dried Xanthosoma I j „ . I '/- ,;  j
leaf !
3 days ferm. Xanthosoma 
leaf
1 day ferm . Xanthosoma 
stem
3 days ferm. Xanthosoma 
stem
7 days ferm. Xanthosoma 
stem
sun-dried |Thevetia fruit
oven-dried Azadirachta 
leaf
oven-dried Zingiber stem
oven-dried Ipomea stem
1 day ferm. Ipomea stem
57
Fig.3.3.1 ATTRACTANT RESPONSES OF BULINUS TRUNCATUS TO 
SOME PROCESSED BIOACTIVE PLANT MATERIALS.
TEST MATERIAL
University of Ghana                              http://ugspace.ug.edu.gh
ATTRACTANT INDEX.
i i
O  N> 4^ CD 00
3 days ferm Ipomea 
stem
7 days ferm. Ipomea 
stem
sun-dried Carica 
papaya leaf
3  days ferm. Carica 
papaya leaf
oven-dried Manihot 
leaf
3 days ferm. Manihot 
leaf
1 day ferm. Manihot 
root
3 days ferm. Manihot 
root
7 days ferm. Manihot 
root
□ □
58
ATTRACTANT RESPONSES OF BULINUS TRUNCATUS TO SOME PROCESSED
BIOACTIVE PLANT MATERIALS.
TEST MATERIAL
University of Ghana                              http://ugspace.ug.edu.gh
Table 3.3.2
ARRESTANT RESPONSE OF BULINUS TRUNCATUS TO SOME
PROCESSED BIC>ACTIVE MATER ALS.
TF^IT MATFR1AI SC IEN TIFIC A n n , T S .1II V F \  I I  F S
NAME MEAN t-value MEAN t-value
INDEX (p<0.05) INDEX (p<0.05)
FRUITS:
Sun-dried milk bush Thevetia nerifolia 1.4 1.3500 1.0 1.0402
LEAVES:
Cassava; Manihot esculenta
oven-dried -2 . 2 -2.5946 0 . 0 0 . 0 0 0 0
3 davs fermented 1.8 2.0641 2 .8 3.4112*
Cocoyam; Xanthosoma maffafa
sun-dreid 4.0 4.0000* 1 1 0.9638
3 days fermented 1.1 1.2334 2.3 2.9672*
Neem, oven-dried Azadirachta indica -3.3 -3.0419** -0.4 -0.4474
Pawpaw; Carica papaya
sun-dried - 1.8 -1.4684 -2 .1 -2.0243
3 days fermented 2 . 6 2.1794* 1 0 0.8587
STEMS:
Cocoyam; Xanthosoma maffafa
1 day fermented 3.6 3.1887* 0.3 0.2629
3 days fermented -0.5 -0.4051 4.3 5.5736*
7 davs fermented -0.3 -0.3243 9.5 4.1977*
Ginger, oven-dried Zingiber officinale -2 . 6 -2.9620** -3.7 -4.3744**
Sweet potato; Ipomea batatas
oven-dried 2 .1 2.5071* 0 . 6 0.4381
1 day fermented 1 .8 2 . 1 0 0 0 2.4 3.4478*
3 days fermented 0 .8 1.7098 1.9 2.4852*
7 days fermented -0.4 -1.1648 4.0 4.0306*
ROOTS:
Cassava; Manihot esculenta
1 day fermented 1 .0 1.0731 0.4 0.6308
3 days fermented - 1.8 -2.3077 4.3 3.7330*
7 days fermented -0 . 6 -0.5901 6.4 7.4453*
Note : * = Significant arrestants ** = Significant repellents.
59
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ARRESTANTINDEX
O  M  4^ CO 03
sun-dried 
Xanthosoma leaves
3 days term 
Xanthosoma leaves
1 day ferm 
Xanthosoma stem
3 days ferm. 
Xanthosoma stem
7 days ferm. 
Xanthosoma stem
sun-dried Thevetia 
fruits
oven-dried 
Azadirachta leaves
I
oven-dried Zingiber 
stem
oven-dried Ipomea 
stem U
1 day ferm. Ipomea 
stem
...E
60
n g .3.3.2 ARRESTANT RESPONSES OF BULINUS TRUNCATUS TO SOME PROCESSED
BIOACTIVE PLANT MATERIALS. □ Adults
TEST MATERIAL
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ARRESTANT INDEX
I I
CO K> O - ' - N J C O J ^ O l C T ) ' ^
3 days ferm. Ipomea
7 days ferm. Ipomea
sun-dried Carica papaya 
leaves
3 days ferm. Carica 
papaya leaves
oven-dried Manihot 
leaves
3 days ferm. Manihot 
leaves
1 day ferm. Manihot 
roots
3 days ferm. Manihot 
root
7 days ferm. Manihot 
roots
□
c_ □>
< ac.
3 (/>
CO
(/>
61
ARRESTANT RESPONSES OF BULINUS TRUNCATUS TO SOME PROCESSED BIOACTIVE
PLANT MATERIALS.
TEST MATERIAL
University of Ghana                              http://ugspace.ug.edu.gh
cocoyam leaves and 1 day fermented cocoyam stem were found to be statistically significant 
arrestants with respective response indices o f 2.1,4.0 and 3 . 6 . In the case o f the juveniles, 7days 
fermented cocoyam was found to be the strongest statistically significant arrestant with a response
index of 9.5. Other significant arrestants for the juvenile snails were sweet potato root tuber ( 1 day,
3 and 7 days fermented ), 3 days fermented cassava leaves, cassava root tubers ( 3 and 7 days 
fermented) and 3 days fermented cocoyam stem tuber
3.3.3 Repellent effects:
The present results show that although some o f the processed materials eftsiiej negative 
responses from both adult and juvenile B. truncatus, most o f  these responses gave no statistically 
significant indices to qualify as repellents. Only oven-dried ginger stem was found to be a significant 
repellent to snails of both ages. The repellent effect o f this material was, however, much stronger on 
the adults than on the juveniles as shown by their respective response indices ( Table 3 .3.2). Oven- 
dried neem leaves were also strong repellent to the adults while on the juveniles the effect was very 
weak.
3.3.4 Response to fermented materials:
With the exception of 3 days fermented cassava root tuber which gave a statistically insignificant 
negative attractant index for the adult snails, the rest o f 12 fermented materials assayed elicited 
positive attractant indices from both adults and juvenile B. truncatus. However, only 5 ( ie 41.7%) 
of these proved to be statistically significant attractants to the adult snails whereas 9 (ie 75%) were
62
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similarly significant as attractants to the juveniles at either p< 0.05, 0.01 or 0.001 confidence limits.
More importantly, responses elicited from the juveniles by the processed materials were generally 
stronger compared to the unprocessed forms as shown by their corresponding response indices 
(Table 3.3.4, Fig 3.3.4). None of these fermented materials was able to elicit statistically significant 
arrestant responses from the adult snails. In fact 5 (41 7%) o f these responses were repellent in 
nature (negative) although their respective indices were not statistically significant enough. Unlike 
the adult snails, all 12 fermented materials elicited positive arrestant responses from the juvenile 
snails out o f which 9 (ie 75%) were statistically significant (Table 3 .3 .4).
The most potent, statistically significant processed material with strong attractant and arrestant effect 
on the adult B . trimcatus is sun-dried cocoyam leaves (attractant index= 3 .8 ; arrestant index = 4.0). 
In the case o f the juveniles, 7days fermented cocoyam stem tuber, with respective attractant and 
arrestant indices o f 9.7 and 9.5, emerged as the strongest fermented bioactive material.
63
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Table 3.3.4
RESPONSE O F liU U N U S TRUNCATUS TO  SO M E FERM EN TED  
BIOACTIVE M ATERIALS._____________________________________
FERMENTED R F . S P O N S P Q
MATERIAL A D  l / l .  T S ./ II V F . N I I . E S
Attr Ind. t- value Arrs. Ind t-value Attr Ind t-value Arrs. Inc . t-value
LEAVES:
Cassava, 3 days 3.5 3.3201 1.8 2.0641 4.3 4.3442* 2 . 8 3.4112*
fermented
Cocoyam, 3 days 1 .6 1.8973 1.1 1.2334 3.6 3.4112* 2.3 2.9672*
fermented
Paw paw ; 3 days 3.1 2.1750 2 . 6 2.1794 2.1 1.5052 1 .0 0.8587
fermented
STEMS:
Cocoyam tuber;
lday fermented 3.8 2.3068* 3.6 3.1887= 2 . 8 1.7742 0.3 0.2629
3 days fermented 3.4 2.3421 * -0.5 -0.4051 8 .2 9 8534* 4.3 5.5736*
7days fermented 2 . 0 1.3942 -0.3 -0.3243 9.7 5.1805* 9.5 4.1977*
Potato tuber;
lday fermented 5.0 3.7268* 1.8 2 . 1 0 0 0 6.5 3.8573* 2.4 3.4478*
3 days fermented 3.9 3.5455* 0 . 8 1.7098 5.1 6.0750* 1.9 2.4852*
7days fermented 2.4 1.7983 -0.4 -1.1648 5.7 4.5894* 4.0 4 0306*
ROOTS:
Cassava tuber;
1 day fermented 4.6 3.2172* 1 .0 1.0731 4.1 3.2966* 0.4 0.6308
3 days fermented - 1.8 -1 3878 - 1.8 -2.3077 5.3 3.2505* 4.3 3.7330*
7 days fermented 0.7 0.4017 -0 . 6 -0.5901 8.5 9.9195’ 6.4 7.4453*
* -  significant attractants and arrestants Attr Ind = Attractant index. Arrs. Ind.= .Arrestant Index
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------ z/~ r-T
TEST MATERIAL
CL
K>
m7Jc/>
“0
0  
z
C/)
m
1m »  
x
CO
cn ->
65
Fig. 3.3.4 CHEMORECEPTION RESPONSE OF ADULT BULINUS TRUNCATUS TO 
SOME FERMENTED BIOACTIVE PLANT MATERIALS.
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TEST MATERRIAL
66
CHEMORECEPTION RESPONSE OF JUVENILE BULINUS TRUNCATUS TO SOME 
FERMENTED BIOACTIVE PLANT MATERIALS.
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3.4 Discussion.
3.4.1 A g e-sp ecific  responses :
The present results clearly indicate that a large number o f  processed materials are highly 
stimulatory to both adult and juvenile B. truncatus (Fig.3.4.1). The extent o f stimulation, however, 
tends to vary with the age of the snails. Thus whereas 66.7% of the processed materials were found 
to either attract and/or arrest the adults to a statistically significant extent, 83 .3% had similar effects 
on the juveniles (Table 3 .3 .1)
With respect to their behavioural responses to processed materials, it is apparent that juvenile B. 
truncatus snails differ from their adults to some extent First, the levels o f stimulation elicited by 
these materials as indicated by their respective response indices were significantly higher for the 
juveniles than for the adults ( Fig. 3 .4.2). Thus, for example, attractant and arrestant indices o f 3 days 
fermented cocoyam tuber for the juvenile snails were 8.2 and 4.3 respectively whereas those for the 
adults were 3.4 and -0.45 . Furthermore, some o f the materials such as 7 days fermented sweet 
potato and 3 days fermented cassava leaves that significantly attracted and arrested the juvenile snails 
had very insignificant effects on the adults. The difference in chemoreception niches between adult 
and juvenile B. truncatus snails as found in the present study on processed materials appears similar 
to those found by Thomas and Assefa (1978) and Thomas et al (1983, 1989) for Biomphalaria 
glabrata, the main South American intermediate host snail, although different snail genera are 
involved and more importantly pure materials with single chemical factors were used .
These workers found that the juveniles o f  Biomphalaria glabrata exhibited a much wider 
chemoreception niche in their reponse towards pure amino acids such as asparagine, glutamine and
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citrulline and some short chain carboxylic acids like propanoic acid, butanoic acid and chloroacetic 
acid To explain these intraspecific differences in chemoreception niche among these snails, they 
postulated that niche diversification has evolved among snails to reduce the intensity o f  intraspecific 
competition for food, shelter,etc Furthermore, these observed differences in niche size may be 
related to differences in feeding habits. As noted by Thomas and Assefa (1979), and Ndifon (1989), 
juvenile snails tend to  feed on a variety o f  microorganisms such as bacteria, epilithic and epiphytic 
algae and appear to be more euryphagous than the adult conspecifics that may often subsist on only 
a few species of aquatic macrophytes . In the natural freshwater environment therefore, juveniles o f 
planorbid snails such as B. truncatus and other related molluscs are stimulated by a much wider 
range o f chemical factors to which they are attracted and utilized as food energy sources than their 
adult conspecifics . This niche diversification is not peculiar to freshwater molluscs alone but it occurs 
at the interspecific level in various aquatic invertebrates (Lenhoff and Lindstedt, 1974, Bardach, 
1975). There is some indication, however, that the behavioural niche o f  most aquatic invertebrates 
may be much narrower than those o f freshwater planorbid snails including B. truncatus.
3.4.2 Effect o f fermented materials:
One other important feature o f the present study is the significantly high attractant and arrestant 
effects o f the fermented materials on the movement o f  B. truncatus . The results indicate that, with 
the exception of fermented pawpaw leaves, all the fermented materials were capable o f significantly 
attracting and arresting either adult and/or juvenile B. truncatus (Table 3.4.2). This result is not 
unexpected Fermentation o f materials from plant origin is a biochemical process leading to 
glycolytic production o f various chemical species by coccoid bacteria which invade these materials
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at the onset o f the process. Prominent among these products are short chain carboxylic acids, 
especially the C2 - Q groups (Patience et al., 1983; Sterry et al., 1985; Daldorph, 1988) . In 
addition, other chemicals like amino acids such as threonine, serine, glutamine, asparagine and 
tryptophan known to be present in plant materials (Jabbar-Muztar et al., 1978) are also likely to be 
released into the external medium during the fermentation process. These materials in their right 
combinations can attract or arrest the snails since they serve either as sources o f  nutrient or 
information in their natural environment (Thomas et al., 1989). An important component o f  the diet 
for pulmonate snails such as Physella acuta, Helisoma duiyi, Lymnciea natalensis, L. stagnalis as 
well as those that serve as hosts to schistosome parasites are decaying plant materials and associated 
short chain carboxylic acids released during anaerobic decomposition (Thomas et al., 1982).
It is important to investigate the chemoresponses o f  other planorbid snails such as Bulinus globosus 
and also Melanoides tuberculate(of the family Melaniidae) towards these fermented bioactive 
natural products. This would help in characterising their chemoreception niches and thereby 
identifying some o f the chemical factors that could be involved in stimulating these snails . At the 
moment, it has been shown that African planorbid snails such as Bulinus globosus are relatively 
unresponsive to some pure carboxylic acids (Thomas et al., 1982) It is also not yet known, at the 
moment, which chemical factors within these fermented natural materials that are responsible for 
stimulating the snails under the present experimental conditions It is possible that, unlike the pure 
carboxylic acids with a single chemical factor, a medley of different chemical species present in these 
natural materials may have a combined effect in attracting and arresting these snails . It may also be 
that the fermenting process simply decomposes the crude material to release the various chemical
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components (eg C\-C 5 carboxylic acids, amino acids, etc ) into the water medium Thus these 
different chemical species, in the appropriate combinations, would then elicit significant attractant and 
arrestant responses from the snails.
The present finding that fermenting materials have a stimulatory effect on B. trimcatus snails also 
lends credence to the observation by Ndifon (1979) that the prosobranch mollusc, LanisWs lybicus, 
and the planorbid, B. globosus, tend to congregate on soaked cassava tubers, a food crop known to 
contain high amounts o f starch and short chain carboxylic ac id s . It is possible that these snails are 
stimulated not only by the amino and carboxylic acids but also the monosaccharide and disaccharide 
units that are also likely products o f plant material fermentation (Ihekoronye and Ngoddy, 1985). 
Many sugars such as maltose, maltotriose, xylose have been observed to attract and arrest some 
planorbid snails such as Bulinus rohlfsi, B. globosus, Biomphalaria glabrata to various extents 
(Kpikpi and Thomas, 1993, Thomas, 1986, 1989).
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CHAPTER 4.
RELATIVE EFFECTIVENESS OF UNPROCESSED AND PROCESSED 
BIOACTIVE MATERIALS AS ATTRACTANTS, ARRESTANTS AND 
REPELLENTS.
An important feature o f the materials used in the present study as indicated by their response indices 
is the change in their stimulatory effects on B. truncatus after processing With the exception o f 
three test materials, ie oven-dried sweet potato tuber, 7 days fermented cocoyam tuber and fresh 
cocoyam leaves, where some processed states had the same effects on the snails as the unprocessed 
forms, the effectiveness o f all the other test materials was altered to a large extent by the various 
processing methods .
4.1 Effect o f  processed Leaves :
The present results show that adult B. truncatus are less responsive towards processed leaves. 
Thus both attractant and arrestant response indices for the adult snails decreased by at least 11.1% 
when the cocoyam, cassava, and pawpaw leaves were processed by either sun-drying, oven-drying 
or fermentation (Tables 4.1.1 and 4 .1 .2). This argument is, however, weakened by the fact that 
both attractant and arrestant responses of the adult snails towards cassava leaves increased by at least 
33.3% when these leaves were fermented for 3 days . Although responses o f juvenile conspecifics 
towards unprocessed leaves is relatively weak, this further reduces by at least 92% when these leaves 
were either sun-dried or oven-dried. Sun-dried cassava leaves could neither attract nor repel 
juvenile B. truncatus snails although in the unprocessed form it proved to be a significant
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Attractant Response of Adult Bulinus truncatus to some
RESPONSE INDEX % CHANGE
TEST MATERIAL UNPROCESSED PROCESSED IN
Sun-drv Oven-drv Fermentation RESPONSE
lda\ 3da\ >7dav<
COCOYAM : 
Leaves 5.6 3.8 -32.14
5.6 1.6 -71.43
Stem tuber 0.4 3.8 +1166.67 
0.4 3.4 +871.43 
0 4 ? 0 +471 43
CA SSA V A : 
Leaves 2 . 2 -2.4 -209.09
2 . 2 3.5 +59.09
Root tuber 5.2 4 t -11.54
5.2 -IS -134.62
5.2 0.7 -86.54
SWEET POTATC 
Stem tuber 5.0 2 . 8 -44.00
5.0 5.0 0 . 0 0
5.0 3.9 -2 2 . 0 0
5.0 2 4 -52.00
PAW PAW : 
Leaves 6.7 -0 . 6 -108.96
6.7 3.1 -53.73
MilLK BUSH : 
Fruits 2.9 3.1 +6.90
GINGER : 
Stem tuber -7.2 -4.2 -41.67
NEEM : 
Leaves 2.3 -3.3 -239.13
Vo increase in response — (  response index o f processed X 100 )  - 100
response index o f  unprocessed
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Attractant Response of Juvenile Bulinus truncatus to some
i a n i e 4 . i /  unD ro c e sse n  a im  i i u ic s s c u  o iu d in v c  i «
RESPONSE INDEX %  C H A N G E
TF.ST MATF.RIAL UNPROCESSEL PROCESS!■J) IN
RESPONSE
3 da vs 7<lavs
COCOYAM : 
Leaves 1.4 0.1 -92.86 
1.4 3.6 + 157.14
Stem tuber 0.4 2 .8 +600.00 
0.4 8 .2 +1950.00 
0 4 9 7 -1325
CASSAVA: 
Leaves 2.3 0 . 0 - 1 0 0 . 0
2.3 4.3 +86.96
Root tuber 2.9 4.1 +141.38 
2.9 5.3 +182.76 
7 9 S -°9 3  10
SWEET POTATO: 
Stem tuber 6.4 1.3 -79.20
6.4 6.5 +4.00
6.4 5.1 -18.40
6.4 5.7 -8.80
PAWPAW : 
Leaves 3.5 -1.7 -148.57 
i * ' -40 00
MILK BUSH : 
Fruits 0.1 2.3 + 2 2 0 0
G IN G ER: 
Stem -9.3 -2 .1 -77.42
NEEM : 
Fruits 0 . 0 -2 . 0 -2 0 0 . 0
% increase in response = ( response index o f  Processed x 100 ) - 100
response index o f Unprocessed
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attractant and arrestant (Tables 4.1.2 and 4.2.2). Pawpaw leaves, which were potent attractants 
and moderate arrestants in the unprocessed state tend to become repellents after sun-drying 
though this repelling property seems to be mild towards juvenile conspecifics. Fermenting the 
leaves o f cassava and cocoyam for 3 days appears to increase their attractant and arrestant effects 
on the juveniles tremendously. Thus response towards fermented cassava leaves for snails o f  both 
ages increased by about 12-87% while in the case o f cocoyam leaves, fermenting increased the 
effectiveness to about 109-157% . Carbohydrate, and hence sugar, content o f  most leaves 
including cassava, cocoyam and pawpaw are known to be as low as 6-7mg per lOOmg weight 
whereas the level o f  mineral salts such as calcium and some amino acids are about 6-60mg lOOmg 
weight (Ihenkoronye and Ngoddy, 1985).
In the natural freshwater environment, snails are known to actively take up calcium and some other 
ions such as iron and magnesium (Thomas, 1973; Bovaird, 1959). Calcium forms an important 
component o f snail shells and are also essential in reproduction (Jahan-Parwar,1975). Thus the 
presence o f calcium in these leaves may be responsible for the observed responses o f  the snails 
towards them. It is also possible that these mineral ions in the unprocessed leaf may be relatively 
inacessible to  the snails as they are more likely to form complexes with other compounds 
Processing o f these leaves by drying and fermentation leads to breakdown o f some o f these 
complexes (Nalewajko & Schindler, 1976) and thus making the calcium ions more available and 
hence the increased movement towards these processed leaves . It would be o f considerable interest 
to investigate these hypotheses further .
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Arrestant Response of Adult Bulinus truncatus to some 
T ab le  4 .2 1 lln >rocessed and Processed Bioactive Materials.______
R e s p o n s e  In d e x %  CHANGE
T f s t  M atkriai. I’ROCFSSFD IN
SiiTwlrv Oven-drv Femienta ion RESPONSE
■<
COCOYAM : 
Leaves 4.5 4.0 - 1 1 .1 1  
4 5 1 1 -7  " '
Stem tuber -0.3 3.6 + 142.20 
-0.3 -0.5 +80.00 
- 0  3 -I, < 0  0 0
CA SSA V A : 
Leaves 2 . 0  -2 .2 -212.82 
? 0 -3  3 3 3
Root tuber 3.2 1.0 -68.75
3.2 - 1.8 -157.14
3.2 -0  h - 1 1 '-.
SWEET POTATO : 
Stem tuber 2.9 2 .1 -26.32 
2.9 1 .8 -36.84 
2.9 0 . 8 -71.93 
. 2 9 -114 04
PAWPAW : 
Leaves 5.2 - 1.8 -133.65 
5 ? -so nn
MILK BUSH 
Fruits 2.4 1.4 -40 43
GINGER : 
Stem -4.7 -2 . 6 -44.68
NEEM : 
Leaves 0.3 -3.3 -1183 33
% increase in response = ( response index o f Processed x 100 ) - 100
response index o f Unprocessed
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Arrestant Response of Juvenile Bulinus truncatus to some
R F S P O N SF  IN DF/X % CHANGE
TF.ST M A T E R IA L S UNPROCESSED PROCFSSF.n IN
Sim-drv Oven-dry Fermentation RESPONSE
lda\ 3 davs 7davs
COCOYAM
Leaves 1.1 l . i 0 .0 0  
1.1 2.3 + 109.09
Stem tuber 1.2 0.3 -79.17
1.2 4.3 +258.33
1.2 9.5 +691.67
C ASSAVA: 
Leaves 2.5 0 . 0 - 1 0 0 . 0 0  
2.5 2 . 8 + 1 2 . 0 0
Root tuber 0 .1 0.4 +300.00
0 .1 4.3 +4200.00
0.1 6.4 +6300.00
SWEET PO T A T O : 
Stem tuber 2.7 0 . 6 -79.25
2.7 2.4 -11.32
2.7 1.9 -30.19
2.7 4,0 +49.06
PAWPAW :
Leaves 1.7 -2 .1 -223.53 
1 7 1 0 -44 1?
MILK BUSH : 
Fruits 1.8 1 .0 -47 22
GINGER : 
Stem -2 . 2 -3.65 +65.91
NEEM : 
Leaves 0 . 0 -0.4 -35.00
% increase in response = ( response index o f  Processed X 100 ) - 100
response index o f Unprocessed
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4.2 Effects o f processed stem tubers :
Attractant and arrestant responses o f  both adult and juvenile B. truncal us towards oven-dried 
sweet potato stem tubers appears to have been reduced relative to those o f  the unprocessed form. 
Extent of reduction, however, is much greater for the juveniles (-79 17 - 79 20%) than for the adults 
(-26.32 - 44 00%) Responses o f  these snails towards cocoyam greatly improved after 
fermentation In the case o f the adult snails, attractant response increased by at least 471% while 
for the juveniles it was 600% . There is an exception to this trend; the stem o f ginger in both the 
processed and unprocessed forms proved to be strong repellent (p<0.05) for snails o f  both ages. 
The effectiveness o f this repelling property however, decreased by about 50% for the adult snails 
while it increased by 77% in the case o f the juvenile conspecifics after oven-drying
4.3 Effects o f  processed roots :
Unprocessed cassava root tubers appear to be highly stimulatory to juvenile B. trimcatus Attractant 
responses to cassava roots tubers increased tremendously for adult snails after fermentation by about 
140-293% while there was a 69-120% decrease in arrestant response after processing. In the case 
of the juveniles, an even greater increase in arrestant response o f about 300-6300% was observed 
when the cassava tubers were processed by fermentation Attractant response also increased 
although to a much lower extent than that o f the arrestant
4.4 Processed Repellents:
The present results show that the ability o f bioactive materials to repel B. trimcatus snails appear to
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have been lost or significantly reduced after processing Ginger stem, a strong repellent in the 
unprocessed state, had its repellent effects reduced by about 42% when processed by oven drying.
Furthermore, some materials such as cocoyam tuber, which proved to be a weak repellent for the 
adult conspecifics in the unprocessed form, had this property reduced by at least 142% and become 
a significant arrestant (p<0.05) to the adult snails after 1 day fermentation (Table 4 .2 .1 ). On the 
other hand, some bioactive materials such as cassava and pawpaw leaves, became weak repellents 
(p<0.05) to snails o f both ages after processing whereas in the unprocessed state they proved to be 
strong attractants and arrestants .
The present results also point to neem leaves as possessing some repellent effects on both adult and 
juvenile B. trimcatus. In the unprocessed form these leaves appear to be neutral to  the juvenile snails 
but weak attractants and arrestants to the adults at p< 0.05 confidence lim it. After oven-drying , 
they showed some significant repellent effects (p<0.05) on snails o f both ages with these effects 
being much lower on the juvenile conspecifics .
The implication o f neem plant in the present work as possessing some repelling effect on B. trimcatus 
is o f interest. The repellent properties o f  all parts o f  the neem plant against terrestrial organisms 
especially insects is well known (Amason et a l , 1988, Ketkar et al ,1976). The most important active 
ingredients in the leaves o f  neem plant responsible for the observed repellent effects on most 
terrestrial organisms are terpenoids and some other complex oils which are known to be inactive at 
higher temperatures due to structural decomposition (Olaifa et al., 1986). Further work needs to be 
done towards identification of the compounds responsible for the observed repellent activity o f  the 
neem leaves against these freshwater bulinid snails It is possible that
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some other unknown compounds that are not affected by high temperatures may be responsible for 
the repellent action o f  these leaves
4.5 Comparing processed and unprocessed Malerials :
An interesting observation is apparent when the response indices o f the processed materials are 
examined vis a vis those o f the unprocessed (Tables 4.1.1 and 4.1.2) . The results show that 
processed materials tend to be much less stimulatory as attractants and arrestants for adult B. 
trimcalus snails whereas they elicit very strong responses from the juveniles. Thus the unprocessed 
materials are more strongly stimulatory to the adult snails than the processed forms . In effect, in 
the present result, the highest attractant and arrestant indices (8 . 8  and 7.3 respectively) are 
generated by ripe mango (unprocessed) when tested on the adult conspecifics . On the other hand, 
processed materials were found to elicit the highest responses from the juvenile snails. In fact the 
fermented forms o f  these materials have been found to be most strongly stimulating . Thus 7 days 
fermented cocoyam tuber, which generated attractant and arrestant indices o f 9.7 and 9.5 
respectively, proved to be the strongest processed bioactive natural product for juvenile B. trunccitus 
in the present bioassay study . Fresh sugar cane was, however, an exception . Although it was 
tested in the unprocessed form only, it proved to be the most potent attractant and arrestant 
(p<0 .0 0 1 ) for the juvenile snails .
These findings lend credence to the observation by Dogbey (1995) that boiled pawpaw fruits (a 
processed bioactive natural material) elicited the highest significant attractant response from 
juvenile B. tnmcatus whereas the unprocessed materials such as sugarcane and sweet potato tubers
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were the most significant stimulants for the adult conspecifics in a similar bioassay study It is quite 
interesting to note that in other chemoreception studies involving other planorbid snail responses 
towards organic molecules like carbohydrates and amino acids (Thomas and Assefa,1979, Thomas 
et al., 1989; Kpikpi and Thomas, 1991,1993; Kpikpi et a l , 1995) the juveniles most often exhibit 
much wider chemoreception niche for these materials than the adults. Sugars such as maltose, 
fructose, sucrose and also short-chain carboxylic acids like lactate, acetate and propanoate are 
known to be normal intermediate and end-products o f  anaerobic fermentation . This occurs when 
anaerobic microbes, especially bacteria, utilise carbohydrate sources in glycolytic energy production 
(Ihenkoronye and Ngoddy, 1985) . In the normal freshwater environment o f  the snails these 
products are known to be released by senescing and decaying macrophytes such as Lemna 
paucicostata and Ceratophylum demersum (Patience et al., 1983a ; Sterry et al., 1985 ; Daldorph. 
1988) into the medium. There is also evidence that some amino acids such as glutamate, aspartate, 
proline and hydroxyproline are released from living freshwater macrophytes into the external medium 
(Watt, 1966; Watzel and Manny, 1972 ; Bardach,1975) These organic molecules, most o f  which 
form a normal component of the dissolved organic matter (DOM), serve as chemical cues which may 
function as kairomones by serving as sources o f information for food and shelter for these snails and 
other aquatic invertebrates and some vertebrates (Carefoot. 1982, Thomas et a l . 1982. 1985; Croll. 
1983; Kemenes et al., 1986) It was also suggested that sugars and carboxylic acids released from 
fermenting and decaying materials in the freshwater habitat o f snails as well as amino acids normally 
present in the medium may also function as pheromones for the location o f  conspecifics. Another
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function of these molecules is that they facilitate the uptake o f heavy metals such as iron or copper 
which are needed for the synthesis o f  respiratory pigments (Jorgenson, 1976; Thomas et al ,1983). 
Thus exogenous molecules o f plant origin occur commonly in the aquatic environment o f both 
marine and freshwater ecosystems (Degens, 1970;) influencing the behavior and physiology o f  many 
aquatic organisms including pulmonate snails (Potts, 1967, Stephens, 1972 ; Gilbertson & Jones, 
1972). DOM and their sugar components are more likely to serve as food to the juvenile planorbid 
snails since their radulae may not be as well developed for scraping the surfaces o f  macrophytes and 
other floating plants for food as in the case o f  the adults (Thomas et al., 1983, 1989). This could 
probably account for the observation that juvenile B. trunccitus were more responsive towards 
processed materials since these were already in a state o f decomposition . It is possible that this 
wider response niche exhibited by the juveniles could be an early neonatal behavior that has been 
retained, when they used to feed entirely on DOM Also, adult chemoreception may become reduced 
due to high degree of discrimination in their response towards chemical factors as a result o f learning 
(Kpikpi & Thomas, 1992) . Audesirk and Audesirk (1985) gave evidence which suggests that 
pulmonate snails and other gastropods are capable o f classical Pavlovian learning after relatively 
few tria ls .
4.6 Effectiveness o f  processing techniques :
Response indices from the present study and those found by other workers (Agudogo, 1992; 
Dogbey, 1995) for some processed natural materials suggest that the effectiveness o f  bioactive natural 
products to attract and/or arrest B. trimcalus depends on the techniques used in processing
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them Thus one form o f a given processed material may prove to be a significant attractant and/ 
or arrestant whereas another form o f it may be a significant repellent
The various processing methods employed in the present work and those used by previous workers 
towards the search for significant bioactive natural materials for use as attractants and arrestants are 
discussed below
4.6.1 Sun-drying and fermentation :
It appears that bioactive materials processed by sun-drying are more effective attractants to the 
adult B. trimcatus snails than the fermented forms. Thus in the present results, sun-dried cocoyam 
leaves proved to be stronger attractants to the adult snails than the 3 days fermented cocoyam leaves 
(response indices were 3.8 and 1.6 respectively). On the other hand, fermented bioactive materials 
tend to be more potent attractants to the juvenile conspecifics than the sun-dried forms. Whereas 
the sun-dried cocoyam leaves generated attractant index o f  0.1 for the juvenile snails 3 days 
fermented cocoyam generated 3.6 (Table 4.6) .
4.6.2 Oven-drying and Fermentation :
Response indices o f oven-drying and fermented forms of bioactive materials as found in the present 
study suggest that the latter forms are more potent attractants to B. trimcatus snails o f  all ages than 
the former ones Thus whereas oven-dried cassava leaves elicited a negative response from the adult 
snails and no response from the juveniles, response indices o f  the 3 days fermented
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forms were 3.5 and 4.3 for the adult and juvenile snails respectively . Similarly, fermented sweet 
potato tubers stimulated both adult and juvenile B. truncatus snails more strongly than the oven-dried 
forms (Table 4.6). However, response index o f 7 days fermented sweet potato tubers for the adult 
snails was much lower than that o f the oven-dried form
It is not clear at the moment, why sun-dried cocoyam leaves, for example, tend to stimulate adult B. 
truncatus snails to a significantly greater extent (p<0.05) than the fermented forms It may be that 
the large amounts o f  minerals such as calcium and iron in these leaves (Ihenkoronye & Ngoddy, 
1985) may be responsible for attracting these adult snails even in the sun-dried state .
Fermented forms o f  bioactive plant materials used in the present work proved to be most strongly 
stimulating , especially to the juvenile snails than the sun-dried and oven-dried forms . This is not 
unexpected . Fermentation o f materials from plant origin gives rise to anaerobic decomposition to 
release a medley o f  chemical factors such as sugars, carboxylic and amino acids most o f  which 
could attract and/ or arrest the snails to various extents (see Chapter 3 .4 o f  present s tu d y ).
4.6.3 Boiling and Fermentation:
A comparison o f  the results o f the present study and those of previous workers (Agudogo, 1992 ; 
Dogbey,1995) shows that o f the two processing techniques, fermentation enhances the attractant 
effects o f  bioactive natural materials on the juvenile B. truncatus than boiling . Furthermore, the 
extent o f these effects tends to increase with the period o f fermentation . Thus whereas no 
significant response was elicited from juvenile snails by boiled cassava tubers, the fermented forms 
of this material generated significant response indices o f 4 .1, 5 .3 and 8 5 respectively for 1, 3 and 7 
days fermentation at p<0 05 confidence limit . In the case o f  cocoyam tubers, w hereas the boiled
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Table 4.6.
Relative efficacy o f  different forms o f  bioactive materials under various processing  
techniques as shown by their respective response indices.___________________________
P P ^ P O M Q F  T 1ST n  T r  F. s
T E S T A I)  U L  T S J U V E N I L E S
M A T E R IA L S H Od B. Fermenta ion S d O d B Ferrnentation
ID 3E I D ID 3D 7D
Cassava leaves - -2.4 - 3.5* -o .c - 4.3*
Cassava tubers - - 4.6*(iS)) 4.6* - 1.8 0.7 - 2.3 4.1* 5.3* 8.5*
3.1**(!)
Cocoyam leaves 3.8* 1 .6 0.1 3.6*
Cocoyam tubers 2 . 1(@) 3.8* 3.4* 2 . 0 -7.4(**) 2 . 8 8 .2 *** 9.7
4.0(1) (!)
Sweet potato tubers 2 .8 1 .8 (@) 5.0s 3.9s 2.4 1.3 -0.3(3) 6.5* 5.1* 5.7*
Note: S.d = Sun-dried; O.d = Oven-dried, B.= Boiled
*, **, *** = Significant response indices at p< 0.05, 0.01 and 0.001 confidence limits 
respectively.
(**) = Significant repellent index at p<0.01 confidence limit.
(@) = data from Dogbey, 1995.
(!) = data from Agudogo, 1992.
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forms were significant repellents (p<0.01) the 3 and 7 days fermented forms were significant 
attractants at p<0 001 significant level
Unlike the juveniles, no sharp difference in chemoresponse of the adult snails was observed for boiled 
and fermented forms o f cassava tubers . Thus both forms o f this material generated significant 
attractant indices for the adults at either p<0.01 or 0.05 confidence limits (Table 4.6). Fermenting 
cassava tubers beyond 1 day, however, tends to destroy its attractant effects on adult B. truncatus 
snails . In the case o f  cocoyam and sweet potato tubers, the boiled forms failed to  elicit any 
significant attractant response from the adults whereas the 1 and 3 days fermented forms were 
significant attractants at p<0.05 confidence lim it. Fermentation o f these materials for up to 7 days, 
however, renders them incapable o f stimulating the snails to any significant ex ten t.
4.6.4 Boiling and Oven-drying:
It is apparent from the results shown in Table 4.6 that, compared to the oven-dried forms, boiled 
forms o f bioactive natural products are not capable o f  eliciting any significant attractant response 
from the adult B. truncatus snails . Thus whereas oven-dried sweet potato tubers elicited 
statistically significant response index o f  2.8 (p<0.05) from the adults the response index o f  the 
boiled form was insignificant. Both forms o f this material were, however, insignificant as attractants 
to the juvenile conspecifics .
4.7 Best processing technique :
It appears that among the four processing techniques so far employed in the search for potent 
bioactive natural materials, fermentation seems to be the most promising method for enhancing
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the attractant and arrestant effects o f natural materials Oven-drying and boiling are more likely 
to render the materials as repellents . The extent o f fermentation to be used, will, however, depend 
on the age group of the target snails Thus whereas fermenting some materials (eg. cocoyam tubers) 
up to a period o f 7 days improves their attractant effects on the juvenile snails the same technique 
is likely to repel the adult conspecifics .
4.8 Suggestions fo r  further work:
Results from the present study suggest the need for further research in this direction to investigate 
the chemoresponses o f B. trimcatus and other planorbid snails towards the various forms o f bioactive 
materials processed under these techniques . This would help to provide comprehensive 
information about the chemoreception niches o f various schistosome host snails with respect to 
natural materials . The importance o f  this information is obvious . It would help to narrow down 
considerably, the search for natural bioactive products for use as attractants and arrestants in the 
manufacture o f  the envisaged controlled release formulations .
Other promising processing techniques that could be employed in further research include ;
1 Combination o f two or more bioactive materials It is possible that a higher response index may 
be produced when more than one known bioactive materials are combined in various proportions 
to form a single product
2. Using two or more processing techniques to obtain a single material For example, sun-dryins 
followed by boiling ; oven-drying followed by fermentation, etc It is likely that the repellent
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property o f  a material may be considerably reduced when subjected to more than one processing 
methods successively .
Sugarcane has been found to be a most promising potent bioactive material both in the present 
study (for juvenile B. truncatus) and in a previous work (for adult B. truncatus) reported by Dogbey 
(1995) . It would be o f considerable interest to investigate the effects o f the processed forms o f  
this bioactive materials on planorbid snails using different processing techniques such as 
fermentation and boiling .
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C H A P T E R  5.
EFFECTIVENESS OF ATTRACTANTS AND ARRESTANTS UNDER  
SIMULATED NATURAL ENVIRONMENTAL CONDITIONS.
5.1 INTRODUCTION
Most o f the efforts at developing controlled release formulations for selective removal o f 
schistosome host snails have been directed towards identification o f  bioactive materials using 
laboratory based bioassays (Thomas and Assefa, 1979; Thomas et al., 1980, 1983,1985. 1986, 1989; 
Daldorph and Thomas, 1988; Kpikpi, 1990; Kpikpi and Thomas, 1992,1993; Dogbey, 1995).
It is envisaged that known snail toxicants incorporated into such attractants and arrestants 
together with appropriate phagostimulants serving as feeding incitants could be used to 
selectively remove schistosome host snails and thereby reducing the adverse effects on non-target 
organisms associated with molluscicides in current use (Thomas and Assefa, 1979).
Having identified these materials it is necessary to devise a method o f finding out how effective they 
can perform when applied in the field In response to this need. Kpikpi (1990) and Dogbey (1995) 
individually tested some o f the various bioactive materials they found in their bioassays under a 
simulated natural environmental condition (SNC) Each o f  these workers devised snail trapping 
units made o f bamboo stems (Bamhusa vulgaris), since these are readily available in the areas 
where schistosomiasis is endemic.
Beside finding out how long the material could remain effective when applied in the field, the test
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under SNC also acts as a further screening procedure to select only the most effective and 
appropriate materials for field testing and application and thereby reducing cost.
For these reasons, the significant attractants and arrestants identified in the bioassay tests as reported 
in chapters 2 and 3 were selected and screened further under SNC using trapping devices made 
of discarded plastic materials
5.1.1 CRITERIA FOR CHOICE OF MA TERIALS TESTED UNDER SNC.
Selection o f bioactive materials for test under SNC was based on the criteria that :
1. They exhibit significantly high bioactivity, ie each material must generate statistically significant 
attractant or arrestant response index o f  not less than 4.0 from either adult or juvenile snails or 
both.
2. Relative cost o f the material must be such that it can be affordable at all times by the local 
communities that might need it for the control purposes
3. The material must be readily available and have only a few alternative uses in the areas where 
it occurs in abundance.
Based on the above criteria, the following materials shown in table 5.1 below were selected for 
further screening under the SNC
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Table 5.1. BIOACTIVE MATERIALS SELECTED FOR SNC TEST.
FRUITS LEAVES STEMS ROOTS
ripe mango fresh cocoyam leaves fresh sugarcane 1 day fermented 
cassava tuber
ripe cashew fresh sweet potato 7 days fermented 
tuber cassava tuber.
ripe miraculin 1 day fermented 
Sweet potato tuber
water melon 3 days fermented 
cocoyam tuber
7 days fermented 
cocoyam tuber
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5 2 M A T E R IA L S A N D  M E T H O D S
5.2.1 SNAIL BREEDING:
About 1900 Bui inns trimcatus snails were bred over a period o f 2 months using the methods 
described in Chapter 2.2.1 The juvenile snails were transfered into a
6 6  x 38 x 21cm tank filled with 42.6 litres o f  tap water The snails were routinely fed with fresh 
lettuce leaves every two days. The medium in which the snails were kept was changed once every 
week.
5.2.2 SIMULA TED NA TURAL ENVIRONMENTAL CONDITION (SNC)
A concrete tank of volume 1,108 litres was used for this purpose The preparation was similar to 
that described by Dogbey ( 1995). The bottom o f the tank was covered with sand, obtained from 
the bottom sediment o f  lake Weija, to a depth o f  3cm and 60 litres o f  tap water added.
Macrophytes such as Ceratophylhmi demersum, Pistia sp, and Nymphaea sp. were added to  cover 
nearly two-third o f the surface o f  the water.
The water in the tank was kept at a temperature o f  29 ±  1°C with a pH range o f  5 ,8 - 6  7.
1200 young adult B. trimcatus snails o f at least 6  weeks old were introduced into the aquarium and 
allowed to acclimatise for 7 days. Two large leaves of fresh lettuce were added to the aquarium every 
other day during the period.
The snails were deprived of the lettuce food ration for a period o f 24 hours prior to the experiment.
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5.2.3 TRAP DESIGN-.
This is a modified version o f a device used by Ayeh-Kumi (1996) as an experimental cage
It consists o f transparent plastic spring water bottle ( ‘ ASTEK INSU’ bottles). The top portion o f  
the bottle was carefully cut off to obtain a cylindrical container o f  height 2 0 cm and diameter 8 6 cm 
Two holes (7x8cm) were made at opposite sides o f the container, one being 3 cm above the base and 
the other 4cm from the cut top end o f  the container By means o f  carpenter’s glue, a nylon net (o f 
mesh size 1 0mm2) was used to cover the holes. Two other holes were made 0.5 cm from the top, 
one on opposite side o f the container for suspending the traps in the aquarium by means o f  string 
( Plate 5.2.3, Appendix 7).
5.2.4. TEST MATERIALS-.
The twelve bioactive materials tested nuder the SNC were those found to  produce statistically
significant response indices o f  at least 4.0 for either adult or juvenile B. truncatus in the bioassay 
tests. The procedure for fermentation o f materials was the same as described in Chapter 3.2.11(3). 
Each material was cut into cylindrical forms with diameter similar to that o f  the low er portion o f  the 
trap. Test materials in the form o f leaves were suspended inside the trap by means o f  a short 
stick through a small hole at the bottom o f the trap This was to prevent the material from moving 
out of the trap when placed in the water
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5.2.5 THE SNC EXPERIMENT:
The inside o f the traps was cleaned by washing thoroughly with tap water prior to  and after 
testing each material The traps were set by placing appropriate quantities o f  test material into the 
trap to make it 1/4 full (ie 5cm full). A small piece o f  stone (an inert material) was placed in each 
trap to act both as a sinker and a control material The traps were set by suspending them vertically 
in the water by means o f  a piece o f  string hung over a wooden grid placed across the aquarium 
tank. The open end o f the traps was 2cm below the water surface (Plate 5.2.4, Appendix 8 ).
For each test material, ten test traps and ten control traps were set. Each test trap was alternated 
with a control trap. This is to ensure that each snail has equal chances o f  moving towards a test 
or control trap. The experiments were performed under a photoperiod o f  12 hours o f  light and 
12  hours o f darkness.
The number o f  snails caught in each trap was counted and recorded after 24 hours for each test 
material. Snails found in and on any trap are deemed to have been caught by that trap. Student 
t-tests (Bailey, 1981) were used to determine the levels o f significance between numbers o f  snails 
caught in test and control traps. A trapping index (ie. total number o f  snails in test traps - total 
number o f snails in control traps) was computed for each test material used The data were used 
to evaluate the efficiency / effectiveness o f  the bioactive material in trapping B. trimcatus snails 
under the SNC experiments.
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5.3 R E SU L T S :
5 3 1 NUMBER OF SNAILS CAUGHT:
The present results show that, apart from the ripe cashew test traps, all traps containing test materials 
caught significantly more snails than their corresponding controls (p< 0.05) (Table 5.3.1). The five 
materials that trapped significantly high numbers o f snails under the present SNC experiments 
include fresh sugarcane > 7 days fermented cocoyam tuber > 1 day fermented cassava tuber > ripe 
mango > fresh cocoyam leaves (Fig. 5.3.1). Total number o f snails caught by test traps was 990 
whereas that for controls was 384
5 3 2 UNPROCESSED MATERIALS UNDER SNC TEST:
The results show that a total o f  571 snails were caught by the seven unprocessed materials tested. 
This represents 57.7% o f the total number o f  snails that entered the traps. The four most strongly 
bioactive unprocessed materials that caught significantly high numbers o f  snails are 
fresh sugarcane > fresh cocoyam leaves > ripe mango > fresh sweet potato. With a total o f  150 
snails in the test traps fresh sugarcane emerged as the strongest unprocessed bioactive material in 
the present SNC tests (Fig. 5.3.2).
5 3 3 PROCESSED MATERIALS:
All the five processed materials tested in the present experiments were in their fermented forms. 
The results show that o f  the total o f 506 B. truncatus snails that entered the test traps containina
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Table 5.3.1
NUMBER OF BULINUS TRUNCATUS SNAILS ENTERING TRAPS
T O T  AI N H M R F R  O F  SNAFUS IN 10 TRAPS
TEST MATERIAL TEST CONTROL TRAPPING INDEX t-value (d <0.05)
UNPROCESSED
FRU ITS :
Cashew 45 33 12 1.2451 (n.s)
Mango 93 28 65 3.6558**
Miraculin 42 19 23 2.4081*
Water melon 72 21 51 3.3966
LEAVES:
Fresh cocoyam leaves 105 44 61 5.4487***
STEM:
Fresh sugarcane 150 38 1 1 2 5.4679***
Sweet potato tuber 64 23 41 4.0354**
PROCESSED
1 day fermented sweet potatc 74 20 54 2.5861*
1 day ferm. cassava tuber 110 33 77 3.2154**
3 days ferm. cocoyam tuber 8 6 29 57 2.8108**
7 days ferm. cassava tuber 87 42 45 2.5399**
7 days ferm. cocoyam tuber 149 55 94 3.7737**
Note : ns = not significant
* ** *** _  sjgnjficant numbers o f snails at 0.05, 0.01 and o.OOl confidence limits
respectively.
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TEST MATERIAL
cp
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processed materials, 149 (ie.29.4%) were found in those traps containing 7 days fermented cocoyam 
tuber whereas those with 1 day fermented tuber caught 110 (217% ) and those with 7 days 
fermented cassava tubers and 3 days fermented cocoyam tuber caught 87 and 8 6  snails respectively. 
Thus the three processed bioactive materials that caught the highest numbers o f  snails include 7 
days fermented cocoyam tuber > 1 day fermented cassava tuber > 7 days fermented cassava tubers. 
The least number o f snails was caught by 3 days fermented cocoyam tuber and 1 day fermented 
sweet potato test traps.
Thus the order o f  effectiveness o f the processed bioactive materials from the present SNC tests is 
7 days fermented cocoyam tuber > 1 day fermented cassava tuber > 3 days fermented cocoyam tuber
> 1 day fermented sweet potato > 7 days fermented cassava tuber (Fig.5.3.3).
5 3 4 BIOACTIVE FRUITS:
It appears that fruits tested under the present SNC experiments caught much fewer snails than other 
bioactive materials. Thus, the total number o f  snails caught by test traps containing fruits were 
relatively lower. Among the four fruits tested, ripe mango emerged as the most potent with a total 
number of 93 snails caught by test traps containing this material Other bioactive fruits that caught 
significantly high numbers o f snails were water melon and ripe miraculin The total number o f 
snails caught by test traps containing ripe cashew was statistically insignificant (p>0.05) compared 
to those caught by the control traps Thus ripe cashew is not a significantly strong bioactive material 
under the present SNC experiments (Fig. 5.3.4).
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TRAPPING INDEX
1 day ferm. Ipomea
1 day ferm Manlhot tubers
7 days ferm. Manihot 
tubers
3 days ferm. Xanthosoma 
tubers
7 days ferm. Xanthosoma 
tubers
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Fig.5.3.3 TRAPPING EFFICIENCY OF SOME PROCESSED BIOACTIVE PLANT 
MATERIALS UNDER SIMULATED NATURAL CONDITIONS.
TEST MATERIAL
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TEST MATERIAL
100
Fig. 5.3.4 TRAPPING EFFICIENCY OF SOME BIOACTIVE FRUITS UNDER 
SIMULATED NATURAL CONDITIONS.
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5 3 5 TRAPPING EFFICIENCY OF BIOACTIVE MATERIALS:
The trapping indices computed for each o f the bioactive materials gives an indication o f how 
effective a bioactive material is likely to function when deployed in trapping devices under field 
conditions.
The results o f the trapping indices show that the five most efficient bioactive materials include 
fresh sugarcane > 7 days fermented cocoyam tuber > 1 day fermented cassava tuber > ripe mango
> fresh cocoyam leaves (Fig.5.3.5). It is important to  note that although sugarcane and 7 days 
fermented cocoyam tuber caught about equal numbers o f  snails, sugarcane proves to be much 
more efficient when used in trapping units (trapping index = 1 1 2 ) than the fermented cocoyam 
(trapping index =94). Also fresh cocoyam leaves tend to catch more snails than ripe mango but 
the latter material has a much higher trapping index (65) than the former (61). Thus ripe mango 
is a more efficient bioactive material than fresh cocoyam leaves.
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TEST MATERIAL
102
Fig. 5.3.5 TRAPPING EFFICIENCY OF SOME SIGNIFICANT BIOACTIVE PLANT 
MATERIALS UNDER SIMULATED NATURAL CONDITIONS.
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5.4 DISCUSSION.
5.4.1 CONCORDANCE WITH BIOASSAY TESTS:
The results o f the present experiments under the SNC show some concordance with some o f
the results o f the bioassay tests reported in chapters 2 and 3 o f  this study. Thus sugarcane and 7 
days fermented cocoyam tuber which were found to be the two most potent processed and 
unprocessed bioactive materials respectively in the bioassay experiments also proved to be the 
materials that most strongly attracted B. trimcatus in the test traps under the SNC experiments. 
This result supports similar SNC test results reported by Dogbey (1995) that sugarcane emerged 
as the strongest bioactive natural product for B. trimcatus, although different trapping units were 
used.
One observation worthy o f note, however, is that whereas in the olfactometer studies, sugar cane 
proved to be much more potent than 7 days fermented cocoyam tuber by virtue o f  its higher 
response indices, the results from the present SNC test indicate that there is no statistically 
significant difference between the total numbers o f  snails caught by test traps containing each o f 
these two bioactive materials (150 and 149 snails for sugar cane and 7days fermented cocoyam tuber 
respectively (Table 5.3.1). A possible factor that may account for this difference in response indices 
during the bioassay test may be the forms in which these materials were used in the tests. As 
pointed out by Dogbey (1995), a directional diffusion gradient is more likely to be created from 
crude bioactive materials presented in their whole or solid forms, such as sugar cane, along which 
the snail could move into the test chamber o f  the olfactometer. On the other hand, diffusion
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gradient emanating from crushed or semi-solid bioactive materials, as in the case o f  7 days 
fermented cocoyam tuber, would tend to be much more diffused and localised within the test 
chambers, making snails to move in all directions within the olfactometer chamber, hence the 
relatively lower response index generated by the fermented cocoyam tuber in the olfactometer 
experiments.
With regards to the efficiency, however, sugar cane emerged as the most efficient trapping material 
(Fig 5 3 .1). Thus it is a better bioactive material than 7 days fermented cocoyam tuber
5.4.2 CONVERSION OF BIOACTIVE INGREDIENTS :
Since about the same numbers o f B. truncatus snails were caught by both sugar cane and 7 days 
fermented cocoyam tuber, it is reasonable to argue that both materials would contain quite similar 
active ingredients responsible for the observed chemoresponse movements o f  the snails.
Mature sugarcane stem is known to contain some 20% sucrose together with fructose and glucose 
as well as small amounts of maltose. Although attractant and arrestant responses o f  bulinid snails 
such as B. globosus and B. rohlfsi towards pure sucrose are not significantly high owing to the 
absence of « (l-4 ) glucosidic bonds in that molecule (Kpikpi, 1993). it is likely that at favourably low 
ph levels, as found in the water medium, equilibrium may shift away from the sucrose molecules as 
a result o f hydrolysis leading to formation o f “ (1-4) glucosidic bonds among the various glucose 
molecules (Stroev, 1986), It is also possible that some other molecules such as carboxylic acids may 
be involved in the chemoresponses o f  the snails towards the sugar cane
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Root and stem tubers such as those o f cocoyam and cassava contain large stores o f polysaccharides 
in the form of starches and amylose . Fermentation processes lead to hydrolysis o f these materials 
with formation o f  intermediate products such as maltose, glucose and some carboxylic acids 
(Ihenkoronye and Ngoddy, 1985) which may be largely responsible for the chemoresponses o f  the 
snails.
5.4.3 FERMENTED MA TERIALS AND FRUITS:
Another point o f  interest in the present results is that traps containing fermented bioactive 
materials appear to catch significantly higher numbers o f  snails than those containing fruits, except 
in the case o f  ripe mango. A possible reason accounting for this observation may be due to  the 
relative calcium levels in these materials. Apart from the large amounts o f  carbohydrates which 
may be utilised by the snails for energy generation (de Zwaan et al., 1976) as well as for location o f  
target organisms in their environment (Thomas, 1982; Croll, 1983; Audesirk and Audesirk, 1985), 
calcium and iron are two elements present in stem and root tubers (Ihenkoronye and Ngoddy, 1985) 
that are o f  physiological importance to aquatic molluscs including B. trimcatus. A lot o f 
experimental evidence indicates that there is a positive correlation between concentration o f 
dissolved calcium and distribution and abundance o f  freshwater snails (Boycott, 1936, Macan, 
1950; Russel-Hunter, 1964) and that the large amounts of calcium needs o f  these snails are obtained 
directly from their foods (Greenaway, 1971a; Jodrey, 1953, Kado,1960; van der Borght,1963, 
Wilbur,1972; Thomas et al., 1974).
Iron, an important element for the synthesis o f  respiratory pigments or enzymes (Taylor,1969, 
Southward and Southward, 1972; Jorgenson. 1976, Ferguson, 1982) is also present in
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greater quantities in these tubers than in ripe fruits (Ihenkoronye,1985). Thus the higher levels o f 
these elements in tubers than in ripe fruits may be responsible for the observed difference in snail 
numbers caught by the respective traps.
It is possible that some other molecules which are presently unknown may also be acting as stimulants 
in addition to the calcium, iron and the carbohydrates. Further work needs to be done to find out 
the other active ingredients in these bioactive materials that are involved in the chemoreception 
profile observed in this study.
One fruit which caught the least number of snails and also proved to be the most ineffective bioactive 
material was ripe cashew. This was not unexpected as it has been shown that cashew , although 
contains some sugars, has significant level o f chemicals with molluscicidal properties (Ayeh-Kumi, 
1996; Webbe and Lambert, 1983). The active ingredients, however, are yet to be identified . 
Fresh sugar cane, 7 days fermented cocoyam tubers and 1 day fermented as well as fresh cocoyam 
leaves and ripe mango can therefore be recommended for field testing .
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C H A P T E R  SIX
GENERAL DISCUSSION
The present chemoreception study o f B. truncatus has revealed some interesting aspects o f  the 
responses o f both adult and juvenile B. truncatus to some unprocessed and processed forms o f 
naturally occurring bioactive materials from common plants. It has also given the efficacy o f  some 
of these materials under simulated natural environmental conditions.
This chapter attempts to summarize these findings, pointing out the implications o f  some o f these 
findings to the control o f the snail hosts o f schistosomiasis. Possible areas o f future research will 
also be pointed out.
6 1 CHOICE OF MATERIALS FOR BIOASSAY TESTS:
The purpose o f these tests was to identify materials from naturally occurring common products 
that can strongly attract or arrest B. truncatus snails (or both). Known snail toxicants could be 
incorporated into these bioactive materials for the manufacture o f controlled release formulations 
that can be used to selectively destroy the schistosome host snails .
Previous work done in this field (Dogbey,1995) influenced to a large extent the selection o f materials 
for the present work. Where one form o f the material was previously tested, a different form o f 
it was chosen for the present study. For example Dogbey (1995) tested the boiled and raw forms 
of sweet potato stem tubers In the present work the raw, fermented and oven-dried
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forms o f sweet potato were tested This is to provide the opportunity to determine which form 
of a bioactive material would be most stimulating to the schistosome host snails. Sugar cane was, 
however, tested in the present work although it has already been tested previously The emphasis 
has been on the availability and cost of the test material Thus all the materials selected are from 
common plants found in areas where schistosomiasis is endemic. For example leaves o f  neem 
plant (Azadirachta indica), fruits o f mango, cashew, water melon, cassava and sweet potato are 
common materials found in these areas and some are even used as food although not staples. The 
expectation is that where a potent bioactive material identified happens to be used as food, the 
active ingredients responsible could be investigated and closely related analogues could be used as 
alternatives .
6.2 BIOASSAY EXPERIMENTS:
6.2.1 UNPROCESSED MA TERIALS:
Adult B. trimcatus snails exhibited a much wider chemoreception niche towards unprocessed 
materials than their juveniles Thus they were significantly attracted , arrested or both towards 15(ie 
42.86%) of the unprocessed materials tested (p<0.05, 0 01 or 0.001) The five most significant 
attractants found for adult B . trimcatus from this study were as follows 
ripe mango > water melon > sugar cane > date palm > fresh pawpaw leaves (Appendices 1-3) 
There were some overlaps between the chemoreception niches o f  adult and those o f  the juveniles 
towards unprocessed materials Thus all the six unprocessed materials that elicited significant
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attractant responses from the juvenile snails were also attractants to the adults at either p< 0.05, 
0.01 or 0.001 confidence limits. These include sugar cane > fresh sweet potato tuber > locust beans
> ripe cashew > fresh lettuce leaves > showers o f gold The extent o f overlap, however, varied 
with each material.
Some overlaps also emerged between the chemoreception niches o f  adult and juvenile snails in 
terms o f arrestant responses. Thus five o f the unprocessed materials were observed to act as 
significant arrestants for both adult and juvenile B. truncatus snails, although the extent o f  these 
arrestant effects varied . These include sugar cane > fresh lettuce leaves > showers o f  gold > 
sweet potato > ripe cashew.
These results lend credence to the findings o f other workers that there were intra and inter- specific 
overlaps in the chemoreception niches of pulmonate snails in their response towards various naturally 
occurring materials (Agudogo, 1992; Ansa, 1994; Dogbey, 1995; Kpikpi,1990).
6.2.2 PROCESSED MATERIALS :
The purpose o f processing materials for the bioassay experiments was to alter the form and texture 
of the materials with a view to enhancing their bioactive properties
Results from this investigation indicate that juvenile B. truncatus respond to a much wider range o f 
processed naturally occurring materials than the adults with some important overlaps. Thus it 
was found that four o f the 23 processed materials significantly attracted both adult and juvenile B. 
truncatus snails at either p< 0.05, 0.01 or 0.001 confidence limits. These include 1 day fermented 
sweet potato tuber > 3 days fermented sweet potato tuber > 1 day fermented cassava root tuber > 
3 days fermented cocoyam tuber (Appendices 4-6) No overlaps were observed between the
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chemoreception niches o f  adults and juveniles o f  these bulinid snails in terms o f their arrestant 
responses to processed materials. In fact the adults were significantly arrested by only 3 processed 
materials : oven-dried sweet potato leaves > sun-dried cocoyam leaves > 1 day fermented cocoyam 
tuber.
The juvenile snails were significantly attracted and arrested by 9 o f  the processed materials all o f 
which happened to be fermented to various extents. These include 7days fermented cocoyam tuber
> 7 days fermented cassava root tuber > 3 days fermented cocoyam and cassava tubers > 7 days 
fermented sweet potato > 3 days fermented cassava leaves > 1 day fermented sweet potato> 3 days 
fermented cocoyam tuber.
It appears that juvenile B. trimcatus snails in the present experiment were more responsive to 
fermented materials than the adults. It is possible that the fermentation process significantly 
altered the texture o f  these materials, and more importantly, shifted the hydrolysis equilibrium 
reactions from polysaccharides such as starches towards oligosaccharides like maltose, maltotriose 
and the formation o f  some carboxylic acids. Planorbid snails have been shown to be significantly 
responsive to these molecules (Thomas, 1986; Kpikpi, 1990; Thomas and Assefa, 1978 ; etc.).
As pointed out by Thomas and Assefa (1979), intraspecific differences in chemoreception niches 
have been evolved to reduce the intensity o f  intraspecific competition for food and other target 
molecules among these snails . Thus in the natural environment food preferences o f the juveniles 
B. trimcatus could be expected to vary from those o f the adults to some extent. Although 
considerable work has been done on the search for significant specific bioactive natural 
materials for use as attractants, arrestants and phagostimulants, there is the need to investigate the
110
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active ingredients responsible for the bioactive effects so far identified. It is also important to study 
the phagostimulation properties o f these materials . The value o f identifying the functional 
molecules in these materials lies in the fact that it would help to narrow down the search to a few 
related natural materials that are likely to contain them. Also if a material is identified as a 
phagostimulant for the snail, it is also likely to  be an attractant and arrestant.
6.3. EVALUATION OF EFFICACY OF BIOACTIVE MATERIAL 
UNDER SIMULATED NATURAL ENVIRONMENTAL CONDITIONS.
As pointed out in chapter 5, this experiment was carried out with the aim o f screening further the 
bioactive materials identified from the bioassay tests. There were two important choices involved 
in this evaluation . These were the choice o f  bioactive materials and the method to be used.
6.3.1 CHOICE OF BIOAC.T1VE MA IE  RIALS:
The characterisation o f the chemoreception profile o f  B. truncatus led to the identification o f  some 
natural plant materials as attractants and arrestants for these snails. Ideally, the most potent o f 
these factors should be selected for the SNC tests A desirable feature o f  such materials should 
be that o f di-bioactivity. Thus materials that acted both as attractants and arrestants should be 
preferred to those which had only a single significant effect on the snails. However, two other 
requirements in addition to the potency o f  the material influenced the final choice o f  materials 
selected for further evaluation. These were cost and availability o f  the test material in the area where 
the disease occurs.
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6.3.2 CHOICE OF TRAPPING DEVICE:
Two main requirements need to be met in the design o f the trapping units The need for the system 
to be able to hold the test material and release them at a slow rate such that some o f the active 
factors could still remain in the traps at the end o f the test period. Secondly, there was the need 
for a rigid framework in which the snails could be trapped (Kpikpi, 1990).
Alternatively, the cost o f the materials also needs consideration Thus discarded plastic containers 
were used for the trapping units . Being non-biodegradable, traps made o f  this material are more 
likely to be used over a longer period than those from plant materials such as bamboo .
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CONCLUSION:
From the bioassay studies using diffusion olfactometers, the conclusions can be drawn that 
Adult B. trimcatus snails exhibit wider chemoreception niche towards unprocessed naturally 
occurring plant materials than the juveniles .
Juvenile B. trimcatus snails exhibit wider chemoreception niche towards processed forms o f these 
materials than the adults.
Response o f  juvenile snails towards fermented materials is more profound than those o f the 
adults.
There were some overlaps in responses o f  adult and juvenile snails towards both processed and 
unprocessed forms o f some bioactive materials except in their arrestant responses towards processed 
materials.
Sugar cane emerged as the most potent, all round bioactive natural material for both adult and 
juvenile B. trimcatus. Ripe mango and 7 days fermented cocoyam tuber were the strongest bioactive 
natural products for adult and juvenile conspecifics respectively.
Processed bioactive materials seem to offer more promising prospects for use as attractants and 
arrestants than unprocessed forms.
In the SNC experiments to test the efficacy o f the 12 selected bioactive materials using trapping 
units made of plastic containers, sugarcane and 7 days fermented cocoyam tuber caught the highest 
number of snails. Sugarcane, however, generated a much higher trapping index value and therefore 
emerged as the most efficient bioactive material.
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Thus sugar cane followed by 7 days fermented cocoyam tubers are most likely to be efficient when 
used as baits in the field.
I 14
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Appendix I UNPROCESSED BIOACTIVE MATERIALS WITH SIGNIFICAN I 
CHEMORECEP TION EFFECTS ON RUUNUS TRUNCATUS. ___________
M E A N  R E S P O N S E ____l]N D IC E S -----------
A n  U L T S . I I I  V R N I L  E S
T E S T  M A T E R IA L Attract, inti. Arrest, ind. Attract, ind. Arrest, ind.
FRUITS:
Anacardium occidental is 5 2 *** 2.4** 3.6*** 1.9**
Ripe Mangifera indica -j 2*** - 1.6 0.9
Citnilus vulgaris g 7*** 6 .8 *** 0.7 2 . 0
Citrus a want ifolia -3.9@ -0.5 -7.9@@@ -2 .8 @@
Synsepalum dulcilicuni 4  g*** 1.6 3.0 3.6*
Phoenix dactylifera 1 -j *** 5 Q*** 1.9 1.8
Theobroma cacao : pod - 0 .2 - -4.9(a)(0)(a)
Beans 3.2** 1.5 0 . 6
Duranta plumieri 3.8* 2 g*** ^ Q***  ̂ 2 ***
Musa paradisiaca 3.0* 0 . 6 0 .6 0 .2
Calotropis procera 3.9* 3.6* -2 .6 @ -2.3@
Azadirachta indica -4.9@ -3.2@@@ -5.0® -3.6@@
Parlaa clappertoniana g 2 *** 0 .2 ^ 0*** 2 .2
ChrysophyHum albicuni -5.2@@ 0.1 -8 .2 @@@ - 1 .0
LEAVES:
Xanthosoma maffafa  ̂ ^*** 4 1.4 1.1
Latuca saliva 4.6*** 4.6** ? J.* *
Carica papaya ^ y*** 3.5 1.7
115
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M F. A N U F S P O N S F ,  I N D I C E S
A D U L T S J T I V F N I L  E S
TEST MATERIAL Attract, ind. Arrest, ind. Attract, ind. Arrest, ind.
STEMS:
Zingiber officinale -7.2@@@ -4.7@@@ -9.3(a}(«}(a> ■2 . 2  @®
Saccharum officinarum 8 5 *** 5 0*** 1 0 .0 *** ^ 'J**.*
Ipomea batatas  ̂ Q*** 2  9 ** 6.3*** 2  7 **
ROOTS:
Manihot esculentus 5 2 *** 3 2 *** -2.9 0.1
j * ** *** statistically significant response indices at 0.05, 0.01 and 0 .0 0 1  probabilities 
respectively.
2 . @@@ = statistically significant repellent indices at 0.05, 0 .0 1  and 0 .0 0 1
probabilities respectively.
1 16
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TEST MATERIAL
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TEST MATERIAL
118
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Appendix 4 PROCESSED BIOACTIVE MATERIALS WITH SIGNIFICANT  
CHEMORECEPTION EFFECTS ON RIJLINUS TRUNCATUS.______________
TEST MATERIALS lv r rA iv  P F S P O N S F  INDICES----
ADULTS JUVENILES
Attract, ind. Arrest, ind. Attract, ind. Arrest., ind.
FRUITS:
Sun-dried Thevetia nerifolici 3.1* - 2.3 -
LEAVES:
Oven-dried Azadirachta indica -3.2® -3.3@@ -2 . 0 -0 .1
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3days ferm Xanthosoma maffafa 1 .6 1.1 3.6** 2.3**
Oven-dried Manihot esculentus -2.4® -2 . 2 0 .0 0 . 0
STEMS:
Oven-dried Zingiber officinale -4.2@@ -2 .6 @@ -2 .1 -3.7@@@
Oven-dried Ipomea batatas 2 .8 ** 2 .1* 1.3 0 . 6
1 day ferm. Ipomea batatas 5.0** 1.8 6.5** 2  4**
3 days ferm. Ipomea batatas 3.5** 0 .8 5.1** 1.9*
7 days ferm. Ipomea batatas 2.4 -0.4 5 7*** 4.0***
1 day ferm. Xanthosoma maffafa 3.8* 3.6** 2 . 8 0.3
3days ferm Xanthosoma maffafa 3.4* -0.5 g 2 ***
7days fermXanthosoma maffafa 2 . 0 -0.3 9  7 *** 9  5 ***
ROOTS:
1 day ferm. Manihot esculentus 4.6** 1 .0 4  y** 0.4
3 day ferm. Manihot esculentus - 1 .8 - 1.8  ̂ 3 ** 4.3**
7 days ferm. Manihot esculentus 0.7 -0 .6 8.5***
* ** *** _ statistcally significant response indices at 0.05, 0.01 and 0.001 probabilities
respectively.
@> @@> @@@ = significant repellents at 0.05, 0.01 and 0.001 probabilities respectively.
119
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TEST MATERIAL
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121
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Appendix 7:
PLATE 5.2.3.
TRAPPING DEVICE USED FOR SNC EXPERIMENT
122
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Appendix 8 .
PLATE 5.2. 4
EXPERIMENT SETUP FOR SNC TESTS. THE TRAPPING 
DEVICES ARE SHOWN IN THE WATER.
123
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