QL430.5 P48 B 14 bit hr C l G362299 University of Ghana http://ugspace.ug.edu.gh STUDIES ON THE USE OF BIOACTIVE MATERIALS IN MOLLUSCAN TRAPS FOR THE CONTROL OF SCHISTOSOME HOST SNAILS BY ISAAC ANLM BAIDOO B.Sc. (Hoas), (Legon) A THESIS SUBMITTED TO THE UNIVERSITY OF GHANA IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF PHILOSOPHY IN ZOOLOGY (APPLIED PARASITOLOGY OPTION) DEPARTMENT OF ZOOLOGY UNIVERSITY OF GHANA LEGON, GHANA. JULY, 1999 University of Ghana http://ugspace.ug.edu.gh Acknowledgement - ......................................... ............................ .......................................... .— 1 Dedication --------------------------- ii Declaration......................................................... ii i List o f F igures----------------------- iv List o f Plates .................................... v List o f T ab les----------------------------------------------------------------------------------------------------- vi Abstract --------------------------------- vii CHAPTER ONE: GENERAL INTRODUCTION ........................- --------- 1 1.1 Schistosom iasis............................................. 1 1.2 Schistosomiasis in Ghana and A frica ------------ 3 1.3 Biology o f the schistosome parasite ------------ 9 1.4 The schistosome intermediate h o s t------------------------------------------------------------------- 10 1.5 Effects o f schistosom iasis----------------------------------------------------------------------------- 12 1.6 Control o f schistosom iasis----------- 13 1.6.1 Chemotherapy-----------------------------------------------------------------------------------------14 1.6.2 Snail host contro l --------------------------------------------------------------------------- 16 1.6.2.1 Environmental control o f the snail h o s t------------------------------------------------------- 16 1.6.2.2 Biological con tro l----------------- 17 1.6.2.2.1 Microbial pathogens, predators, and parasites------------------------------------ 18 1 .6 .2 .2 . 2 Inter-molluscan competition------------------- ----------- -----------------------------2 0 1.6.2.3 The use o f molluscicides ----- 22 1.7 The objectives o f the present study —------------------------------------ 23 CHAPTER TWO: Designing an effective sugarcane trapping unit — -------- 25 2.1 INTRODUCTION -........... ----------- .—25 2.2 MATERIALS AND METHODS - ----------------------------------- -26 2.2.1 Snail breeding —------- ----------------------------------------------------------------------------- 26 2.2.2 Preparation o f aquarium ---------------------------------- ----- ----------------------------------- 27 2.2.3 Preparation of trap s---------------------------------- 28 2.2.3.1 Trap 1 Single units of sugarcane---------------------------------------------------------------- 28 2.2.3.2 Trap 2 Sugarcane peels mat tra p --------------------------------------------------------------- 28 Table o f Contents: University of Ghana http://ugspace.ug.edu.gh 2.2.3.3 Trap 3 Sugarcane grid with p o le s ------- -------------------------------------28 2.2.3.4 Trap 4 Sugarcane grid with ca labash-------------------------------------------------------- 31 2.2.4 The simulated natural conditions experim ents--------------------------- ------------------ 32 2.3 RESULTS -------------------------------------------------------------------------------------------34 2.3.1 Efficacy o f the tra p s ...................... -...................................... 34 2.3.2 Efficiency o f the different designs o f trap s----------------------------------------- 35 2.3.3 Aquarium conditions------------------------------------------------------------------------------- 35 2.4 DISCUSSION------------------------------------------------------------ 48 CHAPTER THREE: Efficacy o f bioactive materials in simulated natural environment -52 3.1 INTRODUCTION------------------------------------------------------------ 52 3.2 MATERIALS AND METHODS — ...........— ---------------------- 53 3.2.1 Snail breeding----------------------------------------------------------------------------------------- 53 3.2.2 Preparation o f test m ateria ls---------------------------------------------------------------------- 53 3.2.3 T rap s----------------------------------------------------------------------------------------------------- 54 3.2.4 Aquarium ----------------------------------------------------- 54 3.2.5 Simulated natural environment experim ents------------------------------------------------ 54 3.3 RESULTS------------------------ --------------------------------------------------------- ---------------55 3.3.1 Efficacy o f test materials in trap s--------------------------------------------------------------- 55 3.3.1.1 Cocoyam (7 days ferm ented)------------------------------------------------------------ 55 3.3.1.2 Cocoyam (1 day ferm ented) --------------------------------- 56 3.3.1.3 Cassava (7 days fermented)----------------------------------------------------------------------- 56 3.3.1.4 Cassava (1 day fermented)------------------------------------------------------------------------56 3.3.1.5 Sweet potato (1 day ferm ented)---------------------------------------- 5 7 3.3.1.6 Sweet potato (7 days ferm ented)------------------------------------------------- 57 3.3.2 Efficiency o f test materials ---------------- -------------------------- ------ ------------------ 3.3.3 Aquarium conditions—------------------------------------------- 5 g 3.4 DISCUSSION--------------------------------------------------- 7 4 CHAPTER FOUR: A combinaation o f bioactive substances and a toxicant -------------78 4.1 INTRODUCTION---------------------------------------------------------------------------------------- - 4.2 MATERIALS AND METHODS----------------------------------------------------------------7 9 University of Ghana http://ugspace.ug.edu.gh 4.2.1 Snail breed ing---------------- ' } 4.2.2 Preparation o f test materials for bioassay te s ts ---------------------------------------------- 80 4.2.3 Diffusion or gradient o lfactometers------------------------------------------------- --------— K1 4.2.4 Bioassay experim ents-------------------------------------------------------------------- ----------- 81 4.3 RESULTS.............................................. — ----------- 87 4.4 DISCUSSION--------------------------------------------- 87 4.5 Simulated natural environment te s ts ..................................— .......... --------- 89 4.5.1 Preparation o f test materials/ traps for S.N.E. experim ents-----------------------------89 4.5.2 The simulated natural environment experim ents--------------------------- 90 4.6 RESULTS - ---------------- 93 4.7 DISCUSSION--------------------------- 102 CHAPTER FIVE: A field evaluation o f the most effective trapping unit and attractant- toxicant combination.................................................. .................................................................104 5.1 INTRODUCTION .............. ------ 104 5.2 The study a reas ................................... ............................................................ .......................105 5.3 MATERIALS AND METHODS - ..................................-................................................ 109 5.3.1 The field experiments----------------------------------------------------------------------------- 109 5.3.1.1 Snail sampling —------------ 109 5.3.1.2 Efficacy o f bioactive substances------------ ------------------------------------------------- 110 5.3.1.3 Efficacy o f attractant-toxicant combination -------------- ------------------------- -------1 10 5.4 RESULTS----------------- ---------- 110 5.4.1 The efficacy o f bioactive substances------------- ----- ----------------- ----------------- 1 10 5.4.2 Efficacy o f attractant-toxicant combination ----------------------------------------- 111 5.5 DISCUSSION--------------------- 126 5.5.1 The efficacy o f bioactive substances---------------- 126 5.5.2 The efficacy o f attractant-toxicant combination -------- 127 CHAPTER SIX: General discussion .............— -------- 130 6.0 Summary o f findings ........... --------- -130 6.1 Summary o f the results on trap design ----------------------------------------------------------- 130 6 . 2 Summary of the results on test o f bioactive substances under simulated natural environment ---------------- ------- ---------------------------------------------------------------------] 32 University of Ghana http://ugspace.ug.edu.gh 6.3 Summary o f the results on combination o f attractant (sugarcane) and toxicant (bayluscide)........................................ -.......... -........................ 133 6.4 Summary o f the results on field evaluation -................-------- 136 6.5 CONCLUSIONS.................................................... -------- 138 6 . 6 RECOMMENDATIONS................................... -................— ------ ------------ ----- —-1 3 9 REFERENCES....................................................... ---------------- 141 University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENT In undertaking these studies, some people have been o f immense help to me in diverse ways, and I deem it expedient to record my sincere gratitude to them. I will begin by expressing my sincere thanks to the Head o f the Department o f Zoology, Dr. David Wilson, Prof. J.K.M. Hodasi who was fomerly the co-ordinator o f the Applied Parasitology programme o f the Department, all the lecturers in the Department and Dr. K.M. Bosompem o f NMIMR (Parasitology unit) for the vital roles that they have played in my postgraduate training. I am particularly indebted to my able supervisor, Dr. J.E.K. Kpikpi. His patience, expert guidance,useful suggestions and unfailing co-operation are what have helped me to complete this work. My association with his entire family has also helped to increase my Christian faith I owe them a great debt o f gratitude for this spiritual growth. I am grateful to the Danish Bilharziasis Laboratory(Denmark) for donating a generous grant which catered for purchases o f all the equipment as well as all other materials needed for the studies. I especially wish to express my sincere thanks to my co­ supervisor, Dr. Henry Madsen (D.B.L. Denmark) who donated an important chemical (Bayluscide) which was used in the studies. I also extend sincere thanks to Mr. Mark Tetteh, a driver and a member o f the ‘City o f God Church’, Accra, who regularly drove me to the study site, and to Master Prince Avomyo who assisted me tremendously on the field. The companionship, encouragement, prayers and fruitful discussions with my course-mates (Ebenezer and George), as well as my colleagues Messrs. George Wiafe (Oceanography Department) and Patrick Ayeh-Kumi (University o f Ghana Medical School), Legon are greatly acknowledged. Finally, to my wife Josephine for her support, my mother Mrs. Salome Baidoo and my father Mr. T.A. Baidoo o f blessed memory for their love, guidance and care given me in my entire education. University of Ghana http://ugspace.ug.edu.gh DEDICATION This work is dedicated to God Almighty for His divine protection and guidance, and to my wife Josephine, my mother Mrs. Salome Adobea Baidoo, and also, in memory o f my late father, Mr. T.A Baidoo who passed away just when this research work was started. ‘Everything that happens in this world happens at the time God chooses. He sets the time for birth and the time for death, the time for planting and the time for pulling up, the time for killing and the time for healing, the time for tearing down and the time for building, He sets the time for sorrow and the time for joy. ’ - Ecclesiastes 3 :1-4 University of Ghana http://ugspace.ug.edu.gh DECLARATION I do hereby declare that except for the references to other people’s work which I have duly acknowledged, this work is a result o f my own original research, and this thesis, either in whole, or in the part has not been presented for another degree elsewhere. (2£ Isaac Anim Baidoo (Author) Dr. J.E.K. Kpikpi (Supervisor) University of Ghana http://ugspace.ug.edu.gh List o f Figures Figure 2 a .............................................. ---------------------------------------------------------------------- Figure 2b ----------------- 43 Figure 2 c ----------------- “ 44 Figure 2 d ----------------------------------------------- 45 Figure 2 e ----------------------------------------------------------------------------------------------------------46 Figure 2 f ----------------------------------------------------------------------------------------------------------- 47 Figure 3a & 3 b — ............................................... ....... .....................................................— 62 Figure 3c & 3 d .................................................................................. -........................................— 63 Figure 3e & 3 f --------------------------------------------------------------------------------------------------- 64 Figure 3 g --------------------------------------- 65 Figure 3h & 31 ..................................................................................-.............................................6 6 Figure 3j & 3 k ......................................................... —67 Figure 31......................... — ............................................... - 6 8 Figure 3m --------------------------------------------------------------------- — ..................... — 69 Figure 3n(i) & 3n(ii)-------------- 70 Figure 3 (o ) .............................................--------------- 71 Figure 3p(i) & 3p (ii)------------------------------------ 72 Figure 3 q ----------------------------------------------------------------------------------------------------------73 Figure 4 a ------------------------------------- 8 6 Figure 4 b ----------------------------------------------------------------------------------------------------------9 7 Figure 4 c .............. ------- 98 Figure 4d -............................................ — --------------- 9 9 Figure 4 e --------------------------- 100 Figure 4 f ........................................ 101 Figure 5 a .......................... -........................................... -108 Figure 5b & 5 c -------------------------------------------------------------------------------------------------118 Figure 5d & 5 e -------------------------------------------------------------------------------------------------1 1 9 Figure 5 f .....................................- ................. ------- 1 20 Figure 5 g ------------------------------ ---------------------------------------------- ------------------------- 121 I V University of Ghana http://ugspace.ug.edu.gh List o f Plates University of Ghana http://ugspace.ug.edu.gh List o f Tables Table 2.1 — .................................. -................................................................................................. 3 7 Table 2 .2 .................................... “ 38 Table 2 .3 ......................................... ------------------- — 39 Table 2 .4 .................................. --------- 40 Table 2 .5 ----------------------------------- -.....................................................................— 41 Table 3 .1 ------------------------------------------- 59 Table 3 .2 ---------------------------------------- 60 Table 3 .3 ......................................................................... ...........................................................-— 61 Table 4a -------------- 85 Table 4 b ---------------------------------------------------------------------------- -----------------------------94 Table 4 c ...................... .................................................................................................................— 95 Table 4 d ................................. 96 Table 5a(i) & ( i i ) ------------------------------------------- -................................112 Table 5b(i) & ( i i ) ....................... .......................................................................................... -— 113 Table 5 c ------------------------------------------------------------ ---------------------------------------------114 Table 5 d ..................................................... -............................... ................... ................... ............115 Table 5 e ................................... —......................................................... 116 Table 5 f ------------------------------------------------------------------------- -...................117 Table 6 .1 ................................................. 131 Table 6 .2 ................................... 135 Table 6 .3 .......................................................................................—...................... 1 37 VI University of Ghana http://ugspace.ug.edu.gh Abstract As an aid to the control o f the schistosome host snails, effective bioactive materials namely cassava, cocoyam, and sweet potato (Domeh, 1998), sugarcane and peels, all in their raw and processed states were tested in simulated natural environment experiments. The experiments were conducted using ‘biopots’ traps designed using a pot with few small windows created on the sides to allow easy diffusion o f test materials to reach the water body to attract the snails It was observed that the materials retained their effectiveness for both Bulinus truncatus and Biomphalaria pfeifferi snails in the following order: cocoyam (1 day fermented) > sweet potato (7 days fermented) > cassava (7 days fermented) > cassava (1 day fermented) > sweet potato (1 day fermented) The effectiveness o f one o f the top attractants for the snails (i.e. sugarcane) was found to be unaffected by the addition o f small quantities o f the toxicant bayluscide (< 1 0 0 .^1, 300(j.l, 500|o.l, and 700|j.l o f 0.6ppm bayluscide per 6.495gm o f sugarcane) when tested under laboratory conditions. Higher quantities, however, reduced the attractant effects o f the bioactive material. Similar findings were obtained when the tests were conducted under field conditions (i.e. < 1,424.5(4.1, 4,274.5^1, 7 ,124.5j4l 0.6ppm bayluscide per 92.579gm o f sugarcane (an attractant) with bayluscide (a toxicant) in the newly developed ‘biopots traps’. The duration o f the traps is a factor to be considered. The details o f the experiments leading to these discoveries and the results are presented in this thesis. vu University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE GENERAL INTRODUCTION 1.1 Schistosomiasis The parasite worm which causes schistosomiasis, also known as bilharzia or bilharziasis was first discovered by German pathologist Dr.Theodor Bilharz while conducting an autopsy in a Cairo hospital (Appleton. 1996). There are now 76 countries in which schistosomiasis is endemic, with more Lhan 600 million people at risk o f infection and some 200 million infected (WHO TDR programme report, 1989). The disease is widely distributed in Africa, Caribean Islands,Latin America, the Eastern Mediterranean, S.E. Asia, and parts o f the Western Pacific region. Schistosomiasis is particularly associated with water development projects such as dams and irrigation schemes because the parasite's intermediate host snails breed in freshwater lakes and streams. In Egypt and the Sudan, where large irrigation systems have existed for many years, schistosomiasis ranks as a public health problem o f the first order (Liese, 1986). After an epidemiological survey, Khallaayoune et al (1995), pointed out that in Morocco, the extensive irrigation networks have contributed to the creation of suitable habitats for the intermediate hosts o f schistosomiasis. They also explained that transmission o f the disease was not restricted to irrigated areas alone but also occurred in natural foci such as temporary swamps (merja), marshy plains and residual water in pre- Saharan palm groves in the south o f the country. The causative agents are trematode flatworm (flukes) o f the genus Schistosoma, transmitted from fresh-water infected snails. The disease has proved very difficult to control. The World Health Organisation has done much to stimulate research into the basic problems related to the control of i University of Ghana http://ugspace.ug.edu.gh schistosomiasis, and now there have been improvement in the efficacy o f drugs available for treating people suffering from the disease. Liese (1986) reported that through UN/WHO sponsored projects, praziquantel which is now the most favored drug for treating schistosomiasis, originally costing US$4 per treatment has been reduced to roughly US$0.32 per treatment. Praziquantel has proved very effective in schistosomiasis control in China, where the drug has been used in the treatment o f more than one million cases (Fu et al, 1988). The drug is now being used by many countries including Ghana to control schistosomiasis. It must be emphasized however, that, snails control, health education and provision o f pipe-borne waters are also very important components o f the control strategy. An integrated approach to control schistosomiasis is thus needed to avoid the risk o f re-infection. To effectively control schistosomiasis the disease needs to be given a top priority in national health policies just as malaria. Akogun (1991), explained that in Nigeria although schistosomiasis is the second most prevalent parasitic disease, the control o f other diseases such as river blindness, sleeping sickness and malaria is given a more serious consideration. Schistosomiasis remains a serious problem in tropical countries although new effective antischistosomal drugs have been produced for treatment, l'he problem can be attributed, at least partly to the reinfection o f treated individuals living in endemic areas as they continue to have contact with infected water bodies during their day to day activities. Chimbari et al (1995). also pointed out that, under normal circumstances not all infected individuals get treated in a community and as such there is always a reservoir through which water bodies can be reinfected. University of Ghana http://ugspace.ug.edu.gh 1.2 Schistosomiasis in Ghana and Africa. The association o f schistosomiasis incidence with development projects in Africa has been documented by many research scientists, (Stephenson, 1947), (W itenberg and Saliternik, 1957), (McCullough, 1965), (Nelson, 1972), (Betterton, 1984), (Liese, 1986), (Madsen et al, 1987). Ouma (1995), stated that the disease was present virtually in all African countries where an estimated 150 million persons are infected. In Africa, dams are constructed for hydro­ electricity and irrigation projects. Unfortunately the creation o f these dams in many countries has provided very suitable environments for the schistosome intermediate host snails. Some o f these occurences that have been reported in some countries are found in. Senegal river basin (Diaw, 1995), irrigation projects all over Morocco (Khallaayoune et al, 1995), Lagdo, Benue Division, Northern province o f Cameroun (Tsafack et al, 1995), irrigated areas in Khartoum state (Hilali et al, 1995). and lake Kariba, which was created to generate hydroelectric power for Zambia and Zimbabwe (Mungomba et al, 1995). In a recent study involving the situation o f potential intermediate hosts o f schistosome parasites in Burkina Faso, it was reported that, o f 496 positive biotopes o f the hosts identified, 40.89% were located in man-made reservoirs, 33.8% in rivers. 19.64% in temporary ponds, 3.44% in irrigation channels and 2.23% in natural lakes (Poda et al. 1997). Public health authorities in such affected African coutries have not taken enough precautions during the onset o f such developmental projects to prevent the spread o f the disease in affected communities. The disease has become endemic in many African countries, and its control has really been a very hard task Nelson (1972), stated thai the Aswan dam in Egypt is an example o f such development in a country where control o f 3 University of Ghana http://ugspace.ug.edu.gh schistosomiasis has been largely ineffective in spite o f a great deal of effort over many years. Appleton (1996), reported that in South Africa, schistosomiasis occurs in most o f Zimbabwe, Swaziland, Mozambique, Northern Province. North west Mpumalanga and Kwazulu-Natal. Witenberg and Saliternik (1957), explained that in Israel, apart from changes in hydrological conditions (for instance creation o f water reservoirs, introduction of wide and complicated irrigation systems, numerous fish ponds), creating habitats for the snails vector, visits by Coptic pilgrims from Egypt, and Egyptian laborers extensively employed in orange groves were important ways of introducing schistosome parasites into Israel. Bilharziasis is endemic throughout most o f the Sudan, and attributed to the development o f the Gezira irrigation scheme (Stephenson, 1947). Schistosomiasis has been found to be endemic in Bomo State (northern Nigeria), due to the construction o f the South Chad irrigation project (Betterton, 1984). A survey conducted in the Federal capital territory in Nigeria (Achillea and Asuma, 1979) on freshwater snails revealed that, on the whole freshwater molluscan fauna was relatively poor. However, there was an indication that schistosomiasis was present at the northwest part o f the capital territory, through the centre towards the southeastern part o f the territory. Bozdech (1972), studied schistosomiasis prevalence in Accra (Ghana) and Kaduna (Nigeria), and reported that the prevalence was higher in Kaduna than in Accra. Schistosomiasis in Ghana has been extensively studied over the years (Papema, 1968, 1965, and 1969), (Odei, 1961, 1965, 1983), (McCullough. 1959, 1962, 1965), (Bozdech, 1973), (Kuma, 1979), (Amankwa et al, 1994). Bilharziasis has been endemic in Ghana for a long time (Odei, 1961). He stated that, the earliest report was in the 1895 A University of Ghana http://ugspace.ug.edu.gh Annual Report o f the Colony o f the Gold Coast, in which it was stated that a patient had been admitted to hospital as a result o f ‘bilharzia haematobia' infection. Paperna (1968, 1969), reported that schistosomiasis was very prevalent among inhabitants o f the local communities at the lower reaches o f the Volta river, in the Eastern region of Ghana, and a few villages o f the inland area o f the Volta Region. He also reported after conducting research among school pupils in Tafo area, Pokuase area and Accra plains. Earlier, Odei (1961) had reported that S. haematobium infection was largely confined to two extensive areas, one in the Central Region o f southern Volta Region, around Ho and Hohoe districts. The prevalence o f schistosomiasis is largely associated with the presence o f aquatic habitats which create congenial atmosphere for the schistosome host snails. In the drier regions o f Ghana the snail habitats are mostly intermittent or seasonal streams, dams, ponds, lagoons, marshes and swamps (Odei, 1965). He explained that, in the secondary forest areas they are mainly swamps, choked and sluggish streams, while in the forest areas they are usually permanent streams and pools. Also, the barraging o f water for fishing, livestock watering and irrigation, may create more snail habitats. It is believed that schistosome parasites existed in some areas of the Volta even long before the dam was constructed. In 1983, Odei reported that the freshwater lagoons in the Osudoku and Battor areas which were associated with the river Volta, and which were filled up during the flood period, were the main sources o f host snails and schistosomiasis transmission in the lower Volta in the pre-Volta dam period. He reported however that, around the same period, that is before Akosombo dam was built across the river Volta in 1964, schistosomiasis was not very common in some communities. This was because conditions prevailing there could not support the University of Ghana http://ugspace.ug.edu.gh establishment of aquatic weeds or snails on the river beds; and that during this period the river Volta did not contain the major transmission foci tor schistosomiasis transmission even though a lot o f fishing activities took place in it. In Ghana urinary and intestinal types of schistosomiasis are both prevalent. McCullough (1965), referring to the prevalence and distribution o f intestinal schistosomiasis in Ghana explained that the disease was confined to Tarkwa and Bogoso in the south-west o f the country, and to a few areas in the north-east around Bawku and at Wiaga near Navrongo. He also stated that the disease could be found at Wa in the northwest and at Nyive and Atikpui villages near Ho in the Volta region. The general prevalence o f the disease can be attributed to the following major factors: (a) creation o f suitable habitats for the snail hosts o f the schistosome parasites, (b) human activities that enhance water contact; this includes occupational, recreational and transport (c) Lack of complete knowledge about the disease among the general public. (d) Lack o f social infrastructure such as latrines and pipe-borne water. A number o f dams that have been established for economic developments such as production o f hydroelectricity and irrigation schemes continue to pose health hazards. There have been reports o f incidence o f schistosomiasis associated with these projects. The Tono irrigation scheme constructed in 1977. in the north-eastern part o f Ghana was an exaample o f an Agricultural developmental programme which resulted in increasing prevalence rate of schistosomiasis in the Kassena Nankana district (Amankwaa et al. 1994). University of Ghana http://ugspace.ug.edu.gh They reported that the Tono scheme area represented an area o f very high endemicity for both urinary schistosomiasis was already endemic in the area prior to the construction o f the scheme, but the prevalence o f intestinal schistosomiasis was related to the irrigated agriculture creating suitable habitats for vector snails. McCullough (1965), suggested that habitats o f Biomphalaria were increasing very fast in Ghana, and that they were man- made. Citing examples o f such instances, he pointed out that near Kintampo, in Ghana, Biomphalaria, together with other molluscs had been found in a series o f burrow-pits bordering a road that was constructed just 10 years ago. Also that, in north-east and north-west o f Ghana, Biomphalaria have become established in dams and fish-ponds, many o f which had been recently constructed. Odei (1983) explained the factors that led to schistosomiasis outbreak after the construction o f both Akosombo and Kpong dams. These dams were established for hydroelectric power. He stated that before the Akosombo dam was constructed, seasonal flooding and scouring o f the bed o f the river Volta by silt-laden flood water prevented establishment o f weeds on the river bed. Also the rate o f flow o f the river prevented the establishment o f host snails in it. In the estuary the influx o f seawater did not favour establishment o f aquatic weeds or snails. He reported that the establishment o f the dam led to the desilting o f the water thereby making it transparent and shallow with a slower rate of flow. This enhanced sunlight penetration and therefore the proliferation of submerged and rooted aquatic macrophytes which the snails mostly associate with. Human activities o f the inhabitants o f the affected communities with the river increased the spread of the disease. The major occupation o f the riparian communities is fishing and clam digging in the river. Some of the inhabitants depend on the river for transport to University of Ghana http://ugspace.ug.edu.gh other villages or communities. Children in such places also enjoy swimming in the water for recreational purpose. The lack o f pipe-borne water in such communities makes them depend on the rivers for water for their domestic use. They also lack social infrastructure such as toilet facilities and therefore continue to contaminate and infest the water bodies with parasites through faecal waste materials and urine when deposited near the water bodies. All these increase the rate o f water contact and for that matter the danger o f getting infected by the schistosome parasites which is found in infested waters. The inhabitants o f some affected communities have not yet received enough education on the mode o f spread o f the disease. In almost all the affected communities along the Volta River, however, a lot o f education by both the ‘Volta Basin Research Project’ (VBRP) and the health sector o f the Akosombo hospital has been going on and together with chemotherapy as well as tocal molluscicidmg, some amount o f progress is being achieved with regards to reduction o f incidence o f the disease. University of Ghana http://ugspace.ug.edu.gh 1.3 Biology of the schistosome parasite The principal schistosomes that affect humans are o f three species. Schistosoma haematobium , Schistosoma mansoni and Schistosoma japonicum. All species have developmental stages in freshwater snail intermediate hosts, from which free-swimming cercariae are shed to penetrate the skin o f the definitive human host when in contact with water. In general, fast-running water is not suitable either for the snail intermediate host or for the cercariae. After successful penetration o f a person’s skin the cercariae transform to schistosomula, which migrate through veins and lymph vessels to the lungs. From there they migrate to the liver, developing into young male and female worms in the portal blood vessels. After 4-6 weeks, mating takes place and the worm-pairs move to their destination, which in urinary schistosomiasis is the vessels o f the bladder and. in the intestinal form, those o f the intestines. Some eggs, produced by the female worm, work their way through the vessel walls and are shed in urine or faeces, completing the life cycle if they reach water, where another free-swimming form (the miracidium). hatched from the eggs, can encounter the snail intermediate host. Many other eggs are retained in the tissues, where they provoke an inflammatory reaction. It is this reaction that is responsible for the disease. The mere presence in the bloodstream of the adult schistosome (which remain joined and continue to shed eggs for several years) does not give rise to a pathological response. However, the degree o f morbidity and the intensity o f infection are determined by the number o f adult worms present in the human host and shedding eggs that ma\ be deposited in organ tissue. o University of Ghana http://ugspace.ug.edu.gh 1.4 The schistosome intermediate host The snails which carry schistosome are members o f the family Planorbidae ( in Africa and South America) or Amnicolidae ( in Asia), and the principal species which act as hosts belong to the following genera in Africa Biomphalaria and Bulinus (aquatic snails); in South America: Biomphalaria (aquatic); in Asia: Oncomelania (amphibious i The bilharzia intermediate host snails occur mostly in permanent water bodies, e.g. rivers, streams, lakes, farm dams, and irrigation canals. Should these dry out. the snails can survive for up to six months by aestivating in the bottom sediments and then emerging when the habitat re-fills. To do this they lower their metabolic rate considerably and, provided the sediments remain aerobic, they survive well. When re-filling occurs, the sediments quickly become anoxic and the aestivating snails resume activity. Being hermaphrodites the snails are capable o f storing sperm from earlier copulations, to use it either for cross-fertilisation with a partner or with their own eggs. Both Bulinus and Biomphalaria have a life span o f about 1 ' / 2 years. Under favorable conditions they reach maturity in about 6 weeks but at times they start egg laying as late as in the 4 lh month of their life. Bulinus lay egg clutches appearing as flat oval packets o f 5-20 eggs arranged in one layer, glued together with a yellowish jelly. An individual Bulinus may lay up to 50 egg clutches during its life. Biomphalaria lays on the average 5 similar egg clutches during its lifetime, each containing up to 30 eggs. The snails lay eggs mostly on stems and undersurface o f leaves of living plants. They may also lay eggs on decaying plants, on fallen leaves and tree branches. Bulinus and Biomphalaria are active day and night. They incessantly crawl on the bottom, on plants and on various objects immersed in water, scraping their radula and swallowing the scrapings rich in vegetable and micro­ University of Ghana http://ugspace.ug.edu.gh organisms and organic debris. They also eat all kinds o f rotting wood. The snails are tolerant o f water varying widely in its physical and chemical characteristics but the optimal temperature is about 25°C, conductivity about 300|is/cm and pH 6 -8 . They are not found in water flowing faster than about 0.3m/sec. In their aquatic habitats the host snails are mostly found associated with some particular aquatic macrophytes (Madsen, 1995). Witenberg and Salitemik (1957), reported that in Israel the main host plant of Bulinus and Biomphalaria was the deciduous Potamogeton nodusus. Others include Ceratophyllum demersum, Nitella translucens, Potamogeton pectinatum, Cynodon dactylon and Panicum spp. They explained that these plants provide shelter, aeration, and sites for oviposition. In a study o f aquatic weeds in the Volta lake o f Ghana, Papema (1969) reported that Ceratophyllum and to lesser extent, Pistia and Scirpus supported large populations o f Bulinus truncatus rohlfsi, and that, the number o f snails greatly declined when the weeds disappeared from many sites in the lake. In another stud) involving lake Kariba, in Zambia, Mungomba et al (1995) reported that the presence of vegetation in general was favorable to the intermediate host snails. They noted that the snails were more abundant in patches with vegetation compared to barren ones. Thomas (1995), suggested that because o f the close mutualistic linkages between certain pulmonate snail hosts o f schistosomiasis and macrophytes, bioengineering measures aimed at snail control should include those directed at the macrophytes. University of Ghana http://ugspace.ug.edu.gh 1.5 Effects of schistosomiasis In humans adult S. haematobium lives in the veins and venules draining the kidneys and urinary system while S. mansoni lives in those draining the intestine. The adult worms do not cause damage to tissues but their eggs. The pathology in schistosomiasis is mainly due to the immunological, and histological reaction o f the host’s tissues to parasite eggs retained in them. Acute schistosomiasis is non-specific febrile syndrome associated with temporary itching. There is also marked eosinophilia and often hepatomegaly or splenomegaly. In general however, those infected with schistosomiasis pass into a chronic stage without the acute stage being particularly noticed. In urinary schistosomiasis, calcification o f eggs deposited in the walls of the ureters and bladder causes localised thickenings which obstruct the flow o f urine from the kidneys, a condition called hydro-nephrosis. Other histopathological features o f urinary bilharzia are bladder ulcers and polyposis and there is also a link with bladder cancer. There is also a relationship between heavy infections in children and impaired growth, psychological development and performance at school. The pathological consequences o f S mansoni infection are enlargement o f the liver and spleen, damage to the intestine and hypertension o f the abdominal and oesophageal veins. The early phases o f egg excretion are associated with general symptoms o f weakness, lassitude, giddiness, weight loss and diarrhoea. According to WHO TDR eleventh programme report (1993). anthropological studies indicate that the disease may have significant social impact, such as associated with haematuria in women. 12 University of Ghana http://ugspace.ug.edu.gh 1.6 Control of schistosomiasis In general, there is no single foolproof method for controlling the disease. There are however different methods that contribute in one way or the other towards reduction of schistosomiasis prevalence. The use o f molluscicides and several other biocontrol methods, for instance, helps in killing the snails, thereby reducing the number o f potential intermediate hosts, and for that matter the number o f infective cercariae. Chemotherapy will reduce the number o f worms present in individuals who are infected. Health education and the provision o f pipe-borne water and toilet facilities can also reduce water contact by the people living in communities where the water bodies are located. Morgan (1977), reported that in St. Lucia, when pipe-borne water supplied to five villages in which the disease was highly prevalent, there was reduction o f human contact by 82 per cent and a corresponding significant reduction in prevalence and incidence o f schistosomiasis. Appleton (1996), pointed out that the current opinion is to aim at reducing morbidity rather than aiming at controlling or eliminating the disease, and this can be achieved through an integrated approach, with all the methods mentioned above being used concurrently. Liese (1986) suggested that the public health services (PHS) arm o f a national health system which is generally responsible for epidemiological surveillance as well as implementation o f disease control strategies with a preventive rather than therapeutic effect, needs to be strengthened to help reduce morbidity of schistosomiasis. In Ghana, starting from an epidemiological research programme in 1971-78, the control strategy included focal mollusciciding, provision o f community water supplies and health education, and selective population chemotherapy using metrifonate. 13 University of Ghana http://ugspace.ug.edu.gh 1.6.1 Chemotherapy The development o f new drugs for effective chemotherapy has been one o f the remarkable achievements towards the reduction o f morbidity o f schistosomiasis. Metrifonate was introduced in the early 1960s and is still widely used, although the required 3-dose treatment leads to logistical problems and difficulties o f compliance for the complete course o f treatment, (Liese, 1986). He stated that hycanthone, introduced in 1965, was the first single dose drug, but has since been superceded by oxamnique (in 1973) and praziquantel (in 1977). Both these drugs provide single oral dose chemotherapy with high rate o f cure. It must be pointed out however, that, metrifonate though cheap, is ineffective against Schistosoma mansoni, and in areas endemic for onchocerciasis, it can induce the violent Mazzoti-type reaction which reduces compliance with the therapy. According to WHO (1993) report, treatment with praziquantel resulted in egg clearance o f 60-90% in infected people, and in a reduction in egg loads o f more than 95% among those who remained with patent infection after treatment. In China, treatment with praziquantel at a dosage o f 60mg/kg, divided into three doses (over 1 day) showed a 97.7% success in treatment (Fu et al, 1988). They reported that the side-effects produced by praziquantel treatment were mild and transient in most cases, and depended on the dosage o f drug given. In their research work, out o f 98% of patients who completed the regimes prescribed, 25-50% were free from any adverse effects. Side-effects mainly involved nervous, digestive and cardiovascular systems. 24.1-49.1 complained o f dizziness, 3.5-27.3%, o f headache, and 12.5-23.7%, o f fatigue. Insomnia, muscular tremor, sweating, numbness in limbs and blurred vision were also 14 University of Ghana http://ugspace.ug.edu.gh noted in a few cases. In a WHO (1989) report, a field trial o f schistosomiasis in Cote d ’Ivoire, using praziquantel brought about the temporary cessation o f S. haematobium transmission, although S. mansoni proved more difficult to control. In spite o f the tremendous successes in the use o f praziquantel there has been a report of an unexpected failure in the use o f the drug, in the findings o f a Senegalese-Dutch study (WHO (1992)). The study which was conducted in a village near Richard Toll, a town on the Senegal River, following an outbreak o f schistosomiasis in the region, showed that only 53 o f the 298 (18 percent) showed cure and there were complaints o f severe side-effeets. According to the report, the head o f the research team, Belgian epidemiologist Bruno Gryssels, believed that this unusual result was not due to schistosome resistance to the drug, but attributed it to an extremely rapid reinfection. The main limitation on the use of praziquantel for control is the high re-infection rate in endemic areas, even after mass treatment, as well as the high cost o f the drug (Gryssels and Polderman, 1991). They therefore recommended that repeated treatment be given where necessary. University of Ghana http://ugspace.ug.edu.gh 1.6.2 Snail host control Snail control is one o f the more feasible means o f interrupting parasite transmission in most areas (Ndamba et al, 1995). Until about 1970, snail host control (mainly by mollusciciding) was the primary procedure for schistosomiasis control (WHO, 1984 report). Snail control may be achieved principally through chemical, environmental or biological means (Madsen, 1990, 1992), (Ndamba et al, 1995). In areas o f intense transmission where chemotherapy is being applied, it is usually describe desirable that snail control activities be coordinated with the treatment regime (WHO report, 1984). According to WHO (1993) report on Tropical Diseases Research, snail control has limited transmission o f schistosomiasis in some places , notably in China, but failed to do so everywhere partly because it is expensive and difficult to sustain. 1.6.2.1 Environmental control o f the snail host It has been reported that the environment in which the schistosome intermediate host snails live can be altered in one way or the other to affect their continuous survival, thereby helping to control these snails (Madsen, 1997; Jordan & Webbe. 1982). These methods o f environmental manipulation include increasing the current speed, fluctuating water level in canals or reservoirs (including drying them out completely), possible reconstruction and re-dimensioning o f canals (cement-lining), and removal o f aquatic plants (Madsen, 1997). By application o f engineering measures, tire speed o f water currents can be increased to exceed 0.3m/s, which is normally the limit at which the snails can cope up with, to make the medium unsuitable for their survival. A possible setback of this method may be erosion of canal banks. In small canals it may be possible University of Ghana http://ugspace.ug.edu.gh to fluctuate water level so that for some period, the snails will face dessication and die. Aquatic plants may also be removed by this method. This may however work for only situations where the snails are found primarily along the banks. In situations where submerged aquatic plants are found at great distances from the shore, water level fluctuations may not significantly affect snail populations, since they will still be covered by some amount o f water at all times. Aquatic macrophytes provide surfaces for snails to graze on and on which to deposit their egg masses. They also protect the snails against high current speeds as well as predators. Removal o f aquatic plants will therefore affect snails population. Limitation o f use o f this method may be a possible erosion o f canal banks. Concrete lining o f canals would help in increasing the current speed within the canals. 1.6.2.2 Biological control Although the use o f molluscicides remains the most effective way o f controlling the schistosome intermediate host snails (McCullough, 1986), the high cost involved in its use as well as its non-specificity to targets (WHO, 1984). thus killing fish and other aquatic organisms, together with its impact on the environment (Andrews, Thyssen and Lorke, 1983) suggest that the use o f biological control will be a very useful alternative to control the snail hosts. Biological control employs the use o f living organisms, whether introduced or otherwise manipulated to reduce the density o f the target species (Madsen, 1995). These organisms may be predators, parasites, pathogens or competitors. In its broadest sense, the use o f pheromones, genetic manipulation and fertility control may 17 University of Ghana http://ugspace.ug.edu.gh also be considered as biological control. Several hundred species, ranging from fish to fungi have been considered as potential competitors or predators (including parasites and pathogens), but their efficiency has rarely been tested outside laboratory model systems (McCullough, 1981). Madsen pointed out that it is highly unlikely that one method o f biocontrol can be used to control the schistosome intermediate host snails, under all conditions. He therefore suggested that all the methods should be tested and sometimes combining some o f the methods if possible. Biological control demands regular inspection o f sites, as well as mass production and introduction o f the control agents. As a result of this, it is mostly viewed as labour intensive. 1.6.2.2.1 Microbial pathogens, predators and parasites Pathogens are microorganisms that often kill their host with subsequent liberations o f millions o f individual microbes (Madsen, 1995). In a WHO (1984) report, invading pathogens may cause atrophy o f organs, aplasia, necrosis and tissue liquification, alterations in tissue enzymes and haemolymph components, metaplasia, hyperplasia and neoplasia. Physiological changes in fecundity, growth, locomotion and feeding behavior may also occur in snails infected with particular pathogens. A number of microbial pathogens from freshwater snails have been described. This includes fungal organisms such as Catenaria sp., which invades and destroys egg masses o f Biomphalaria glabrata; protozoa such as Glaucoma paeedopthora which infects eggs and kills the embryo o f Biomphalaria sp. and Bulinus (Physopsis) sp.. Hartmanella quadriparia which affects reproduction and survival during aestivation o f Biomphalaria I X University of Ghana http://ugspace.ug.edu.gh and Bulinus s p and bacteria such as Bacillus pinotti destroys Biomphalaria glabrata. McCullough (1981) explained that virtually all the mentioned studies were laboratory- based, and no successful field trials had yet been achieved. According to WHO (1984) report, the number o f microbial pathogens described from freshwater snails is relatively small, due to limited interest in this field. Predators o f freshwater snails are identified virtually in every major group o f the animal kingdom (Michelson, 1957). Madsen (1995), however stated that most o f these predators are polyphagous, and pointed out that specific snail predators comprise certain cichlid species larvae o f the sciomyzidae species and certain species o f leeches. McMahon et al (1977) reported that the fish Astatoreochromis alluadi had a significant long term effect on snail populations in artificial dams in Kenya. Other malacophagous fish species include Serranochromis sp.. Tilapia melanopleura and Clarias sp. (McCullough, 1981). Appleton (1996) also cited examples o f predators as fish, notably some barbels and cichlids, insects such as the larvae o f the marsh flies (family Sciomyzidae), the Giant waterbugs (family Belostomitidae ) and leeches o f the famih Glossiphiniidae, but pointed out that although they have all been tested, none has been found suitable for field trials. McCullough (1981) expressed that almost all observations on fish/snail host interactions have been carried out in East and Central Africa, but none could be accepted as conclusive, thus the need for much more detailed investigations. The most prominent parasites o f freshwater snails are trematodes (Madsen, 1995). Trematode infections may decrease the rate of replacement o f snail populations by approximately 10-20% (Brown et al, 1988). It has been reported (WHO, 1984). that Angiostrongylus canlonensis, a rat nematode has been proposed as a biological control 10 University of Ghana http://ugspace.ug.edu.gh agent, but unfortunately it also causes serious human disease in the Pacific region and as such its use has not been recommended. According to McCullough (1981), Ribeiroia marini guadeloupensis, a trematode capable o f sterilizing the snail hosts (Biomphalaria glabrata) is one trematode parasite that has been studied in detail by French scientists in Guadeloupe. He mentioned that due to target specificity as well as it being competitively economical, the method was attractive and deserves to be explored. In comparing the efficiency of Ribeiroia guadeloupensis and Malignities tuberculate (competitor) as control agents for biocontrol, Pointier (1989) found out that the trematode was more efficient in the short term. 1.6.2.2.2 Inter-molluscan competition Competitors are organisms which affect the abundance o f the target species through competition for shared resources (Madsen, 1995). McCullough (1981) stated that the attraction o f the method for snails control is based on the principle o f competitive exclusion/displacement, whereby it is believed that if two species are sufficiently similar in their biological profile, then one (the stronger and hopefully, the introduced species) will eliminate the other weaker (the target species or control its population size). The use of competitors has been considered as the most promising canditates for the biological control o f the pulmonate intermediate hosts (Madsen, 1990), (Appleton, 1996). According to Appleton (1996), WHO (1984), considerable success has been achieved, particularly with three South American ampullariids (Marisa cornuarietis, Pumice glace, and P. paludosa) and two thiariids (Malignities tuberculate and the Asian Tarebia 20 University of Ghana http://ugspace.ug.edu.gh granifera). Other pulmonate snails that have been used in biological control include Helisoma duryi , WHO (1984), McCullough (1981), (Madsen, 1992) and Physa spp (McCullough, 1981). Results of a test performed Pointier et al (1989) showed that there was a rapid colonization of Thiara tuberculate a competitor snail, when it was introduced into two groups o f water-cress beds containing Biomphalaria glabrata; the populations of the competitor snails increased appreciably whilst populations o f B. glabrata declined considerably, when sampling was done after 1 year. Approximately a year later (i.e. approximately 2 years after starting the test), B. glabrata had totally disappeared from samples taken. They explained that a similar result was obtained when B. straminea was used in place o f B. glabrata, and following this success programme, T. tuberculate was introduced into all other water-cress cultures in Martinique. According to WHO (1984) report, results o f medical surveys carried out recently in the Island o f Martinique, have all shown that transmission o f intestinal schistosomiasis has been totally interrupted, and explained that the situation correlated with replacement o f B. glabrata by B. straminea. Pointier and McCullough (1989) stated that, there was no evidence that Thiara snails cause any adverse environmental impact. If more research studies are done on the use o f competitor snails for the control o f schistosome intermediate host snails, it may be very good substitute to the use o f molluscicides, which though very effective has it's own limitations. 21 University of Ghana http://ugspace.ug.edu.gh 1.6.2.3 The use of molluscicides In general there are two forms o f molluscicides, namely synthetic molluscicides, and plant molluscicides. However, Appleton (1995) stated that although many plant molluscicides have been screened, only one, Phytolacca dodecandra or Endod has been adequately tested, although it has not even been registered for use in its country o f origin This implies that most o f the molluscicides that have been used successfully in schistosomiasis control programmes are the synthetic type. Not until 1960. two chemicals were widely used as molluscicides. These were NaPCP and CuS04. NaPCP was particularly' Used in Brazil and Japan, whilst CuS04 was particularly used in Africa. These chemicals however had their defects. NaPCP was unstable in the presence of sunlight and also posed immense health hazards to spray men. The use o f C uS04 required that the vegetation o f the area be cleared before it became effective, and also could not destroy snail eggs. In 1960, the first specially formulated molluscicide. Bayluscide (Bayer 73) was produced. At present it is the only molluscicide which is commercially available (Madsen, 1992). Bayluscide is the ethanoalamine salt o f niclosamide, and with specific gravity o f 1, which is the same as water, it disperses readily when sprayed. Apart from the snails it is also toxic to their eggs and the miracidium and cercariae o f schistosome. The chemical has been used in many control programmes. Webbe (1964) reported that Bayluscide application to the Mirongo River produced a very satisfactory degree o f control o f 5. mansoni transmission. Also, in the Rahid irrigation scheme, Sudan, the use o f the chemical in a focal mollusciciding achieved a great success in control. Madsen (1992) pointed out that the effectiveness in the use of molluscicides depends on water velocity, presence o f submerged or floating University of Ghana http://ugspace.ug.edu.gh aquatic macrophytes, and permanence o f habitats. There are thus different mollusciciding strategies. In flowing watercourses such as natural streams, canals and drains in irrigation schemes the recommended methods include spraying, partial treatment, controlled spillage, focal/contact point treatment, dam-and-flush treatment, and drip-feed dispensing. For lentic water bodies such as farm storage dams and pools in rivers, total volume treatment, focal/contact point treatment and slow-release formulations can be used. As a result o f the high cost o f Bayluscide (i.e. both product and operational cost) most developing countries in which the disease needs to be controlled cannot afford the cost o f its use. It is highly desirable that extensive research-be carried out to rely on production o f plant molluscicides which may be o f a lower cost. Alternatively, new methods involving the judicious use, that is, smaller quantities o f Bayluscide with maximum results must be developed. Currently, much emphasis is on the possibility o f using bioactive substances that can act as attractants, arrestants, or phagostimulants in snail control (Thomas and Assefa (1979), Kpikpi and Thomas (1986), Kpikpi et al (1995), Kpikpi and Thomas (1992, Thomas et al (1985). It is envisaged that a toxicant can be incorporated into these attractants and arrestants to form a slow release system, which essentially will make use o f smaller quantities o f Bayluscide as compared to using Bayluscide alone. 1.7 The objectives of the present study: The present study was undertaken with the following objectives: a) designing an effective schistosome host snail trapping unit with sugarcane. University of Ghana http://ugspace.ug.edu.gh b) performing simulated natural environment experiments to determine the efficacy o f some identified bioactive substances namely, fermented cocoyam (fermented Xanthosoma muffafa), fermented cassava (fermented Manihot esculenta) , and fermented sweet potato (fermented Ipomoea batatas), c) finding out the efficacy o f a combination o f bioactive material and a toxicant (bayluscide) in simulated natural environment tests, for snails control, and d) field trials o f (i) best trapping unit identified (ii) most potent bioactive substance, and (iii) combination o f bioactive material and toxicant. University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO DESIGNING AN EFFECTIVE SUGARCANE TRAPPING UNIT 2.1 Introduction Chemoreception studies involving Biomphalaria glahrata (Thomas et al, 1979), Bulinus rohlfsi (Thomas et al, 1985), Bulinus (Physopsis) globosus (Morelet) and Bulinus rohlfsi (Clessin) (Kpikpi, 1991), suggest that schistosome host snails, like other gastropod molluscs are sensitive to chemicals. This property enables them to detect other organisms or targets in the aquatic medium in which they live. Bioassay tests using diffusion olfactometers, (Kpikpi and Ansa, 1994), (Dogbey, 1995), and (Domeh, 1998). showed that some naturally occurring materials, in their raw or processed forms, were capable of attracting schistosome host snails. They showed that some of these substances actually have properties which seem to keep the snails attached to them for a considerable period (that is, arrested). These bioactive substances were categorised as attractants. arrestants and phagostimulants. Attractants are substances which cause snails to spend significantly more time on their sides as compared with their controls. Those which make the snails to spend significantly more time on them, as compared with controls are deemed to have an arrestant effect (Dogbey, 1995). Phagostimulants are those substances which stimulate feeding behaviour. It has been suggested that these bioactive substances with significantly high attractant and arrestant indices for the schistosome host snails can play a major role in selective removal of the schistosome host snails from an infested aquatic body. These substances can be presented in an aquatic medium in the form o f traps to attract or arrest the snails. 25 University of Ghana http://ugspace.ug.edu.gh Sugarcane (Saccharum officinarum), among other bioactive substances tested emerged as the most potent bioactive substance. When presented as chunks (whole) in bamboo cylinders, it attracted more snails (both Bulinus truncatus and Biomphalaria pfeifferi ) than controls in tests conducted in simulated natural environment tests (Dogbey,1995). The main objective o f the present study was to design a more effective trapping unit with sugarcane (Saccharum officinarum) as the bioactive factor. The different forms of presentation in an aquarium in which the tests were conducted were: (i) sugarcane in single units (ii)sugarcane in grids (designed with poles support) (iii)sugarcane in grids (designed with calabash) (iv) sugarcane peels, woven together to form a mat. All the experiments were performed under simulated natural environment using Bulinus truncatus snails. The details o f the findings are discussed in this chapter. 2.2 MATERIALS AND METHODS 2.2.1 Snails breeding Bulinus truncatus snails used were obtained from Weija lake. They were bred in separate glass tanks filled with tap water and kept in the laboratory where they received approximately 12 hours of artificial light and darkness. The average temperature o f the water medium was 26±1°C. The snails were fed regularly with fresh lettuce leaves. The water was changed weekly and replaced with fresh water. Within a period o f 1 month. 1500 healthy snails (both juveniles and adults) could be obtained for the test. 26 University of Ghana http://ugspace.ug.edu.gh 2.2.2 Preparation of aquarium The aquarium, which was situated outside the laboratory where direct sunlight could be obtained during daytime was constructed with concrete. It has a cylindrical shape, with a diameter o f 130 cm and height of 77 cm, giving a total volume o f 2.01 x 106cm3. The bottom o f the aquarium was filled with soil obtained from the beach o f A Weija lake to a height o f 15 cm. It was filled with tap water to about 2/3 full (0.67x10 cm3) and then aquatic plants (Ceratophyllum demersum ) added to cover about 2/3 o f the total surface. Ceratophyllum affords the snails with shelter and points o f attachment. The system was allowed to generate sufficient oxygen through photosynthesis to aerate the water medium. Plate 2a; Aquarium, situated outside the laboratory in the open 27 University of Ghana http://ugspace.ug.edu.gh 2.2.3 Preparation of traps 2.2.3.1 TRAP 1 SINGLE UNITS OF SUGARCANE This trap was designed using single units o f sugarcane and calabash. The test material was prepared by first peeling o ff the sugarcane and then cutting it into smaller pieces at the nodes o f the sugarcane to obtain the single units. A total o f 20 calabashes of similar shapes and sizes were obtained. The sides o f the calabashes were cut to create four small windows. This was to allow diffusion o f the chemical components o f the test material which was fixed on a stick and firmly placed in the calabash, into the water medium. Each o f these calabashes was then fastened to a pole by strings to act as a support and to enable the test material be placed at a desired depth in the water. 2.23.2 TRAP 2 SUGARCANE PEELS MAT TRAP The sugarcane was first cut into pieces at the nodes, and then carefully peeled. The peels were used to prepare the trap.The peels were wov en together by string to form a mat (Plate 2b(i)). The ends o f each peel mat were then fastened to a pole by means o f string (Plate 2b(ii)). This arrangement allowed the test materials and their controls to be well positioned below the surface o f the water. In a similar manner, controls were prepared by using polystyrene. 2.2.3.3 TRAP 3 SUGARCANE GRID WITH POLES The grid is a combination of several units of sugarcane pieces. This was prepared by cutting the sugarcane at the adjacent joints to obtain pieces, measuring 13 cm in University of Ghana http://ugspace.ug.edu.gh (i) (ii) Plate 2b (i) Sugarcane peels mat Trap; (ii) Sugarcane peels mat Trap fastened to wooden pole supports 29 University of Ghana http://ugspace.ug.edu.gh length. The sugarcane pieces were then peeled, after which each piece was split into two halves. Four o f the half pieces were stuck together by pushing a needle shaped piece of wood through them to form a grid (Plate 2c). The grid measured 18 cm in length. 10 grids were made and the ends o f each grid were fastened to wooden supports, shown in Plate 2b(ii), by strings. This was to help position the test materials in the aquarium. The control traps were made o f polystyrene, and were also placed in the aquarium in the same manner. Plate 2c; Sugarcane grid 30 University of Ghana http://ugspace.ug.edu.gh 2.2.3.4 TRAP 4 SUGARCANE GRID WITH CALABASH The sugarcane grid was prepared using the same method described in Trap 2. However, instead of using poles as support, the whole grid was placed inside a calabash which had windows created on the sides. A string was then tied at two ends at the mouth of the calabash so that the calabashes can be hung on a wooden bar placed across the mouth of the aquarium (Plate 2d). Plate 2d; The Calabash Traps 31 University of Ghana http://ugspace.ug.edu.gh 2.2.4 THE SIMULATED NATURAL CONDITIONS EXPERIMENTS All the experiments were conducted in the aquarium in the open (Plate 2a). At the start o f the experiment, 1000 healthy snails o f Bulinus truncatus were released into the aquarium and a period o f 7 days allowed for them to acclimatize to the aquarium condition, before the tests were performed. The experiments were performed for a duration o f 3 days and 3 nights (i.e. 72 hours). Readings were taken every 12 hours from the start o f experiments. All the test materials and controls were placed in the aquarium, completely submerged below the water surface. For traps that had poles as support, the lower ends o f the poles were pushed into the aquarium, while for the traps without poles, they were hung from wooden bars across the mouth of the aquarium. In order to obtain readings from the experiments, the traps were removed one after another and inspected for snails. These were counted and their numbers recorded. In each experiment 10 experimental traps and 10 control traps were used. The temperature o f the aquarium, water conductivity, dissolved oxygen concentration, and the pH of the water were all monitored throughout the experiments. Paired t-tests (Bailey, 1981) was used to determine whether there were significant differences in number o f snails trapped in controls. The trapping index, which refers to the difference in number o f snails trapped on test and snails trapped on controls gave an idea of the effectiveness of the different designs o f traps. 32 University of Ghana http://ugspace.ug.edu.gh Plate 2e; (i) A set up for S.N.C. experiments. Aquarium containing some calabash Traps Plate 2e; (ii) Aquarium containing sugarcane grid with poles Trap 33 University of Ghana http://ugspace.ug.edu.gh 2.3 RESULTS 2.3.1 Efficacy of the traps Four different designs o f trapping units were used in different experiments. In Traps 1 and 4, which involved calabashes in their designs the number o f snails found in the pots as well as those attaching to the outside part o f the calabashes were considered trapped. In Traps 2 and 3, involving the use o f poles, the numbers o f snails that were found attached to the test materials were the ones considered trapped. In addition the snails that were attached to the pole supports and were found in the vicinity o f the test material were considered trapped. This is because they were believed to within the concentration gradient. The results o f all the experiments have been shown in Table 2.1. In Trap 1, the largest number o f snails trapped was after 60 hours, when 40 snails were counted in the test trap. The least number o f snails trapped was 18, observed after 24 hours. After 60 hours however, the number o f snails found in test trap reduced considerably to 28 snails. In sugarcane peels mat trap (i.e. Trap 2) the largest number o f snails caught in the test traps was 30, occurring after 24 hours and 36 hours after which the number declined till the least (13 snails), was observed after 72 hours. In Trap 3 (sugarcane trap, with poles) the number o f snails caught in the test trap increased from 32 snails to 36 snails between 12 hours and 24 hours. However, the number started declining between 36 hours and 72 hours, till the least number recorded (15 snails) was reached between 60 hours and 72 hours. The sugarcane grid trap, (with pots) which seemed to perform best, trapped 81 snails after 72 hours. It revealed somehow a fluctuating trend, in that there was a rise in 34 University of Ghana http://ugspace.ug.edu.gh number of snails between 12 hours and 24 hours, a tall after 36 hours then an increase after 48 hours, a decrease after 60 hours, and finally an increase after 72 hours. The efficacy o f each trapping unit therefore varied within the test period. In Table 2.3, a statistical analysis o f the results using t-test to compare test and control traps confirms the significant differences o f the traps’ performance for the different periods. 2.3.2 Efficiency of the different designs of traps To compare the efficiency o f the different trapping designs the total number of snails trapped by test traps over the 3-day period, for each design was worked out. In a similar manner, the total number o f snails trapped on control traps was also calculated. The difference between results o f tests and controls, as shown in Table 2.1, gives ihe trapping indices for different trap designs. These values are considered as indicators o f efficiency o f the traps. Trap D (i.e. sugarcane grid trap, with pot) recorded trapping index o f 135 snails which was considered as the highest. Trap A (i.e. single units o f sugarcane trap), recorded a trapping index o f 6 6 , which was the least among the four trap designs. Trap B (sugarcane peels mat trap) had trapping index of 84 while Trap C recorded 96. The results can be arranged as follows: Sugarcane grid trap(with pot) > sugarcane grid trap(with poles) > sugarcane peels mat trap > single units o f sugarcane trap. 2.3.3 Aquarium conditions During the experimental periods, an average temperature o f 27.4°C was recorded in the mornings, 30.4°C in the afternoons and 32°C in the evenings. Using a Water 35 University of Ghana http://ugspace.ug.edu.gh Quality Checker (model U-10), an average water conductivity value o f 102 fiSm/cm" was measured. Average dissolved oxygen was 2.68 mgl"1, whilst average salinity measured was 0.01%. The aquarium water had an average pH o f 8.5. 36 University of Ghana http://ugspace.ug.edu.gh Table 2.1 NUMBER OF SNAILS TRAPPED IN SIMULATED NATURAL ENVIRONMENT EXPERIMENTS FOR DIFFERENT TRAP DESIGNS (1) SUGARCANE (Single units) TRAP TIME (Hours) 12 24 36 48 60 72 TEST TRAPS 19 18 21 28 40 28 CONTROLS 15 17 6 19 16 15 TRAPPING INDEX 4 1 15 9 24 13 (2) SUGARCANE PEEL MATS TIME (Hours) 12 24 36 48 60 72 TEST TRAPS 24 30 30 29 19 13 CONTROLS 12 11 4 4 10 10 TRAPPING INDEX 12 19 26 25 9 oJ (3) SUGARCANE GRID TRAP (WITH POLES) TIME (Hours) 12 24 36 48 60 72 ! TEST TRAPS 32 36 30 29 19 13 CONTROLS 12 17 24 1 2 2 TRAPPING INDEX 2 0 19 6 28 17 11 (4) SUGARCANE GRID TRAP (IN POTS) TIME (Hours) 12 24 36 48 60 72 TEST TRAPS 49 51 35 69 23 81 CONTROLS 29 27 16 48 13 40 TRAPPING INDEX 2 0 24 19 21 10 41 37 University of Ghana http://ugspace.ug.edu.gh Table 2.2 COMPARING A TRAP DESIGN OVER A 3-DAY PERIOD (pooled) TEST TRAPS SINGLE UNITS PEEL MATS GRID WITH POLES GRID WITH POTS 154 145 154 308 CONTROLS 88 61 58 173 TRAPPING ’INDEX 6 6 84 96 135 SIGNIFICANCE LEVEL ** * - *** * = P < 0.05; ** = P < 0.01; * * * = P <0.001 38 University of Ghana http://ugspace.ug.edu.gh Table 2.3 TRAPPING INDICES OF THE DIFFERENT TRAP DESIGNS OF SUGARCANE OVER 72 HOURS TIME (Hours) SINGLE UNITS SUGARCANE PEEL MATS GRID TRAP (WITH POLES) GRID TRAP (IN POTS) T C (T.I) (L.S) T C (T.I) (L.S) T C (T.I) (L.S) T C (T.I) (L.S) 12 19 15 4 24 12 12 - 32 12 2 0 *** 49 29 2 0 * 1 24 18 17 1 30 11 19 ** 36 17 19 ** 51 27 24 * 36 21 6 15 *** 30 4 26 *** 35 24 11 ** 35 16 19 * 48 28 19 9 29 4 25 * 21 1 2 0 - * 69 48 21 - 60 40 16 24 * 19 10 9 * 15 2 12 ** 23 13 2 0 * 72 28 15 13 - 13 10 3 - 15 2 13 ** 81 40 41 - T = NUMBER OF SNAILS TRAPPED BY TEST MATERIALS, C = NUMBER OF SNAILS TRAPPED BY CONTROLS, (T.I) = TRAPPING INDEX = T-C, (L.S) = LEVEL OF SIGNIFICANCE, (* = P < 0.05; ** = P < 0 .0 1 ; *** = P < 0.001). 30 University of Ghana http://ugspace.ug.edu.gh Table 2.4 TRAPPING UNITS AND THEIR SUPPORTS DTN Trapping Unit 1 Trapping Unit 2 Trapping Unit 3 Trapping Unit 4 (hours) T C Ts Cs T C Ts Cs T C Ts Cs T C Ts Cs 12 19 15 5 6 24 12 24 32 32 12 18 14 49 29 0 0 24 18 17 5 7 30 11 12 26 36 17 19 28 51 27 0 0 36 21 6 8 6 30 4 27 30 35 24 10 9 35 16 0 0 ' 48 28 19 5 10 29 14 20 23 21 1 13 2 69 48 0 0 60 40 16 6 8 19 10 40 28 15 2 4 2 23 13 0 0 72 28 15 4 8 13 10 24 10 15 2 4 2 81 40 0 0 Total Number of snails 154 88 33 45 145 61 147 149 154 58 68 57 308 173 0 0 trapped in 3 days Trapping unit 1 = Single units o f sugarcane trap, Trapping unit 2 =Sugarcane peel mats, Trapping unit 3 =Sugarcane grid (with poles) trap, Trapping unit 4 =Sugarcane grid (with pots) trap, T = number o f snails trapped on test trap; Ts = number o f snails on supports o f test traps; C =number of snails trapped on controls, Cs = number o f snails on supports o f controls; DTN = Duration. 40 University of Ghana http://ugspace.ug.edu.gh Table 2.5 A comparison o f trapping indices (Domeh, 1998 & the present studies) Total number o f snails caught in 10 traps Domeh (1998) (transparent plastic bottle traps) Present studies T1 T2 T3 T4 Test traps 150 154 145 154 308 Control traps 38 88 61 58 173 Trapping Index 112 6 6 84 96 135 Significance Level * * * ** * - *** * = P < 0.05; ** = P < 0.01; *** = p <0.001 T1 = Single units T2 = Peel mats T3 = grid with poles T4= grid with pots 41 University of Ghana http://ugspace.ug.edu.gh FI GU RE 2a ; EF FI CA CY OF SU G AR CA NE (S IN GL E UN IT S) U ND ER SI M UL AT ED N A TU R A L EN VI R O N M EN T o££ w §LU Oo□ CNJIs- o CD CO CDco CsJ CsJ Q B dd v iJ i s i iv n s d o ygaiAiniM TI ME (H O U R S) University of Ghana http://ugspace.ug.edu.gh FI G UR E 2b ; EF FI CA CY OF SU G AR CA NE PE EL MA TS UN DE R SI M UL AT ED N A TU R A L E N VI R O N M EN T TI M E (H O U R S ) University of Ghana http://ugspace.ug.edu.gh FI GU RE 2c ; EF FI CA CY OF SU G AR CA NE GR ID TR AP UN DE R SI M UL AT ED NA TU RA L EN VI R O N M EN T a a d d v a i s h v n s j o y a a w r iN University of Ghana http://ugspace.ug.edu.gh FI GU RE 2d ; EF FI CA CY OF SU G A RC AN E GR ID IN PO TS UN DE R SI M UL AT ED N A TU R A L E N V IR O N M E N T o cr i- t LU OI— o □ IS University of Ghana http://ugspace.ug.edu.gh FI GU RE 2e ; A CO M PA RI SO N OF TR AP PI NG IN DI CE S (D O M EH , 19 98 ; & PR ES EN T S TU D IE S ) TR A PP IN G IN D E X University of Ghana http://ugspace.ug.edu.gh FI GU RE 2f ; A CO M PA RI SO N OF TR AP PI NG IN DI CE S FO R DI FF ER EN T TR AP D ES IG N S CL - > < < Q D r-~ < < > - > O O O o O o 0 1 o s$ Q LUHzHI tz LU a: UJ co LL- < < > > < < co co CO CO < < o o Qo LU LU \ - f - Zz LU LU cnm LU LU LL LL CO > - > - < < Q O T— P'- O O h - h- < < h - 1—o o CL CL 1— H LU LU LU LU § § CO CO 1 T T C\J o CD CO CDoo ^r CNJ C\J 00v£> X3QNI O N Id dV id l TI M E (H O U R S ) University of Ghana http://ugspace.ug.edu.gh FI GU RE 3m ; A CO M PA RI SO N OF TR AP PI NG IN DI CE S FO R DI FF ER EN T NA TU RA L B IO A C TI VE SU B ST AN CE S -B IO M PH A LA R IA o o LLJ [U z UJ . 2 q; ct w w Q >-< COw< D _ _ o Q LU UJ h - h - z Z LU LLI (Z cn LU LLI LL LL CO > > < < Q Q T— —-■ —' O o \— h - < < H h - O O Q_ Q_ 1— 1— LU LLI LLI LU 5 § (0 if ) X3QNI O N Id d V y i TI M E (H O U R S ) University of Ghana http://ugspace.ug.edu.gh FIGURE 3n (i) A COMPARISON OF TRAPPING IND ICES SHOW ING THE RESPONSE OF BULINUS SNAILS TO 7 DAYS FERMENTED B IOACTIVE MATER IALS TRAPPING INDICES FIGURE 3n (ii) A COMPARISON OF TRAPPING IND ICES SHOW ING THE RESPONSE OF BULINUS SNA ILS TO 1 DAY FERMENTED B IOACTIVE MATERIALS 7 DAYS FERMENTED SWEET POTATO 7 DAYS FERMENTED CASSAVA 7 DAYS FERMENTED COCOYAM 1 DAY FERMENTED SWEET POTATO 1 DAY FERMENTED CASSAVA 1 DAY FERMENTED COCOYAM o 50 100 150 200 250 t r a p p in g in d ic e s 7 0 University of Ghana http://ugspace.ug.edu.gh FI GU RE 3( o) ; A CO M PA RI SO N OF TR AP PI NG IN DI CE S SH OW IN G TH E RE SP O NS E OF B U LI N U S SN AI LS TO SO ME BI O AC TI VE NA TU RA L M A TE R IA LS TR AP PI NG IN D IC E S University of Ghana http://ugspace.ug.edu.gh FIGURE 3p (i) A COMPARISON OF TRAPP ING IND ICES SHOW ING THE RESPONSE OF B IOMPHALAR IA SNA ILS TO 7 DAYS FERMENTED B IOACTIVE MATER IALS 7 DAYS FERMENTED SWEET POTATO 7 DAYS FERMENTED CASSAVA 0 20 40 60 80 100 120 140 TRAPPING INDICES FIGURE 3p (ii) A COMPAR ISON OF TRAPP ING IND ICES SHOW ING THE RESPONSE OF B IOMPHALARIA SNAILS TO 1 DAY FERMENTED BIOACTIVE MATERIALS 1 DAY FERMENTED SWEET POTATO 1 DAY FERMENTED CASSAVA 1 DAY FERMENTED COCOYAM 0 20 40 60 80 100 120 140 160 TRAPPING INDICES 72 University of Ghana http://ugspace.ug.edu.gh FI GU RE 3q ; A CO M PA RI SO N OF TR AP PI NG IN DI CE S SH O W IN G TH E RE SP O NS E O F BI O M PH AL AR IA SN AI LS TO SO M E BI O AC TI VE NA TU RA L M A TE R IA LS TR A PP IN G IN D IC E S University of Ghana http://ugspace.ug.edu.gh The ability o f the fermented natural bioactive materials, namely fermented products o f cocoyam, cassava, and sweet potato to attract or arrest the schistosome host snails has been studied in diffusion bioassays using olfactometers (Domeh, 1998). Results o f the present studies which were conducted under S.N.E. conditions have revealed a concordance with those o f diffusion bioassays. Each o f the natural bioactive •materials tested in traps caught significantly more snails than the control traps (p < 0.05). and cocoyam (1 day fermented) recorded the highest trapping index for both Bulinus truncatus and Biomphalaria pfeifferi snails (Figures 3o & -3q). This suggests that cocoyam (1 day fermented ) was the most efficient bioactive natural material among the others tested. This result was not unexpected, as Domeh (1998) showed that 1 day fermented cocoyam was among the top three attractants identified during his studies. Observations that were made during the present studies showed that 7 days fermented cocoyam became so soft and got disintegrated into pieces, while 1 day fermented cocoyam did not. This might possibly explain why 1 day fermented cocoyam performed more efficiently than 7 days fermented cocoyam. To support this, it can be postulated here that the diffusion gradient that was created in the water medium, with the test material as the source, to attract the snails (Kpikpi, 1990) was disturbed in the case o f the 7 days fermented cocoyam. This is because as the material got disintegrated and floated out o f the traps, there was little or no diffusion gradient maintained for snails to travel on to the traps, hence the low catch o f snails. It was also observed that 7 days fermented sweet potato which recorded the second highest trapping index for Bulinus truncatus (Figure 3 o) did not also disintegrate during 3.4 DISCUSSION 74 University of Ghana http://ugspace.ug.edu.gh the test period. These seem to support the view that test materials are capable o f establishing a more efficient diffusion gradient to attract snails than crushed forms, as suggested by Dogbey (1995). The observations that were made o f the ability o f fermented bioactive natural materials to attract or arrest snails support the hypothesis put forward by Thomas (1996), He suggested that a link exists between fermented natural materials in aquatic ecosystem ‘and the release o f short-chain carboxylic acids (C2-C 5) and other metabolites by bacteria present in the water. He explained that due to the high biochemical oxygen demand o f the bacteria, they resort to anoxic, glycolytic fermentation with the release o f short-chain carboxylic acids (C2 - C5) to obtain their energy. Essentially, these short-chain carboxylic acids are potentially a valuable source o f energy for detritivorous invertebrates such as snails. In addition, hydrolysed starch and maltose present in cocoyam, cassava, and sweet potato could be a valuable food source for the snails (Thomas et al, 1986). Although all the tested materials belong to the same class o f food (i.e. carbohydrates), there may be variations in the proportions in which their chemical constituents are combined. This may possibly affect their rate o f fermentation in water and consequently the rate o f release of carboxylic compounds together with any other chemical compounds into the aquatic medium. This may account for the differences in the performance o f the different materials tested, as represented by their trapping indices (Figure 3q & Figure 3o ). The generally fluctuating trend o f trapping indices (Figure 31 & Figure 3m) recorded over the test period occurred probably because o f variations in the weather (i.e. 75 University of Ghana http://ugspace.ug.edu.gh whether day or night). All the S.N.C. tests were started at a period prior to darkness (i.e. in the late afternoon). The first set o f results (i.e. 12 hours) were periods o f darkness. Generally, the dark periods corresponded with the troughs on the graphs, while the light periods were represented by the crests. The presence o f light probably came with some amount o f heat to slightly increase the temperature of the water, which might have increased the metabolic activity o f the snails. The snails probably might be more active •during the day than in the night. Secondly, an increase in temperature during daylight might have increased fermentation process (Thomas, 1996), leading to an increase in rate of release of short-chain carboxylic acids (C2 - C5) which the snails depend on for energy. This could be the reason o f different trapping indices recorded for the different periods, although the same test material was used. It can therefore be suggested that, in working out a control programme using attractants or arrestants, better results could be achieved when traps are set during the day than in the night. The bioactive materials that were tested also recorded different times o f peak performance, all occurring during ‘day’ periods rather than "night’ periods (Figures 31 & 3m). Cocoyam had a peak performance at 12-24 hours for both snail species. The peak performance recorded for cassava were during 48 hours (for Bulinus truncatus) and 12-24 hours for Biomphalaria pfeifferi. Sweet potato recorded its peak performance at 48-72 hours for both species. These differences in peak performances might have occurred as a result o f the different rates of fermentation in the different test materials. This essentially determined the rates at which the chemical factors in each of the materials will be released into the aquatic medium. 76 University of Ghana http://ugspace.ug.edu.gh There have been several reports on differences in the chemoreception niches of snails that belong to different species (Thomas et al, 1985; Kpikpi & Thomas, 1992). Trapping indices recorded in the present studies are suggestive o f the fact that the response rate o f Bulinus truncatus to each o f the bioactive materials is higher than Biomphalaria. Whilst the highest trapping index in the tests with B. truncatus was 71, the highest for B. pfeifferi was only 37. It can therefore be suggested that B. truncatus snails ' have better preferences for fermented cocoyam, cassava, and sweet potato than B. pfeifferi. These observations may be very useful in both selective snail sampling and snail control. The test materials are recommended for field trials so that they can be combined with a toxicant to form a ‘slow release system’ to be used in schistosome host snails control. These materials are very common in every market in the country, and can be obtained at all seasons within the year. The only point that seems to be a limitation of their use on a large scale scale for snail trapping is the fact that these materials form a major foodstuff consumed by many people in the country. However, studies conducted by Dogbey (1995) have revealed that the quantity o f the bioactive materials does not affect the trapping index. Smaller quantities can therefore be used in the traps for any snail control programme, so that consumption by humans will not be affected. 77 University of Ghana http://ugspace.ug.edu.gh CHAPTER FOUR A COMBINATION OF BIOACTIVE SUBSTANCES AND A TOXICANT 4.1 Introduction One o f the effective means o f interrupting the life cycle o f the schistosome parasite is by controlling the populations of intermediate host snails (Schiff and Evans, 1977) The use of molluscicides, which has been known and used for so many years, remains an •effective means o f controlling snails population in an infested water body. Unfortunately, the setbacks that accompany the method, notably among them being the indiscriminate destruction o f other non-target aquatic organisms has not- changed. All available molluscicides may have adverse effect on fish and may hamper water productivity (Paperna, 1969). Again, although molluscicides have been used extensively in some endemic situations, they may be expensive and the organisation needed to apply them is complex (Pointier and McCullough, 1989). It has been suggested that the modem approach is not necessarily to indulge in the widespread application o f chemical to natural waterbodies but through focal mollusciciding (Schiff and Evans, 1977). Focal mollusciciding deals with only selected sites such as the water contact sites alone. This reduces the quantity o f molluscicides applied to the water body. The disadvantage o f this method however is that it is not very effective if the water body has a relatively high velocity of flow (Meyer-Lassen et al, 1994). Schiff and Evans (1977) pointed out that focal control o f snails in slow moving, or stagnant aquatic bodies by judicious application o f molluscicides is inexpensive and may prevent major outbreaks o f schistosomiasis. Focal control, using slow-release systems may be an effective means o f controlling the snails with minimum hazards posed in the aquatic medium. Earlier attempts in the use o f 78 University of Ghana http://ugspace.ug.edu.gh slow-release systems have not made use o f any bioactive materials. Various slow-release systems that have been used were formed by incorporating molluscicidal chemicals into a rubber or elastomer matrix during manufacture (Schiff and Evans, 1977). Kpikpi (1997) pointed out that currently, much emphasis is on the search for very potent bioactive substances in terms o f being attractant and arrestant with a toxicant incorporated in them to form a slow release system. It is believed that the system, formed with a potent •bioactive substance will attract the snails, and the toxicant in it will kill the snails. The main objective o f the present study is to combine identified bioactive substances and a molluscicidal chemical (Bayluscide) to find out whether (i) the bioactive substances maintain their attractant and arrestant properties, (ii) the snails that are trapped die as a result o f the molluscicide. The first part o f this investigation involved bioassay tests using diffusion olfactometers, whilst the second part was simulated natural environment experiments in an aquarium. 4.2. MATERIALS AND METHODS 4.2.1 Snail b reed ing Bulinus truncatus snails were used for this experiment. Snails were collected from a parent stock and bred in separate glass tanks measuring 66 x 38 x 21 cm, and filled with tap water. These were kept inside the laboratory at a room temperature o f 26±1°C and approximately 12 hours light and 12 hours darkness each day. The snails were routinel\ fed with lettuce. The water in each of the 10 tanks was changed weekly. As eggs were laid by the snails and hatched, the number o f snails in each glass tank increased. The juvenile snails were selected and put into separate tanks for them to grow. In a period o f 79 University of Ghana http://ugspace.ug.edu.gh two months a lot o f snails could be obtained. Healthy adult snails were then selected and used in both the diffusion bioassay tests and the simulated natural environment experiments. 4.2.2 Preparation of test materials for bioassay tests Pieces o f sugar cane that could fit into the cylindrical chambers within the .olfactometers were prepared. Sugar cane was chosen for this experiment because o f its high attractant indices for both Bulinus and Biomphalaria snails (Dogbey, 1995). Secondly, sugar cane has a spongy nature which appears to have the capability o f retaining chemical properties, and to release them slowly. With the aid o f a sharp pointed needle, small holes were created longitudinally through the mid portion o f each piece of sugar cane. Each hole measured about 2/3 o f the full length o f each sugar cane piece. Four different treatments were given to the test materials as follows: (I) sugar cane pieces each with a single hole; (II) sugar cane pieces each with 3 holes; (III) sugar cane pieces each with 5 holes, and (IV) sugar cane pieces each with 7 holes. With the aid o f a micropipette o f capacity, (50-200pl), lOOpl o f 0.6ppm that was prepared in the laboratory from dilution o f a measured quantity o f powdered bayluscide, was carefully poured into each hole. These were allowed to stand for a period so that the chemical will permeate the matrix of the sugar cane to mix well with chemicals o f sugar cane. According to Meyer-Lassen et al (1994), 0.6ppm bayluscide concentration is capable o f killing both Bulinus and Biomphalaria snails in water that is stagnant. 80 University of Ghana http://ugspace.ug.edu.gh 4.2.3 Diffusion or Gradient olfactometers The olfactometers used consisted o f a rectangular central chamber measuring 7.8 by 1.9 by 2.0cm, joined at each end to a cylindrical chamber measuring 2.4cm in diameter and 2.0cm in depth. A perspex block consisting o f 20 olfactometers was used (Plates 4a & 4b). * 4.2.4 BIOASSAY EXPERIMENTS The olfactometers were thoroughly washed with hot water and detergent and rinsed several times with tap water before and after each experiment. Tap water was poured into each chamber at the start o f each experiment. The test materials and controls were placed in the cylindrical chambers, with one test material and one control oppositely placed in each central chamber. A snail was placed in the middle o f each chamber. The position o f each assay snail was noted at 2.5 minute intervals for 30 minutes. Snails that were found on the test side were considered positive, +, and recorded. Snails that were found on the side of the controls were scored negative, - . In some instances the snails were found attached to the tests or controls. These were considered arrested, and were scored as (+), for tests and (-) for controls. The controls were sugar cane pieces without bayluscide. 20 controls and 20 test materials were used. In the first test the test materials had a single hole, and contained 1 OOjj.1 o f 0.6ppm bayluscide. The experiment was performed for three more times with varying quantities o f the bayluscide. After the 30 minute tests, the snails were allowed, in each case to remain in the set up to find out whether they will die after a period, since none o f the snails died in the process of the tests. This was to be sure that the chemical molecules of 81 University of Ghana http://ugspace.ug.edu.gh the toxicant were also diffusing into the surrounding water. The results were analysed to determine the levels o f significance o f any difference in bioactivity by using paired t-test (Bailey, 1981). 82 University of Ghana http://ugspace.ug.edu.gh Plate 4b; A set up for the diffusion bioassay experiments 83 University of Ghana http://ugspace.ug.edu.gh 84 University of Ghana http://ugspace.ug.edu.gh Table 4a; COMBINATION OF SUGAR CANE & TOXICANT (DIFFUSION BIOASSAYS) Statistical analysis using t-test. Test in 20 olfactometers MEAN ATTR. INDEX LEVEL OF SIGNIFICANCE MEAN ARR. INDEX LEVEL OF SIGNIFICANCE Sugar cane + lOOjul of 0.6ppm bayl. (i.e. 1 hole per test material) 1.3579 - 0.1028 - Sugar cane + 300|il of 0.6ppm bayl. (i.e. 3 holes per test material) 0.8992 - 1.6810 - Sugar cane + 500jil of 0.6ppm bayl. (i.e. 5 holes per test material) 1.0771 - 0.0245 - Sugar cane + 700jal of 0.6ppm bayl. (i.e. 7 holes per test material) 2.0269 - 2.5595 - * = P < 0.05, ** = P < 0 .0 1 . *** = P < 0.001 (ATTR. = Attractant; ARR. = Arrestant; bayl. = bayluscide) 85 University of Ghana http://ugspace.ug.edu.gh FI GU RE 4a : EF FI CA CY OF A CO M BI NA TI O N OF AN A TT R A C TA N T (S ug ar ca ne ) AN D A TO X IC A N T X X LU LU Q Q Z z— h- H z Z < < 1— \— o <(J) LU 01CZ 1— a: < < □ CO 10 c\j vO CO in 10 o University of Ghana http://ugspace.ug.edu.gh 4.3 RESULTS Results o f the bioassay tests using diffusion olfactometers indicated that attractant and arrestant properties o f sugar cane were maintained when 0.6ppm o f bayluscide, in various amounts was added. O f the four different tests which were performed, only the fourth test using the largest quantity o f bayluscide (7 x lOOpl) produced a significant change in the arrestant effect o f the sugar cane. The first 3 tests namely, (I) sugar cane with only one hole and lOOpl o f the toxicant, (II) 3 holes and (3 x lOOpl) o f the toxicant, (III) 5 holes and (5 x lOOpl) o f the toxicant, produced no significant differences in attractant and arrestant indices, as can be seen from Table 4a. The fourth test also gave the highest attractant and arrestant indices (Figure 4a). It was discovered that in each of the tests, all the snails died within two hours. 4.4 DISCUSSION The present results have made it possible to identify the extent o f tolerance o f the snails to attractants that have some amounts o f toxicant (0.6 ppm bayluscide) combined with them. In the separate experiments involving additions o f lOOpl, (3 x lOOpl), and (5 x lOOpl), there was no significant difference (p < 0.05) in the time spent by the snails on the side of the test materials, as compared with the time spent on the side o f 'test materials and toxicant’. This implies that addition o f the toxicant factor in various quantities o f lOOpl, (3 x lOOpl), and (5 x 100j.il) per 6.495 gm o f the sugar cane did not alter attractant effects of the sugar cane. However, using (7 x lOOpl) o f the toxicant, there was a difference in the time spent by snails attached to the test material (i.e. arrested) as compared with time spent attached (i.e. arrested) to 'test materials and toxicant' 87 University of Ghana http://ugspace.ug.edu.gh (controls). This difference was stastically significant (p < 0.05). This means that the arrestant effect o f the sugar cane was altered when (7 x 100(_il) o f the toxicant was added to 6.495 gm o f the sugar cane. This suggests that the quantity o f toxicant can have an effect on the bioactive substance. Large quantities o f the toxicant can probably produce a very significant change in attractant and arrestant properties. This may be due to the faci that with large volumes o f the toxicant, its molecules may be encountered by the snails in 'the aquatic medium more frequently than the attractant molecules. The fact that none of the assay snails died in the process, that is within the 30 minutes period o f the experiments indicates that the sucrose molecules o f the sugar cane were probably diffusing out faster than that o f the toxicant into the surrounding water medium to create a diffusion gradient. The snails were attracted to move up the chemical gradient using their chemoreceptors (Kpikpi, 1990). It seems very likely that within the test period (i.e. 30 minutes), not much o f the toxicant molecules were encountered by the assay snails since the molecules probably had not yet diffused into the surrounding water medium. However, it was evident after 2 hours that the molecules o f the toxicant had diffused into the water and the snails were killed. The slow rate o f diffusion of the toxicant molecules was probably due to the position in the sugar cane test pieces at which it was introduced. The toxicant was actually introduced into the middle portion o f the sugar cane test materials, and might have taken some time to pass through the matrix o f sugar cane to reach the outside water environment. University of Ghana http://ugspace.ug.edu.gh 4.5 Simulated Natural Environment Tests 4.5.1 PREPARATION OF TEST MATERIALS/ TRAPS FOR SIMULATED NATURAL ENVIRONMENT EXPERIMENTS. The sugar cane grid trap, already described in the previous chapter was used. Each grid was prepared by joining 4 pieces o f sugar cane that have already been split into two (Plate 4c). Each o f the grids was then fixed inside a calabash trap with windows created on the sides (Plate 4d) after the toxicant had been added. Bayluscide toxicant was used in combination with the test materials. The same concentration (i.e. 0.6ppm of bayluscide) used in the olfactometer diffusion assays was used in varying quantities in separate experiments. The quantities o f the bayluscide needed to be added to each sugar cane grid was calculated by simple proportion, based on the combination used in the olfactometer diffusion tests. This determined the number of holes to be created in each test material. Small holes were then created in the sugar cane pieces by means o f a giant needle. By means o f a micropipette 1 OOjal was measured and carefully emptied into each hole. These test materials were then allowed to stand for a period o f 30 - 60 minutes for the chemical to diffuse into the spongy nature o f the sugar cane. They were then placed in the prepared calabash traps and were ready for use. In the first test, 1424.9(al o f 0.6ppm bayluscide was added to 92.549gm o f sugar cane (i.e. weight o f each sugar cane grid). In the second test the quantity of bayluscide was increased to 4,274.5(j.l (i.e. 3 x amount in the first test). In the third test, 7,124.5(0.1 o f 0.6ppm bayluscide (i.e. 5 x amount in the first test) was used. Sample o f calculation o f bayluscide used- Range o f weights o f sugar cane pieces in olfactometer = 6.501 - 6.489gm 89 University of Ghana http://ugspace.ug.edu.gh Average weight = 6.495gm, this weight corresponded with lOOpl o f 0.6ppm bayluscide (i.e. fo r 1 hole). Range o f weights o f sugar cane grids fo r S.N.E. experiments = 89.099 - 95.999gm Average weight = 92.549gm. Thus, quantity o f bayluscide needed fo r 92.549gm o f sugar cane = 92.549/6.495 x 100 = 1,424.9pi, and this implies that 3-4 holes be created in each o f the 4 sugar cane pieces form ing the whole grid. Following similar calculations it was realised that, fo r the second test a total o f 4,274.5p i o f bayluscide will be added to the sugar cane grid, which means that 10-11 holes be created in each o f the 4 pieces form ing a grid. Also, in the third experiment 7,124.5 p i o f bayluscide was to be added to each sugar cane grid, which means that 17-18 holes be created in each o f the 4 pieces. 4.5.2 THE SUMULATED NATURAL ENVIRONMENT EXPERIMENTS 10 test traps and 10 control traps were set up in the aquarium. Each trap was placed in the aquarium water, slightly submerged and suspended from a wooden bar placed at the mouth o f the aquarium (Plate 4f). The control traps were similar sugar cane grid traps without the toxicant. All the test traps were arranged on one half o f the aquarium whilst the control traps were also placed on another half. One week prior to the beginning o f the experiments, 800 adult snails were selected and introduced into the aquarium to get them acclimatised to the aquarium conditions. They were fed routinely with lettuce leaves, but were denied o f food 24 hours before each experiment. The traps were inspected every 12 hours. At each inspection, the traps were removed from the aquarium individually and the snails that were found inside the traps, as well as snails attached to the outside part o f the calabashes were considered trapped. They were counted and recorded. Suspected killed snails in each trap were also counted; and to be very sure the snails were dead they were removed and placed in water to find out whether they will recover. Dead snails were also identified by change o f colour o f the snails or inability to bubble in 5% potassium hydroxide (Okafor, 1990). Three different sets of 90 University of Ghana http://ugspace.ug.edu.gh experiments were performed in the aquarium separately, with varying quantities o f 0.6ppm bayluscide combined with the sugar cane. 91 University of Ghana http://ugspace.ug.edu.gh Plate 4e; Traps ready for use Plate 4f; Traps placed in aquarium, suspended by met i strings tied to wooden bars at mouth of the aquarium 92 University of Ghana http://ugspace.ug.edu.gh 4.6 Results The numbers o f snails trapped after every 12 hours for each o f the 3 different experiments have been depicted in Figures 4b, 4c, and 4d. Table 4c shows the total number o f snails trapped and recorded over a 3-day period. The amount o f the toxicant was varied in the 3 different experiments to find out its lethal effect on the snails as they were attracted by the attractant. It can be observed that the test, involving the use o f sugar xane (92.579gm) and 7,124.5(^1 o f the toxicant (i.e. 0.6ppm bayluscide) recorded the highest number o f snails trapped (i.e. 231 snails). Out o f this, 15 snails forming 6.44% of the total snails were killed. This was followed by the test involving sugar cane (92.579gm) and 1,424^1 of the toxicant. A total o f 211 snails were trapped, out o f which none was killed. In the third test, in which sugar cane and 4,274.5fil o f the toxicant was used, a total o f 196 snails was recorded as snails trapped. Out o f this number. 16 snails forming 8.33% were killed. The control traps which were made up o f the same quantity of sugar cane (i.e. 92.579gm) but no toxicant added, in similar traps revealed a trend of results that was similar to those of the test traps. Observations that were made revealed that none of the snails died in any of the control traps. A comparison o f the number of snails trapped in test traps and those o f the control traps shows that there was not much difference in the snails trapped (Figure 4f). A statistical analysis (Table 4d) suggests that with the exception o f the test involving the use of 1,424jal o f bayluscide, in which results after 24 hours and 60 hours gave a significant difference (p < 0.05), all the other tests showed no significant difference in number o f snails trapped in test traps as compared with control traps. 93 University of Ghana http://ugspace.ug.edu.gh Table 4b; PERCENTAGE OF SNAILS KILLED BY COMBINATION OF SUGAR CANE (92.579gm) & VARIOUS AMOUNTS OF 0.6ppm BAYLUSCIDE IN SIMULATED NATURAL ENVIRONMENT EXPERIMENTS ____________________ TEST DURATION (HOURS) T Tk K% C Ck K% SUGAR CANE 12 24 0 0 29 0 0 24 39 0 0 24 0 0 (92.579gm) & 36 29 0 0 37 0 0 0.6ppm .BAYLUSCIDE 48 35 0 0 36 0 0 60 46 0 0 38 0 0 (1,424.9^1) 72 38 0 0 45 0 0 SUGAR CANE 12 34 11 32.4 27 0 0 (92.579gm) & 24 19 5 26.2 23 0 0 0.6ppm 36 34 0 0 39 0 0 BAYLUSCIDE 48 29 0 0 35 0 0 (4,274.5)^1) 60 42 0 0 39 0 0 72 38 0 0 38 0 0 SUGAR CANE 12 50 14 28.0 50 0 0 (92.579gm) & 24 40 1 2.50 37 0 0 0.6ppm 36 25 0 0 37 0 0 BAYLUSCIDE 48 21 0 0 28 0 0 (7,124.5j_il) 60 45 0 0 49 0 0 72 52 0 0 62 0 0 T = Total number of snails trapped in test traps; Tk = Total number o f snails found killed in test traps; K% = percentage o f snails killed; C = Total number o f snails trapped in control traps; Ck = Total number o f snails found killed in control traps. 94 University of Ghana http://ugspace.ug.edu.gh Table 4c; PERCENTAGE OF SNAILS KILLED BY COMBINATION OF SUGAR CANE & VARIOUS AMOUNTS OF BAYLUSCIDE IN 3 DAYS OF SIMULATED NATURAL ENVIRONMENT EXPERIMENTS. DURATION TEST/TREATMENT T Tk K% C Ck K% T.I. 3 DAYS Sugar cane (92.579gm) & l,424|il o f 0.6ppm bayluscide 211 0 0 209 0 0 2 3 DAYS Sugar cane (95.579gm) & 4,274^1 o f 0.6ppm bayluscide 196 16 8.33 201 0 0 5 3 DAYS Sugar cane (92.579gm) & 7,124.5|j.l o f 0.6ppm bayluscide 231 15 6.44 263 0 0 32 T = Total number o f snails trapped in test traps; Tk = Total number o f snails found killed in test traps; K% = percentage o f snails killed; C = Total number o f snails trapped in control traps; Ck = Total number o f snails found killed in control traps; T.I. = Trapping index. * = P < 0.05; ** = p <0.01; *** = P<0.001 95 University of Ghana http://ugspace.ug.edu.gh Table 4d; NUMBER OF SNAILS TRAPPED IN A SIMULATED NATURAL ENVIRONMENT EXPERIMENTS INVOLVING A COMBINATION OF AN ATTRACTANT & A TOXICANT. DURATION TRAPPING LEVEL OF TEST (HOURS) INDEX SIGNIFICANCE (1) Sugar cane (92.549gm) & 12 -5 - 1,424^1 o f 0.6ppm o f bayluscide 24 15 * 36 -8 - 48 1 - 60 12 * 72 -7 - (2) Sugar cane (92.549gm) & 12 7 ~ - 4,274.5^.1 of 0.6ppm o f bayluscide 24 -4 - 36 -5 - 48 -6 - 60 3 - 72 0 - (3) Sugar cane (92.549gm) & 12 0 - 7,124.5(il) o f 0.6ppm bayluscide 24 3 - 36 - 1 2 - 48 -7 - 60 -4 - 72 - 1 0 - * = P < 0.05; ** = P < 0.01; *** = P<0 .001 96 University of Ghana http://ugspace.ug.edu.gh FI GU RE 4b ; EF FI CA CY OF CO M BI N A TI O N OF 92 .5 49 gm SU GA R CA NE & 14 24 .9 ul O F 0.6 pp m BA YL US CI DE IN S. N .E . EX PE R IM EN T o DC fc |LU O I— O □ □ CMr^ - o CD 00 r-~ CDco CM c\j Q 3d d v y i s h iv n s j o y3aiAini\i TI ME (H O U R S ) University of Ghana http://ugspace.ug.edu.gh FI GU RE 4c ; EF FI CA CY OF CO M BI N AT IO N OF 92 .5 49 gm SU GA R CA NE & 4, 27 4. 5u l BA YL US CI DE IN S. N .E . E X P E R IM E N T ocr J Ij 6 4 4 *** 48 11 26 37 1 2 3 3 4 * * * 60 5 18 23 -» 6 9 14** 72 7 17 24 4 4 8 16** BUL - Bulinus truncatus; BIOM. = Biomphalaria pfeifferv, Trp = Trapping * = P < 0.05; ** = P < 0.01; *** = P < 0.001. 113 University of Ghana http://ugspace.ug.edu.gh Table 5c; TABLE SHOWING SOME PARAMETERS MEASURED DURING PRELIMINARY SURVEY Quadrat Avge water Depth/cm PH Conductivity (ms/cm) Avge Temp.(°C) Avge Oxy. Conc. (mg/1) Total number o f snails counted BUL BIOM 1 30.2 6.1 0.3421 28.7 2.3 6 9 2 28.6 5.8 0.4105 29.2 3.4 4 5 3 22.5 5.8 0.4212 30.1 -3.8 4 3 4 24.0 5.9 0.3853 30.3 3.9 10 10 5 24.2 6.0 0.3287 30.1 4.1 6 13 Avge = average; temp. = temperature; oxy. = oxygen; conc. = concentration; BUL = Bulinus truncatus\ BIOM= Biomphalariapfeifferi. Quadrats 1, 4, and 5 were water contact sites. University of Ghana http://ugspace.ug.edu.gh Table 5d; TOTAL NUMBER OF SNAILS TRAPPED AT THE TWO CONTRASTING MICRO-ENVIRONMENTS BY USE OF THE TWO BIOACTIVE SUBSTANCES. EXPERIMENT TOTAL NUMBER OF SNAILS TRAPPED IN 3 DAYS TEST CONTROL BUL BIOM TOTAL BUL BIOM TOTAL EFFICACY OF 1 DAY FERMENTED COCO YAM JIN LESS POPULATED AREA) 79 52 131 27 38 65 EFFICACY OF 1 DAY FERMENTED COCOYAM (IN DENSE POPULATION AREA) 161 143 304 77 61 138 EFFICACY OF 7 DAYS FERMENTED SWEET POTATO (LESS DENSE POPULATION AREA) 25 21 46 3 9 12 EFFICACY OF 7 DAYS) FERMENTED SWEET POTATO (IN DENSE POPULATION AREA) 46 137 183 13 22 35 BUL - Bulinus truncatus; BIOM = Biomphalaria pfeifferi. 115 University of Ghana http://ugspace.ug.edu.gh Table 5e; EFFICACY OF SUGAR CANE-BAYLUSCIDE COMBINATION AS AN ATTRACTANT/ ARRESTANT KILLER Dur (hrs) NUMBER OF SNAILS TRAPPED NUMBER OF SNAILS KILLED PERCENTAGE OF SNAILS KILLED (%) Test Control Test Control Test ControlBu Bi T Bu Bi T Bu Bi T Bu Bi T 12 16 25 41 30 14 44 6 8 14 0 0 0 34.39 0.00 24 16 10 26 15 6 21 4 2 6 0 0 0 23.07 0.00 36 21 7 28 15 10 25 0 0 0 0 0 0 0.00 0.00 48 15 4 19 6 14 20 0 0 0 0 0 0 0.00 0.00 60 17 6 23 6 12 18 0 0 0 0 0 o ' 0.00 0.00 72 12 7 19 14 4 18 0 0 0 0 0 0 0.00 0.00 Bu = Bulinus truncatus; Bi = Biomphalaria pfeifferi; T = total o f both snails; Dur = duration University of Ghana http://ugspace.ug.edu.gh Table 5f; Statistical analysis to determine whether combination o f toxicant has a significant effect on attractant/ arrestant potency o f sugar cane. Duration (hours) Number o f snails in test traps Number o f snails in control traps Trapping Index 12 41 44 -3 24 26 21 5 36 28 25 3 48 19 20 -1 60 23 18 5 72 19 18 1 * = P <0.05; ** = p < 0.01; *** = P<0~.001 117 University of Ghana http://ugspace.ug.edu.gh NU M BE R OF SN AI LS TR AP PE D IN 20 TR AP S | TO TA L NU M BE R OF SN AI LS TR AP PE D IN 20 TR A PS F I G U R E 5 b ; E F F I C A C Y O F 1 D A Y F E R M E N T E D C O C O Y A M ( I N L E S S D E N S E P O P U L A T I O N A R E A ) BULINUS BIOMPHALARIA TOTAL TRUNCATUS PFEIFFERI □ TEST QCONTROL| FIGURE 5c; EFFICACY OF 1 DAY FERMENTED COCOYAM (IN DENSE POPULATION AREA) BULINUS BIOMPHALARIA TOTAL TRUNCATUS PFEIFFERI 118 University of Ghana http://ugspace.ug.edu.gh F I G U R E 5 d ; E F F I C A C Y O F 7 D A Y S F E R M E N T E D S W E E T P O T A T O ( I N L E S S D E N S E P O P U L A T I O N A R E A ) BULINUS BIOMPHALARIA TOTAL TRUNCATUS PFEIFFERI FIGURE 5e; EFFICACY OF 7 DAYS FERMENTED SWEET POTATO (IN A DENSE POPULATION AREA) □ TE ' □ CO1 ROL □ TES □CONTROL < q: Q LU£L Q.nN University of Ghana http://ugspace.ug.edu.gh FI GU RE 5g ; KI LL IN G EF FE CT OF SU GA R CA NE - BA YL U SC ID E C O M B IN A TI O N AT W E IJ A s i i v n s j o aaaiA iriN TI M E (H O U R S ) University of Ghana http://ugspace.ug.edu.gh (i) Canoes for fishing docked at a water contact site ( i i ) A m arke t scene at a w a te r c o n ta c t s ite 122 University of Ghana http://ugspace.ug.edu.gh (iii) Fish traders waiting to buy fish Plate 5a; Some o f the activities that take place at the water contact sites at Weija lake 1 2 3 University of Ghana http://ugspace.ug.edu.gh Plates jo oc 3c, a section ot the shore o f Weija lake showing some o f the traps 1 2 4 University of Ghana http://ugspace.ug.edu.gh Plate 5d; Student setting snail traps at Weija lake Plate 5e; A section showing the east bank o f the Weija lake 1 2 5 University of Ghana http://ugspace.ug.edu.gh The present results show that significantly more snails were caught in test traps than in control traps, when both fermented raw cocoyam and sweet potato were tested in the Weija lake. These results revealed a concordance with results obtained in simulated natural environment experiments using the same test materials and trap design (Chapter 3), and results o f diffusion bioassay experiments (Domeh, 1998). Similar observations were made by Dogbey (1995) when he tested the efficacy, o f some bioactive materials under S.N.E. and also in the field. Bioactive substances that were used created a diffusion gradient around them with the substance as the source when placed in the aquatic medium, and the snails were attracted along this gradient and subsequently trapped (Kpikpi, 1990). Response to chemical substances in general by the schistosome host snails has been extensively studied (Thomas & Assefa, 1979; Thomas et al, 1985; Thomas et al, 1983; Kpikpi & Thomas. 1992). The present results showed that the test traps were effective in both dense population area, and less dense population area of the snails. For each o f the two bioactive substances tested the test traps caught more snails than the control traps. This was in agreement with results obtained in the simulated natural environment experiments. However, whilst the values o f trapping indices for the tests in the dense population area were almost all statistically significant (p < 0 .0 1 ). about half o f the trapping indices recorded for the area of less dense population were not significant (p > 0.05) (Table 5a & 5b). This seems to suggest that the traps performed better in a dense population area than in a less dense population area. 5.5 DISCUSSION 5.5.1 THE EFFICACY OF BIOACTIVE SUBSTANCES 126 University of Ghana http://ugspace.ug.edu.gh Again, results from the studies indicated that there was no remarkable change in the attractant potency o f the bioactive materials over the 3-day period. This was in agreement with results o f S.N.E. experiments. In the test involving the use o f 1 day fermented cocoyam (conducted in dense population area o f snails) the largest number o f snails trapped was recorded even after 72 hours (3 days) (Table 5a, ii). '5.5.2 THE EFFICACY OF ATTRACTANT-TOXICANT COMBINATION In the second aspect o f the present studies in which sugar cane was combined with a toxicant to assess its efficacy in attracting snails as well as killing them, results show that there was no statistically significant difference (p > 0.05) in number o f snails caught in test traps as compared with control traps. These results are in agreement with those of simulated natural environment experiments using the same bioactive materials and trap design (Chapter 4). This might probably be due to the strong attraction o f sugar cane for the snails. This seemed to be strong enough to supersede negative effects of bayluscide (0.6 ppm). The lake water might have been able to reach into the tiny spaces in the sugar cane where the chemical was poured and might have diluted its concentration with time, such that its potency to kill snails had reduced. This might explain why only a small percentage o f the snails that were trapped were killed (12 hour and 24 hour periods only) with none killed in the subsequent periods. The use o f calabash for the trap design was very suitable in terms o f its ability to hold the test materials in the aquatic medium and to release the chemical slowly (partly through the windows created on the sides) into its immediate environment. Secondly, it has a m University of Ghana http://ugspace.ug.edu.gh wide aperture to allow snails to enter, as well as a rigid framework such that snails can be trapped inside it. Weija lake was considered suitable and chosen for the present studies because o f the history o f schistosomiasis in the surrounding communities as well as the availability o f schistosome host snails in the water, which was found to be true during the preliminary study. The sites chosen for traps location were influenced by the availability o f aquatic vegetation, notably Ceratophyllum demersum with which the snails associate most (Papema, 1969; Odei, 1983; Madsen et al, 1987). Preliminary studies involving snails sampling revealed that the snails population at the water contact sites was higher than the non-contact sites (Table 5c). Water contact sites are mainly sites where people wash clothes or utensils, collect water for domestic purposes, bathe or swim. In a study carried out on snails survey in the Niger basin, Madsen et al (1987) observed a close link between water contact sites and occurrence o f the schistosome host snails. They remarked that human water contact activities create favourable biotopes for the snails, for example by increasing the food resources o f the habitat. This important observation at the water contact sites should be kept in mind when setting up a snail control programme. Results o f the present studies indicate that it is possible to remove schistosome host snails selectively by using traps with bioactive materials as baits. When sugar cane was combined with 0 .6 ppm bayluscide, it was discovered that the quantity o f the toxicant at the given concentration did not have much effect on the attractant /arrestant potency, but only few snails were killed for a limited period o f 24 hours. It may be very useful to remove the traps after every 24 hours to add some more o f the toxicant. This 128 University of Ghana http://ugspace.ug.edu.gh means a daily inspection o f traps and application o f more toxicant. The advantage in using the described trapping unit is that it is easily affordable locally, and its application, that is the setting o f traps along the banks o f the lake is already a common practice by the fishermen and the people living in the communities around infested water bodies. Sweet potato and cocoyam are foodstuffs that are very common in Ghana. The use o f the traps can therefore be patronised easily. University of Ghana http://ugspace.ug.edu.gh CHAPTER SIX GENERAL DISCUSSION 6.0 Summary of findings The present results show that calabash which can be easily obtained in some villages in Ghana, at a low cost can be used to design a trapping system to catch schistosome host snails using bioactive materials as baits. Kpikpi (1990) designed a similar trap using bamboo which was also used by Dogbey (1995) in both simulated natural environment experiments and also in field tests on the Weija lake. From these studies, it was shown that cocoyam, cassava, and sweet potato, all in their raw fermented states attracted more snails than their controls. These naturally occurring materials have therefore been documented as bioactive materials or known to contain bioactive factors for these snails It was also found that limited quantities (in the range o f 1,424^1 - 7,124.5(^1) o f 0.6ppm bayluscide did not reduce the attractant effects o f sugar cane; on the contrary snails were still attracted and killed in the traps over a 24-hour period. In the present chapter an attempt will be made to summarise the findings o f the overall studies and to point out how this will contribute to solving the problem associaied with the extensive application o f toxicants in water bodies to control the snails. 6.1 Summary of the results on trap design The main objective o f this test was to design an effective trapping unit to trap the schistosome host snail. Sugar cane was selected for this test because o f its outstanding potency as an attractant/arrestant material (Dogbey. 1995). The traps were as follows: n o University of Ghana http://ugspace.ug.edu.gh (i) single units o f sugar cane trap, (ii) sugar cane peels mat trap, (iii) sugar cane grid trap-with calabash. Results obtained from simulated natural environment experiments involving each o f these traps revealed a significant difference (p < 0.05) between tests and controls, with regard to the number o f snails trapped. The highest total number o f snails trapped over the 3 days period o f test (308 snails) was when the sugar cane grid trap (with calabash) was used as shown in Table 6.1. The design o f the calabash trap was * such that it somehow formed an enclosure with few small windows created on the sides. This design might have contributed to the trapping efficiency o f the unit, since snails that were attracted or arrested could be prevented to some extent from moving away easily. In other traps there were no such enclosures to prevent the snails from moving away easily. That might have accounted for the observed results as shown in the table. Secondly, the diffusion gradient created by the sugar cane in the water medium (Kpikpi, 1990) could have dispersed more easily in the traps without enclosures than the one in which the sugar cane was partially enclosed as was done with the calabash trap. Essentially, it was along the diffusion gradient that the snails were attracted. Table 6.1 Total number o f snails trapped over 3 days in different trap designs in simulated natural environment experiments & their trapping indices. ________________ Trap types A B C D Total number of snails trapped in 3 days 154 145 154 308 Trapping Index 6 6 ** 84* 96* 1 3 5 *** * = P < 0.05; ** = P < 0.01; * * * = p <0.001 University of Ghana http://ugspace.ug.edu.gh A = single units o f sugar cane trap B = sugar cane peels mat trap C = sugar cane grid trap (with poles) D = sugar cane grid trap (with calabash). 6.2 Summary of the results on test of bioactive substances under simulated natural environment. The results from the simulated natural environment experiments revealed that all the *test materials, raw cocoyam, cassava, and sweet potato (fermented for 1 day and 7 days) when used in the calabash traps caught more snails than the control traps. The difference was found to be statistically significant (P < 0.05) for each o f the materials. This revealed a concordance with diffusion bioassay studies conducted by Domeh (1998) using the above mentioned test materials. It was discovered however that, in experiments where cassava or cocoyam (fermented for 7 days) was used the number o f snails trapped reduced after 48 hours. After 48 hours it was observed that the test materials had become so soft in water that they easily got disintegrated and fell into the aquarium water in bits. Since each o f these bits was capable of attracting snails, the snails somehow eventually got scattered along different diffusion gradients set up by the scattered bits o f bioactive material. This might have accounted for the low numbers o f snails caught in the test traps after 48 hours. Results showed that sweet potato ( 7 days fermented) did not become as soft as cassava or cocoyam fermented for 7 days did. As a result o f this, more snails were still attracted /arrested and caught by the test traps after 48 hours. In increasing order o f effectiveness o f these bioactive materials to attract or arrest the snails, cassava (7 days fermented) < sweet potato (1 day n? University of Ghana http://ugspace.ug.edu.gh fermented) < cassava (1 day fermented) < sweet potato (7 days fermented) < cocoyam (1 day fermented). In general, more Bulinus truncatus snails were trapped than Biomphalaria pfeifferi. This difference was probably due to the differences in their chemoreception niches, as already reported in some previous studies (Thomas et al, 1979; Thomas & Assefa, 1978; Kpikpi, 1991; and Kpikpi & Thomas, 1993). The two different species o f snails had different response rates to the bioactive materials. 6.3 Summary of the results on combination of attractant (sugar cane) and toxicant (bayluscide) Results o f the first part o f these studies which involved diffusion bioassay tests in olfactometers revealed that the schistosome host snails could still be attracted by sugar cane when limited amounts o f 0.6 ppm bayluscide was combined. There was no significant difference in attractant and arrestant indices (p > 0.05) for sugar cane- bayluscide combination used as test, as compared with sugar cane alone used as control for almost all the four different experiments conducted. It was however observed that as the quantity o f bayluscide used to combine with the sugar cane increased to 700|il, there was a difference in attractant/ arrestant index. This was statistically significant (p < 0.05). This suggested that the quantity o f the toxicant combined to the sugar cane to achieve success in attracting the snails could be critical. However, the fact that none o f the snails died within the 40 minutes test period until 2 hours later after the test when left in the olfactometers showed that the sugar cane molecules probably diffused into the surrounding water medium taster than the molecules o f the toxicant was introduced. University of Ghana http://ugspace.ug.edu.gh Similar results were obtained when the same studies were conducted under simulated natural environment experiments. This formed the second part o f the studies. In the S.N.E. experiments the test and control materials were placed in the calabash traps. There was no significant difference in number of snails caught (p > 0.05) in test traps as compared with those caught in control traps. This suggested that the attractant/ arrestant potency o f sugar cane did not change when limited amounts o f 0 . 6 ppm bayluscide was added. With regard to the killing effect o f the sugar cane- bayluscide combination, it was discovered that proportions o f snails killed out o f the number trapped were not very high (Table 6.2) and occurred only during 12 and 24 hour periods.-Results show that in all, 8.33% of the snails trapped were killed in the second experiment when 4,274.5|il o f the bayluscide was combined with the sugar cane. Likewise, 6.44% o f the snails trapped were killed in the third experiment when 7,124.5|al o f the bayluscide was used. None of the snails died in experiment 1, when l,424.9pl o f the bayluscide was combined with the sugar cane. These observations suggest that the quantity o f bayluscide to be used effectively could be critical. This was in agreement with results o f the diffusion bioassay experiments. The low percentages o f snails killed could be attributed to the dilution of the toxicant that might have occurred in the aquarium water. For the same reason it could be that the concentration of the bayluscide after 24 hours became so low that it could not kill the trapped snails. Removal of the test traps after every 24 hours to add some more bayluscide might be highly recommendable. I'M University of Ghana http://ugspace.ug.edu.gh Table 6.2 Percentage o f snails killed in S.N.E. experiments involving the use o f sugar cane-bayluscide combination. Experiment Total number of snails trapped (test) Number o f snails killed (test) Percentage killed (%) (test) Total number o f snails trapped (control) Number o f snails killed (control) Percentage killed (%) (control) 92.579 gm sugar cane & l,424.9(il of 0 .6ppm bayluscide 211 0 0 209 0 0 92.579 gm sugar cane & 4,274.5^1 of 0 .6ppm bayluscide 196 16 8.33 201 0 0 92.579 gm sugar cane & 7,124.5^.1 of 0 .6ppm bayluscide 231 15 6.44 263 0 0 University of Ghana http://ugspace.ug.edu.gh 6.4 Summary of the results on field evaluation The field work which was conducted at Weija lake near Accra, produced results that were in agreement with results obtained at the laboratory. The best trapping design identified previously in S.N.E. experiments in the laboratory together with the two top bioactive materials, also identified in a similar manner in the laboratory were tested on the field. The choice o f the sites for the situation o f the test traps was influenced by the presence o f aquatic weeds, such as Ceratophyllum demersum with which the snails are known to be closely associated along the shoreline. The tests were conducted in two contrasting micro-environments, namely ‘a dense population 'area’ and 'a less dense population area’, determined from a preliminary survey. Results obtained from the evaluation o f the bioactive materials namely, raw cocoyam (1 day fermented) and raw sweet potato (7 days fermented) showed that more snails were caught in the test traps than in the control traps. The difference was statistically significant (p < 0.05). This conformed with results obtained in S.N.E. experiments at the laboratory. Similar results were obtained by Dogbey (1995) when he tested some bioactive materials in the same lake using a different trapping unit designed by Kpikpi (1990). Results in the less dense population area showed that although all the test traps caught more snails than the controls, during some few traps inspection periods (i.e. 12-hour, 36-hour, & 72-hour periods for fermented cocoyam, and 36-hour, 48-hour, & 60-hour periods for fermented sweet potato) the difference was found not to be statistically significant (p > 0.05). With regard to the test involving a combination o f sugar cane and 0.6 ppm bayluscide, results again revealed a concordance with what was obtained in S.N.E. experiments. There was no significant difference (p > 0.05) in snails caught in test traps as compared 136 University of Ghana http://ugspace.ug.edu.gh with those o f control traps. This suggested that the quantity o f bayluscide added did not adversely affect the attractant potency o f the sugar cane. It was also observed thai. 34.39% o f the snails trapped after 12 hours were killed by the toxicant, whilst 23.07% were killed after the next 12 hours (i.e. 24 hours). None o f the snails died in the subsequent traps inspection periods (Table 6.3). This again suggested that the unit was capable of killing snails trapped only over a 12 to 24 hour period. The lake water 'probably diluted the concentration o f the toxicant to some extent that it could no longer kill the snails. To restore the killing effect o f the attractant-toxicant combination after 24 hours, it would be very necessary that after 24 hours the traps are removed and more bayluscide added. Table 6.3 Percentage o f snails killed in sugar cane-bayluscide combination experiments Time (hours) Number o f snails caught in test traps Number o f snails killed in test traps Percentage o f snails killed (%) 12 41 14 34.39 24 26 6 23.07 36 28 0 0 48 19 0 0 60 23 0 0 72 19 0 0 137 University of Ghana http://ugspace.ug.edu.gh 6.5 Conclusion According to the results obtained from the present studies, it can be concluded that cassava, cocoyam, and sweet potato, all in their raw fermented states were capable of attracting the schistosome host snails, B. truncatus and B. pfeifferi, when tested in simulated natural environment experiments. Cocoyam (1 day fermented) and sweet potato (7 days fermented) however emerged as the top two attractants. Results from field ’experiments conducted at Weija, on the lake showed that these top two attractants were effective in attracting the snails. Both cocoyam and sweet potato are food materials that are common in Ghana, and can easily be obtained in almost all-the market centres. They can be obtained at all times within the year to be used for snail trapping. A combination of sugar cane and some limited amounts o f 0 . 6 ppm bayluscide and used in the calabash trap could still attract snails which were trapped and killed. Although the percentages o f snails killed were comparatively small the highly selective manner in which they were killed is o f considerable interest. One major objective o f this study was to find ways of killing schistosome host snails without killing non-target organisms. The present findings show that this is now possible. Moreover, the pollution of the aquatic body could be reduced significantly if this method or improved versions are adopted rather than blanket mollusciciding regimes. The fact that the potency o f the attractant-toxicant combination could last for only 24 hours means that a daily inspection of the traps for some fresh bayluscide to be added would be necessary. Since the trapping system makes use o f only little amounts o f bayluscide one can conclude that the use of this method to control the snails will not only be at an affordable cost but will be environmentally friendly. 138 University of Ghana http://ugspace.ug.edu.gh 6.6 Recommendations In the light o f the findings made in the present studies the following points are recommended: 1. Since traps setting, normally for fish and other aquatic organisms are already a common practise by the fishermen and the other inhabitants o f communities around such water bodies, the use o f the schistosome host snail traps can easily be integrated into the work pattern o f the people. They however must be educated to be aware o f the need to control the snails so that the idea will be accepted and practiced willingly by them. This will essentially reduce the high cost o f labour-normally associated with applications o f snail toxicants to control snails. 2. Calabashes, together with the bioactive materials can be obtained in almost any market in Ghana at an affordable cost; so this trapping unit can easily be constructed locally .at anytime since the components are always available. Care must be taken so that the calabashes will not crack so that they can be reused for a long period. The quantities o f the bioactive materials used in the traps generally are small and will not affect human consumption. Again since very small amounts o f bayluscide needs to be used, the cost o f using this method is less expensive and will cause little or virtuallv no harm to other aquatic organisms. 3. It will be necessary to combine the toxicant with other bioactive materials that have been already identified to ascertain their efficacy with regard to ability to attract and kill. 139 University of Ghana http://ugspace.ug.edu.gh 4. There will also be the need to conduct a regular chemical analysis o f the water at the sites where the traps are positioned to find out whether these traps will at any point in time cause any pollution in the water. 140 University of Ghana http://ugspace.ug.edu.gh REFERENCES: Amankwaa, J.A., Bloch, P., Mayer-Lassen, J., Olsen, A., Christensen, N.O. (1994) Urinary and intestinal schistosomiasis in the Tono Irrigation Scheme, Kassena/ Nankana District, Upper East Region, Ghana. Trop. Med. Parasit. 4 (45), 319-323. Andrews, P., Thyssen, J. & Lorke, D. (1983) The biology and toxicology of molluscicides, Bayluscide. Pharmacology and Therapeutics 19, 245-295. Ansari, N. (1971) Parasites and progress. 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