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The response of miracidia and cercariae of Bunodera mediovitellata to light and to gravity Kennedy, Murray James 1974

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c7 THE RESPONSE OF MIRACIDIA AND CERCARIAE OF BUNODERA MEDIOVITELLATA TO LIGHT AND TO GRAVITY, by Murray James Kennedy B.Sc, University of British Columbia, 1971 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS -FOR THE DEGREE OF MASTER OF SCIENCE In the Department of Zoology We accept this thesis as conforming to the required standard The University of British Columbia April, 1974 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s m a y b e g r a n t e d b y t h e H e a d o f my D e p a r t m e n t o r b y h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t b e a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, C a n a d a D a t e i i ABSTRACT This thesis investigates the effect of light, and to a lesser extent of gravity, on the distribution of the two free-living larval stages (miracidia and cercariae) of the digenetic trematode Bunodera mediovitellata. Test tubes, with various portions blackened, were illuminated by a horizontal white light, to determine the photoresponse and georesponse of miracidia and cercariae. Four-arm test chambers were used to determine the lowest light intensity at which miracidia and cercariae reacted. This intensity proved to be the same for both miracidia and cercariae even though miracidia are photonegative and cercariae are photopositive. Two-arm test chambers, illuminated with monochromatic light, were used to determine which wavelength(s) the larval stages were responding to. Cercariae showed a single response peak at 550Anm. while miracidia showed two peak's; one at 550 run. and the other at 650 nm. The second peak may be due to a screening effect by the pigment which surrounds the photoreceptor. The experimental results support the hypothesis that be-havioural responses of free-living miracidia and cercariae to environmental stimuli guide? them to the general area of their next host and thereby increase! the chance of host-parasite contact. i i i The photonegative and geopositive behaviour of miracidia of B.mediovitellata would keep them on or near the bottom of the pond and i n the general habitat of the next host, Pisidium casertanum. Cercariae are photopositive and either geopositive or are very weak swimmers. Their behaviour would keep them on the bottom of the pond i n the prefered habitat of the next probable host, an insect nymph or a crustacean. A model depicting the possible theoretical combinations of photoresponse with georesponse was constructed. A survey of the l i t e r a t u r e was undertaken to f i n d l a r v a l stages with known photo-response and georesponse. The responses of these larvae were tabulated and compared to the model. In each case, the responses of the larvae supported the hypothesis that a complementary response to l i g h t and gravity should occur more frequently i n nature than antagonistic reponses or a response to either l i g h t or gravity alone. iv TABLE OF CONTENTS Page TITLE PAGE i ABSTRACT i i TABLE OF CONTENTS i v LIST OF FIGURES v i LIST OF TABLES v i i ACKNOWLEDGEMENTS v i i i INTRODUCTION 1 MATERIALS AND METHODS 6 Collecting and Maintaining Miracidia 6 Collecting and Maintaining Cercariae 6 TESTING OF PHOTORESPONSE AND GEORESPONSE 6 Materials and Methods 6 Results 7 Miracidia 7 Cercariae 8 RESPONSES TO WHITE LIGHT 9 Materials and Methods 9 Results 14 Miracidia 14 Cercariae 15 RESPONSES TO MONOCHROMATIC LIGHT 20 Materials and Methods 20 V Miracidia 23 Cercariae 26 DISCUSSION 28 THEORETICAL DISCUSSION 34 SUMMARY 39 LITERATURE CITED 40 v i LIST OF FIGURES Figure Page 1. Test Chamber Design 11 2. Percent of miracidia of Bunodera mediovitellata found in covered side-arm of test chambers exposed to white light 16 3. Percent of cercariae of Bunodera mediovitellata found in the exposed side-arm of test chambers exposed to white light 18 4. Percent of miracidia found in covered arm (monochromatic light) 21 5. Percent of cercariae found in exposed arm (monochromatic light) v i i LIST OF TABLES Table Page 1. Vertical distribution of miracidia subject to white light and gravity 12 2. Vertical distribution of cercariae subject to white light and gravity 13 3. Distribution 6f miracidia (white light) 17 4. Distribution of cercariae (white light) 19 5. Distribution of miracidia (monochromatic light) .... 22 6. Distribution of cercariae (monochromatic light) .... 25 7. Theoretical Table 28 v i i i ACKNOWLEDGEMENTS I would like to thank Dr. J.R. Adams and Dr. D.G.S. Wright for their kind assistance during the study and for c r i t i c a l l y reading the manuscript. I would also like to thank Drs. P.A. Dehnel, N.R. Li l y , D.J. Randall, and S. Shaw for c r i t i c a l l y reading the manuscript. I am further indebted to Wayne and Eda Reid, John Marsh, and Bob and Linda Barsaloux for their assistance in col-lecting f i e l d samples and to Linda Barsaloux for typing the thesis. 1. Introduction Digeneans (flukes) have complex l i f e cycles involving two inter-mediate hosts. Adult flukes commonly inhabit intestines and other organs of the vertebrate body (Noble and Noble, 1971) with eggs leaving the host through the intestine. Eggs usually enter water, where they are either eaten by a f i r s t intermediate host, or, more commonly, hatch. After hatching in the water, a free-swimming miracidium emerges from the egg. A miracidium must locate and penetrate the appropriate f i r s t intermediate host before using up i t ' s own energy supply, for i t does not feed. With but one excep-tion, the f i r s t intermediate host is a mollusc, usually a gastropod, occasionally a lamellibranch or a scaphopod. In the exceptional case the f i r s t intermediate host i s an annelid. After penetrating the mollusc, miracidia undergo a developmental and proliferative process giving rise to cercariae. Cercariae leave the molluscan host and lead a brief free-living existence during which time a second i n -termediate host or place of encystment must be found. The second in-termediate host may be a mollusc, vertebrate, crustacean or larval insect. Many digeneans therefore have two free-living stages which must locate a new host or place of encystment in order to perpetuate the parasites' l i f e cycle. These free-living stages are subjected not only to environmental conditions which may prevent transmission (temperature, water velocity) but also to predators, some of which live as commensals on molluscs (Khalil, 1961; Michelson, 1964). The 2. success of parasite transmission could be enhanced i f the parasites' free-swimming stages respond to environmental factors in such a way that (1) the time spent searching for a host is reduced and (2) the probability of host parasite contact i s increased by selective para-site dispersal towards the hosts* preferred habitat. Two main views exist concerning the mechanisms involved in miracidium-mollusc contact. The f i r s t view i s that miracidia locate their host purely by chance (Malek, 1950; Mattes, 1926; Stunkard, 1943) while the other view suggests miracidia respond to environmental stimuli which direct them to the general area of the molluscan host, after which they are attracted by a chemical(s) given off by the host (Faust, 1924; Etges and Decker, 1963; Maclnnis, 1965; Chemin, 1970; D.G.S. Wright, 1971; and others). This pattern of host-finding be-haviour provides a solution to the problem of the successful comple-tion of the free-living phase of the trematode life-cycle, since i t further reduces the element of chance which i s inherent in any ex-planation based on random encounters between host and parasite in nature. At present we have only limited knowledge of miracidial be-haviour in response to environmental stimuli. Most recorded observa-tions have been made under conditions with inadequate controls (D.G.S. Wright, 1971). Wright (1959) and later Schiff (1968) suggest that with some modifications miracidial behaviour f a l l s into three phases: 3. Phase 1: Physical stimuli such as light and/or gravity bring miracidia into the general area where the intermediate host may be found. Phase 2: Chemical stimuli attract the miracidia into the immediate area of the host. Phase 3: Physical and chemical structure of the host surface may induce the miracidia or cercariae to penetrate. The response of miracidia to physical stimuli has been described for several species. Negative geotaxis and positive phototaxis of miracidia of Schistosomatium douthitti have been demonstrated by McMullen and Beaver (1945). Wright (1971), using an equal energy spectrum, demonstrated further theeeffect of monochromatic light on the behaviour of S.douthitti. Takahashi et.al. (1961) used a r t i f i -c i a l light of different intensities, and different temperatures to demonstrate a positive phototaxis in miracidia of Schistosoma  japonicum. Miracidia of Fasciola hepatica show a photopositive response and those of F_. gigantica a photonegative response (Yasuraoka, 1954). Thus, miracidia of F.hepatica are brought to the surface and within the general environment of their amphibious lymnaeid hosts while the larvae of F.gigantica remain in deeper water where their chances of finding their benthic lymnaeid hosts are enhanced. 4. Isseroff and Cable (1968) showed that the miracidia of Philopthalmus megalurus change their photoresponse with age. Newly hatched miracidia are photopositive and later become photonegative. The miracidia attempt to penetrate their molluscan host only after becoming photonegative. Other work on the response of miracidia to light has dealt with the hatching of ova (Maldonado et.al. 1950; Roberts, 1950; Standen, 1951) and infection rates of miracidia in molluscs under f i e l d condi-tions. (Upatham, 1973) or in the laboratory (Chernin and Dunaven, 1962; Schiff, 1968; Upatham, 1972; and Webb, 1966), using various ratios of miracidia to mollusc. Phototactic behaviour of cercariae tends to be positive in species whose next host is a vertebrate and negative i f the host i s a bottom-dwelling invertebrate (Cable, 1972). Haas (1969) working with Diplostomum spathaceum demonstrated the existence of 2 phases in the cercarial swimming behaviour. A passive phase involving a period of sinking through the water alternates with an active phase which i n -volves active swimming back toward the surface of the water. Haas (1969) further demonstrated that the response of the cercariae to stimulation by light depended, among other things, on whether the stimulation occured during the active or passive phase, and whether the change of light intensity i s positive or negative. Increased light intensity during the passive or active phase lengthened the swimming period of cercariae. Decreased intensity in the passive phase releases an active swimming phase. However, shadowing during the active phase inhibited swimming. 5. Other work on the response of cercariae to light has dealt with the periodicity of emergence of cercariae from their snail host (Giovannola, 1936; Olivier, 1951; Luttermoser, 1955; Chernin, 1964; and Asch, 1972). Changes in the swimming behaviour of miracidia near snails and snail faeces was used by Wajdi (1966) as an indication of attraction. He observed that miracidia of Schistosoma mansoni were attracted to light in the absence of snails but not with snails present. However, Etges and Decker (1963) tested the effects of light and gravity on miracidia of S.mansoni, and showed that these factors were more power-ful stimuli in determining the orientation of the miracidia than the chemical ones produced by their molluscan host Australorbis glabratus. The present study concentrated on the photoresponses of the lar-val stages of Bunodera mediovitellata for two main reasons: 1) Bunodera mediovitellata belongs to the family Allocreadiidae most of the members of which characteristically use a b i -valve for the f i r s t intermediate host rather than a snail. The behaviour of miracidia which enter clams has not been studied before. 2) The presence of two free-living larval stages in the l i f e cycle (miracidia and cercaria) would enable a comparison to be made between the two stages. 6. Materials and Methods Collecting and Maintaining Miracidia: Sticklebacks (Gasterosteous aculeatus) containing flukes of Bunodera  mediovitellata (Zimbaluk et Roytman, 1965) were collected from a pond in Queen Elizabeth Park, Vancouver, B.C. In the laboratory fish were fed frozen brine shrimp, and maintained at 15°C ± 1°C. Flukes were dissected from the sticklebacks' intestine and placed in a 0.1% Courtland solution (Wolf, 1963) and kept at 20°C ± 1°C Flukes laid a l l of their eggs (250-400 eggs per fluke) within six hours under these conditions. Hatched miracidia having a mean age of 12 hours were subsequently collected using a 1 ml. tuberculin syringe with a 26 G needle. Collecting and Maintaining Cercariae: Pisidium casertanum were collected from Tin Can Creek in Musqueum Park, Vancouver, B.C. The clams were brought back to the laboratory where they were maintained in finger bowls at 20°C under a light intensity of 88 lux. and a 12 hr:12 hr photoperiod. The bowls were checked daily for cercariae. Those which emerged were pipetted from the bowls in lots of 25 and used the same day in photoresponse experiments. Testing of Photoresponse and Georesponse: Glass test tubes measuring 93 mm. high by 25 mm. wide were used. They were covered with black tape in such a way as to determine the re-lative influence of light and gravity on the distribution of miracidia and cercariae, in the following manner. The f i r s t test tube was com-pletely covered with black tape. A hood made of black paper and covered 7. with tape was also used to put over the mouth of the test tube so that no light could enter the tube. The second test tube was lef t untaped, while the third test tube had the top 2/3 covered with black tape. A hood was also made for this tube. The last test tube had the bottom 2/3 covered with black tape. These test tubes stood vertic a l l y and were exposed to a horizontal light beam supplied by a Bausch and Lomb micro-scope lamp with a light intensity of 1400 lux at 30 cm. from the light source. This was the distance at which a l l the test tubes were placed. The test tubes were f i l l e d with 0.1% Cortland solution and either miracidia or cercariae were pipetted into the middle part of the tube. The top 1/3 of each test tube was designated A, the middle 1/3 B, and the bottom 1/3 C. Counting of the test animals was done by pipetting the Cortland solution in each location: A, B, C into separate petri dishes and counting the number of test animals in each location with the aid of an Olympus dissecting microscope. Each test condition was run for 30 minutes at 20°C and repeated five times. The response of miracidia and cercariae to light also observed by placing 25 larvae into tissue culture dishes (60 mm.x 15 mm.) f i l l e d with 0.1% Cortland solution and shining light from the above Bausch and Lomb microscope lamp onto them from various angles. Photoresponse and Georesponse: Results 1) Miracidia: Nearly a l l miracidia were found in the lower one third of the test tubes in a l l experiments of test conditions 1, 2, and 4; and in the upper 8. one third in test condition 3 (Table, 1). Very few miracidia were found in the middle one third of a l l test conditions. Test conditions (T.C.) number 1 and 2 suggest that a georesponse i s present in the miracidia, and that no reversal of georesponse occurs in the presence of light. T.C. 3 demonstrated that the miracidia have a negative photoresponse, the negative photoresponse being stronger than the positive georesponse. T.C. number 4 was designed to see i f a positive photoresponse was pre-sent. When the miracidia were observed in the tissue culture dishes, i t was seen that they moved directly away from the source of light, and are therefore phototactic as defined by Fraenkel and Gunn (1961). A photokinetic response did not seem to be present, that i s there was not an increase in the rate of turning of miracidia when exposed to an increase in light intensity. An increase in the rate of swimming with increase in light intensity was not tested. Illuminating the miracidia from the tope caused them to dive to the bottom of the dish and illumina-tion from the bottom, made them move to the surface of the dish. The miracidia moved in straight lines, even when illuminated evenly, until they reached the side of the dish, after which they followed the wall around and did not move away from the wall until the illumination on the wall side was greater than that on the opposite side in which case i t moved away from the brighter light. 2. Cercariae: Cercariae moved to the bottom of the test chambers under a l l experimental conditions. They did not move to position A of T.C. 4 9. despite their positive photoresponse (See P. 15 for photoresponse re-sults) . This suggested that either their tendency to sink to the bot-tom i s stronger than their tendency to move toward light or, the cercariae are not able to swim very far above the bottom of the test tube. Cercariae showed both a photokinetic and a phototactic behaviour when observed in the culture dishes. When light was shone on to resting cercariae they began an active swimming phase; when light was shpne.on actively swimming cercariae this seemed to prolong the swimming phase. Decreasing the light intensity on swimming cercariae tended to inhibit swimming motions, while decreasing the light intensity on resting cercariae did not in i t i a t e swimming. A phototactic response was also noted in illuminated cercariae. Cercariae moved toward the light source. This was achieved by a series of swims with intermittent rests until the cercariae reached the side of the dish nearest the source of illumination. Once the cercariae reached the wall side of the dish -ik»y rested. There then followed a period where swimming alternated with resting. The cercariae always remained near the side of the chamber closest to the source of illumination. This spontaneous alternating swimming and resting phase was also observed in cercariae that were exposed to even illumination. At no time did the cercariae move more than five millimeters off of the bottom of the dish. Response to White Light: Materials and Methods: Behavioural responses of miracidia to white light were tested. 10. in a four-arm chamber constructed of lucite ( f i g . l ) . A bank of four fluorescent lights was supported by a cage, constructed of adjustable metal shelf r a i l i n g , in such a way as to allow the light bank to be raised and lowered to achieve different light intensities. As well, each fluorescent tube could be turned on or off in order to adjust light intensities. The light shone directly down on the test chambers. Light intensities were measured with a Gossen Lunasix 3 light meter. Twenty-five miracidia were pipetted into the center well of the test chamber. The chamber was placed in a blackened box with one arm of the test chamber projecting out through a hole in the box so that only that arm was directly exposed to the light. Two chambers were run at a time for a total of 30 minutes at 20°C. Four experimental r e p l i -cations were made for each of ten light intensities between 0 and 22,000 lux except 0.3 lux and 0.35 lux which were repeated eight times. Two controls were used to test for random distribution in the chambers. The entire chamber in the f i r s t control was exposed to over-head light. The chambers of the second control were placed in a black box and no light allowed to enter. Twenty-five miracidia per chamber were used for the controls. Two chambers for each control were run simultaneously for thirty minutes at 20°C. A total of ten repeats were run for each control. The percent of miracidia found in the covered arm in experimental chambers after the test period, was calculated by dividing the number of miracidia found in the covered arm by the total number of miracidia 11. Test Conditio n Locat ion D i s t r ibut ion of M i r a c i d i d / E x p t . F Condition E, E 3 E 5 PI A 0 0 0 0 3 1 N B 0 0 4 0 0 I 1 C 2 6 2 8 1 3 1 7 1 9 A 0 0 0 0 0 2 B 1 0 0 0 0 C 2 0 1 0 2 0 1 6 1 0 3 i i -1 A B 2 6 3 1 9 0 1 2 0 1 5 0 2 0 0 L C 1 4 1 7 5 r A 0 0 0 1 0 A B 1 0 0 0 0 1 C 2 1 9 1 8 1 7 8 Table 1: The v e r t i c a l d i s t r i b u t i o n of miracidia subjected to horizontal i l l u m i n a t i o n with white l i g h t , and to gravity. The test chambers were maintained at 20°C. Black shading represents the area of the tube covered by black tape and the white area i s the uncovered area ex-posed to ill u m i n a t i o n . 1 3 . Test Conditio n Location D i s t r ibut ion of C e r c a r i a e ^ Expt. J* x Condition Ei E 3 ^4 £ 5 1 Mr A B C 0 0 1 5 0 0 1 4 0 0 1 5 0 0 1 5 0 0 1 5 A 0 0 0 0 0 2 B 0 0 0 0 0 C 1 5 1 5 1 5 1 1 1 5 3 A B 0 0 0 0 0 0 0 0 0 0 C 1 5 1 5 1 5 1 5 1 3 • A 0 0 0 0 0 4 m B C 0 1 5 0 1 5 0 15 0 1 5 0 1 5 T a b l e 2: T h e v e r t i c a l d i s t r i b u t i o n o f c e r c a r i a e s u b j e c t e d t o h o r i z o n t a l i l l u m i n a t i o n w i t h w h i t e l i g h t , a n d t o g r a v i t y . T h e t e s t c h a m b e r s w e r e m a i n t a i n e d a t 2 0 ° C . B l a c k s h a d i n g r e p r e s e n t s t h e a r e a o f t h e t u b e c o v e r e d b y b l a c k t a p e a n d t h e w h i t e a r e a r e p r e s e n t s t h e u n c o v e r e d a r e a e x p o s e d t o h o r i z o n t a l i l l u m i n a t i o n . 14. that moved from the center well and were recovered alive. The figure was then converted to percent. The percent cercariae found in the ex-posed arm was calculated in the same way. Figures 2 and 3 were then constructed using this percentage. Cercariae were tested in the same manner as the miracidia except that three arms were exposed to white light. The fourth arm was covered by pushing i t through a hole in a blackened box. At the completion of each experiment the arms were sealed off at the center well and the number of animals i n each arm and in the center well counted. The Chi-square test was applied to the number of miracidia (or cercariae) which had moved from the center well. Results 1) Miracidia: The number of miracidia found in the side arms of any one chamber did not diff e r significantly in the ten control chambers held at zero lux, or in the ten control chambers held at 88 lux (Table 3). More miracidia were found in the covered arm when the exposed arms were sub-jected to light intensities between 0.35 lux and 22,000 lux (X 2 = P< 0.05) (Table 3). Miracidia were randomly distributed in the test chamber at light intensities between 0 lux and 0.3 lux (Figure 2). Increasing the light intensity f a l l i n g on the exposed arms from 0.35 lux to 22,000 lux did not significantly increase the number of miracidia recovered from the covered arm. Results 2 ) Cercariae: Cercariae di f f e r from miracidia in being photopositive. However, the intensity at which cercariae begin to show a significant movement toward light (Fig. 3; Table 4) i s the same as the intensity at which miracidia show a significant movement away from light (Fig. 2 ) . 1 6 . 2 3 4 Light Intensity (Lux) F i g . 2: P e r c e n t a g e o f m i r a c i d i a o f B u n o d e r a m e d i o v i t e l l a t a f o u n d i n t h e c o v e r e d s i d e - a r m o f t e s t c h a m b e r s e x -p o s e d t o w h i t e l i g h t f o r 3 0 m i n u t e s a t 20°C. ± 1°C. M i r a c i d i a l r e s p o n s e d i d n o t c h a n g e a t i n t e n s i -t i e s g r e a t e r t h a n 5.5 l u x ( S e e T a b l e 3 ) . Random d i s t r i b u t i o n . 1 7 . l i g h t A r m No. In N o . No. moved / f o u n d I n t e n s i t y ( L u x ) C e n t e r dead f r o m in covered a rm X ' P C o v e r e d 1 2 3 W e l l t e n t e r orrr 0 2 2 1 7 19 2 0 3 1 9 7 8 2 8 2 0 - 3 N S 0 - 2 5 2 0 19 1 8 2 0 4 1 9 7 7 2 5 -9 0 0 N S 0 - 3 4 4 4 0 4 4 4 2 2 2 8 1 7 0 2 5-9 0 - 1 N S 0 3 5 1 1 0 1 0 1 4 1 2 0 5 4 1 4 6 7 5 - 3 1 4 8 - 0 S 0-4 6 7 4 6 6 0 1 7 8 3 8 0 - 7 1 0 2 - 6 s 0 - 7 6 0 4 5 7 0 2 4 7 6 7 8 9 8 8 - 4 5 2-8 7 2 6 6 5 . 0 11 8 9 8 2 -0 1 1 0 8 S 5-5 5 8 4 5 4 0 2 9 7 1 8 1 - 7 9 0 - 8 s 11 5 2 4 5 3 1 3 5 6 4 8 1 - 3 8 1 0 s 1 6 7 1 5 5 6 0 1 3 8 7 8 1-6 1 1 0 - 6 s 88 4 3 8 4 5 3 3 7 6 0 7 1 - 7 5 2 - 1 s 1 7 5 5 8 8 1 6 1 2 6 7 3 7 9 - 5 8 0 - 7 s 3 5 0 5 2 5 3 3 0 3 7 6 3 8 2 -5 8 2-9 s 2,8 0 0 8 3 1 4 1 1 9 9 0 9 2 - 2 1 6 2 - 7 s 2 2 , 0 0 0 8 2 3 3 0 0 1 2 8 8 9 3 - 2 1 6 3 - 6 s C O N T R O L S 0 5 4 5 3 5 1 5 2 1 5 2 5 2 1 0 2 5 - 7 0 - 0 N S 88 5 7 5 3 5 9 5 2 1 3 1 6 2 2 1 2 5 -8 0 1 N S T a b l e 3: D i s t r i b u t i o n o f m i r a c i d i a o f B u n o d e r a m e d i o v i t e l l a t a i n t e s t c h a m b e r s w i t h o n e a r m c o v e r e d a n d t h r e e a r m s e x p o s e d t o w h i t e l i g h t f o r 3 0 m i n u t e s a t 20°C. P e r -c e n t f o u n d i n c o v e r e d a r m i s c a l c u l a t e d o n t h e t o t a l n u m b e r o f m i r a c i d i a r e c o v e r e d a l i v e f r o m a l l t h e s i d e a r m s . S i g n i f i c a n c e l e v e l P < 0.05 N.S. = n o t s i g n i f i c a n t 1 2 3 4 Light Intensity (Lux) 5 Percentage of cercariae of Bunodera mediovitellata found i n the exposed side-arm of test chambers ex-posed to white l i g h t for 30 minutes at 20°C ± 1°C. Cercarial response did not change at i n t e n s i -t i e s greater than 5.5 lux. (See Table 4). Random d i s t r i b u t i o n . 1 9 . l i g h t A r m N o . In No . No. moved X * c e n t e r dead f r o m i n expo&ed P l n t en s 11 y l u x E x p o s e d 1 2 3 w e l l c en te r arm a r m 0 2 6 22 25 2 2 2 3 9 5 2 7 4 0-2 N S 0-25 2 I 20 19 2 3 9 8 8 3 2 5-3 0 N S 0-30 48 52 4 2 4 2 0 1 6 8 4 2 6 0 0-9 N S 0-3 5 1 22 1 2 1 0 1 0 7 3 9 154 79-2 18 1-1 S 0-4 5 7 5 5 3 27 3 0 7 0 81-4 8 9-2 s 0-7 6 5 6 4 6 0 19 8 1 8 0-2 9 8-4 s 2-8 69 4 3 7 0 1 7 8 3 8 3-1 111-7 s 5-5 7 1 5 6 5 1 1 2 87 8 1-6 1 1 1-0 5 11 5 2 5 3 4 3 3 3 64 8 1-3 8 1-0 S 16 63 3 4 6 0 2 4 7 6 82-9 101-9 s 88 7 8 6 7 7 0 2 9 8 79-6 1 1 6-8 s 1 7 5 6 9 6 5 4 1 1 5 84 82-9 . 1 09-7 s 3 5 0 6 0 ' 5 6 4 2 2 3 7 5 8 0-0 90-3 s 2,8 0 0 8 0 3 4 2 0 1 1 8 9 8 9-9 4 9-3 s 2 2,0 0 0 83 4 0 3 1 9 9 0 92-2 16 2-7 s C O N T R O L S 0 54 5 6 57 53 3 2 7 22 0 24-5 0 N S 88 58 6 2 53 50 5 2 2 2 2 3 2 6 0 0-1 N S Table 4: Distribution of cercariae of Bunodera mediovitellata i n test chambers with three arms covered and on arm exposed to white l i g h t for 30 minutes at 20°C. Per-cent found i n exposed arm i s based on the t o t a l number of cercariae recovered a l i v e from a l l side arms. Significance l e v e l P<0.05 N.S. = not s i g n i f i c a n t . S = s i g n i f i c a n t . Responses to Monochromatic Light: Materials and Methods: Responses of miracidia to monochromatic light were tested using a grating monochromator with a 150 W. Xenon light source giving a specified band pass of 10 nanometers. The lamp was operated with a regulated power supply producing 150 W. at 20V. DC. and 7.5 amps. Coming infrared (651069) and ultraviolet (650-52) cut-off f i l t e r s , placed in front of the lamp, restricted energy to the visible spectrum To control light intensity from the monochromator, the light beam was collumated and passed through two circular, inconel coated, neu-t r a l density wedges. A light intensity of 0.23 it .05yu-W/cm2. was used as i t was found to be the lowest intensity that would e l i c i t s a maxi-mal response from the larvae. The light intensity was measured with a Hewlett Packard Models 8330A/8334A radiant flux meter system. The behavioural responses of miracidia subjected to monochromati light were tested in a straight chamber constructed of lucite (Fig.l). The beam from the monochromator was adjusted so that i t passed hori-zontally through one half of the test chamber. The other half of the test chamber was shielded by placing a blackened sleeve over i t . Twenty-five miracidia (or cercariae) were pipetted into the middle of the test chamber. Two chambers were run at a time. Each run lasted 30 minutes at 20°C. After 30 minutesaipl'asticene wedge was inserted to separate the exposed and sheltered halves of the chamber. The number of test animals found in each half was then counted. Spectral responses of miracidia and cercariae were tested at 25 nm. intervals 21. i i l 400 450 500 550 Wavelength (nm) 600 650 700 "JFig. 4: Percentage of miracidia of Bunodera mediovitellata found i n covered side-arm of test chambers exposed to mono-chromatic l i g h t for 30 minutes at 20°C ± 1°C. Random d i s t r i b u t i o n . Wave No. of Miracidia in No. No. Total °/0 found length exposed covered dead recovered in covered X2 (nm) arm a rm arm 4 00 50 50 0 100 1 00 5 00 0 425 50 4 8 2 98 1 00 49 -0 o 4 50 49 4 7 2 96 1 00 4 9-0 0-2 4 7 5 52 48 0 100 1 00 4 8-0 0-1 50 0 50 49 1 99 1 00 49-5 0 5 2 5 28 72 0 100 100 720 9-7* 5 5 0 1 98 1 99 1 00 9 9-0 * 475 575 26 72 2 9 8 100 73-5 10-8 6 00 4 0 5 9 1 99 1 00 5 9-6 1-8 62 5 50 4 9 1 99 1 0 0 4 9.5 0 6 50 1 5 85 0 1 0 0- 1 0 0 85-0 * 24'5 675 39 59 2 9 8 100 60-2 2-0 700 4 6 47 7 93 1 00 5 0-5 0 Table 5: Distribution of miracidia of Bunodera mediovitellata in test chambers with one arm covered and one arm ex-posed to an equal energy spectrum (irradiance = 0.2lj^/m2) for 30 minutes at 20°C. Percent found in covered arm is based on the total number of miracidia recovered alive from the test chambers. The total i s from 4 replicates each containing 25 miracidia. * Significance level P<0.05 Other *X values not significant. 23. from 400 to 700 nm. inclusive. Four repeats were used for each wave-length tested. Two control chambers were run for each wavelength tested. Both arms of the control chambers were exposed to the light beam or both shielded. Monochromatic Light Response: Results 1) Miracidia: The distribution of miracidia in the test chambers at wave-lengths 400, 425, 450, 475, 500, 600, 625, 675 and 700 nm. did not diffe r significantly tfr«m\ that expected of a random distribution. The total number of miracidia found in the arm subjected to the above wavelengths equalled or slightly exceeded the number of miracidia found in the covered arm at the same wavelength (Table 5). However, significantly more miracidia, than that expected by chance alone, were found in the covered arm at wavelengths 525, 550, 575, 600, and 650 nm. (Fig.4; Table 5). Thus, miracidia exhibited two peak res-ponses to monochromatic light. The greatest response was found at a wavelength of 550 nm. from which 99% of the miracidia moved away, and were subsequently found in the covered arm. The second peak res-ponse was at 650 nm. where 85% of the miracidia moved away from this wavelength. Miracidia also moved away from wavelengths 525 and 575, but the response was not as great (72% and 73.5% respectively) as that found at 550 and 650 nm. Movement of miracidia in the illuminated and blackened control chambers was random (Table 5). An equal number of miracidia was 24. 100 - , 8<H 6 0 H 4 0 -2<H 4 0 0 4 5 0 500 550 600 6 5 0 7 0 0 Wovelength (nm) Fig. 5: Percentage of cercariae of Bunodera mediovitellata found in exposed side-arm of test chambers exposed to mono-chromatic light for 30 minutes at 20°C + 1°C. Random distribution. 25. Wove No- of cercariae in No. No. Total 7 found ' O length exposed covered dead recovered in exposed X2 (nm) arm a rm arm 4 0 0 47 4 8 5 95 1 0 0 4 9-5 0 4 25 5 0 4 7 3 9 7 1 0 0 5 1-5 0-T 4 5 0 4 8 44 8 92 1 0 0 5 2-2 0-1 4 7 5 4 6 43 1 1 89 1 0 0 5 1-7 0-1 5 00 52 43 5 95 1 0 0 52-0 0'4 5 25 56 2 8 1 6 8 4 1 0 0 6 6-7 4-7 5 5 0 9 6 4 0 10 0 1 0 0 96-0 * 42-3 575 8 0 1 6 4 96 1 0 0 8 3-3 21-3 600 60 40 0 00 1 0 0 60-0 2-0 6 2 5 44 43 1 3 8 7 1 0 0 5 0-6 0 6 50 4 8 5 2 0 10 0 l o o 4 8-0 0 675 4 8 52 0 100 1 0 0 48-0 0 700 4 5 4 7 8 92 1 0 0 4 8-9 0 Table 6: Distribution of cercariae of Bunodera mediovitellata in test chambers with one arm covered and one arm exposed to an equal energy spectrum (irradiance = 0.23yuW/m2) for 30 minutes at 20°C. Percent found in covered arm is based on the total number of cercariae recovered alive from the test chambers. The total i s from 4 replicates each con-taining 25 cercariae. * Significance level P<0.05 Other "X1 values not significant. 26. found in both halves of the control chambers. 2) Cercariae: The number of cercariae recovered in the exposed arm did not di f f e r significantly from random when the exposed arm was subjected to wavelengths of 400, 425, 450, 475, 500, 525, 625, 650, 675, or 700 nm. However, significantly more cercariae were found in the ex-posed arm at wavelengths 550, 575 and 600 nm. ('Fig.5; Table 6). Ninety-six percent of the cercariae exposed to wavelength of 550 nm. were recovered in the irradiated arm. Eighty-percent of the cer-cariae were recovered in the exposed arm at 575 nm. and 60% at 600 nm. Migration of cercariae in the control chambers was random (Table 6). 27, t a r v o e Mirocidio Schistosomatium doutliitti Schistososoma mansonl Fasciola hepatica Schistosoma japonicom Schistosoma haematobium Bunodera mediovitellata Photoresponse Georesponse Host location in water — surface + + bottom Reference McMullen & Beaver 1945 Maclnnis 196S Yasuraoka 1954 Tokahashi 1961 McMullen & Bauman 1949 Wajdi 1972 Onchomiracidio Diplorchis ranae Ozak i 1935 Cercar iae Trichobilharzia ocellata Gigantobilharzia huttoni Heterobilharzia omerieono Cercariae littorinalinae Cercariae elongata + + + + + surface after Lee 1962 Table 7: Possible combinations of Photoresponses and Georesponses in nature. 28. Discussion Since the lives of miracidia and cercariae are so short i t i s important that the responses of the cercariae be as closely related as possible in a biologically advantageous way to those of the inter-mediate host, so that the maximum chance of contact may results. The experiments with the miracidia of B.mediovitellata des-cribed here demonstrate the presence of two behaviour patterns which would assist in host location. One of the operative stimuli was light (Fig.2). When offered a choice of light and shade, the miracidia moved into the shaded portion of the test chamber. This movement away from light occured at a l l light intensities above 0.35 lux. Below 0.35 lux the miracidia were found randomly distributed in the test chamber. The response of miracidia to very low light intensi-ties would probably serve to keep the miracidia on the bottom of a body of water at a l l times of the day. If a situation arose where the light intensity dropped below 0.35 lux then the georesponse of the organism would keep i t on the bottom of a body of water. Both the positive georesponse and the negative photoresponse would serve to d i -rect the miracidia to the microhabitat of i t ' s natural host Pisidium  casertanum. It was also shown that light is a stronger stimulus than gravity when the two were used in an antagonistic way (Table 1, test condition 3). However, under natural conditions the two responses would comple-ment one another. 29. Miracidia then are probably directed to their host by a number of responses to stimuli which may act together or in a sequence. These responses would lead the miracidium to those microhabitats where hosts are most likely to be found or even to the host i t s e l f . Miracidia of B.mediovitellata have a negative photoresponse and a positive georesponse. This is different from behaviour observed in Schistosoma mansoni (Chernin and Dunavan, 1962), Schistosoma japonicum (Takahashi et.al., 1961), Schistosomatium douthitti (Yasuraoka, 1954). The above four species exhibit a positive phototaxis accompanying a nega-tive geotaxis. The miracidia of these species penetrate intermediate hosts which live on or near the surface of a body of water. Takahashi's (1961) work also indicates that miracidia of S.japonicum exhibit the same phototactic and geotactic behaviour as that found in i t ' s intermediate host Oncomelania nosophora elucidated by Webbe and Msangi (1958), and Wright (1956). These authors showed that the intermediate host of S.japonicum congregates near the surface on the underside of water l i l y leaves. However, the miracidia of Fasciola gigantica show a photonegative response (Yasuraoka, 1954). Thus miracidia of this species move to the bottom of the pond where their chances of finding their lymnaeid hosts are enhanced. Isseroff and Cable (1968) showed that the miracidia of Philophthalmus megalurus change their photoresponse with age. Newly hatched miracidia are photopositive and later become photonegative. The authors suggest that the i n i t i a l photopositive behaviour serves as a dispersal mechanism, for miracidia attempt to penetrate their molluscan host only after 30. becoming photonegative. The wavelengths to which miracidia of B.mediovitellata most strongly reacted were 550 nm. and 650 nm. (Fig.4). This i s the f i r s t time any miracidia h<*y« been shown to exhibit two peak responses. In the literature there i s l i t t l e discussion of the effect of monochromatic light on behaviour of miracidia. In a study by Wright (1971), miracidia of S.douthitti were exposed to an equal energy spectrum of monochromatic light. The wavelength which attracted miracidia was the blue-green range (550 nm.). The wavelengths which penetrate deepest into fresh water are 475 to 500 nm. (Mills, 1972), but this depends on such things as dissolved and suspended matter which may lead to a shift of maximum transmittance towards 550 nm. (Jerlov, 1971). Wrights' findings were similar to the observations made on other invertebrates whose photoreceptors have not been de-monstrated. Light reactions for these animals have maximum sen-s i t i v i t y between 470 nm. and 530 nm. which f a l l s off more rapidly towards the longer wavelengths than the shorter ones (Steven, 1963). Goldsmith (1965) states three possible hypothesis for the presence of a sensitivity peak in the red end of the spectrum: 1) Presence of a red receptor 2) Neural Interaction hypothesis 3) Screening hypothesis. The f i r s t hypothesis b r i e f l y stated argues for the presence of a re-ceptor with a maximum sensitivity in the red end of the spectrum. The second hypothesis suggests the presence of an inhibitory inter-31. action between receptor cells containing different pigments and therefore having different spectral sensitivities. The third hypo-thesis, and the one favoured by Goldsmith, claims that the red peak!; in the spectral sensitivity function i s caused by the presence of red screening pigments which absorb; a l l but long wavelengths. This leakage of long wavelengths through the pigment screen potentiates a response. Very l i t t l e work has been done to show the ultrastructure of photoreceptors in cercariae and miracidia (Isseroff, 1964; Pond and Cable, 1966). Both miracidia and cercariae show differences in the de-gree of pigmentation as well as the number of receptors. The maximum number of receptors per eyespot that has been found is two. The photopositive behaviour of cercariae of B.mediovitellata would result in orienting the non-pigmented portion of the screening-cup toward light. Light would enter the photoreceptor directly and so we would not expect to get a response at the red end of the spec-trum due to screening. However, the photonegative behaviour of miracidia of B.mediovitellata would result in light passing through the pigmented cup before reaching the photoreceptor. This may have given rise to the response at the red end of the spectrum. The presence of a red receptor or the possibility of a neural interaction i s not ruled out. A study correlating the behavioural] responses of larvae to monochromatic light with eyespot morphology may enable us to determine which hypothesis i s operative. 32. Cercariae show a horizontal phototactic movement towards light but not a vertical one. Cercariae were observed to remain on or near the bottom of test tubes in experiments, designed to test for a photo-response and a georesponse (Table 2). This suggested that cercariae exhibit either a strong geopositive response which i s stronger than their tendency to move toward light (Test condition 4, Table 2) or, cercariae are weak swimmers and are unable to swim in a vertical fashion for very long. Whatever the case may be, the cercarial be-haviour would effectively keep i t on or near the bottom of the pond and in the more illuminated areas. Cercariae would be found on top of debris such as leaves and sticks rather than underneath them. The known second intermediate hosts of the Allocreadiidae are a l l bottom dwelling insect nymphs oudcrustaceans which tend to be found in areas more exposed to light (Ward and Whipple, 1918). The be-haviour of the cercariaj then would keep i t in the area of the pond where i t has the greatest opportunity of encountering i t ' s host. Cercariae also show a photokinetic response in thattthey are stimulated to swim when light f a l l s on them. As well, cercariae also show a spontaneous swimming behaviour alternating with a resting period in evenly illuminated light. This behaviour may act to at-tract hosts so that host-parasite contact is further ensured. The phenomenon of photoresponses in cercariae has not been much studied (Smyth, 1966). It is generally believed that only those cercariae that possess eyespots are photopositive (Smyth, 1966; Erasmus, 1972; Lee, 1962). Cercariae showing a photopositive response w i l l not 33. necessarily be found at or near the surface of a body of water. Cercariae of B.mediovitellata are photopositive but they are more like l y to be found near the bottom of the pond. Until now i t has been thought that in natural conditions a positive phototactic orientation to light results generally in the concentration of cercariae in the upper levels of the pond, and this i s thought to have some sig-nificance in aiding host-parasite contact. A negative geotaxis in the cercariae of Opistioglyphe ranae was described by Styczynska-Jurewicz (1961). He showed that for the f i r s t 4-8 hours after emergence from the snail Lymnaea stagnalis, cercariae swim towards and near the water surface as the result of strong negative geotaxis. After 8 hours the cercariae sank to the bottom of the tank and eventually died. In a later paper Styczynska-Jurewicz (1962) suggested that the cercarial behaviour pattern was correlated with the behaviour of tadpoles (in which they normally encyst) which were confined to the surface waters because of the poor oxygen content of the deeper layer. 34. Theoretical Discussion The existence of nine possible combinations involving photo-response and georesponse can be predicted for l a r v a l trematodes (Table 7 ). These combinations form three groups which depend on the location of the host under natural conditions. I f the intermedi-ate host i s a bottom dweller, such as a clam, we would expect the; larvae to exhibit one of the following combinations of responses: Type 1), A photonegative and a geopositive response Type 2) A photonegative response and no georesponse Type 3) No photoresponse but a geopositive response. A l l of these responses would direct the larva into the general area of the host. Once i n the general area of the host a chemotaxis or rheotaxis may direct the f i n a l contact. A Type 1 response would probably give the strongest directing cues and so i n nature we would expect selection for t h i s type of res-ponse. The photoresponses and georesponses of only three trematodes known to have a bottom dwelling intermediate host have been s u f f i c i e n t l y worked out to f i t into t h i s theoretical discussion. a) Schistosoma haematobium b) Dip1orchis ranae c) Bunodera mediovitellata The miracidia for S.haematobium, B.mediovitellata and the onchomiracidia for D.ranae a l l exhibit the Type 1 response and so support the argument of a selection for a Type 1 response. The larval stages of other trematodes which have bottom dwelling hosts have had their photoresponses tested but not their georesponses (Yasuraoka, 1954; Isseroff and Cable, 1968). These larvae also exhibited a photonegative behaviour. Although the georesponses of these larvae must be tested before they can be used in the theoretical table, their known behaviour is not incompatible with the model. Three combinations of photoresponse and georesponse are possible for parasitic larvae which must penetrate a surface dwelling host. Type 4) A photopositive and a geonegative response Type 5) A photopositive response and no georesponse Type 6) No photoresponse but a geonegative response A Type 4 response should be the most effective in directing the larva to the general habitat of the next host, and so, under natural condi-tions there should be a selection favouring the evolution of a Type 4 response. The photoresponses and georesponses of ten species of larval trematodes (which must penetrate a surface dwelling host) have been sufficiently worked out to f i t into the theoretical table. Of these, four species of miracidia and five of cercariae, exhibit the Type 4 response (Table 7). The other species is a non-swimming cercariai of Asymphylodera amnicolae which has a curious form of negative geotaxis and no photoresponse, and so f i t s a Type 6 response. Cercariae of this species emerge from their snail host, climb to the top of the snail and cling to i t ' s tentacles. This behaviour pattern appears to be related to ease of infection of the second intermediate host, which are uninfected individuals of the same snail (Smyth, 1966). 36. Although responses of Type 2, 3, 5, and 6 may exist in nature they should not be as heavily favoured as Type 1 and 4 which have two directing forces operating to guide the larvae to their host. Some authors make the claim that a photoresponse i s the most important directing force (Etges and Decker, 1963) and data from the experiments with miracidia seem to support this argument. However, this application requires some caution in nature. In standing, clear water a photoresponse may in fact dominate the movement of the larvae. However, i f the miracidiwmfinds i t s e l f in muddy water so that light does not penetrate very well, the georesponse of the larvae may be the i n i t i a l single most important response directing the larvae. As well, i f the larva finds i t s e l f in clear running water, a rheoresponse may i n i t i a l l y be more important in directing the larva. Larvae which have been tested for a rheoresponse tend to swim against the current at an angle to the current (Yasuraoka, 1952). This behaviour would take the animal to the margins of the stream, and to slower running water or even to s t i l l pools, after which a photoresponse and/or georesponse could provide the directing cues. It is therefore im-portant to consider the type of habitat that the larvae i s found in before overstressing the major importance of any single response of a parasite to i t ' s environment. The three remaining responses: Type 7) A photopositive and a geopositive response Type 8) A photonegative and a geonegative response Type 9) No photoresponse or georesponse 37. are less likely to be found in nature. Type 7 and Type 8 are anta-gonistic responses and should they arise would tend to hinder host location unless they occured in a sequential fashion. In the case of a Type 7 response, the larva may i n i t i a l l y respond photoposi-tively. This may serve to distribute the larvae over a wide area. When the larva gets older, tKe^photoresponse may be dampened or even shut off and a geopositive response dominate the movement of the larvae. Alternatively, the larva with a Type 7 response may have a surface dwelling host which is found at or near the surface of the water only during the morning and evening. Both larva and host move to the surface of a body of water during the part of the day when the light intensity i s not very great. At this stage the larva is photopositive and exhibits no georesponse. As the light intensity increases the photoresponse may be dampened or shut off and a posi-tive georesponse takes over and directs the larva to the bottom of the pond. The degree to which one response dominates the other could be graded so that the larva did not suddenly move to the bottom of the pond but rather gradually moved to the bottom as the georesponse gained dominance. In this way, the larva would follow the host to a more suitable depth. The Type 8 response may work in the reverse of a Type 7 res-pose. If larva with a Type 9 response occur they should tend to distribute themselves randomly throughout the water and depend on accidental ingestion by the host. An alternative adaptation for larvae with a Type 9 response would be to suppress the free li v i n g stage and, in the case of miracidia, hatch only on being ingested 38. by the host. Eggs which settle on the bottom of the pond would be ingested by the host as i t foraged for food. Such an adaptation i s seen in Clonorchis sinensis and many members of the genus Opisthorchis (Schell, 1970). 39. SUMMARY 1. The effect of light, and to a lesser extent of gravity, on the dis-tribution of the two free-living larval stages (miracidia and cercariae) of the digenetic trematode Bunodera mediovitellata was investigated. 2. Miracidia exhibited a positive georesponse while a georesponse for cercariae was not conclusively shown. Light was the stronger stimulus directing the movement of miracidia when they were exposed to light and gravity in an antagonistic way. 3. Miracidia were shown to move away from white light at intensities greater than or equal to 0.35 lux. Cercariae moved toward white light at the same intensities. 4. Miracidia showed two peak responses to monochromatic light. The f i r s t peak at 550 nm. i s probably the wavelength which acts on the photopigment directly and results in the negative photoresponse. The second peak at 650 nm. may be caused by light passing through a screening pigment before reaching the photoreceptor. 5. The experimental results for miracidia and cercariae of B.mediovitellata supports the hypothesis that the behavioural res-ponses of free-living stages of trematodes to environmental stimuli increases their chance of host-parasite contact. 6. A theoretical table was constructed to show the possible combinations of photoresponse and georesponse which could occur in nature. 40. LITERATURE CITED Asch, H.L. and Drane, W., 1965. Schistosoma mansoni: Analysis of the kinetics of deather of parasitic organisms by employing a t t r i -tion of cercariae as a model. Expl. Parasit., 31: 284-289. Cable, R.M., 1972. Behaviour of digenetic trematodes. i n : Behavioural aspects of parasite transmission ed. E. U. Canning and C. A. 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