OF FASCIOLA GIGANTICA FROM LYMNAEA NATALENSIS

SEVERAL OBSERVATIONS CONCERNING CERCARIAL SHEDDINGS

OF FASCIOLA GIGANTICA FROM LYMNAEA NATALENSIS

RÉSUMÉ : LES ÉMISSIONS CERCARIENNES DE FASCIOLA GIGANTICA CHEZ LYMNAEA NATALENSIS. A PROPOS DE QUELQUES OBSERVATIONS.

INTRODUCTION

T

here are two rhythms o f cercarial shedding o f Fasciola hepatica from Lymnaea truncatula : a circadian rhythm o f parasite shedding during the night and an infradian rhythm with a periodicity o f 7 days (Audousset et al., 1 9 8 9 ) . T h e latter rhythm d o e s n o t o c c u r w i t h c e r c a r i a l s h e d d i n g s from F.

gigantica w h e n the parasites are shed from L. trun- catula, an abnormal intermediate host o f this n e m a - tode ( R a k o t o n d r a v a o and Rondelaud, 1991)·

In v i e w o f t h e s e results, the question arises w h e t h e r this infradian rhythm o c c L i r s with cercarial sheddings o f F. gigantica from its natural intermediate host in Africa , L. natalensis. Cercariae o f this trematode are already k n o w n for their shedding at night ( K w o et al., 1970 ; Albaret et al., 1 9 8 0 ) ; h o w e v e r , there are n o other data in the literature c o n c e r n i n g the e x i s t e n c e o f a n o t h e r rhythm. T h e p u r p o s e o f the present study w a s to a n s w e r this question by reporting results o f an e x p e r i m e n t a l infection o f L. natalensis b y F. gigan- tica u n d e r laboratory conditions.

* Laboratoire d'Histopathologie Parasitaire, Faculté de Médecine.

** and Laboratoire de Parasitologie, Faculté de Pharmacie, 2 rue du Docteur Raymond Marcland. F-87025 Limoges Cedex.

*** Département de Recherches Zootechniques et Vétérinaires, Division de Parasitologie, BP4, Tananarive, Madagascar.

Send correspondence to : Dr D. Rondelaud, Faculté de Médecine, F-87025 Limoges Cedex.

La production des cercaires par Lymnaea natalensis et leur transfor- mation en métacercaires sont pour la plupart nocturnes, avec 70.7 % des kystes dénombrés entre 19 heures et 1 heure. Il n'y a pas de rythme infradien dans la distribution numérique des moyennes journalières par rapport à la durée de l'expérience ou de la période patente. Un quart des limnées émettent leurs cercaires en une seule vague et les autres les produisent en 2 à 13 temps. Les kystes flottants représentent 35 % du nombre total des métacercaires et leur nombre diminue au cours des jours qui constituent les deux premières vagues d'émission. Des localisations aberrantes de méta- cercaires ont été notées à l'intérieur de la coquille lorsque le mol- lusque meurt.

MATERIALS AND METHODS

SNAILS AND PARASITE

The c o l o n y o f L. natalensis o r i g i n a t e d from natural watercress b e d s located at Ambanidia a n d at V o h i s a r i k a in t h e p r o v i n c e o f T a n a n a r i v e ( M a d a g a s c a r ) . T h e adults w e r e transpor- ted t o F r a n c e a n d p l a c e d in c o v e r e d a q u a r i a ( a t 23° C) w h e r e they laid eggs. T h e s e eggs gave rise to the 4-mm snails that w e r e used in this experiment.

T h e F. gigantica eggs w e r e regularly collected from h e a v i l y i n f e c t e d c a t t l e g a l l b l a d d e r s at t h e m a i n s l a u g h t e r h o u s e in T a n a n a r i v e ( M a d a g a s c a r ) . T h e y w e r e filtered through several sieves, w a s h e d , and pla- c e d in total darkness for 2 0 days at 20° C.

EXPERIMENTAL PROTOCOL

T h e 4 8 3 snails w e r e e a c h e x p o s e d for 4 hours to 2 F. gigantica miracidia in a 35-mm diameter petri dish (Falcon) with 2-3 ml o f water from the breeding contai- ner. Twenty-five snails that were not subjected to the parasites were used as controls. T h e snails were subse- q u e n t l y raised in c o v e r e d c l o s e d - c i r c u i t aquaria, 5 snails per liter o f water. T h e aquaria were placed in an air-conditioned room at 23° C. For 12 hours per day (7 a.m. to 7 p.m.), an electric light provided light o f 3,000 lux intensity at the surface o f the containers. T h e snails were fed fresh lettuce ad libitum.

O n the 30th day o f the e x p e r i m e n t , the 3 4 8 survivors and 25 controls w e r e isolated in 3 5 - m m petri dishes DA COSTA .C.*, DREYFUSS G.**, RAKOTONDRAVAO***, RONDELAUD D.*

Summary :

The shedding of cercariae from Lymnaea natalensis and their trans- formation into metacarcariae occurred mainly at night, and 7 0 . 7 % of the cysts were counted between 7 p.m. and 1 a.m.

There is no infradian rhythm in the numerical distribution of the daily mean values as related to experiment duration or patent per- iod duration. One quarter of the snails shed their cercariae in a single wave and the others in 2 to 13 waves. Floating cysts repre- sent 3 5 % of the total number of metacercariae and their number decreased during the two first waves of shedding. Aberrant meta- cercarial localizations were noted on the inner surface of the shell when the snail died.

KEY WORDS : Cercaria. Cercarial shedding. Fasciola gigantica. Lymnaea natalensis. Metacercaria.

MOTS CLES : Cercaire. Emission cercarienne. Fasciola gigantica. Lymnaea natalensis. Métacercaire.

Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1994011039

DA COSTA С , DREYFUSS G., R A KOTO ND RA V AO, RONDELAIJD D.

Fig. 1. - Cercarial sheddings of F. gigantica

i) the number of survivors over time (la) and snails which shed cercariae (lb) as related to experiment duration ; ii) daily distribution of cercariae from the beginning of the experiment (lc) and that of the patent period (Id).

4 0

R C A R I A L S H E D D I N G S O F FASCIOLA GIGAMJCA F R O M LYMNAMA NATA

with 2-3 ml of water and a piece o f lettuce (0,5 c m²) per recipient. T h e dishes were placed in the same air-condi­

tioned room where the original colony was raised. Each day the snails were monitored from 2 p.m. to 4 p.m., during which time the water was changed and maintai­

ned at the same temperature, the lettuce replaced, and the fixed or floating metacercariae counted until snail death. At this time the shell height was measured.

A s p e c i a l c o u n t w a s p e r f o r m e d e v e r y s i x h o u r s (1 a.m.. 7 a.m., 1 p.m., 7 p.m.) for 15 days to deter­

mine the time o f maximal shedding.

T w e n t y floating cysts w e r e immersed in B o u i n ' s fixa­

tive for histological study. Serial sections, 5 pm thick, w e r e obtained and stained using Harris' hematoxylin and modified G a b e ' s trichrome.

PROCESSING OF DATA

T h e v a l u e s o b t a i n e d from t h e total m e t a c e r c a r i a l counts w e r e averaged and standard deviations deter­

m i n e d for e a c h o b s e r v a t i o n d a t e , s p e c i f y i n g t h e c o l o n y utilized and the particular day o f the patent period. T h e m e a n daily values w e r e then subjected to the autocorrelation test ( B r o o m , 1 9 7 9 ) to determine w h e t h e r there was periodicity in cercarial shedding.

A w a v e o f cercarial shedding was defined as a period w h e r e at least 10 c e r c a r i a e w e r e s h e d o v e r o n e or several successive days without interruption. T h e c h a ­ racteristics o f these waves o f shedding w e r e studied by Anova (Stat-Itcf, 1 9 8 8 ) .

RESULTS

GENERAL CHARACTERISTICS OF INFECTION

2

0 6 snails s h e d cercariae and the other 142 snails died without emitting parasites. In snails that s h e d p a r a s i t e s , t h e life s p a n w a s 9 8 . 9 ± 30.7 days : in snails without cercarial shedding it w a s 66.7 ± 2 3 . 4 days, and 132.4 ± 3 1 . 5 days in controls.

T h e post-mortem shell height was respectively 8.6 ± 1.5 mm, 8 + 2 mm, and 12.4 ± 2.7 mm.

In snails that s h e d cercariae, the m e a n day o f o n s e t o f the patent period w a s day 57 o f the experiment, with individual v a l u e s r a n g i n g from day 3 2 to day 1 0 5 . The duration o f the patent period w a s 41 ± 3 0 . 9 days, with e x t r e m e s o f 1 and 135 days. A total n u m b e r o f 3 5 , 4 5 6 cercariae was o b t a i n e d o f w h i c h 6 4 . 9 % o f the cysts w e r e fixed o n substrate, 3 5 % w e r e floating, and 0 . 0 5 9 % w e r e found free o n the floor o f the recipient.

PERIODICITY OF SHEDDING

There w e r e 2 0 6 survivors until day 4 6 o f the experi­

ment, after which the n u m b e r o f survivors gradually

decreased until day 1 3 1 . T h e numbers leveled off from day 131 to day 143 (at 22-24 snails) ; the last snails died on day 175 (Fig. l a ) . Until day 4 5 , there were no more than 10 snails that shed cercariae ; this number then increased to a maximum b e t w e e n days 6 8 and 79 (up to 77 snails on day 7 4 ) . Subsequently, the number d r o p p e d b e l o w 10 snails per day from day 122 and c e a s e d o n day 165 w h e n shedding stopped (Fig. l b ) . The distribution o f cercariae throughout the experiment was irregular with daily peaks which did not e x c e e d 4 0 parasites per snail with sometimes very wide standard d e v i a t i o n s . M e a n m a x i m a l v a l u e s o c c u r e d b e t w e e n days 62 and 67 o f the experiment (18-23 parasites per snail per day) and subsequently decreased progressi­

vely until shedding stopped on day 162 (Fig. l c ) . Examination o f the m e a n daily values in relation to the patent period (Fig. I d ) revealed that the highest m e a n v a l u e s o c c u r e d b e t w e e n d a y 2 a n d day 11 (with 9 to 17 cysts per snail per day) ; they subse­

quently d e c r e a s e d progressively until they c e a s e d on day 112 o f the patent period despite the survival o f several snails until day 135.

Analysis o f these m e a n daily values using the auto­

c o r r e l a t i o n test did n o t d e m o n s t r a t e an infradian rhythm in the distribution o f m e a n values as related to e x p e r i m e n t d u r a t i o n o r d u r a t i o n o f t h e p a t e n t period (results not s h o w n ) .

T h e majority o f the cercariae w e r e shed throughout t h e n i g h t ( 7 0 . 7 % d u r i n g t h e first half, 7 p . m . to 1 a.m. ; 2 0 . 4 % during the s e c o n d half). During the day the p e r c e n t a g e s w e r e respectively 1 and 7.9 %·

WAVES OF SHEDDING

Twenty-five percent o f L. natalensis shed their cercariae in a single wave. Most o f these snails died a few days after the onset o f the patent period, while the others died after longer periods (after day 115 and more).

S e c o n d , third, and fourth w a v e s o c c u r r e d respectively in 18.2 %, 16.5 % and 10.6 % o f the snails. T h e remai­

ning snails ( 2 9 . 4 % ) s h e d their parasite in 5 to 13 w a v e s (results not s h o w n ) .

M e a n w a v e duration w a s 2.7 to 4 . 9 days. T h e first interwave period was l o n g e r than s u b s e q u e n t o n e s : 4.6 to 10.9 days instead o f 2.3 to 7.1 days (Fig. 2 a ) . T h e n u m b e r o f cysts per w a v e did not e x c e e d o n e hundred and often ranged b e t w e e n 5 0 and 7 0 meta­

c e r c a r i a e (Fig. 2 b ) . Fluctuation o f t h e m e a n values w e r e o b s e r v e d a c c o r d i n g t o t h e n u m e r i c a l w a v e order. Comparison o f these m e a n values using Anova did not d e m o n s t r a t e any significant difference bet­

w e e n the durations o f waves, those o f interwaves, as well as the cercariae production per w a v e .

T h e c h r o n o l o g y o f these waves was studied in snails t h a t s h e d c e r c a r i a e in 3 a n d 4 w a v e s ( F i g . 3 ) .

>Λ COSTA С DRF.YFl ISS ι . RAK< >T< INI >R W Л' ). Ri >N1 HI \l 1*1»

C e r c a r i a l s h e a d i n g s w e r e i r r e g u l a r o v e r t i m e . T h e w a v e s f o l l o w e d e a c h o t h e r to o n e - d a y intervals o r greater lengths o f time.

FLOATING CYSTS

180 snails (87.3 % ) shed cercariae which b e c a m e floa­

ting cysts. T h e s e cysts represented 35 % o f the total number o f metacercariae ; however, there was substan­

tial individual variation, and if only snails that shed 500 parasites or m o r e are c o n s i d e r e d , the minimum and m a x i m u m p e r c e n t a g e s w e r e r e s p e c t i v e l y 4 . 5 % and 81 % (with 8 2 7 floating cysts out o f a total o f 1,014).

T h e s e cysts float due to the p r e s e n c e o f an outer ring that is f o r m e d b y the outer layer o f the external wall

Fig. 2. - Characteristics of waves of shedding : i) wave and interwave duration in numerical order (2a) ;

ii) the number of metacercariae per wave (2b). The first interwave (*) corresponds to the period from day 1 of the patent period to the onset of the first wave of shedding. The results are presented in relation with a) the wave number per snail group, and b) the numerical wave order.

of the cyst and contains air-filled l a c u n a e w h i c h dis­

sociate the fibrils o f this layer. T h e three other layers are not involved in this ring formation. T h e total dia­

m e t e r w a s 3 4 6 ± 4 2 . 5 μ η ι a n d t h e h e i g h t w a s 1 2 8 . 4 ± 2 8 . 6 p m o n t h e serial s e c t i o n s . T h e ring's r a d i u s w a s 6 0 . 3 ± 2 8 . 4 μηι a n d its t h i c k n e s s w a s 16.3 ± 4 . 8 p m at the b a s e o f the cyst.

If the n u m b e r o f floating and fixed cysts that w e r e col­

l e c t e d during t h e first t w o w a v e s o f s h e d d i n g are considered (Fig. 4 ) , it was found, despite several diver­

gent figures in the percentages over time, that the floa­

ting cysts b e c a m e less and less numerous during the first w a v e : from 4 9 . 8 % o n the first day to 7.1 % on the tenth (Fig. 4 a ) . Inversely, the p e r c e n t a g e o f fixed cysts i n c r e a s e d in a c o r r e s p o n d i n g m a n n e r . T h e r e w e r e similar findings for the s e c o n d w a v e o f shedding (Fig. 4 b ) : a similar decrease occurred, with only 12 % o f f l o a t i n g c y s t s o n t h e t e n t h d a y o f t h e w a v e . E x a m i n a t i o n o f the other w a v e s also s h o w e d a n u m e ­ rical d e c r e a s e in floating cysts, h o w e v e r , t h e r e w a s g r e a t e r v a r i a t i o n o f t h e results b e g i n n i n g with t h e fourth w a v e o f shedding (results not s h o w n ) .

Fig. 3· - Distribution of the waves of shedding and their duration in snails which shed cercariae in 3 waves (3a) or 4 waves (4b).

4 2

CERCARIA! SHEDDINGS OF FASCIOLA аюлтсл FROM LYMNAEA NATAŒNSIS

Fig. 4. - Distribution of floating and fixed cysts in relation with the shedding day for the first wave of shedding (4a) and the second (4b).

ABNORMAL LOCALIZATIONS OF CYSTS

With snail death, abnormalities in the formation and l o c a l i z a t i o n o f t h e m e t a c e r c a r i a e w e r e o b s e r v e d . Many fixed cysts w e r e found o n the inner surface o f the shell near the peristome. Rarely, other m e t a c e r c a - riae had implanted o n the b o t t o m o f the petri dish, in the immediate vicinity o f the dead snail. Occasionally, dead cercariae w e r e found.

21 cysts w e r e f o u n d o n t h e b o t t o m o f t h e d i s h e s . T h e y did not adhere to the wall ( o r to faeces), and did not p o s s e s s a ring.

DISCUSSION

T

he m e a n number o f metacercariae obtained for e a c h L. natalensis ( 1 7 2 . 1 ) c o r r e s p o n d s to the r a n g e o f v a l u e s r e p o r t e d b y C h e r u i y o t a n d

W a m a e (1990) with a Kenyan colony o f the same snail w h e n it was infected by F. gigantica (68.4 per snail infected in the laboratory, 103 to 385.5 w h e n it is natu- rally infected). On the other hand, there was a discre- p a n c y with o u r results c o n c e r n i n g t h e n u m b e r o f floating cysts. Our percentage (35 % ) corresponds with that reported by Rakotondravao and Rondelaud (1991) in L. truncatula that was infected by the same trema- tode (31.6 % floating cysts) ; however, it was definiti- vely h i g h e r than the figures cited by Albaret et al.

(1980), and Cheruiyot and W a m a e (1990) w h o repor- ted respectively 25 and 1.6 %. O t h e r authors such as Alicata (1953) or H a m m o n d (1974) did not c o m m e n t o n any w i d e variation. T w o probably c o m p l e m e n t a r y h y p o t h e s e s c a n e x p l a i n this d i s c r e p a n c y . T h e first confirms the a p p r o x i m a t i v e p e r c e n t a g e value obtai- n e d b y R a k o t o n d r a v a o a n d R o n d e l a u d (1991) and suggests that these experimental conditions are parti- cularly favourable for the formation o f floating cysts.

T h e s e c o n d e m p h a s i z e s the individual variability bet- w e e n t h e s n a i l s e m p l o y e d o r e v e n c o l o n i e s a s demonstrated by the p e r c e n t a g e s obtained by Esclaire et al. (1989) in three c o l o n i e s o f L. truncatula infec- ted b y F. hepática (1.4 to 8.2 % floating cysts).

T h e majority o f sheddings occurs at night, with most parasites being shed between 7 p.m. and 1 a.m. This first result concords with the observations o f Kwo et al.

(1970), Albaret et al. (1980), and Salimov and Azimov (1983). On the other hand, the time o f shedding differs from that reported by Rakotondravao and Rondelaud (199D, where the latter stated that shedding predomi- nantly occurred around 1 or 2 a.m. This discordance can not b e explained by experimental conditions since they were identical, neither by the malagasy strain o f the trematode which was also identical. It is thus logi- cal to seek another explanation. T w o hypotheses have b e e n provided by authors studying schistosomes and their snail hosts. T h e first hypothesis involved the role played by the species o f pulmonate snail utilized, sug- gesting that the latter may control to a degree the shed- ding o f its parasite (Asch, 1972 ; Anderson et al, 1976).

T h e s e c o n d h y p o t h e s i s states that shedding is only parasite dependent, at least under normal environmen- tal conditions. A tremadote's cercariae retain their own rhythm o f shedding, regardless o f the compatible snail host that it parasitizes (Mouahid and T h e r o n , 1986 : Mouahid et al, 199D. Additional studies are therefore necessary to determine the role played by the snail in the shedding o f its cercariae.

T h e a b s e n c e o f an infradian rhythm in daily s h e d - dings differs from that d e s c r i b e d b y Audousset et al.

(1989) c o n c e r n i n g s h e d d i n g o f F. hepática f r o m L. truncatula. This d i s c r e p a n c y must b e interpreted cautiously. It is i n d e e d p o s s i b l e to regard this as a s p e c i f i c result o f o u r t r e m a t o d e . H o w e v e r , a n o t h e r working hypothesis c a n not b e dismissed. L. natalen-

DA COSTA С , DREYFUSS G., RAKOTONDRAVAO, RONDELAUD D.

sis has, in fact, b e e n recognized by Boray ( 1 9 7 8 ) as a species that has a normal parasite-host relationship. It should b e remembered, however, that F. gigantica has only recently b e e n detected into Madagascar (Daynès, I 9 6 0 ) , and that infection due to the parasite has spread over the entire island and has b e c o m e a major problem to the livestock industry (Touratier, 1988). T h e question arises whether the a b s e n c e o f rhythm represents an, as yet, incomplete compatibility b e t w e e n F. gigantica and the snail populations, despite the present frequency o f this epizootic disease. This hypothesis is supported by Rondelaud ( 1 9 9 3 ) w h e n he observed that the characte- ristics o f Fasciola infection are modified in populations of L. truncatula when the frequency o f natural encoun- ters with F. hepática is not the same.

The presence o f many floating cysts at the beginning o f each wave was a n e w finding. No hypothesis can b e put forth to explain this finding especially since most of the experimental factors did not influence floating cyst formation (Vareille-Morel and Rondelaud, 1 9 9 1 ) . Throughout our study, no dead larvae w e r e observed after leaving a live snail or during cyst formation. In view o f these observations, the hypothesis put forth by the latter authors concerning lesions that arise in cerca- riae which form these cysts during passage through the perianal region o f the snail does not appear to apply to the results obtained with F. gigantica.

T h e finding o f cysts fixed to the inside o f the shell when the snail died requires another explanation. T h e presence o f dead cercariae at that moment and/or rare cysts in the immediate vicinity o f the snail suggests that the larvae have "expended" all their energy migrating through the snail's tissues which partially retract at the time o f death, and no longer have the strength to swim and attach to their usual substrate. T h e p r e s e n c e o f many cercariae b e l o w the mantle wall at the time o f the snail's death also supports this hypothesis.

ACKNOWLEDGEMENTS

T

h e co-author from Madagascar (Rakotondravao) is grateful to G T Z - P E P A for sponsoring his par- ticipation to the work.

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Accepté le 2 janvier 1994

44 Mémoire