shed seven

Article available at http://www.parasite-journal.org or http://dx.doi.org/10.1051/parasite/1994014401
FASCIOLA HEPATICA : A STUDY OF THE SHEDDING OF CERCARIAE
FROM LYMNAEA TRUNCATULA RAISED UNDER CONSTANT CONDITIONS
OF TEMPERATURE AND PHOTOPERIOD
DREYFUSS G.* and RONDELAUD D.**
Summary :
Investigations on the shedding of cercariae of Fasciola hepatica
were carried out in Lymnaea truncatula in order to verify the existence of a low-frequency periodicity in the numerical distribution of
metacercariae per snail and per day when the snails are raised
under controlled conditions. Preadult L. truncatula were thus collected in the field, individually exposed to two miracidia, and subsequently raised until their death under constant temperature (20°C)
and photoperiod (12 h/12 h diurnal rhythm). The 102 snails shedding parasites produced 2 4 , 3 2 5 metacercariae of which 5 % were
floating cysts. The daily production of cercariae was maximal
during the first 3 0 days of the patent period, subsequently decreased until day 114, and ceased on day 124. No infradian-type
rhythm was noted in the daily distribution of mean values. The snails
shed their cercariae in one to 14 waves ; 2 0 . 6 % and 15.7 % of
the snails produced their parasites in four and five periods respectively. The authors suggest that the seven-day periodicity found by
Audousset et al. ( 1989) in the daily distribution of cercariae produced by three colonies of L. truncatula raised in seminatural conditions must be attributed only to the influence of environmental
factors.
Resume : FASCIOLA HEPATICA : ÉTUDE DES ÉMISSIONS CERCARIENNES
CHEZ LYMNAEA TRUNCALATA ÉLEVÉE SOUS DES CONDITIONS CONSTANTES DE
TEMPÉRATURE ET DE PHOTOPÉRIODE
Des observations sur les émissions cercariennes de Fasciola hepatica ont été réalisées chez Lymnaea truncatula afin de vérifier l'existence d'une périodicité avec une basse fréquence dans la
distribution numérique des métacercaires par mollusque et par jour
lorsque les limnées sont élevées sous des conditions contrôlées. Des
mollusques préadultes ont été ainsi prélevés sur le terrain, exposés
individuellement à deux miracidiums et maintenus par la suite
jusqu'à leur mort sous des conditions constantes de température
(20°C) et de photopériode(12heures diurnes). Les 102 limnées ont
fourni 24.325 métacercaires avec 5 % de kystes flottants,Laproduction journalière est maximale dans les 30 premiers jours de la
période patente et diminue ensuite jusqu'au 1 1 4 jour pour cesser
au 124 jour. Il n'y a pas de rythme de type infradien dans la distribution journalière des moyennes. Les mollusques ont émis leurs parasites sur 1 à 14 vagues, avec 20,6 % et 15,7 % des limnées
produisant leurs cercaires en quatre et cinq périodes. Les auteurs
suggèrent que la périodicité de sept jours trouvée par Audousset et
al. (1989) dans la distribution journalière des cercaires produites
par trois colonies de L. truncatula élevées dans des conditions semiKEY WORDS : cercaria. metacercaria. cercarial shedding. Fasciola hepanaturelles doit être attribuée à l'influence des seuls facteurs du
tica. lymnaea truncatula.
milieu.
MOTS CLES : cercaire. mélacercaire. émission cercarienne. Fasciola hepatica. Lymnaea truncatula.
C
e r c a r i a e o f Fasciola
hepatica
are shed
from
the host snail in o n e or several times during
the patent p e r i o d . Studies o n this s h e d d i n g
h a v e already b e e n carried out in different
Lymnaea
s p e c i e s b y several authors. Roberts ( 1 9 5 0 ) f o u n d that
the infected snails regularly s h e d their c e r c a r i a e o v e r
a w e e k a n d m o r e w h e r e a s Krull ( 1 9 3 3 ) , Kendall a n d
McCullough ( 1 9 5 1 ) , B o u i x - B u s s o n et al. ( 1 9 8 5 ) reported that c e r c a r i a l e m i s s i o n w a s d i s c o n t i n u o u s
shedding
waves
Audousset
et al.
separated
by periods
with
of rest.
( 1 9 8 9 ) confirmed the existence o f
t h e s e s h e d d i n g w a v e s b y studying
o f infected Lymnaea
truncatula
three p o p u l a t i o n s
that w e r e
subjected
t o v a r i a b l e daily t e m p e r a t u r e s r a n g i n g b e t w e e n 8 °
and 2 2 ° C. In addition, their o b s e r v a t i o n s d e m o n s t r a t e d that s h e d d i n g o c c u r r e d with an i n f r a d i a n - t y p e
* Laboratoire de Parasitologie, Faculté de Pharmacie.
** Laboratoire d'Histopathologie Parasitaire, Faculté de Médecine.
2 rue du Docteur Raymond Marcland. F-87025 Limoges Cedex.
Send correspondence to : Dr. D. Rondelaud, Faculté de Médecine,
87025 Limoges Cedex. Fax : 55.43.58.01.
Parasite, 1994, /, 401-404
e
e
rhythm with a periodicity o f s e v e n days. In view o f
these last results, the question arises w h e t h e r the characteristics o f shedding o f cercariae are related t o
e n v i r o n m e n t a l c h a n g e s ( e . g . temperatures or p h o t o p e r i o d ) or if they w e r e d u e to internal rhythm in L.
truncatula.
T o a n s w e r this question, w e thus studied
shedding in L. truncatula
c o l l e c t e d in the field a n d
raised u n d e r c o n s t a n t c o n d i t i o n s in o r d e r to a s s e s s
the part o f e n v i r o n m e n t a l d e t e r m i n i s m in s h e d d i n g
rhythms.
Preadult snails measuring 4 ± 0.2 m m in height w e r e
c o l l e c t e d in May a n d J u n e in t w o m e a d o w s at Le
Treuil in the c o m m u n e o f Limoges-Landouge, department of Haute-Vienne (France). This colony was
known
to b e devoid o f any trematode
infection
b e c a u s e o f regular sampling at t h e site a n d histological e x a m i n a t i o n in 7 0 % o f the snails c o l l e c t e d . T h e y
were transported
t o t h e l a b o r a t o r y in i s o t h e r m a l
c o n d i t i o n s a n d acclimatized for at least 4 8 hours in
standard aquaria b e f o r e b e i n g s u b j e c t e d to the e x p e riment. T h e eggs o f F. hepatica
Note de recherche
were collected
from
401
DREYFUSS G. and RONDELAUD D
Fig. 1. - Cercarial sheddings of F. hepatica from
truncatula : la) numerical distribution of snails with shedding during the patent
period; lb) numerical distribution of metacercariae per snail and per day during the patent period; 1c) correlogram of the cercarial shedding data in fig. lb. Autocorrelation coefficients are plotted at one day intervals.
Table I. Distribution of snails producing cercariae in relation to the number of waves of shedding.
402
Note de recherche
Parasite, 1994, 1, 401-404
CERCARIAL SHEDDINGS OF FASCIOLA HEPATICA FROM LYMNAEA TRUNCATULA
heavily infected cattle gallbladders at the slaughterh o u s e o f Limoges. T h e y w e r e incubated for 20 days
at 20° C in total darkness according to the m e t h o d
described by Ollerenshaw ( 1 9 7 1 ) .
T w o b a t c h e s w e r e constituted. Twenty-five controls
w e r e not e x p o s e d to the p a r a s i t e . T h e o t h e r 2 6 7
s n a i l s w e r e i n d i v i d u a l l y e x p o s e d in 3 5 - m m petri
dishes to two miracidia o f E. hepatica
for four hours
at 20° C. T h e y w e r e then raised in aquaria for 30
days, with five snails per liter o f water. T h e recipients
w e r e p l a c e d in an air-conditioned room under the
following conditions : a constant temperature of 20°
C, and an artificial light source of 3,000 lux intensity
at the surface of the aquaria in a 1 2 h / 1 2 h diurnal
r h y t h m . T h e s n a i l s w e r e f e d l e t t u c e ad
libitum
(Hourdin et al., 1993). On day 30, the 23 controls and
the 140 survivors from the experimental group were
individually monitored in 3 5 - m m petri dishes with 2-3
ml o f w a t e r p e r r e c i p i e n t and a p i e c e o f l e t t u c e
( 0 . 5 c m ) . T h e petri dishes w e r e kept in the s a m e
conditions as the aquaria. T h e y w e r e c h e c k e d daily in
the morning, b e t w e e n 10 and 12 a.m. for maximal
shedding of cercariae occurred during the night. T h e
water present in the dishes is then c h a n g e d a n d a
p i e c e o f lettuce is added if necessary until the first
emission of cercariae. At the onset o f shedding, the
daily surveillance w a s pursued, however, a count was
performed of the fixed metacercariae, floating cysts.
This protocol was followed until snail death.
2
T h e individual n u m b e r s o f m e t a c e r c a r i a e r e c o r d e d
daily from the infected snails were averaged and their
s t a n d a r d d e v i a t i o n s d e t e r m i n e d . T h e m e a n daily
values w e r e then analyzed by a method of detecting
activity rhythms ( B r o o m , 1 9 7 9 ; T h é r o n , 1981), such
as the autocorrelation test. W e have also studied the
n u m b e r of shedding waves during the patent period.
Each w a v e is characterized by the regular emission o f
10 cercariae at least during o n e or several days, and
is separated from the subsequent w a v e by an interw a v e o f o n e day or m o r e .
O n e - h u n d r e d - t w o snails shed parasites, yielding a freq u e n c y o f 7 4 %. Their post-mortem height was 5.7 ±
0.6 mm (as c o m p a r e d with 7.2 ± 1.2 mm in controls).
T h e patent period b e g a n on day 44 (± 7.8 days) and
lasted 4 6 ± 27.6 days. T h e total n u m b e r of metacercariae shed by all L. truncatula
w a s 2 4 , 3 2 5 of which
2 3 , 0 5 8 w e r e fixed and 1,267 w e r e floating, yielding a
percentage of 95 % and 5 % respectively.
There w e r e 102 snails which shed parasites (fig. l a ) on
the first day, and 56 on the second day. Their number
s u b s e q u e n t l y d e c r e a s e d until day 7 6 to four s n a i l s ;
afterwards, shedding involved o n e to four snails until
day 114. It was thereafter irregular and ceased on day
124.
Parasite, 1994, 1, 401-404
Changes in the mean numbers of metacercariae per
snail and per day are shown in figure l b . T h e values
were greater during the first 30 days of the patent
period and subsequently decreased until day 114 despite the p r e s e n c e of several isolated increases.
Subsequently the numbers w e r e low or zero until day
124.
Correlogram of fig. l c d o e s not s h o w any low-frequency periodicity in the production of F. hepatica cercariae. There was no infradian-type rhythm when the
L. truncatula
are raised under controlled conditions.
Table I lists the 102 snails in relation with the n u m b e r
o f shedding periods. Fourteen waves w e r e recognized in t h e s e snails. Four or five s h e d d i n g periods
were most frequently e n c o u n t e r e d (20.6 % and 15.7
% o f the snails respectively). Each of the other percentages did not e x c e e d 9 %.
T h e p r e s e n c e of an infradian-type rhythm in cercarial
shedding has already b e e n described in several trematode-bearing snails. Periods of high and low prod u c t i o n in the daily distribution o f c e r c a r i a e have
b e e n reported with a periodicity of 35 days and more
in three Schistosoma
species from Biomphalaria
glabrata ( T h é r o n , 1 9 8 1 ) , Bulinus
globosus
(Fryer a n d
Probert. 1988), B. truncatus
( K e c h e m i r and Théron,
1989), and Planorbarius
metidjeusis
(Mouahid and
T h é r o n . 1986, 1 9 8 7 ) . Another infradian-type rhythm
was also found by Audousset et al. ( 1 9 8 9 ) in the daily
d i s t r i b u t i o n o f F. hepatica
cercariae produced by
three colonies of L. truncatula
raised in seminatural
conditions (daily temperature varying b e t w e e n 8° and
22° C) but its periodicity w a s only seven days.
O u r data demonstrate the a b s e n c e o f an infradiantype rhythm in the emission of E. hepatica
cercariae
w h e n L. truncatula
is raised under constant conditions. However, the experimental design (short acclimatization o f snails a n d daily m a n i p u l a t i o n s ) may
interfere with biological rhythm so that our results
s h o u l d b e t a k e n as p r e l i m i n a r y . A n o t h e r r e p o r t
(Dreyfuss and Rondelaud, 1994) gives also the s a m e
result in L. tomentosa
infected b y the same trematode
and raised at 23° C. LJnder these conditions, there is a
discordance b e t w e e n these data and those reported
by Audousset et al. ( 1 9 8 9 ) . O n e explanation would
b e to attribute the rhythm found by Audousset et al.
to the experimental conditions used by these authors
during their observations on shedding o f cercariae. If
this assumption is true, then o n e or several external
factors may influence this periodicity (temperature,
l i g h t , . . . ) , as w a s p r e v i o u s l y d e m o n s t r a t e d d u r i n g
observations by Kendall and McCullough ( 1 9 5 1 ) , and
by P ê c h e u r ( 1 9 6 6 ) c o n c e r n i n g other characteristics o f
e m i s s i o n o f F. hepatica
c e r c a r i a e . Further o b s e r v a tions w e r e thus necessary to determine the nature o f
Note de recherche
403
DREYFUSS G. and RONDELAUD D.
the e n v i r o n m e n t a l f a c t o r ( s ) w h i c h might affect this
periodicity.
W h e n i n f e c t e d L. truncatula
are raised under
constant conditions, there is an increase in the number o f shedding waves (a total o f 1 4 ) as in the number o f snails w h i c h p r o d u c e their cercariae over
several periods. T h e s e results d o not c o n c o r d with
t h e n u m b e r o f w a v e s (five o r s e v e n ) r e p o r t e d b y
Audousset
et al. ( 1 9 8 9 ) in L, truncatula
or by
Dreyfuss and Rondelaud ( 1 9 9 4 ) in L. t o m e n t o s a (at a
constant temperature o f 2 3 ° C ) . T h e r e is likewise a
d i s c r e p a n c y in t h e f r e q u e n c y in w h i c h snails s h e d
their parasites in a single wave : 7.8 % in o u r study,
38.1 % and 5 0 % respectively for the previously cited
authors. T h e s e c h a n g e s in wave pattern c a n only b e
ascribed to the constant conditions and the lower
breeding temperature used during o u r experiment.
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Parasite, 1994, 7, 401-404