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The Stress of Lymnaea truncatula Just before Miracidial Exposure with Fasciola hepatica Increased the Prevalence of Infection
Experimental Parasitology 99, 49–51 (2001) doi:10.1006/expr.2001.4609, available online at http://www.idealibrary.com on
RESEARCH BRIEF The Stress of Lymnaea truncatula Just before Miracidial Exposure with Fasciola hepatica Increased the Prevalence of Infection
M. Abrous,*,† D. Rondelaud,* and G. Dreyfuss† Laboratoire d’Histopathologie Parasitaire, Faculte´ de Me´decine, and †Laboratoire de Parasitologie, Faculte´ de Pharmacie, 2, rue du Docteur Raymond Marcland, 87025 Limoges Cedex, France
Abrous, M., Rondelaud, D., and Dreyfuss, G. 2001. The stress of Lymnaea truncatula just before miracidial exposure with Fasciola hepatica increased the prevalence of infection. Experimental Parasitology 99, 49–51. Single-miracidium infections of Lymnaea truncatula with Fasciola hepatica were carried out under laboratory conditions to determine whether the stress of snails just before miracidial exposure had any influence on the prevalence of Fasciola infection, redial burden, and cercarial shedding. Three methods, i.e., the fasting of L. truncatula for 3 days in water filtered through a Millipore membrane, the effect of 6–8⬚C water for 15 min, or the immersion of L. truncatula in a detergent solution at low concentration for 15 min, were used to stress snails. Enhanced susceptibility of snails to F. hepatica infection was noted in stressed groups (93–96% vs 48–50% in controls). The number of free rediae did not show any variation in controls as well as in stressed groups, except for fasted snails in which free rediae were significantly fewer. No differences in cercarial production between controls and the cold group were noted. Fasting, cold shock, or detergent exposure prior to exposure to F. hepatica miracidia might have weakened the snails so that they were not as efficient in avoiding miracidial penetration, thus leading to higher infection rates. 䉷 2001 Academic Press Index Descriptors and Abbreviations: Fasciola hepatica; Lymnaea truncatula; cercaria; miracidium; prevalence; stress.
The physiologic state of snails before miracidial exposure may have consequences on trematode larval development. Using the system Lymnaea truncatula/Fasciola hepatica, Rondelaud (1994, 1995) demonstrated that the infection rate in snails and the redial burden were significantly lower if miracidial exposure occurred on day 2 following the end of an experimental desiccation or followed treatment with a sublethal dose of cupric chloride. In contrast, the consequences of these factors were limited if miracidial exposure occurred on day 9 after physical or chemical injury. The role of snail physiologic state on the success of infection was also revealed by dual infections of L. truncatula with two different trematodes. If the time interval between the exposure of snails to Paramphistomum daubneyi and the exposure to F. hepatica was less than 4 h, the prevalence of infection with the former trematode, the latter, or both was high (Chipev et al. 1985; Augot et al. 1996). In contrast, if the time between exposures was 24 h or more, the larval development of the former trematode often inhibited that of the latter (Chipev et al. 1985). In view of successful dual infections of L. truncatula when short times before dual exposures were used, one might wonder what the consequences of snail stressing were on the success of trematode infection when physical or chemical injury of snails occurred just before miracidial exposure. To answer this question, an experiment was performed by subjecting L. truncatula to a stress (of variable type) just before each snail was exposed to a single miracidium of F. hepatica and by studying the redial burden and cercarial shedding. The population of L. truncatula was living in a road ditch at Masvaudier, commune of Courcelles, Department of Creuse, central France. A total of 1000 preadult snails, 4 ⫾ 0.2 mm tall, were collected in June from this population and kept for 48 h at room temperature (20⬚C) before the experiment. Eggs of F. hepatica were collected from the gallbladders of heavily infected cattle. They were incubated for 20 days at 20⬚C, in complete darkness.
The success of a trematode infection in a freshwater snail is dependent on at least three principal factors: the age of the snail at exposure, the temperature of the water in which the miracidium and the snail are located, and snail nutrition, which determines a more or less fast growth of the shell throughout infection (Smyth and Halton 1983). However, all factors influencing snail infection are not still known.
0014-4894/01 $35.00 Copyright 䉷 2001 by Academic Press All rights of reproduction in any form reserved.
49
50
ABROUS, RONDELAUD, AND DREYFUSS
TABLE I Characteristics of Infection in Controls and in Three Groups of Lymnaea truncatula Subjected to a Stress before Miracidial Exposure with Fasciola hepatica (1 Miracidium/Snail) Snails infected by F. hepatica and dissected at day 30 pe Parameter
Control group
Fasted group
Cold-treated group
Detergent-treated group
No. of snails at exposure No. of surviving snails at day 30 pe No. of snails harboring larval forms at day 30 pe Prevalence of infection (%)a No. of free rediae per infected snail (means ⫾ SD)
100 60 30 50.0 24.8 ⫾ 5.9
100 65 63 96.9 15.4 ⫾ 6.7
100 91 84 92.3 24.0 ⫾ 5.1
100 89 85 95.5 26.4 ⫾ 6.2
a
Prevalence of infection (number of snails harboring larval forms/number of surviving snails at day 30 postexposure).
Four groups of 100 snails each were used to study the effect of stress on the survival of uninfected snails at day 30 postexposure (pe). Snails from the first group were not subjected to stress (unstressed and uninfected controls), whereas snails from the three other groups were subjected to a 3-day fast in spring water, sterilized and filtered through a 3-m Millipore membrane (fasted and uninfected controls); exposure to cold water at 6–8⬚C for 15 min (cold-treated and uninfected controls); or exposure to a detergent solution (Paic Citron, Colgate-Palmolive, Courbevoie, France) at low concentration (4 drops in 3 L of spring water) for 15 min (detergent-treated and uninfected controls). Snails were subsequently raised for 30 days in polypropylene boxes 1 m by 55 cm and 15 cm high, with 50 snails per box (Augot et al. 1996). Boxes were maintained under constant conditions of 20⬚C ambient temperature and diurnal photoperiod of 3000- to 4000-lux light intensity. At day 30 pe, the survivors from each group were counted. Four other groups of 100 snails each were used to study the effects of stress on the redial burden of F. hepatica in L. truncatula. One group of snails was not subjected to stress prior to single-miracidium infections (infected controls). In contrast, in the other three groups, the protocol for stressing snails was similar to that used for the corresponding three groups of controls. As the cold or detergent treatments had a temporary narcotizing effect on L. truncatula, at the end of the stress period the snails from the three groups were placed in normal spring water, without food for 1 h at 20⬚C, before each was subjected to a single miracidium of F. hepatica for 4 h in a 35-mm-diameter petri dish (fast, cold, and detergent groups of infected snails). At day 30 pe, the surviving L. truncatula from these four groups were dissected to determine the number of snails harboring F. hepatica larval forms and to count free rediae. The last two groups of 100 snails each were used to study the cercarial shedding of F. hepatica. Snails from the first group were subjected only to single-miracidium infections (control group), while the L. truncatula from the other group were subjected to cold water at 6–8⬚C for 15 min before each was exposed to a single miracidium (cold group). Controls and stressed snails were raised for 30 days at 20⬚C using the aforementioned protocol. At day 30 pe, each surviving snail was placed in a 35-mm-diameter petri dish, containing 2–3 mL of spring water and a piece of steeped lettuce. These dishes were maintained at 20⬚C. Each day, a metacercarial count was performed and the water in the dish was changed until snail death. Routine postmortem dissection of snail cadavers was performed to distinguish uninfected snails (without larval forms) from infected snails that died without shedding.
The parameters studied were the survival rate of snails at day 30 pe, the prevalence of Fasciola infection, the number of free rediae per snail dissected at day 30 pe, the length of time between exposure and the first cercarial shedding, the duration of shedding, and the total number of cercariae produced by each snail. Comparison tests of experimental frequencies and one-way analysis of variance were used to establish levels of significance. Snail survival at day 30 pe was high in the first four groups of uninfected controls. It was 99% in unstressed controls, 84% in fastfed controls, 99% in cold-treated controls, and 98% in detergent-treated controls (data not shown). No significant difference between these survival rates was noted, whatever the mode of comparison. Table I gives the results found in the groups of infected snails when dissected at day 30 pe. The survival rates recorded in the cold and detergent groups were significantly higher (P ⬍ 0.001) than those from the control and fasting groups. In contrast, there were no significant differences between the former rates (cold and detergent groups) and the latter percentages. The prevalence of infection recorded in controls was significantly lower (P ⬍ 0.001) than those from the three groups subjected to stress. The redial burden in the fasted group was significantly lower (F ⫽ 4.33, P ⬍ 0.01) than the redial burdens from the three other groups. In contrast, there were no significant differences in the redial burdens of F. hepatica between controls, the cold group, and the detergent group. Table II gives the results found in snails studied for the cercarial shedding of F. hepatica. Snail survival rate at day 30 pe was significantly higher (P ⬍ 0.001) in the cold group than in controls. A similar finding was recorded for the prevalence of infection, which was significantly higher (P ⬍ 0.001) in the former group. The time between exposure and the first cercarial shedding, the duration of the shedding period, and the mean number of cercariae shed per snail did not show any significant variation between the two groups. The only difference concerned the total number of cercariae per group: it was higher in the cold group, as the number of cercaria-shedding snails was greater in the former group. The survival rates found in the control and fasted groups of infected snails fall within the ranges of percentages that Rondelaud and Barthe (1982, 1987) had already reported for the survival of L. truncatula at day 30 pe when subjected to single-miracidium infections with F. hepatica. The increased survival found in the cold- and detergenttreated groups of infected snails was more difficult to interpret and two perhaps complementary explanations might be proposed to interpret this result. The first was the nature of these two factors and the
51
STRESS OF Lymnaea truncatula AND INFECTION
TABLE II Characteristics of Infection in Controls and in One Group of Lymnaea truncatula Subjected to a Stress before Miracidial Exposure with Fasciola hepatica (1 Miracidium/Snail) Parameter
Control group
Cold group
No. of snails at exposure No. of surviving snails at day 30 pe No. of infected snails that died without sheddinga No. of cercaria-shedding snails Prevalence of infection (%)b Time between exposure and the first cercarial shedding (days): means ⫾ SD Duration of shedding period (days): means ⫾ SD
100 64 7 24 48.4 57.3 ⫾ 8.2 17.1 ⫾ 4.3
100 88 25 60 96.5 59.4 ⫾ 5.3 20.2 ⫾ 6.3
Total number of cercariae produced by the snails from the group No. of cercariae per cercaria- shedding snail: means ⫾ SD
2168 90.3 ⫾ 76.3
5976 99.6 ⫾ 64.8
a
Dissection of cadavers just after the death of snails. Prevalence of infection (number of cercaria-shedding snails ⫹ number of infected snails that died without shedding/number of surviving snails at day 30 postexposure). b
conditions under which the snails were subjected to stress, which were aggressive for snails, as demonstrated by the survival rates of corresponding uninfected controls and those given in Table I. The second was the better capacity of snails to ensure the larval development of F. hepatica when the stress occurred just before miracidial exposure. If the latter explanation might be considered valid, this better capacity would occur in the first 2 weeks pe, as the highest mortality rate of infected controls was noted during this period. Enhanced susceptibility of L. truncatula to infection was noted in the three stressed groups of infected snails when the time interval between snail stress and miracidial exposure was short. To explain this result, the most valid hypothesis would be to assume that starvation, cold shock, or detergent exposure had weakened snails so that they were not efficient at avoiding miracidial penetration and subsequent development of sporocysts. However, an indirect effect of these factors on the snail’s internal defense system via a temporary inhibition cannot be excluded. The number of F. hepatica free rediae did not show any significant variation in controls as in stressed groups, except for snails subjected to a 3-day fast in which free rediae were fewer. The limitation in the size of the redial burden in the latter snails agrees with the reports by Rondelaud (1994): according to this author, the number of free rediae clearly decreased in snails subjected to a 10-day desiccation before infection (18–25 rediae per snail at day 30 pe vs 43 rediae per snail in infected controls). This decrease in redial burden must be considered a consequence of snail fasting, as the nutriments within the snail were scarce and, consequently, induced a delay in the larval development of F. hepatica. In contrast, except for the total number of cercariae shed from infected snails, the other characteristics of cercarial shedding were the same in controls as in the cold group. The stress applied to snails just before miracidial exposure had no influence on the cercarial shedding of F. hepatica, with the probable exception of snails subjected to fasting, because the number of free rediae was lower than in the other groups of stressed snails. It is difficult to determine what biological significance snail stressing may have in the field in the interaction of L. truncatula and F. hepatica. In contrast, under laboratory conditions, a cold shock applied just before miracidial exposure will be useful in the experimental production
of F. hepatica metacercariae, as it will increase the prevalence of experimental infection and, subsequently, the total number of cercariae shed from infected snails.
REFERENCES Augot, D., Abrous, M., Rondelaud, D., and Dreyfuss, G. 1996. Paramphistomum daubneyi and Fasciola hepatica: The redial burden and cercarial shedding in Lymnaea truncatula submitted to successive unimiracidial cross-exposures. Parasitology Research 82, 623–627. Chipev, N., Vassilev, I., and Samnaliev, P. 1985. Interactions between Paramphistomum c.f. daubneyi Dinnik, 1962 and Fasciola hepatica L. in successive cross-invasions of Lymnaea (Galba) truncatula. Helminthologia 20, 80–88. Rondelaud, D. 1994. Fasciola hepatica: The infection rate and the development of redial generations in Lymnaea truncatula exposed to miracidia after experimental desiccation and activation in water. Journal of Helminthology 68, 63–66. Rondelaud, D. 1995. The characteristics of redial generations in Lymnaea truncatula exposed to Fasciola hepatica miracidia after poisoning by sublethal doses of cupric chloride. Veterinary Research 26, 21–26. Rondelaud, D., and Barthe, D. 1982. Les ge´ne´rations re´diennes de Fasciola hepatica L. chez Lymnaea truncatula Mu¨ller. A propos des effets de plusieurs facteurs. Annales de Parasitologie Humaine et Compare´e 57, 245–262. Rondelaud, D., and Barthe, D. 1987. Fasciola hepatica: e´tude de la productivite´ d’un sporocyste en fonction de la taille de Lymnaea truncatula. Parasitology Research 74, 155–160. Smyth, J. D., and Halton, D. W. 1983. “The Physiology of Trematodes.” Cambridge Univ. Press, Cambridge, UK. Received 12 October 1998; accepted with revision 13 February 2001
RESEARCH BRIEF The Stress of Lymnaea truncatula Just before Miracidial Exposure with Fasciola hepatica Increased the Prevalence of Infection
M. Abrous,*,† D. Rondelaud,* and G. Dreyfuss† Laboratoire d’Histopathologie Parasitaire, Faculte´ de Me´decine, and †Laboratoire de Parasitologie, Faculte´ de Pharmacie, 2, rue du Docteur Raymond Marcland, 87025 Limoges Cedex, France
Abrous, M., Rondelaud, D., and Dreyfuss, G. 2001. The stress of Lymnaea truncatula just before miracidial exposure with Fasciola hepatica increased the prevalence of infection. Experimental Parasitology 99, 49–51. Single-miracidium infections of Lymnaea truncatula with Fasciola hepatica were carried out under laboratory conditions to determine whether the stress of snails just before miracidial exposure had any influence on the prevalence of Fasciola infection, redial burden, and cercarial shedding. Three methods, i.e., the fasting of L. truncatula for 3 days in water filtered through a Millipore membrane, the effect of 6–8⬚C water for 15 min, or the immersion of L. truncatula in a detergent solution at low concentration for 15 min, were used to stress snails. Enhanced susceptibility of snails to F. hepatica infection was noted in stressed groups (93–96% vs 48–50% in controls). The number of free rediae did not show any variation in controls as well as in stressed groups, except for fasted snails in which free rediae were significantly fewer. No differences in cercarial production between controls and the cold group were noted. Fasting, cold shock, or detergent exposure prior to exposure to F. hepatica miracidia might have weakened the snails so that they were not as efficient in avoiding miracidial penetration, thus leading to higher infection rates. 䉷 2001 Academic Press Index Descriptors and Abbreviations: Fasciola hepatica; Lymnaea truncatula; cercaria; miracidium; prevalence; stress.
The physiologic state of snails before miracidial exposure may have consequences on trematode larval development. Using the system Lymnaea truncatula/Fasciola hepatica, Rondelaud (1994, 1995) demonstrated that the infection rate in snails and the redial burden were significantly lower if miracidial exposure occurred on day 2 following the end of an experimental desiccation or followed treatment with a sublethal dose of cupric chloride. In contrast, the consequences of these factors were limited if miracidial exposure occurred on day 9 after physical or chemical injury. The role of snail physiologic state on the success of infection was also revealed by dual infections of L. truncatula with two different trematodes. If the time interval between the exposure of snails to Paramphistomum daubneyi and the exposure to F. hepatica was less than 4 h, the prevalence of infection with the former trematode, the latter, or both was high (Chipev et al. 1985; Augot et al. 1996). In contrast, if the time between exposures was 24 h or more, the larval development of the former trematode often inhibited that of the latter (Chipev et al. 1985). In view of successful dual infections of L. truncatula when short times before dual exposures were used, one might wonder what the consequences of snail stressing were on the success of trematode infection when physical or chemical injury of snails occurred just before miracidial exposure. To answer this question, an experiment was performed by subjecting L. truncatula to a stress (of variable type) just before each snail was exposed to a single miracidium of F. hepatica and by studying the redial burden and cercarial shedding. The population of L. truncatula was living in a road ditch at Masvaudier, commune of Courcelles, Department of Creuse, central France. A total of 1000 preadult snails, 4 ⫾ 0.2 mm tall, were collected in June from this population and kept for 48 h at room temperature (20⬚C) before the experiment. Eggs of F. hepatica were collected from the gallbladders of heavily infected cattle. They were incubated for 20 days at 20⬚C, in complete darkness.
The success of a trematode infection in a freshwater snail is dependent on at least three principal factors: the age of the snail at exposure, the temperature of the water in which the miracidium and the snail are located, and snail nutrition, which determines a more or less fast growth of the shell throughout infection (Smyth and Halton 1983). However, all factors influencing snail infection are not still known.
0014-4894/01 $35.00 Copyright 䉷 2001 by Academic Press All rights of reproduction in any form reserved.
49
50
ABROUS, RONDELAUD, AND DREYFUSS
TABLE I Characteristics of Infection in Controls and in Three Groups of Lymnaea truncatula Subjected to a Stress before Miracidial Exposure with Fasciola hepatica (1 Miracidium/Snail) Snails infected by F. hepatica and dissected at day 30 pe Parameter
Control group
Fasted group
Cold-treated group
Detergent-treated group
No. of snails at exposure No. of surviving snails at day 30 pe No. of snails harboring larval forms at day 30 pe Prevalence of infection (%)a No. of free rediae per infected snail (means ⫾ SD)
100 60 30 50.0 24.8 ⫾ 5.9
100 65 63 96.9 15.4 ⫾ 6.7
100 91 84 92.3 24.0 ⫾ 5.1
100 89 85 95.5 26.4 ⫾ 6.2
a
Prevalence of infection (number of snails harboring larval forms/number of surviving snails at day 30 postexposure).
Four groups of 100 snails each were used to study the effect of stress on the survival of uninfected snails at day 30 postexposure (pe). Snails from the first group were not subjected to stress (unstressed and uninfected controls), whereas snails from the three other groups were subjected to a 3-day fast in spring water, sterilized and filtered through a 3-m Millipore membrane (fasted and uninfected controls); exposure to cold water at 6–8⬚C for 15 min (cold-treated and uninfected controls); or exposure to a detergent solution (Paic Citron, Colgate-Palmolive, Courbevoie, France) at low concentration (4 drops in 3 L of spring water) for 15 min (detergent-treated and uninfected controls). Snails were subsequently raised for 30 days in polypropylene boxes 1 m by 55 cm and 15 cm high, with 50 snails per box (Augot et al. 1996). Boxes were maintained under constant conditions of 20⬚C ambient temperature and diurnal photoperiod of 3000- to 4000-lux light intensity. At day 30 pe, the survivors from each group were counted. Four other groups of 100 snails each were used to study the effects of stress on the redial burden of F. hepatica in L. truncatula. One group of snails was not subjected to stress prior to single-miracidium infections (infected controls). In contrast, in the other three groups, the protocol for stressing snails was similar to that used for the corresponding three groups of controls. As the cold or detergent treatments had a temporary narcotizing effect on L. truncatula, at the end of the stress period the snails from the three groups were placed in normal spring water, without food for 1 h at 20⬚C, before each was subjected to a single miracidium of F. hepatica for 4 h in a 35-mm-diameter petri dish (fast, cold, and detergent groups of infected snails). At day 30 pe, the surviving L. truncatula from these four groups were dissected to determine the number of snails harboring F. hepatica larval forms and to count free rediae. The last two groups of 100 snails each were used to study the cercarial shedding of F. hepatica. Snails from the first group were subjected only to single-miracidium infections (control group), while the L. truncatula from the other group were subjected to cold water at 6–8⬚C for 15 min before each was exposed to a single miracidium (cold group). Controls and stressed snails were raised for 30 days at 20⬚C using the aforementioned protocol. At day 30 pe, each surviving snail was placed in a 35-mm-diameter petri dish, containing 2–3 mL of spring water and a piece of steeped lettuce. These dishes were maintained at 20⬚C. Each day, a metacercarial count was performed and the water in the dish was changed until snail death. Routine postmortem dissection of snail cadavers was performed to distinguish uninfected snails (without larval forms) from infected snails that died without shedding.
The parameters studied were the survival rate of snails at day 30 pe, the prevalence of Fasciola infection, the number of free rediae per snail dissected at day 30 pe, the length of time between exposure and the first cercarial shedding, the duration of shedding, and the total number of cercariae produced by each snail. Comparison tests of experimental frequencies and one-way analysis of variance were used to establish levels of significance. Snail survival at day 30 pe was high in the first four groups of uninfected controls. It was 99% in unstressed controls, 84% in fastfed controls, 99% in cold-treated controls, and 98% in detergent-treated controls (data not shown). No significant difference between these survival rates was noted, whatever the mode of comparison. Table I gives the results found in the groups of infected snails when dissected at day 30 pe. The survival rates recorded in the cold and detergent groups were significantly higher (P ⬍ 0.001) than those from the control and fasting groups. In contrast, there were no significant differences between the former rates (cold and detergent groups) and the latter percentages. The prevalence of infection recorded in controls was significantly lower (P ⬍ 0.001) than those from the three groups subjected to stress. The redial burden in the fasted group was significantly lower (F ⫽ 4.33, P ⬍ 0.01) than the redial burdens from the three other groups. In contrast, there were no significant differences in the redial burdens of F. hepatica between controls, the cold group, and the detergent group. Table II gives the results found in snails studied for the cercarial shedding of F. hepatica. Snail survival rate at day 30 pe was significantly higher (P ⬍ 0.001) in the cold group than in controls. A similar finding was recorded for the prevalence of infection, which was significantly higher (P ⬍ 0.001) in the former group. The time between exposure and the first cercarial shedding, the duration of the shedding period, and the mean number of cercariae shed per snail did not show any significant variation between the two groups. The only difference concerned the total number of cercariae per group: it was higher in the cold group, as the number of cercaria-shedding snails was greater in the former group. The survival rates found in the control and fasted groups of infected snails fall within the ranges of percentages that Rondelaud and Barthe (1982, 1987) had already reported for the survival of L. truncatula at day 30 pe when subjected to single-miracidium infections with F. hepatica. The increased survival found in the cold- and detergenttreated groups of infected snails was more difficult to interpret and two perhaps complementary explanations might be proposed to interpret this result. The first was the nature of these two factors and the
51
STRESS OF Lymnaea truncatula AND INFECTION
TABLE II Characteristics of Infection in Controls and in One Group of Lymnaea truncatula Subjected to a Stress before Miracidial Exposure with Fasciola hepatica (1 Miracidium/Snail) Parameter
Control group
Cold group
No. of snails at exposure No. of surviving snails at day 30 pe No. of infected snails that died without sheddinga No. of cercaria-shedding snails Prevalence of infection (%)b Time between exposure and the first cercarial shedding (days): means ⫾ SD Duration of shedding period (days): means ⫾ SD
100 64 7 24 48.4 57.3 ⫾ 8.2 17.1 ⫾ 4.3
100 88 25 60 96.5 59.4 ⫾ 5.3 20.2 ⫾ 6.3
Total number of cercariae produced by the snails from the group No. of cercariae per cercaria- shedding snail: means ⫾ SD
2168 90.3 ⫾ 76.3
5976 99.6 ⫾ 64.8
a
Dissection of cadavers just after the death of snails. Prevalence of infection (number of cercaria-shedding snails ⫹ number of infected snails that died without shedding/number of surviving snails at day 30 postexposure). b
conditions under which the snails were subjected to stress, which were aggressive for snails, as demonstrated by the survival rates of corresponding uninfected controls and those given in Table I. The second was the better capacity of snails to ensure the larval development of F. hepatica when the stress occurred just before miracidial exposure. If the latter explanation might be considered valid, this better capacity would occur in the first 2 weeks pe, as the highest mortality rate of infected controls was noted during this period. Enhanced susceptibility of L. truncatula to infection was noted in the three stressed groups of infected snails when the time interval between snail stress and miracidial exposure was short. To explain this result, the most valid hypothesis would be to assume that starvation, cold shock, or detergent exposure had weakened snails so that they were not efficient at avoiding miracidial penetration and subsequent development of sporocysts. However, an indirect effect of these factors on the snail’s internal defense system via a temporary inhibition cannot be excluded. The number of F. hepatica free rediae did not show any significant variation in controls as in stressed groups, except for snails subjected to a 3-day fast in which free rediae were fewer. The limitation in the size of the redial burden in the latter snails agrees with the reports by Rondelaud (1994): according to this author, the number of free rediae clearly decreased in snails subjected to a 10-day desiccation before infection (18–25 rediae per snail at day 30 pe vs 43 rediae per snail in infected controls). This decrease in redial burden must be considered a consequence of snail fasting, as the nutriments within the snail were scarce and, consequently, induced a delay in the larval development of F. hepatica. In contrast, except for the total number of cercariae shed from infected snails, the other characteristics of cercarial shedding were the same in controls as in the cold group. The stress applied to snails just before miracidial exposure had no influence on the cercarial shedding of F. hepatica, with the probable exception of snails subjected to fasting, because the number of free rediae was lower than in the other groups of stressed snails. It is difficult to determine what biological significance snail stressing may have in the field in the interaction of L. truncatula and F. hepatica. In contrast, under laboratory conditions, a cold shock applied just before miracidial exposure will be useful in the experimental production
of F. hepatica metacercariae, as it will increase the prevalence of experimental infection and, subsequently, the total number of cercariae shed from infected snails.
REFERENCES Augot, D., Abrous, M., Rondelaud, D., and Dreyfuss, G. 1996. Paramphistomum daubneyi and Fasciola hepatica: The redial burden and cercarial shedding in Lymnaea truncatula submitted to successive unimiracidial cross-exposures. Parasitology Research 82, 623–627. Chipev, N., Vassilev, I., and Samnaliev, P. 1985. Interactions between Paramphistomum c.f. daubneyi Dinnik, 1962 and Fasciola hepatica L. in successive cross-invasions of Lymnaea (Galba) truncatula. Helminthologia 20, 80–88. Rondelaud, D. 1994. Fasciola hepatica: The infection rate and the development of redial generations in Lymnaea truncatula exposed to miracidia after experimental desiccation and activation in water. Journal of Helminthology 68, 63–66. Rondelaud, D. 1995. The characteristics of redial generations in Lymnaea truncatula exposed to Fasciola hepatica miracidia after poisoning by sublethal doses of cupric chloride. Veterinary Research 26, 21–26. Rondelaud, D., and Barthe, D. 1982. Les ge´ne´rations re´diennes de Fasciola hepatica L. chez Lymnaea truncatula Mu¨ller. A propos des effets de plusieurs facteurs. Annales de Parasitologie Humaine et Compare´e 57, 245–262. Rondelaud, D., and Barthe, D. 1987. Fasciola hepatica: e´tude de la productivite´ d’un sporocyste en fonction de la taille de Lymnaea truncatula. Parasitology Research 74, 155–160. Smyth, J. D., and Halton, D. W. 1983. “The Physiology of Trematodes.” Cambridge Univ. Press, Cambridge, UK. Received 12 October 1998; accepted with revision 13 February 2001