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Single-species infections of Echinostoma caproni cercariae in pulmonate snails and concurrent infections of E. caproni and Echinostoma trivolvis cercariae in Biomphalaria glabrata
International Journal for Parasitology 28 (1998) 595-597
RESEARCH
NOTE
Single-species infections of Echinostoma caproni cercariae in puhnonate snails and concurrent infections of E. caprod and Echinostoma trivolvis cercariae in Biomphalaria ghbrata Betsy A. Frazer and Bernard Fried* Department
of Biology,
Lafayette
College,
Easton,
PA 18042.
U.S.A.
Received 24 July 1997; accepted 14 November 1997
Abstract
Single-species infections of Echinostoma caproni cercariae in various pulmonate snails showed that a higher Percentage of cercariae encysted successfullyin Biomphalaria glabrata than in other snail speciesand that the pementage encystment was significantly greater in B. glabrata than in either Helisoma trivolvis or Lymnaea elodes. There were signi~t differences in cyst diameter, thickness of outer and inner cysts, and diameter of excretory concretions b&wepm E. caproni and Echinostoma trivolvis metacercariae. These morphological differences helped to distinguish each species bf cyst in concurrent cercarial infections of E. caproni and E. trivolvis in B. giabrata. Moreover, in concurrent it&e&ions E. trivolvis localised only in the saccular kidney, whereas E. caproni occurred in both the saccular kidney and the Pericardium. 0 1998 Australian Society for Parasitology. Published by Elsevier Science Ltd. Key words: Echinostoma caproni; Echinostoma glabrata; Helisoma trivolvis; Lymnaea elodes
trivolvis;
Cercariae; Single-species infections; Concurrent infections; Biomphalaria
Cercariae of 37-collar-spined Echinostoma show broad specificity for their second intermediate snail hosts. Studies on the allopatric species Echinostoma trivolvis and Echinostoma caproni have indicated that numerous gastropods serve as both natural and experimental second intermediate hosts for these echinostomes (see reviews in [ 1,2]). Anderson and Fried [3] showed that Biomphalaria glabrata is a convenient experimental host for cercarial penetration and encystment of E. trivolvis (referred to as Echinostoma revolutum in that study). They also studied cercarial encystment in -__ *Corresponding author. Tel: (610) 250-5463. Fax: (610) 2506557. e-mail: friedb@,lafvax.lafayette.edu.
snails other than B. glabrata and found a higher percentage of encystment in planorbid rather than physid snails. Preference for some second intermediate snail hosts was also demonstrated by McCarthy and Kanev [4] for cercariae of the echinostome Pseudoechinoparyphitun echkatum. Relatively littie quantitative inform&o& is available on cercarial encystment of E. capranl in second intermediate snail hosts. A purpose of this study was to examine cercarial encystment of E. caproni in B. glabrata (Puerto Rican M-line, a&no strain), Helisoma trivolvis (Colorado and Pennsylvania strains) and Lymnaea elodes. ExperimeWal studies on concurrent infections in snails with echinostome cercariae are not available. Therefore, a second purpose of this study was to examine concurrent infec-
SOO20-7519/98 %19.00+0.00 io 1998 Australian Society for Parasitology. Published by Elsevier Science Ltd. Printed in Great Britain PZI: SOO20-7519(97)00206-3
596
B.A. Frazer
and B. Fried / International
tions of E. trivolvis and E. caproni in B. glabrata with the intention of documenting encystment and habitat preferences in these infections. Cercariae of E. caproni were obtained from experimentally infected B. glabrata and used within 2 h post-emergence as described in Anderson and Fried [3]. To determine infectivity in different species of pulmonates, 10 snails of each species, l-3 mm in shell diameter for the planorbids and 3 mm in shell height for L. elodes, were placed singly in individual 1J-cm wells in a multi-well chamber containing 1.5 ml of artificial spring water [5] per well and 10 cercariae of E. caproni. The snails were left in the wells for 24 h. At 24 h p.i., the snails were examined by removing the shell under a dissecting microscope and crushing the body between a cover slip and a glass slide. Encysted metacercariae were then counted in the kidney-pericardium region. To observe interspecific morphological differences between E. caproni and E. trivolvis cysts, measurements of cyst diameter, outer cyst wall thickness, inner cyst wall thickness and diameter of the excretory concretions were made on 10 cysts of each species from experimental cercarial infections in B. glabrata. Cyst measurements were made as described in Fried and Reddy [6]. For studies on concurrent infections of E. trivolvis and E. caproni cercariae, the same protocol was used as described for E. caproni infections, except that a known number of cercariae of each species was placed in each well containing a 4% mm shell diameter B. glabrata. This size of snail was used because it was relatively easy to observe encysted metacercariae in the snail’s kidney versus the pericardium. A previous study [7] on cercarial encystment of E. trivolvis in H. trivolvis (Colorado strain) showed that snail size (age) did not affect the percentage which encysted in that host-parasite model. Two inocula, one of five E. trivolvis and five E. caproni cercariae and the other of one E. trivolvis and one E. caproni cercariae were used. Snails were transferred to finger bowls (10 snails/bowl) 24 h p.i. and fed lettuce ad libitum with water changed every third day and then were necropsied at 2 and 4 weeks pi. The shell was removed and observations were made on cysts in the kidney-pericardium region of the snails. The mean number f S.E.M. of E. caproni cysts in
Journalfor
Parasitology
28 (1998)
595-597
snails each exposed to 10 cercariae (n = 10 for each snail species) was 9.0 + 0.4 for B. glabrata; 6.5 + 0.5 for H. trivolvis (PA); 6.6 + 0.6 for H. trivolvis (CO) and 2.0 +0.7 for L. elodes. Encystment in B. glabrata was significantly greater (Student’s t-test, P < 0.05) than in any other experimental pulmonate used, with most cysts recovered in the pericardial cavity. Measurements (in pm) of the cyst structures studied always showed significant interspecies differences (Student’s t-test, PcO.05). Thus, the cyst diameter of E. caproni (155 f 1.7) was greater than that of E. trivolvis (1465 1.5); the outer cyst of E. trivolvis was thicker (12.2k0.4) than that of E. caproni (7.5kO.2). The inner cyst of E. caproni (2.5 LO.2) was thicker than that of E. trivolvis (1.8 kO.2). The diameter of the excretory concretions of E. caproni (9.1+ 0.4) was greater than that of E. trivolvis (5.7 + 0.2). These findings helped to distinguish the echinostome species in concurrent infections in B. glabrata. Table 1 shows recovery data from concurrent infections with E. trivolvis and E. caproni cercariae. Experiments from single-species cercarial infections in B. glabrata showed habitat differences in cercarial encystment. Cysts of E. trivolvis preferred the saccular kidney, whereas those of E. caproni preferred the pericardium. In concurrent infections in B. glabrata, the preferred site of encystment of E. trivolvis and E. caproni differed; E. trivolvis encysted only in the saccular kidney whereas E. caproni encysted in both the saccular kidney and pericardium. When an inoculum of one cercaria of each species was used, the pericardium was always the preferred site of encystment for E. caproni at 2 and 4 weeks p.i. There is a higher percentage of encystment of E. caproni in B. glabrata than in either strain of H. trivolvis or L. cloaks. In the field, species of Biomphalaria are natural second intermediate hosts of E. caproni; H. trivolvis and L. elodes are not used since these snails are native to North America and do not occur in endemic areas of E. caproni. Concurrent infections in snails with different species of echinostome cercariae merit further studies. They have not been studied previously because of difficulties in distinguishing cysts of closely related echinostome species in snails. Without mor-
B.A. Frazer
Table 1 Mean number+S.E.M. Echinostoma
trivolvis
and B. Fried / International
Journal
for Parasitology
28 (1998)
of encysted metacercariae in kidney or pericardium of Biomphalaria and Echinostoma caproni Recovery data
No. of cercariae used
E. trivolvis
595-597
597
g&rata
exposed concurrently with
--
I-.
.-.- _-. _. -.--...
-_---.._---
EC
Pericardium
Kidney
Pericardium
5 1
0 0
4.OkO.7 0.8kO.2
1.0+0.1
3.0+0.ti
0.8kO.2
0
5
5
0
1
1
0
3.OkO.7 0.8kO.2
2.OkO.4 o.s+o.2
2.010.6 0
Et 2 weeks
Kidney
p.i.
5 1 4
.__- -~
E. caproni
weeks pi.
Et= Echtiostoma n = five trials for
trivolvis;
EC= Echinostoma
caproni
each experiment.
phological information on individual species of echinostome cysts based on maintaining known species in the laboratory, it is difficult to distinguish 37collar-spined Echinostoma species solely on cyst morphology. Indeed, Fujino et al. [8] developed specific primers for the detection of genomic DNA of E. trivohis and E. caproni because the encysted metacercariae of these species were difficult to distinguish. Our study indicates that E. cuproni and E. trivolvis can be distinguished in experimental infections in B. glabrata on the basis of differences in cyst morphology and cyst location.
[2] Fried B, Huffman J. The biology of the intestinal trematode Echinostoma caproni. Adv Par&to1 199@38:31 l-368. [3] Anderson JW, Fried B. Experimental infection of P&a heterostropha,
[4]
[S]
[6]
[7]
References [1] Huffman JE, Fried B. Echinostoma Parasitol 1990;29:215-269.
[8] and echinostomiasis. Adv
Helisoma
trivolvis
a~& BieJrrplbaiaria
&&ratu
(Gastropoda) with Echinostoma revohrhun (Trematoda) cercariae. J Parasitol 1987;73:49-54. McCarthy AM, Kanev I. Ps~~i~~ypfi~ eddatum (Digenea: Echinostomatidae): exPerimenti observations on cercarid specificity toward second intermediate hosts. Parasitology 1990,100:423-428. Ulmer MJ. Notes on rearing of snails in the laboratory. In: Mach&s AJ, Voge M, editors. Experiments and techniques in parasitology. San Francisco: WH Freeman. 1970:143144. Fried B, Reddy A. Comparative studies on excystation and morphological features of meof Eckinos#oma paraensei and E. caproni. Int J Pat&to1 1997;27:%99-901. Schmidt KA, Fried B. Experimental infection Helisoma rrivolvis (Colorado strain) snails with cercariae of Echinostoma trivolvis. Int J Parasitol 19%;26:287-289. Fujino T, Zhiliang W, Nagano I, T&&&i Y, Fried B. Specific primers for the detection of genomic DNA of Echinostoma trivolvis and E. caproni (Trematoda: Echinostomatidae). Mol Cell Probes 1997;11:77-80.
RESEARCH
NOTE
Single-species infections of Echinostoma caproni cercariae in puhnonate snails and concurrent infections of E. caprod and Echinostoma trivolvis cercariae in Biomphalaria ghbrata Betsy A. Frazer and Bernard Fried* Department
of Biology,
Lafayette
College,
Easton,
PA 18042.
U.S.A.
Received 24 July 1997; accepted 14 November 1997
Abstract
Single-species infections of Echinostoma caproni cercariae in various pulmonate snails showed that a higher Percentage of cercariae encysted successfullyin Biomphalaria glabrata than in other snail speciesand that the pementage encystment was significantly greater in B. glabrata than in either Helisoma trivolvis or Lymnaea elodes. There were signi~t differences in cyst diameter, thickness of outer and inner cysts, and diameter of excretory concretions b&wepm E. caproni and Echinostoma trivolvis metacercariae. These morphological differences helped to distinguish each species bf cyst in concurrent cercarial infections of E. caproni and E. trivolvis in B. giabrata. Moreover, in concurrent it&e&ions E. trivolvis localised only in the saccular kidney, whereas E. caproni occurred in both the saccular kidney and the Pericardium. 0 1998 Australian Society for Parasitology. Published by Elsevier Science Ltd. Key words: Echinostoma caproni; Echinostoma glabrata; Helisoma trivolvis; Lymnaea elodes
trivolvis;
Cercariae; Single-species infections; Concurrent infections; Biomphalaria
Cercariae of 37-collar-spined Echinostoma show broad specificity for their second intermediate snail hosts. Studies on the allopatric species Echinostoma trivolvis and Echinostoma caproni have indicated that numerous gastropods serve as both natural and experimental second intermediate hosts for these echinostomes (see reviews in [ 1,2]). Anderson and Fried [3] showed that Biomphalaria glabrata is a convenient experimental host for cercarial penetration and encystment of E. trivolvis (referred to as Echinostoma revolutum in that study). They also studied cercarial encystment in -__ *Corresponding author. Tel: (610) 250-5463. Fax: (610) 2506557. e-mail: friedb@,lafvax.lafayette.edu.
snails other than B. glabrata and found a higher percentage of encystment in planorbid rather than physid snails. Preference for some second intermediate snail hosts was also demonstrated by McCarthy and Kanev [4] for cercariae of the echinostome Pseudoechinoparyphitun echkatum. Relatively littie quantitative inform&o& is available on cercarial encystment of E. capranl in second intermediate snail hosts. A purpose of this study was to examine cercarial encystment of E. caproni in B. glabrata (Puerto Rican M-line, a&no strain), Helisoma trivolvis (Colorado and Pennsylvania strains) and Lymnaea elodes. ExperimeWal studies on concurrent infections in snails with echinostome cercariae are not available. Therefore, a second purpose of this study was to examine concurrent infec-
SOO20-7519/98 %19.00+0.00 io 1998 Australian Society for Parasitology. Published by Elsevier Science Ltd. Printed in Great Britain PZI: SOO20-7519(97)00206-3
596
B.A. Frazer
and B. Fried / International
tions of E. trivolvis and E. caproni in B. glabrata with the intention of documenting encystment and habitat preferences in these infections. Cercariae of E. caproni were obtained from experimentally infected B. glabrata and used within 2 h post-emergence as described in Anderson and Fried [3]. To determine infectivity in different species of pulmonates, 10 snails of each species, l-3 mm in shell diameter for the planorbids and 3 mm in shell height for L. elodes, were placed singly in individual 1J-cm wells in a multi-well chamber containing 1.5 ml of artificial spring water [5] per well and 10 cercariae of E. caproni. The snails were left in the wells for 24 h. At 24 h p.i., the snails were examined by removing the shell under a dissecting microscope and crushing the body between a cover slip and a glass slide. Encysted metacercariae were then counted in the kidney-pericardium region. To observe interspecific morphological differences between E. caproni and E. trivolvis cysts, measurements of cyst diameter, outer cyst wall thickness, inner cyst wall thickness and diameter of the excretory concretions were made on 10 cysts of each species from experimental cercarial infections in B. glabrata. Cyst measurements were made as described in Fried and Reddy [6]. For studies on concurrent infections of E. trivolvis and E. caproni cercariae, the same protocol was used as described for E. caproni infections, except that a known number of cercariae of each species was placed in each well containing a 4% mm shell diameter B. glabrata. This size of snail was used because it was relatively easy to observe encysted metacercariae in the snail’s kidney versus the pericardium. A previous study [7] on cercarial encystment of E. trivolvis in H. trivolvis (Colorado strain) showed that snail size (age) did not affect the percentage which encysted in that host-parasite model. Two inocula, one of five E. trivolvis and five E. caproni cercariae and the other of one E. trivolvis and one E. caproni cercariae were used. Snails were transferred to finger bowls (10 snails/bowl) 24 h p.i. and fed lettuce ad libitum with water changed every third day and then were necropsied at 2 and 4 weeks pi. The shell was removed and observations were made on cysts in the kidney-pericardium region of the snails. The mean number f S.E.M. of E. caproni cysts in
Journalfor
Parasitology
28 (1998)
595-597
snails each exposed to 10 cercariae (n = 10 for each snail species) was 9.0 + 0.4 for B. glabrata; 6.5 + 0.5 for H. trivolvis (PA); 6.6 + 0.6 for H. trivolvis (CO) and 2.0 +0.7 for L. elodes. Encystment in B. glabrata was significantly greater (Student’s t-test, P < 0.05) than in any other experimental pulmonate used, with most cysts recovered in the pericardial cavity. Measurements (in pm) of the cyst structures studied always showed significant interspecies differences (Student’s t-test, PcO.05). Thus, the cyst diameter of E. caproni (155 f 1.7) was greater than that of E. trivolvis (1465 1.5); the outer cyst of E. trivolvis was thicker (12.2k0.4) than that of E. caproni (7.5kO.2). The inner cyst of E. caproni (2.5 LO.2) was thicker than that of E. trivolvis (1.8 kO.2). The diameter of the excretory concretions of E. caproni (9.1+ 0.4) was greater than that of E. trivolvis (5.7 + 0.2). These findings helped to distinguish the echinostome species in concurrent infections in B. glabrata. Table 1 shows recovery data from concurrent infections with E. trivolvis and E. caproni cercariae. Experiments from single-species cercarial infections in B. glabrata showed habitat differences in cercarial encystment. Cysts of E. trivolvis preferred the saccular kidney, whereas those of E. caproni preferred the pericardium. In concurrent infections in B. glabrata, the preferred site of encystment of E. trivolvis and E. caproni differed; E. trivolvis encysted only in the saccular kidney whereas E. caproni encysted in both the saccular kidney and pericardium. When an inoculum of one cercaria of each species was used, the pericardium was always the preferred site of encystment for E. caproni at 2 and 4 weeks p.i. There is a higher percentage of encystment of E. caproni in B. glabrata than in either strain of H. trivolvis or L. cloaks. In the field, species of Biomphalaria are natural second intermediate hosts of E. caproni; H. trivolvis and L. elodes are not used since these snails are native to North America and do not occur in endemic areas of E. caproni. Concurrent infections in snails with different species of echinostome cercariae merit further studies. They have not been studied previously because of difficulties in distinguishing cysts of closely related echinostome species in snails. Without mor-
B.A. Frazer
Table 1 Mean number+S.E.M. Echinostoma
trivolvis
and B. Fried / International
Journal
for Parasitology
28 (1998)
of encysted metacercariae in kidney or pericardium of Biomphalaria and Echinostoma caproni Recovery data
No. of cercariae used
E. trivolvis
595-597
597
g&rata
exposed concurrently with
--
I-.
.-.- _-. _. -.--...
-_---.._---
EC
Pericardium
Kidney
Pericardium
5 1
0 0
4.OkO.7 0.8kO.2
1.0+0.1
3.0+0.ti
0.8kO.2
0
5
5
0
1
1
0
3.OkO.7 0.8kO.2
2.OkO.4 o.s+o.2
2.010.6 0
Et 2 weeks
Kidney
p.i.
5 1 4
.__- -~
E. caproni
weeks pi.
Et= Echtiostoma n = five trials for
trivolvis;
EC= Echinostoma
caproni
each experiment.
phological information on individual species of echinostome cysts based on maintaining known species in the laboratory, it is difficult to distinguish 37collar-spined Echinostoma species solely on cyst morphology. Indeed, Fujino et al. [8] developed specific primers for the detection of genomic DNA of E. trivohis and E. caproni because the encysted metacercariae of these species were difficult to distinguish. Our study indicates that E. cuproni and E. trivolvis can be distinguished in experimental infections in B. glabrata on the basis of differences in cyst morphology and cyst location.
[2] Fried B, Huffman J. The biology of the intestinal trematode Echinostoma caproni. Adv Par&to1 199@38:31 l-368. [3] Anderson JW, Fried B. Experimental infection of P&a heterostropha,
[4]
[S]
[6]
[7]
References [1] Huffman JE, Fried B. Echinostoma Parasitol 1990;29:215-269.
[8] and echinostomiasis. Adv
Helisoma
trivolvis
a~& BieJrrplbaiaria
&&ratu
(Gastropoda) with Echinostoma revohrhun (Trematoda) cercariae. J Parasitol 1987;73:49-54. McCarthy AM, Kanev I. Ps~~i~~ypfi~ eddatum (Digenea: Echinostomatidae): exPerimenti observations on cercarid specificity toward second intermediate hosts. Parasitology 1990,100:423-428. Ulmer MJ. Notes on rearing of snails in the laboratory. In: Mach&s AJ, Voge M, editors. Experiments and techniques in parasitology. San Francisco: WH Freeman. 1970:143144. Fried B, Reddy A. Comparative studies on excystation and morphological features of meof Eckinos#oma paraensei and E. caproni. Int J Pat&to1 1997;27:%99-901. Schmidt KA, Fried B. Experimental infection Helisoma rrivolvis (Colorado strain) snails with cercariae of Echinostoma trivolvis. Int J Parasitol 19%;26:287-289. Fujino T, Zhiliang W, Nagano I, T&&&i Y, Fried B. Specific primers for the detection of genomic DNA of Echinostoma trivolvis and E. caproni (Trematoda: Echinostomatidae). Mol Cell Probes 1997;11:77-80.