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Mating behaviour of Echinostoma caproni and E. trivolvis in concurrent infections in hamsters
Pergamon
Mating Behaviour of Echinostoma caproni and E. trivolvis in Concurrent Infections in Hamsters PAUL M. NOLLEN* Department of Biological Sciences, Western Illinois University, Macomb, IL 614.55, U.S.A. (Recked
28 June
1996; trccepted
16 Augusr
1996)
Abstract-Nollen P. 1997. Mating behaviour of Echinostoma cuproni and E. frivoluis in concurrent infections in hamsters. International Journal for Parasitology 27: 71-75. Young adults of Echinostomn caproni and E. triuolvis were recovered from hamsters. Some worms of each species were exposed to 13H]tyrosine for 1 h to label sperm, and these were transplanted singly to uninfected hamsters with various combinations of unexposed worms of either the opposite species or both species. Worms recovered 5 days later were serially sectioned, processed for autoradiography and observed for the location of radioactive sperm. Interspecies mating was detected when E. cuproni was the sperm donor and E. triuoluis the recipient, but not the converse. The crossinsemination rate during interspecies mating was very low (13%) when compared to the normal rate of E. caproni intraspecies mating (52%). When single donor adults of either E. cuproni or E. trivolvis had a choice of both recipient species, no interspecies mating took place, but both self- and cross-inseminated in a nonrestrictive mating pattern typical of echinostome species. After transplantation, both species localized in their normal habitat within the hamster intestine. However, 25% of opposite species recoveries were found within 1 cm of each other, making interspecies mating a possibility. Copyright % 1997 Australian Society for Parasitology. Published by Elsevier Science Ltd. Key words:
mating:
Echinostoma
intraspecies
caproni;
mating;
fkhinostoma
self-insemination;
concurrent cross-insemination.
triwk:i.s;
A close genetic relationship has been found between several species of Echinostomu within the 37-collarspined group (Morgan & Blair, 1995: Sloss et al., 1995). At least 3 species commonly maintained in the laboratory, E. cuproni, E. trivolvis and E. puraensei, will grow readily in hamsters. All are recognized as separate species on the basis of electrophoretic and DNA analysis. The mating behaviour of these species was found to be non-restrictive, where adults selfinseminate when alone and both self- and crossinseminate in groups (Nollen, 1990, 1993, 1996a). In a recent report, Nollen (1996b) found that E. cuprorti and E. paraensei in concurrent infections of mice would not cross-inseminate, no matter which species served as the sperm donor. Both species would self- and cross-inseminate in the presence of the other species without interspecies mating. This mating barE-mail:
PM-NolIen@
autoradiography:
interspecies
rier is further evidence that E. caproni and E. paraensci are distinct species and incapable of hybridization. Sloss et al. (1995) found E. cuproni and E. tricolris to be more genetically related to each other than either to E. paraensei. Since both E. caproni and E. tricolvis can be maintained in hamsters in the laboratory. studies were initiated to see if these 2 species would trade sperm. Previous work with concurrent infections of these 2 species found that mice were not good hosts for E. trivolak (Iorio et al., 1991). However, concurrent infections of E. cuproni (formerly E. liei) and E. trivokis (formerly E. revolutum) in hamsters were successful (Huffman et al., 1988, 1992). Both species exhibited some interspecies attraction when tested in an in vitro system (Fried & Haseeb, 1990). Herein sperm were labeled with [3H]tyrosine in a donor worm and then followed by autoradiography, a method used in previous mating studies (Nollen, 1983). Labeled sperm found in the seminal receptacle of the donor adult indicated self-insemination, while labeled sperm found in the seminal receptacle of an
INTRODUCTION
* Fax: 309 298-2270:
infectlons:
wiu.edu. 71
P. M. Nollen
12
unlabeled recipient worm indicated cross-insemination. Single labeled worms of both species were transplanted to uninfected hamsters, either with unlabeled adults of the opposite species or with both species to determine patterns of interspecies or intraspecies mating. MATERIALS
AND
METHODS
Syrian golden hamsters (Harlan-Sprague-Dawley), 56weeks old, were infected with 60 metacercarial cysts of either E. caproni or E. trivolvis. The E. caproni strain was obtained from Dr Bernard Fried, Lafayette College, Easton, PA, U.S.A. and has been maintained here in a laboratory cycle since 1989. Cysts of E. trivolvis were sent directly from Dr Fried’s laboratory and were originally collected from naturally-infected snails. After 10 days of growth. adults of both species were recovered and stored in Hedon-Fleig’s saline at 39°C. Some adults of both species were exposed in Hedon-Fleig’s saline to 100 &i ml- ’ [3H]tyrosine (5 1 Ci mmol-‘, ICN Pharmaceuticals) for 1 hand then transplanted singly with 4-10 untreated adults of the opposite or both species, depending on the numbers available. Details of transplant to the small intestine of hamsters are given by Nollen (1993). Worms were recovered 5 days after transplant and fixed in alcohol-formalin-acetic acid. Those adults found within 1 cm of each other were processed as a group. For autoradiography, worms were embedded in paraffin, serially sectioned at 12pm, deparaffinized in xylene and coated with NTB-2 nuclear emulsion (Eastman Kodak). After 3 weeks of dark storage in foil-covered slide boxes, the slides were developed for 3 min in Kodak D-19, stained with haematoxylin, and observed for the position of silver grains.
RESULTS
Most of the E. caproni adults were recovered clustered in the ileum of the hamster small intestine, while E. trivolvis were found scattered throughout the jejunem and ileum. In all the transplants 25% (35/139) of the worms were found within 1 cm of the opposite species, indicating that complete species separation did not take place after transplantation. Worm recovery and insemination data are given in Table 1. When single, labeled E. caproni were transplanted with 4-9 unlabeled E. trivolvis, all but one of the 6 sperm donor worms self-inseminated. Three of these 6 adults also carried on interspecies mating by inseminating 5 of 39 E. trivolvis. Of these 5 worms, 2 were not recovered within 1 cm of the E. caproni sperm donor worm. When 1 labeled E. caproni was transplanted with unlabeled worms of both species, no interspecies mating was observed. However, labeled E. caproni selfinseminated and cross-inseminated with the unlabeled E. caproni in the hamster intestine. When using E. trivolvis adults as the labeled sperm donors, no interspecies mating with 28 unlabeled E. caproniin 5 experiments was observed (Table 1). How-
ever, 3 of the 5 labeled E. trivolvis adults self-inseminated. When the labeled E. trivolvis were transplanted with unlabeled worms of both species, interspecies mating did not occur with adults of E. caproni. Here, 6 of 11 labeled E. trivolvis self-inseminated and also cross-inseminated with 17 of the 60 possible unlabeled E. trivolvis. In this series of experiments, several unlabeled E. trivolvis were found with radioactive sperm in their seminal receptacles, even though they were not close (within 1 cm) to the sperm donor. DISCUSSION
The process of transplantation did not affect the ability of these species to find their natural habitat within the hamster’s intestinal tract. Adults of E. caproni were found clustered in 1 or 2 areas of the ileum. while adults of E. trivolvis were found in groups, but scattered throughout the jejunem and ileum. None were found as far forward as the duodenum. This matches the recovery pattern of these 2 species by Huffman et al. (1992) in concurrent laboratory infections. With this pattern of habitat selection, 25% were still found in close contact (within 1 cm) with the opposite species. This was mostly due to the random location of E. trivolvis. Even though E. caproni and E. trivolvis were recognized as valid species in a previous morphological study (Christensen et al., 1990) E. caproni will mate with E. trivolvis when given the chance, but not the converse (Table 1). Adults of E. caproni carried out an unrestricted mating pattern with E. trivolvis by selfinseminating itself and cross-inseminating with the opposite species. However, when the E. caproni sperm donor adults were given the choice of unlabeled adults of both species, they never carried out interspecies mating, but did exhibit the unrestricted insemination pattern with their own species. In a few cases unlabeled worms had radioactive sperm in their seminal receptacles and no labeled worm was identified in their recovery group. Thus, there must be some migration of donor worms within the intestinal habitat. This was most evident when E. trivolvis was the labeled sperm donor. Insemination rates from this study were compared to those from reports on single-species mating in Table 2. A study by Nollen (1990) showed that in single infections the self-insemination rate of E. caproni adults was 71%, while in groups 63% self-inseminated and 52% cross-inseminated. In concurrent infections with unlabeled E. trivolvis, E. caproni self-inseminated at a slightly higher rate (83% vs 71%), but crossinseminated at a lower rate (13% vs 52%). Thus, interspecies mating happens less readily than intraspecies mating. Donor worms of E. caproni when
Mating in concurrent infections of Echinoston~ spp. Table I-Insemination in concurrent infections of Echinusfomu ~riz~lz~is (ET) and Erhi~OSIOWZU cuproni (EC) labeled with tritiated tyrosine and returned to uninfected hamsters with unlabeled worms Insemination”
Number of worms/experiment Labeled ET EC
Unlabeled ET EC
I 1 1 1 I I
4 5
Totals Percentages
39
6 7
8 9
Self
1 1 1 1 I I
I I I 0 1 1 5
6
Cross ET
EC
0 0 0 1 2 3 5 83(5!6)
I I I 1 Totals Percentages I 1 I 1 1 1 I
4 4 4
Totals Percentages
46
1 1 I I 1 1 I I Totals Percentages
Number of experiments
5 6 7 I
4
1
5
2
6 8 28
1 I 5
1
I
2 3
2
1 1 0 3 14
1 I
2 4
2
1 1 0
5 6 6
I 7 60
4 3 4 26
0 0 0 0 0
1 2 1 1 1 9
I I I 1 1
0 1 0 I 1 0
3
2
1 11
I
2
13(5/39)
6 55(6/11)
60(3:5)
0(0/28)
0 0 0 0 0 0 0
0 2 1 3 I 0 0
0,'46 44(4!9)
7 50(7!14)
3
0 0 0 0 0 0 0 0
I 0 6
I 1 6 3 17 65(17!'60)
0;26
“Number of adults inseminated/total number of adults are shown in parentheses.
given the chance to mate with either species, selfinseminated less readily than in either single species group infections or concurrent infections (44% vs 63% and 83%), but cross-inseminated at approximately the same rate (50% vs 52%). Adults of E. trivoiuis werefound to self-inseminate
in singleinfections at 55%, but in groups self- and cross-insemination rateswere 58% and 14%, respectively (Nollen, 1993;Table2). In concurrentinfections with unlabeledE. caproni, self-insemination rateswere similar to thosefound in single-species group infections (58% vs 60%). When a choice of unlabeled adults of E. caproni and E. trivolvis was given to the E. trivolvis donor worms,self-insemination rateswere similar
(58% vs 55%)
but cross-insemination
rates
were markedly different (14% vs 65%). Although samplesize on most of the self-inseminationdata is small, cross-inseminationsample sizes are large enough to give more validity to the comparisons. Higher cross-inseminationrates in concurrent infections when E. trivohis wasthe donor are puzzling. It is improbable that thesespecieswould ever be found overlappingin the natural setting.Echinostoma caproni was describedfrom snails in Madagascar (Richard, 1964).while E. trivolvis (formerly E. rewhum) is found in varioushabitatsand hostsin North America. In spiteof the ability of E. caproni to inseminate E. trivolvis, molecular studiesusing isoeiectro focusing, electrophoresisand DNA techniquesindicate that they are sufficiently geneticallyseparatedto
74
P. M. Nollen Table 2-Percent insemination of Echinostoma triz&is (ET) and Echinos~or~~ caproni (EC) in single species and concurrent infections Percent insemination” Labeled ET EC
X X X X
Unlabeled ET EC
X X X X
X X x X x
x X x
Self ET 63 (S/S)” 71 (5/7)b 83 (5/6) 44 (4/9) 58 (7/12) 55 (6/l 1)’ 60 (3/5) 55 (6/l 1)
cross EC 52 (15/29)”
13 (5/39) 0 (O/46) 14 (S/35) 0 (O/28) 65 (17/60)
50 (7/14)
0 WW
“Number of adults inseminated/total number of adults are shown in parentheses.
bNollen (1990). ‘Nollen (1993).
beconsideredvalid species (Kristensen& Fried, 1991; Slosset al., 1995:Morgan & Blair, 1995). When 3 speciesof 37-collar-spinedechinostomes were comparedby electrophoresis,E. caproni and E. trivolvis were found geneticallycloser to each other than to E. paraensei (Slosset al., 1995).A similar geneticcomparisonusing rDNA ITS sequencedata found E. trivolvis and E. paraensei closerto eachother than to E. caproni. The fact that E. caproni will carry on interspeciesmating with E. trivolvis as reported here,bolstersthe closerelationshipof these2 species found by the electrophoretic data. No interspecies matingwasfound betweenE. caproni and E. paraensei indicating that they are valid species(Nollen, 1996b), an observationalsomadeby Slosset al. (1995),Morgan & Blair (1995)and Meece& Nollen (1996). A concurrent mating study utilizing the eyefluke speciesPhilophthalmus megalurus and Phi~ophthalmus gralli, hasdemonstratedinterspecies mating between 2 closely related digeneans(Nollen, 1984). Philophthalmus megalurus and P. gralli were found to have different electrophoreticcharacteristicsand are consideredto be separatespecies(Nollen et al., 1985). When P. megalurus and P. gralli weretransplantedin concurrent infectionsin chicken’seyes,P. megalurus would matewith P. gralli at levelssimilarto the crossinseminationrates with other P. megalurus. When given the choice of either P. megalurus or P. gralli, the donor P. megalurus cross-inseminated with both species,but the rate was lower with P. gralli. Philophthalmus gralli adults would not carry out interspeciesmating, but exhibited the typical restrictive mating pattern (in groupsthey readily cross-inseminate but never self-inseminate) with their own species even in the presenceof the oppositespecies. A major difference between
mating in concurrent
infections of echinostomeswhen compared to eyeflukes is that no interspeciesmating wasfound when a choiceof the samespecieswas given to the donor echinostomeadult. Also, the rate of cross-insemination of the oppositespecieswasmuch lower in E. caproni (13%) when compared to P. megalurus (72.6%) (Nollen, 1984). No evidenceof fertilization betweenE. caproni and E. trivolvis, e.g., radioactive E. caproni spermin E. trivolvis eggs,was found in this study. Thus, no conclusion can be reached on whether hybridization would be possiblebetweenthese2 species.Suspected natural hybrids between hermaphroditic digeneans havebeendescribed(Nollen, 1983).Accountsof natural and laboratory-producedhybrids betweenschistosomespecieshave beensummarizedby Basch(1991). So far no attemptsto producehybrids in the eyefluke and echinostomespeciesusedin concurrent mating studieshave beenreported. REFERENCES Basch P. F. 1991. Schistosomes: Development, Reproduction and Host Relations, pp. 28-31. Oxford University Press, New York. Christensen N. 0. & Fried B., Kanev I. 1990. Taxonomy of 37-collar-spined Echinostoma (Trematoda: Echinostomatidae) in studies on the population regulation in experimental rodent hosts. Angewandte Parasitologie 31: 127-130. Fried B. & Haseeb M. A. 1990. Intra- and interspecies chemo-attraction in Echinostoma caproni and E. trivolvis in vitro. Journal of the Helminthological Society of Washington 57: 72-73. Huffman J. E. & Alcaide A., Fried B. 1988. Single and concurrent infections of the golden hamster, Mesocricetus auratus, with Echinostoma revolutum and Eehinostoma liei (Trematoda: Digenea). Journal of Parasitology 74: 606 608.
Mating
in concurrent
infections
Huffman J. E.. Murphy P. M.&Fried B. 1992. Superimposed infections in golden hamsters infected with Echinostoma caproni and Echinostoma trivolvis (Digenea: Echinostomatidae). Journal of the Helmintholoctical Society of’washington 59: 1621. Iorio S. L.. Fried B. & Hosier D. W. 1991. Concurrent infections of Echinostoma caproni and Echinostoma tricolcis in ICR mice. International Journal for Parasitology 21: 715-717. Kristensen A. R. & Fried B. 1991. A comparison of Echinastoma caproni and Echinostoma trivolvis (Trematoda: Echinostomatidae) adults using iscelectric focusing. Journul of Parasitology 71: 496498. Meece J. K. & Nollen P. M. 1996. A comparison of the adult and miracidial stages of Echinostoma paraensei and E. caprani. International Journal,for Parasitology 26: 3143. Morgan J. A. T. & Blair D. 1995. Nuclear rDNA ITS sequence variation in the tremdtode genus Echinostoma: an aid to establishing relationships within the 37-collarspined group. Parasitology 111: 609-615. Nollen P. M. 1983. Patterns of sexual reproduction among parasitic platyhelminthes. Parasitology 86: 999120. Nollen P. M. 1984. Mating behavior of Philophthalmusmegalurus and P. gralli in concurrent infections of chicks. International Journal,fbr Parasitology 14: 71-74.
of
Echinostomcr
spp.
Nollen P. M. 1990. Echinostoma caproni-mating behavior and the timing of development and movement of repro ductive ceils. Journal of Parasitology 76: 784-789. Nollen P. M. 1993. Echinostoma trivolris-mating behavior of adults raised in hamsters. Parasitology Research 79: 13&132. Nollen P. M. 1996. The mating hebaviour of Ed~hit~~~f~~~~~ paraensri grown in mice. Journal af Helmintholag~~ 70: 43 45. Nollen P. M. 1996. Mating behaviour of Echinostwnu c~zpmi and E. paraensei in concurrent infections in mice. ./r/zirntri of Helminthology 70: 133-I 36. Nollen P. M., Leslie J. F., Cain G. D. & MacNab R K. 1985. A comparison of Texan and Hawaiian strains of the avian eyefluke, Philophthalmus gralli, with a cautionary note on the importation of exotic animals. Jartrnal of Pwtr sitology 71: 618C624. Richard J. 1964. Trematodes d‘oiseaux de Madagascar (Note III). Especies de la famille Echinostomatidae Poche 1926. Annales de Parasitolagie Humuine et Comparw 39: 607 620. Sloss B., Meece J. & Roman0 M., Nollen P. 1995. The genetic relationships between Echinostoma caproni, E. paraensr/. and E. trivolvis as determined by electrophoresis. J~LII~~~J af’Helmintholog~ 69: 243246.
Mating Behaviour of Echinostoma caproni and E. trivolvis in Concurrent Infections in Hamsters PAUL M. NOLLEN* Department of Biological Sciences, Western Illinois University, Macomb, IL 614.55, U.S.A. (Recked
28 June
1996; trccepted
16 Augusr
1996)
Abstract-Nollen P. 1997. Mating behaviour of Echinostoma cuproni and E. frivoluis in concurrent infections in hamsters. International Journal for Parasitology 27: 71-75. Young adults of Echinostomn caproni and E. triuolvis were recovered from hamsters. Some worms of each species were exposed to 13H]tyrosine for 1 h to label sperm, and these were transplanted singly to uninfected hamsters with various combinations of unexposed worms of either the opposite species or both species. Worms recovered 5 days later were serially sectioned, processed for autoradiography and observed for the location of radioactive sperm. Interspecies mating was detected when E. cuproni was the sperm donor and E. triuoluis the recipient, but not the converse. The crossinsemination rate during interspecies mating was very low (13%) when compared to the normal rate of E. caproni intraspecies mating (52%). When single donor adults of either E. cuproni or E. trivolvis had a choice of both recipient species, no interspecies mating took place, but both self- and cross-inseminated in a nonrestrictive mating pattern typical of echinostome species. After transplantation, both species localized in their normal habitat within the hamster intestine. However, 25% of opposite species recoveries were found within 1 cm of each other, making interspecies mating a possibility. Copyright % 1997 Australian Society for Parasitology. Published by Elsevier Science Ltd. Key words:
mating:
Echinostoma
intraspecies
caproni;
mating;
fkhinostoma
self-insemination;
concurrent cross-insemination.
triwk:i.s;
A close genetic relationship has been found between several species of Echinostomu within the 37-collarspined group (Morgan & Blair, 1995: Sloss et al., 1995). At least 3 species commonly maintained in the laboratory, E. cuproni, E. trivolvis and E. puraensei, will grow readily in hamsters. All are recognized as separate species on the basis of electrophoretic and DNA analysis. The mating behaviour of these species was found to be non-restrictive, where adults selfinseminate when alone and both self- and crossinseminate in groups (Nollen, 1990, 1993, 1996a). In a recent report, Nollen (1996b) found that E. cuprorti and E. paraensei in concurrent infections of mice would not cross-inseminate, no matter which species served as the sperm donor. Both species would self- and cross-inseminate in the presence of the other species without interspecies mating. This mating barE-mail:
PM-NolIen@
autoradiography:
interspecies
rier is further evidence that E. caproni and E. paraensci are distinct species and incapable of hybridization. Sloss et al. (1995) found E. cuproni and E. tricolris to be more genetically related to each other than either to E. paraensei. Since both E. caproni and E. tricolvis can be maintained in hamsters in the laboratory. studies were initiated to see if these 2 species would trade sperm. Previous work with concurrent infections of these 2 species found that mice were not good hosts for E. trivolak (Iorio et al., 1991). However, concurrent infections of E. cuproni (formerly E. liei) and E. trivokis (formerly E. revolutum) in hamsters were successful (Huffman et al., 1988, 1992). Both species exhibited some interspecies attraction when tested in an in vitro system (Fried & Haseeb, 1990). Herein sperm were labeled with [3H]tyrosine in a donor worm and then followed by autoradiography, a method used in previous mating studies (Nollen, 1983). Labeled sperm found in the seminal receptacle of the donor adult indicated self-insemination, while labeled sperm found in the seminal receptacle of an
INTRODUCTION
* Fax: 309 298-2270:
infectlons:
wiu.edu. 71
P. M. Nollen
12
unlabeled recipient worm indicated cross-insemination. Single labeled worms of both species were transplanted to uninfected hamsters, either with unlabeled adults of the opposite species or with both species to determine patterns of interspecies or intraspecies mating. MATERIALS
AND
METHODS
Syrian golden hamsters (Harlan-Sprague-Dawley), 56weeks old, were infected with 60 metacercarial cysts of either E. caproni or E. trivolvis. The E. caproni strain was obtained from Dr Bernard Fried, Lafayette College, Easton, PA, U.S.A. and has been maintained here in a laboratory cycle since 1989. Cysts of E. trivolvis were sent directly from Dr Fried’s laboratory and were originally collected from naturally-infected snails. After 10 days of growth. adults of both species were recovered and stored in Hedon-Fleig’s saline at 39°C. Some adults of both species were exposed in Hedon-Fleig’s saline to 100 &i ml- ’ [3H]tyrosine (5 1 Ci mmol-‘, ICN Pharmaceuticals) for 1 hand then transplanted singly with 4-10 untreated adults of the opposite or both species, depending on the numbers available. Details of transplant to the small intestine of hamsters are given by Nollen (1993). Worms were recovered 5 days after transplant and fixed in alcohol-formalin-acetic acid. Those adults found within 1 cm of each other were processed as a group. For autoradiography, worms were embedded in paraffin, serially sectioned at 12pm, deparaffinized in xylene and coated with NTB-2 nuclear emulsion (Eastman Kodak). After 3 weeks of dark storage in foil-covered slide boxes, the slides were developed for 3 min in Kodak D-19, stained with haematoxylin, and observed for the position of silver grains.
RESULTS
Most of the E. caproni adults were recovered clustered in the ileum of the hamster small intestine, while E. trivolvis were found scattered throughout the jejunem and ileum. In all the transplants 25% (35/139) of the worms were found within 1 cm of the opposite species, indicating that complete species separation did not take place after transplantation. Worm recovery and insemination data are given in Table 1. When single, labeled E. caproni were transplanted with 4-9 unlabeled E. trivolvis, all but one of the 6 sperm donor worms self-inseminated. Three of these 6 adults also carried on interspecies mating by inseminating 5 of 39 E. trivolvis. Of these 5 worms, 2 were not recovered within 1 cm of the E. caproni sperm donor worm. When 1 labeled E. caproni was transplanted with unlabeled worms of both species, no interspecies mating was observed. However, labeled E. caproni selfinseminated and cross-inseminated with the unlabeled E. caproni in the hamster intestine. When using E. trivolvis adults as the labeled sperm donors, no interspecies mating with 28 unlabeled E. caproniin 5 experiments was observed (Table 1). How-
ever, 3 of the 5 labeled E. trivolvis adults self-inseminated. When the labeled E. trivolvis were transplanted with unlabeled worms of both species, interspecies mating did not occur with adults of E. caproni. Here, 6 of 11 labeled E. trivolvis self-inseminated and also cross-inseminated with 17 of the 60 possible unlabeled E. trivolvis. In this series of experiments, several unlabeled E. trivolvis were found with radioactive sperm in their seminal receptacles, even though they were not close (within 1 cm) to the sperm donor. DISCUSSION
The process of transplantation did not affect the ability of these species to find their natural habitat within the hamster’s intestinal tract. Adults of E. caproni were found clustered in 1 or 2 areas of the ileum. while adults of E. trivolvis were found in groups, but scattered throughout the jejunem and ileum. None were found as far forward as the duodenum. This matches the recovery pattern of these 2 species by Huffman et al. (1992) in concurrent laboratory infections. With this pattern of habitat selection, 25% were still found in close contact (within 1 cm) with the opposite species. This was mostly due to the random location of E. trivolvis. Even though E. caproni and E. trivolvis were recognized as valid species in a previous morphological study (Christensen et al., 1990) E. caproni will mate with E. trivolvis when given the chance, but not the converse (Table 1). Adults of E. caproni carried out an unrestricted mating pattern with E. trivolvis by selfinseminating itself and cross-inseminating with the opposite species. However, when the E. caproni sperm donor adults were given the choice of unlabeled adults of both species, they never carried out interspecies mating, but did exhibit the unrestricted insemination pattern with their own species. In a few cases unlabeled worms had radioactive sperm in their seminal receptacles and no labeled worm was identified in their recovery group. Thus, there must be some migration of donor worms within the intestinal habitat. This was most evident when E. trivolvis was the labeled sperm donor. Insemination rates from this study were compared to those from reports on single-species mating in Table 2. A study by Nollen (1990) showed that in single infections the self-insemination rate of E. caproni adults was 71%, while in groups 63% self-inseminated and 52% cross-inseminated. In concurrent infections with unlabeled E. trivolvis, E. caproni self-inseminated at a slightly higher rate (83% vs 71%), but crossinseminated at a lower rate (13% vs 52%). Thus, interspecies mating happens less readily than intraspecies mating. Donor worms of E. caproni when
Mating in concurrent infections of Echinoston~ spp. Table I-Insemination in concurrent infections of Echinusfomu ~riz~lz~is (ET) and Erhi~OSIOWZU cuproni (EC) labeled with tritiated tyrosine and returned to uninfected hamsters with unlabeled worms Insemination”
Number of worms/experiment Labeled ET EC
Unlabeled ET EC
I 1 1 1 I I
4 5
Totals Percentages
39
6 7
8 9
Self
1 1 1 1 I I
I I I 0 1 1 5
6
Cross ET
EC
0 0 0 1 2 3 5 83(5!6)
I I I 1 Totals Percentages I 1 I 1 1 1 I
4 4 4
Totals Percentages
46
1 1 I I 1 1 I I Totals Percentages
Number of experiments
5 6 7 I
4
1
5
2
6 8 28
1 I 5
1
I
2 3
2
1 1 0 3 14
1 I
2 4
2
1 1 0
5 6 6
I 7 60
4 3 4 26
0 0 0 0 0
1 2 1 1 1 9
I I I 1 1
0 1 0 I 1 0
3
2
1 11
I
2
13(5/39)
6 55(6/11)
60(3:5)
0(0/28)
0 0 0 0 0 0 0
0 2 1 3 I 0 0
0,'46 44(4!9)
7 50(7!14)
3
0 0 0 0 0 0 0 0
I 0 6
I 1 6 3 17 65(17!'60)
0;26
“Number of adults inseminated/total number of adults are shown in parentheses.
given the chance to mate with either species, selfinseminated less readily than in either single species group infections or concurrent infections (44% vs 63% and 83%), but cross-inseminated at approximately the same rate (50% vs 52%). Adults of E. trivoiuis werefound to self-inseminate
in singleinfections at 55%, but in groups self- and cross-insemination rateswere 58% and 14%, respectively (Nollen, 1993;Table2). In concurrentinfections with unlabeledE. caproni, self-insemination rateswere similar to thosefound in single-species group infections (58% vs 60%). When a choice of unlabeled adults of E. caproni and E. trivolvis was given to the E. trivolvis donor worms,self-insemination rateswere similar
(58% vs 55%)
but cross-insemination
rates
were markedly different (14% vs 65%). Although samplesize on most of the self-inseminationdata is small, cross-inseminationsample sizes are large enough to give more validity to the comparisons. Higher cross-inseminationrates in concurrent infections when E. trivohis wasthe donor are puzzling. It is improbable that thesespecieswould ever be found overlappingin the natural setting.Echinostoma caproni was describedfrom snails in Madagascar (Richard, 1964).while E. trivolvis (formerly E. rewhum) is found in varioushabitatsand hostsin North America. In spiteof the ability of E. caproni to inseminate E. trivolvis, molecular studiesusing isoeiectro focusing, electrophoresisand DNA techniquesindicate that they are sufficiently geneticallyseparatedto
74
P. M. Nollen Table 2-Percent insemination of Echinostoma triz&is (ET) and Echinos~or~~ caproni (EC) in single species and concurrent infections Percent insemination” Labeled ET EC
X X X X
Unlabeled ET EC
X X X X
X X x X x
x X x
Self ET 63 (S/S)” 71 (5/7)b 83 (5/6) 44 (4/9) 58 (7/12) 55 (6/l 1)’ 60 (3/5) 55 (6/l 1)
cross EC 52 (15/29)”
13 (5/39) 0 (O/46) 14 (S/35) 0 (O/28) 65 (17/60)
50 (7/14)
0 WW
“Number of adults inseminated/total number of adults are shown in parentheses.
bNollen (1990). ‘Nollen (1993).
beconsideredvalid species (Kristensen& Fried, 1991; Slosset al., 1995:Morgan & Blair, 1995). When 3 speciesof 37-collar-spinedechinostomes were comparedby electrophoresis,E. caproni and E. trivolvis were found geneticallycloser to each other than to E. paraensei (Slosset al., 1995).A similar geneticcomparisonusing rDNA ITS sequencedata found E. trivolvis and E. paraensei closerto eachother than to E. caproni. The fact that E. caproni will carry on interspeciesmating with E. trivolvis as reported here,bolstersthe closerelationshipof these2 species found by the electrophoretic data. No interspecies matingwasfound betweenE. caproni and E. paraensei indicating that they are valid species(Nollen, 1996b), an observationalsomadeby Slosset al. (1995),Morgan & Blair (1995)and Meece& Nollen (1996). A concurrent mating study utilizing the eyefluke speciesPhilophthalmus megalurus and Phi~ophthalmus gralli, hasdemonstratedinterspecies mating between 2 closely related digeneans(Nollen, 1984). Philophthalmus megalurus and P. gralli were found to have different electrophoreticcharacteristicsand are consideredto be separatespecies(Nollen et al., 1985). When P. megalurus and P. gralli weretransplantedin concurrent infectionsin chicken’seyes,P. megalurus would matewith P. gralli at levelssimilarto the crossinseminationrates with other P. megalurus. When given the choice of either P. megalurus or P. gralli, the donor P. megalurus cross-inseminated with both species,but the rate was lower with P. gralli. Philophthalmus gralli adults would not carry out interspeciesmating, but exhibited the typical restrictive mating pattern (in groupsthey readily cross-inseminate but never self-inseminate) with their own species even in the presenceof the oppositespecies. A major difference between
mating in concurrent
infections of echinostomeswhen compared to eyeflukes is that no interspeciesmating wasfound when a choiceof the samespecieswas given to the donor echinostomeadult. Also, the rate of cross-insemination of the oppositespecieswasmuch lower in E. caproni (13%) when compared to P. megalurus (72.6%) (Nollen, 1984). No evidenceof fertilization betweenE. caproni and E. trivolvis, e.g., radioactive E. caproni spermin E. trivolvis eggs,was found in this study. Thus, no conclusion can be reached on whether hybridization would be possiblebetweenthese2 species.Suspected natural hybrids between hermaphroditic digeneans havebeendescribed(Nollen, 1983).Accountsof natural and laboratory-producedhybrids betweenschistosomespecieshave beensummarizedby Basch(1991). So far no attemptsto producehybrids in the eyefluke and echinostomespeciesusedin concurrent mating studieshave beenreported. REFERENCES Basch P. F. 1991. Schistosomes: Development, Reproduction and Host Relations, pp. 28-31. Oxford University Press, New York. Christensen N. 0. & Fried B., Kanev I. 1990. Taxonomy of 37-collar-spined Echinostoma (Trematoda: Echinostomatidae) in studies on the population regulation in experimental rodent hosts. Angewandte Parasitologie 31: 127-130. Fried B. & Haseeb M. A. 1990. Intra- and interspecies chemo-attraction in Echinostoma caproni and E. trivolvis in vitro. Journal of the Helminthological Society of Washington 57: 72-73. Huffman J. E. & Alcaide A., Fried B. 1988. Single and concurrent infections of the golden hamster, Mesocricetus auratus, with Echinostoma revolutum and Eehinostoma liei (Trematoda: Digenea). Journal of Parasitology 74: 606 608.
Mating
in concurrent
infections
Huffman J. E.. Murphy P. M.&Fried B. 1992. Superimposed infections in golden hamsters infected with Echinostoma caproni and Echinostoma trivolvis (Digenea: Echinostomatidae). Journal of the Helmintholoctical Society of’washington 59: 1621. Iorio S. L.. Fried B. & Hosier D. W. 1991. Concurrent infections of Echinostoma caproni and Echinostoma tricolcis in ICR mice. International Journal for Parasitology 21: 715-717. Kristensen A. R. & Fried B. 1991. A comparison of Echinastoma caproni and Echinostoma trivolvis (Trematoda: Echinostomatidae) adults using iscelectric focusing. Journul of Parasitology 71: 496498. Meece J. K. & Nollen P. M. 1996. A comparison of the adult and miracidial stages of Echinostoma paraensei and E. caprani. International Journal,for Parasitology 26: 3143. Morgan J. A. T. & Blair D. 1995. Nuclear rDNA ITS sequence variation in the tremdtode genus Echinostoma: an aid to establishing relationships within the 37-collarspined group. Parasitology 111: 609-615. Nollen P. M. 1983. Patterns of sexual reproduction among parasitic platyhelminthes. Parasitology 86: 999120. Nollen P. M. 1984. Mating behavior of Philophthalmusmegalurus and P. gralli in concurrent infections of chicks. International Journal,fbr Parasitology 14: 71-74.
of
Echinostomcr
spp.
Nollen P. M. 1990. Echinostoma caproni-mating behavior and the timing of development and movement of repro ductive ceils. Journal of Parasitology 76: 784-789. Nollen P. M. 1993. Echinostoma trivolris-mating behavior of adults raised in hamsters. Parasitology Research 79: 13&132. Nollen P. M. 1996. The mating hebaviour of Ed~hit~~~f~~~~~ paraensri grown in mice. Journal af Helmintholag~~ 70: 43 45. Nollen P. M. 1996. Mating behaviour of Echinostwnu c~zpmi and E. paraensei in concurrent infections in mice. ./r/zirntri of Helminthology 70: 133-I 36. Nollen P. M., Leslie J. F., Cain G. D. & MacNab R K. 1985. A comparison of Texan and Hawaiian strains of the avian eyefluke, Philophthalmus gralli, with a cautionary note on the importation of exotic animals. Jartrnal of Pwtr sitology 71: 618C624. Richard J. 1964. Trematodes d‘oiseaux de Madagascar (Note III). Especies de la famille Echinostomatidae Poche 1926. Annales de Parasitolagie Humuine et Comparw 39: 607 620. Sloss B., Meece J. & Roman0 M., Nollen P. 1995. The genetic relationships between Echinostoma caproni, E. paraensr/. and E. trivolvis as determined by electrophoresis. J~LII~~~J af’Helmintholog~ 69: 243246.