Experimental Parasitology 121 (2009) 163–166
Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Polystoma gallieni: Experimental evidence for chemical cues for developmental plasticity Mathieu Badets a,*, Jérôme Boissier a, Philippe Brémond b, Olivier Verneau a a
UMR 5244 CNRS-EPHE-UPVD, Biologie et Ecologie Tropicale et Méditerranéenne, Parasitologie Fonctionnelle et Evolutive, Université Via Domitia, 52 Avenue Paul Alduy, 66860 Perpignan Cedex, France b UR 016 CENETROP, Santa Cruz de la Sierra, Bolivia, IRD, Ambassade de France en Bolivie, 128 bis rue de l’Université, 75351 Paris 07, France
a r t i c l e
i n f o
Article history: Received 15 July 2008 Received in revised form 13 October 2008 Accepted 28 October 2008 Available online 5 November 2008 Keywords: Monogenea Polystomatidae Polystoma gallieni Amphibia Hyla meridionalis Developmental plasticity Host-derived signals
a b s t r a c t Among monogeneans that display direct life cycles, plastic developmental strategies may have been selected to counter the lack of transmission opportunities. Within amphibian polystomatids, some species of the genus Polystoma develop into two different phenotypes depending on the host physiological stage to which free swimming larvae attach. When oncomiracidia infest old tadpoles, they develop slowly and migrate during host metamorphosis towards the bladder where they reach maturity. On the other hand when larvae infest young tadpoles, they develop rapidly into neotenic phenotypes that reproduce in the branchial chamber. These alternative developments are explored through experimental infestations with Polystoma gallieni, the specific polystome of the stripeless tree frog Hyla meridionalis. When oncomiracidia were left for 6 h in water in which young tadpoles had been previously maintained for one day, they mainly developed into the neotenic phenotype regardless of the tadpole stage they encountered later. This indicates that P. gallieni could collect released host-derived signals before physical contact with its host. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction Whether chemical or physical, environmental cues collected by animals during development can trigger phenotypic plasticity, thus enhancing fitness within unpredictable ecosystems (see Relyea, 2003). Widely investigated in prey–predator interactions, a large diversity of signals may also mediate modifications of the ontogenetic development in host-parasite associations and maximize parasite transmission efficiency (Combes et al., 2002; Thomas et al., 2002). Indeed, parasites experience through their life cycles both definitive and intermediate host species as well as transitional environments, among which they may perceive many more signals than previously suspected (Thomas et al., 2002; Lagrue and Poulin, 2007). For instance, some parasitic nematodes that have been extensively studied, may switch their development on receipt of host-derived, transitional and environmental signals (see Hawdon and Shad, 1991). Within the Platyhelminthes, the Digenea may also either interrupt development when the intermediate host detects the presence of predators, namely their definitive host species, or adopt progenesis when no predator-related physiological response is mediated (Lagrue and Poulin, 2007). Among the Monogenea in which host specificity has been suspected to be, at least in part, mediated through host chemical signals (see Buchmann and Lindenstrøm, 2002 for a review; Ohashi et al., 2007), the link between * Corresponding author. Fax: +33 4 68 66 22 81. E-mail address:
[email protected] (M. Badets). 0014-4894/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2008.10.013
alternative development mechanisms and environment has never been explored. The Monogenea encompasses more than 25,000 species (Cribb et al., 2002) that show a direct life cycle involving one single host species. Monogeneans mainly infest gills or the skin of actinopterygian and chondrichthyan fishes and to a lesser extent the bladder, pharyngeal and palpebral cavities of sarcopterygians. With about 150 species classified in 21 genera, the Polystomatidae is the most diversified family among monogeneans, infesting amphibious tetrapods like amphibians and freshwater turtles. In response to the various ecological and reproductive patterns occurring within amphibians, a large diversity of developmental and behavioural strategies may have been shaped to maximize transmission efficiency among polystomatids (Kearn, 1994; Whittington, 1997; Tinsley, 2004). For instance Protopolystoma xenopodis, which is found from the permanently aquatic African clawed toad Xenopus laevis, reproduces regularly throughout the year. On the other hand, Pseudodiplorchis americanus that infests the desert-dwelling Couch’s spadefoot toad, Scaphiopus couchii, reproduces on only two or three nights a year. Due to the short reproduction period of its host in extreme environments, P. americanus has adopted ovoviviparity and lays fully developed eggs that hatch immediately during the host’s mating assembly, thus increasing transmission efficiency (Tinsley, 1983). While most polystomes retain the typical direct life cycle of monogeneans, some species of Polystoma, which infest almost exclusively anuran hosts of the Neobatrachia, display a singular
164
M. Badets et al. / Experimental Parasitology 121 (2009) 163–166
developmental plasticity (Combes, 1967, 1968; Maeder, 1973; Murith et al., 1977, 1978; Murith, 1979, 1981; Kok and du Preez, 1989, 1998; Kok, 1990). The life cycle is completed either on the gills of the tadpole or inside the bladder of the adult frog, leading to morphologically very different adult parasites. During the host breeding period, bladder worms lay eggs that develop into aquatic swimming larvae (oncomiracidia) infesting the branchial cavities of tadpoles. If larvae attach to young tadpoles, i.e. tadpoles less than 10–13 days old, they develop into neotenic phenotypes reaching sexual maturity after about 3 weeks. They then reproduce on the tadpole gills and die during host metamorphosis (Gallien, 1935; Williams, 1961; Combes, 1968). By contrast, if larvae attach to old tadpoles, i.e. tadpoles older than 10–13 days, they develop much more slowly and migrate to the bladder during host metamorphosis. They reach maturity when the hosts reproduce for the first time, about 2–3 years later, as another well developed phenotype or bladder form (Gallien, 1935; Williams, 1961). Therefore, depending on the physiological stage of the tadpoles to which parasite larvae attach, Polystoma is able to develop into two different phenotypes, both of them being adapted to maximize transmission in relation to host behaviour and ecology in temporary freshwater environments (Williams, 1961; Combes, 1968). Instances of unexpected parasite development following experimental infestations were reported in the 1980s whilst studying the biology of Polystoma integerrimum that infests the common frog Rana temporaria (Brémond, unpublished observations). When tadpoles of different age classes were brought together and infested simultaneously, some parasites developed into the neotenic phenotype on old tadpoles as though they infested young tadpoles. It was suspected that the presence of young tadpoles during infestation experiments could trigger neotenic development regardless of the age of tadpoles that subsequently acted as hosts. In the present study, we investigated whether neotenic development requires physical contact between host and parasite. We reared tadpoles of the stripeless tree frog Hyla meridionalis and eggs of its specific parasite Polystoma gallieni in order to perform experimental infestations. We infested old tadpoles with two different samples of parasite larvae, one sample being previously left in spring water, and the other in water in which young tadpoles had been maintained. We measured the proportion of both resulting parasite phenotypes in each experiment and consider the possible host signal that could induce parasite development.
twice a week. As parasite eggs may bind to frog eggs, the parasite eggs were removed when present. After hatching, tadpoles were immediately divided into samples of 30 individuals in tanks containing six litres of spring water. They were reared at 23 °C under artificial daylight and fed abundantly with commercial fish food (Tetra-min) and boiled lettuce before and after infestation experiments. 2.2. Infestation experiments The experimental infestation procedure described below was designed to test whether chemical cues produced by tadpoles could influence parasite development before physical contact between host and parasite. Two infestation experiments were conducted, both of which involved two control and one trial samples of 30 tadpoles each. For experiment 1, the two control samples involved young and old tadpoles aged 1 and 14 days, respectively. The trial sample involved tadpoles aged 14 days. For experiment 2, the two control samples consisted of young and old tadpoles aged 7 and 24 days, respectively. The trial sample involved tadpoles aged 24 days. All control tadpoles were individually exposed over 6 h to five oncomiracidia previously left over 6 h in spring water. On the other hand, the trial tadpoles were infested following the same procedure but with oncomiracidia that had previously been kept for 6 h in water, in which young tadpoles had been maintained for 24 h. These tadpoles were 1 day old in experiment 1 and 7 days old in experiment 2. All infestation experiments were performed in 10 cl round plastic dishes filled with 5 cl of spring water. Afterwards, tadpoles were reared for 7 days, thus allowing first stages of parasite development. No tadpole mortality was observed. Finally tadpoles were anesthetized with MS 222 (1%) for a few minutes and sacrificed. They were examined for the presence of parasites; if present, parasite phenotype was determined. According to Gallien (1935), Williams (1961) and Combes (1968), the neotenic development pathway involves fast developing haptoral suckers and digestive tracts with brown or red contents due to the intense blood feeding activity of the gill parasites. On the other hand, the bladder development pathway involves slow developing internal organs and haptoral suckers with two growing hamuli located between the first pair of suckers. Fisher’s exact tests were performed to compare the distribution of the parasitic phenotypes, i.e. neotenic versus bladder developmental pathways.
2. Materials and methods 3. Results 2.1. Host and parasite sampling and rearing conditions Fieldwork was conducted during spring 2007 in a temporary pond in the vicinity of Opoul, which is a small village located in Southern France (Province of Pyrénées Orientales, 42°520 N, 2°520 E; altitude 160 m). Hyla meridionalis adults were collected by hand at night using flashlights (Ministerial authorisation no. 07/168/AUT delivered to Olivier Verneau for animal capture). During captivity, frogs were maintained individually in plastic tanks and fed every day with flies and crickets. Infected frogs were detected by the presence of parasite eggs released within host urine. Polystome eggs were recovered once a day by pipetting under binocular microscope and incubated at 23 °C in Petri dishes with commercial mineral water according to empirical recommendations of Combes (1968). Following that procedure, about 90% of eggs hatched after 12–15 days. After hatching, parasite larvae were divided into two samples and left for 6 h in spring water or in water in which young tadpoles less than 7 days old had been maintained for 1 day. Frog spawn obtained from natural mating in the field was incubated at 20 °C in oxygenated spring water, which was renewed
Results are shown in Fig. 1. In both experiments, control samples show as expected that young tadpoles aged 1 and 7 days only displayed parasites developing towards the neotenic phenotype, while old tadpoles aged 14 and 24 days preferentially displayed parasites developing towards the bladder phenotype. The fact that one 14 day old and two 24 day old tadpoles showed parasites developing towards the neotenic form was unexpected. Nevertheless former experimental infestations using old tadpoles of the same cohort displayed no neotenic parasitic larvae (data not shown). In each experiment, the distribution of the parasite phenotypes differs significantly between the two control samples (Fischer’s exact test, p < 0.0001 for experiments 1 and 2). Conversely, there were more tadpoles in the trial samples displaying parasites developing towards the neotenic phenotype than in the control samples involving tadpoles of the same age (Fischer’s exact test, p = 0.0056 and p < 0.0001 for experiments 1 and 2, respectively). In experiment 1, we reported a tadpole from the trial sample with two parasites, one of which followed the neotenic pathway and the other the bladder pathway. When this tadpole
M. Badets et al. / Experimental Parasitology 121 (2009) 163–166
Experiment 1
28 20
20 11
10 11
0 Control 1 1 day old tadpoles
1
Control 2 14 day old tadpoles
0 Trial 1 * 14 day old tadpoles
Experiment 2
23
20
18 12 8 0 Control 3 7 day old tadpoles
3
4
Control 4 24 day old tadpoles
2 Trial 2 24 day old tadpoles
Fig. 1. Groups of tadpoles following experimental infestations. Black boxes: tadpoles bearing parasites that developed towards the neotenic phenotype. Grey boxes: tadpoles bearing parasites that developed towards the bladder-destined phenotype. White boxes: uninfected tadpoles. Experiment 1 involved 1 day old and 14 day old tadpoles while experiment 2 involved 7 day old and 24 day old tadpoles. Tadpoles were individually exposed to either five oncomiracidia of Polystoma gallieni previously exposed for 6 h to spring water (control samples) or to oncomiracidia exposed for 6 h to water that had contained young tadpoles, 1 day old in trial 1 and 7 days old in trial 2. One tadpole had two parasites, one of which followed the neotenic pathway and the other the bladder pathway. Thus this tadpole was counted twice.
165
3, Fig. 1). In contrast, when larvae were brought into contact with old tadpoles aged 14 days or more, they developed mostly into bladder-destined parasites (controls 2 and 4, Fig. 1). Oncomiracidia that were exposed to water that had previously held young tadpoles were more likely to undergo neotenic development, regardless of the age of tadpoles subsequently infected (trials 1 and 2, Fig. 1). These results demonstrate that Polystoma larvae are sensitive to tadpole-derived chemicals in the water, which reveal the host’s physiological stage without the requirement for physical contact. Also, previous infestation experiments involving tadpoles aged 10–13 days produced parasites with an expanded intermediate phenotype, namely the ‘‘metagyrodactyloide” phenotype, which was described as ‘‘arrested neotenic development” in the early stages of development (Gallien, 1935; Combes, 1968). Because these larvae were unable to switch to the bladder-destined phenotype, they all died before host metamorphosis. Thus products released by young tadpoles probably act as determinants in the early steps of neotenic development and are likely to be precise and concentrated to take effect. Several studies conducted on fish monogeneans have revealed that mucopolysaccharides and carbohydrates extracted from skin and mucous cells could potentially play a part in the interaction and specificity within host–parasite associations (Buchmann, 1998; Buchmann and Bresciani, 1998). It has also been shown that deciliation of oncomiracidia, which normally occurs after attachment on the host, could be governed by carbohydrates extracted from fish epithelium (Yoshinaga et al., 2000; Ohashi et al., 2007). Because different types of molecules may be released during the 1st day of tadpole development, identification of the developmental induction signals is difficult. However, the frog–Polystoma interaction could provide new opportunities to investigate host-derived cues within the Monogenea that act as the intimate link between developmental plasticity and changing environment. Acknowledgments
was removed from the analysis, the difference between trial and control samples remained statistically significant (Fischer’s exact test, p = 0.0054).
We are grateful to Daniel James Phillips for his help in collecting frogs. We also thank one anonymous reviewer for constructive and very helpful remarks and Vanessa Messmer for improving the English.
4. Discussion References Because polystome transmission as well as frog reproduction are restricted to freshwater environments and to only a few months of the year in the rainy season, the genus Polystoma through its alternative development, is the single example among the Monogenea that shows an opportunistic and plastic strategy to increase the number of infective larvae in relation to host habits (Combes, 1968). In this host–parasite association, both partners experience a race against the clock. Tadpoles have to metamorphose before the pond dries up by the end of June, while neotenic parasites must develop quickly and reproduce long enough before host metamorphosis in order to give rise to a second generation of free swimming infective larvae, which themselves have to find another host before the pond dries up. Thus, temporal limitations in the completion of host and parasite life cycles may have constrained Polystoma to trigger neotenic development only on tadpoles less than 10–13 days old. In fact, larvae that attach to tadpoles that have passed this physiological threshold would not have time to reproduce as neotenic forms before host metamorphosis. Hence, larvae that attach to old tadpoles always grow up into immature parasites, migrate to the bladder during the host’s metamorphosis and reach maturity a few years later. Our results show that neotenic development was always involved when parasite larvae were brought experimentally into contact with young tadpoles less than 7 days old (controls 1 and
Buchmann, K., Lindenstrøm, T., 2002. Interactions between monogenean parasites and their fish hosts. International Journal for Parasitology 32, 309–319. Buchmann, K., 1998. Some histochemical characteristics of the mucus microenvironment in four salmonids with different susceptibilities to gyrodactylid infection. Journal of Helminthology 72, 101–107. Buchmann, K., Bresciani, J., 1998. Microenvironment of Gyrodactylus derjavini on rainbow trout Oncorhynchus mykiss: association between mucous cell density in skin and site selection. Parasitology Research 84, 17–24. Combes, C., 1967. Recherches sur les formes néoténiques de Polystomatidae (Monogenea). Présence d’une forme néoténique chez Polystoma gallieni Price, 1938. Bulletin de la Société Neuchâteloise des Sciences Naturelles 90, 205–214. Combes, C., 1968. Biologie, écologie des cycles et biogéographie de digènes et monogènes d’amphibiens dans l’est des Pyrénées. Mémoire du Muséum National d’Histoire Naturelle, Série A Zoologie, Fascicule Unique, 1–195. Combes, C., Bartoli, P., Théron, A., 2002. Trematode transmission strategies. In: Lewis, E.E., Campbell, J.F., Sukhdeo, V.K. (Eds.), The Behavioural Ecology of Parasites. CAB International, Wallingford, pp. 1–12. Cribb, T.H., Chisholm, L.A., Bray, R.A., 2002. Diversity in Monogenea and Digenea: does lifestyle matter? International Journal for Parasitology 32, 321–328. Gallien, L., 1935. Recherches expérimentales sur le dimorphisme évolutif et la biologie de Polystoma integerrimum Fröhl. Travaux de la Station Zoologique de Wimereux 12, 1–181. Hawdon, J.M., Shad, G.A., 1991. Developmental adaptations in nematodes. In: Aeschlimann, A., Bolis, L., Toft, C.A. (Eds.). Parasite–host Associations, Oxford University Press, pp. 247–298. Kearn, G.C., 1994. Evolutionary expansion of the Monogenea. International Journal for Parasitology 24, 1227–1271. Kok, D.J., 1990. Polystoma umthakathi (Monogenea): establishment, mortality and reproduction of neotenic parasites in experimentally infected Natalobatrachus bonebergi tadpoles. South African Journal of Zoology 25, 26–30.
166
M. Badets et al. / Experimental Parasitology 121 (2009) 163–166
Kok, D.J., du Preez, L.H., 1989. Polystoma australis (Monogenea): development and reproduction in neotenic parasites. South African Journal of Zoology 24, 225–230. Kok, D.J., du Preez, L.H., 1998. The relative importance of bladder versus neotenic stages of Polystoma marmorati and P. umthakathi in natural frog populations in South Africa. Journal of Helminthology 72, 117–121. Lagrue, C., Poulin, R., 2007. Life cycle abbreviation in the trematode Coitocaecum parvum: can parasites adjust to variable conditions? Journal of Evolutionary Biology 20, 1189–1195. Maeder, A.-M., 1973. Monogènes et Trématodes parasites d’Amphibiens en Côte d’Ivoire. Revue Suisse de Zoologie 80, 267–322. Murith, D., 1979. Identité des larves de polystomes (Monogenea) parasites du têtard de Dicroglossus occipitalis (Günther) en Côte-d’Ivoire. Zeitschrift für Parasitenkunde 59, 187–194. Murith, D., 1981. Contribution à l’étude de la systématique des polystomes (Monogènes, Polystomatidae) parasites d’Amphibiens Anoures de basse Côted’Ivoire. Revue Suisse de Zoologie 88, 475–503. Murith, D., Vaucher, C., Combes, C., 1977. Coexistence de la néoténie et du cycle interne chez un Polystomatidae (Monogenea). Comptes Rendus de l’Académie des Sciences, Paris 284, 187–190. Murith, D., Miremad-Gassmann, M., Vaucher, C., 1978. Contribution à l’étude des polystomes d’amphibiens du Cameroun. Revue Suisse de Zoologie 85, 681–698.
Ohashi, H., Umeda, N., Hirazawa, N., Ozaki, Y., Miura, C., Miura, T., 2007. Purification and identification of a glycopotrein that induces the attachment of oncomiracidia of Neobenedenia girellae (Monogenea, Capsalidae). International Journal for Parasitology 37, 1483–1490. Relyea, R.A., 2003. How prey respond to combined predators: a review and an empirical test ecology 84, 1827–1839. Thomas, F., Brown, S.P., Sudkhedo, M., Renaud, F., 2002. Understanding parasites strategies: a state-dependent approach? Trends in Parasitology 18, 387–390. Tinsley, R.C., 1983. Ovoviviparity in platyhelminth life-cycles. Parasitology 86, 161– 196. Tinsley, R.C., 2004. Platyhelminth parasite reproduction: some general principles derived from monogenans. Canadian Journal of Zoology 82, 270–291. Whittington, I.D., 1997. Reproduction and host-location among the parasitic platyhelminthes. International Journal for Parasitology 27, 705–714. Williams, J.B., 1961. The dimorphism of Polystoma integerrimum (Frölich) Rudolphi and its bearing on relationships within the Polystomatidae: part III. Journal of Helminthology 35, 181–202. Yoshinaga, T., Nagakura, T., Ogawa, K., Wakabayashi, H., 2000. Attachment-inducing capacities of fish tissue extract on oncomiracidia of Neobenedenia girellae (Monogenea, Capasalidae). Journal of Parasitology 86, 214–219.