Site hosted by Angelfire.com: Build your free website today!
label1.gif (1092 bytes)

The effect of Trematode parthenitae on the growth of Onoba aculeus, Littorina saxatilis and L. obtusata (Gastropoda: Prosobranchia).

A.M. Gorbushin* and I.A. Levakin
WEB version of the paper published in J.Mar.Biol.Ass.U.K. (1999), 79, 273-279
Department of Cytology and Histology, Biology & Soil Science Faculty, St. Petersburg State University, Universitetskaya nab., 7/9, St. Petersburg, 199034, RUSSIA. *Corresponding author E.-mail: gorbushin@yahoo.com
To determine whether modern conceptions predict snail's growth responses to trematode infestation a field growth study of Onoba aculeus, Littorina saxatilis and L. obtusata infected with two closely related Microphallidae trematodes was conducted in the White Sea. In each of the three host-parasite combinations studied trematode infection causes inhibition of snail reproduction. However, in defiance of the classical interpretation of the gigantic growth of infected snails as a side effect of parasitic castration, the presented study failed to demonstrate that Microphallus piriformes causes gigantism in Littorina. The infection tended to stunt growth in L. obtusata and had no significant effect on growth rate of L. saxatilis. In contrast, gigantic growth was observed in O. aculeus infected with M. pseudopygmaeus. Considering that both trematode species are very similar biologically, the discrepancy is attributed to differences in the life history of the snail's hosts. Onoba aculeus is a relatively short-lived snail (2.5 - 3 y). The life span of L. saxatilis and L. obtusata is much longer (up to 9-11 y). These findings agree with previously reported 'energetic' hypothesis that predicts growth alterations in accordance with life history variations of the snail species.

Introduction.

Trematode parthenogenetic generations (sporocysts and rediae) infecting snails have been shown to cause changes in host growth rate (Sousa, 1983; Minchella, 1985; Gorbushin, 1997). Several studies have provided evidence of stunting effect of trematode infections (Moose, 1963; Sturrock & Sturrock, 1970; Sousa, 1983; Fernandez & Esch, 1991) whereas others have shown increased growth rates among infected specimens (Rothschild & Rothschild, 1939; McCleland & Bourns, 1969; Meuleman, 1972; Sluiters et al., 1980; Wilson & Denison, 1980; Mouritsen & Jensen, 1994, Huxham et al., 1995; Gorbushin, 1997). The phenomenon is usually connected with inhibition of snail reproduction (castration) (Sousa, 1983; Minchella, 1985) and it has been demonstrated that the growth rate correlates with the level of parasitic castration of a snail host (Gorbushin, 1997).
The existing hypotheses - 'adaptive' (Minchella,1985) and 'nonadaptive' (Sousa,1983) - attempting to explain the alterations in host growth operate with life history variations of snail species and predict different growth responses to infestation in semelparous and iteroparous molluscs. Most of the observations forming the basis of those views involved comparisons of the growth of healthy snails with ones infected with single species of parthenitae. Few researchers have adopted the approach of Sousa (1983) - comparing a growth response of certain snail host following infestation with different trematode species. However, this approach looks quite productive. For example, it has been demonstrated that the growth response of infected Hydrobia ulvae varies significantly with the trematode species. Most probably due to its lower pathogenicity, sporocistoid parthenitae induce increased host growth in contrast to redioid ones which cause no effect on growth rate (Gorbushin, 1997). This phenomenon produces the serious objection to both 'adaptive' and 'nonadaptive' hypotheses. In short, the differences between growth responses of infected semelparous and iteroparous snails may not be the result of different life histories of ones but that of different pathogenicity of parthenitae parasiting in these groups of hosts.
One way of attacking this problem are comparative studies of growth responses of short-lived and long-lived snail hosts following the infestation with the same or biologically similar trematode species. The growth study of Littorina saxatilis (Olivi, 1792) (Prosobranchia: Littorinidae), L. obtusata (Linnaeus, 1758) (Prosobranchia: Littorinidae), Onoba aculeus Gould, 1841 (Prosobranchia: Onobidae) infected with trematodes of 'pygmaeus' group (Microphallidae) was undertaken to use this approach therefore testing the principle idea of both concepts - significant role of host life history variations in affecting the phenomenon of growth alteration in infected snails.

Materials and methods.

Study systems. The investigation was carried out in the Chupa inlet, Kandalaksha Bay of the White Sea (Russia) in late August 1996. All three prosobranch species studied are dioecious and direct developers; show habitat selection with regard to the type of substrate but coexist at some extension of intertidal zone.
Onoba aculeus - small-sized species, height up to 5 mm. It inhabits the low level of the intertidal zone and is common on muddy gravel covered by fucoids. The species is involved in Fucus vesiculosus + Ascophillum nodosum biocenosis and reaches a population density of up to 6570 ind. m-2. An oviparous species. Recruits come from July to September (Scarlato, 1987). The longevity is 2.5 to 3 years (unpublished data). In the studied area O. aculeus is intermediate host of two trematode species - Microphallus pseudopygmaeus (Microphallidae) and Notocotylus sp. (Notocotylidae) (unpublished data).
Littorina obtusata is middle-sized snail, shell height up to 12 mm. An oviparous species. Recruits appear in August- September (Scarlato, 1987). The longevity in the White Sea is 8 to 11 years (Sergievsky et al., 1991).
Littorina saxatilis- shell height up to 12 mm. An ovoviviparous species. Recruits appear in August - September (Scarlato, 1987). ). The longevity in the White Sea is 7 to 9 years (Sergievsky et al., 1991).
The principle characteristics of ecology, population structure and trematode fauna of both periwinkle species in the White Sea is described by Granovitch 1992, Sergievsky et al., 1997. All the three snail species have no seasonal migrations and overwinter in anabiosis, forming aggregations beneath stones and sea grass.
Two closely related trematode species - Microphallus piriformes (Odner, 1905) Galaktionov, 1983 and M. pseudopygmaeus (Galaktionov, 1980) belong to the most specialized genus of the evolutionary advanced Microphallidae family (Deblock, 1977; Galaktionov & Dobrovolsky, 1987). They are very similar morphologically and ecologically. Due to minor morphological differences both were accounted as Microphallus pygmaeus (Levinsen, 1881) by the early 1980's (Galaktionov, 1980, 1983, 1984). These species have identical life cycles: parthenogenetic generations (sporocysts) develop in marine intertidal gastropods; there is no second intermediate host and metacercariae mature inside a daughter sporocysts; the definitive host - sea birds (usually Somateria mollissima) - can only become infected by eating an infected snail with mature metacercariae; molluscs are infected by swallowing trematode eggs. The development of the parasites inside the snail up to invasion stage occupies 1.5-2.0 months (Galaktionov, 1980, 1983, 1984).

map1.gif (8930 bytes)

Figure 1. Map of sampling locations (see the text).

Collection of animals. Littorina saxatilis and L. obtusata were sampled in Kruglaya Bay (Figure 1, loc. 1). The korga nearby Levin Navolok (a korga is the local name for the kind of bank exposed during low tide; Figure 1, loc. 2) was used for sampling of O. aculeus. Molluscs were sampled qualitatively in the middle horison of the fucoid belt during low tide. 126 L. saxatilis were collected handly from upper and lateral surfaces of several stones, 133 L. obtusata were washed off from seaweeds cut off from the stones. 75 Onoba aculeus were obtained by sieving removed sediment (muddy sand) lying between stones through sieves retaining snails larger than 0.5 mm. The sampled specimens were size-biased since mainly the larger individuals were collected in order to maximize the probability of finding infected ones.

Measurements and dissection. As a natural mark of beginning (late May) of snail growth season a last winter growth interruption line on a snail shell was used. The mechanism of it's formation on a shells of the White Sea intertidal snails is described by Gorbushin, 1993. The following measurements were taken from the snails' shell (Figure 2): shell diameter at the level of the last winter growth interruption line (d0) (Figure 3 A - C) and shell angle gain (D j ) from the last winter interruption line to the rim of the shell aperture. Because the specimens were collected almost at the end of snail growth season (Autumn frosts are possible already in early September), angle gain from an identified last winter-grade line on the shell should be considered as an annual growth. All measurements were taken under the stereomicroscope using a standard ocular provided with a micrometer and an ocular angle meter. For angle growth measurement, the shell was vertically oriented so that the collumelar axis was perpendicular to the plane of measurements. The error of the diameter measurement did not exceed 0.05 mm and the angle gain was measured in Littorina sp. and O. aculeus with p /32 and p /12 accuracy respectively.

Figure 2.

Figure 2. The diagram of a snail shell measurements (see the text).

Figure 3a

Figure 3b

 

Figure 3c

Figure 3. Photographs of (l) winter-grade lines on a shells of (A) Littorina saxatilis, (B) L. obtusata and (C) Onoba aculeus. 'uninf' - 2-y-old healthy individual; 'inf' - the same aged individual infected with Microphallus pseudopygmaeus.

Following measurements, the shells were crushed and the specimens were sexed according to the presence or absence of penis. Also the size of the penis was estimated by measuring the length from base to the tip under the stereomicroscope. The abdominal soft parts of the specimens were subsequently dissected and examined for trematode infection. Trematode species were identified according to Galaktionov (1980, 1983, 1984). During examination the sexual maturity of the individuals was also determined. Each snail was examined for the presence of sexual products in the gonads (eggs in female and active spermatozoids in males).
Two-tailed t-test was used for comparison between mean penis sizes of infected and uninfected L. saxatilis and L. obtusata males. The proportion of males in Onoba aculeus population studied was low so the statistical analysis for this species was not performed.
Since growth rate decreases with snail size (see results), comparison of growth rates between infected and uninfected specimens was carried out by applying a paired two-tailed t-test on pairs of snails with similar shell diameter at the last winter line (d0). Pairs of snails were chosen randomly from the data base using computer algorithm. Due to possible growth rate differences between sexes only individuals with similar sex were paired. In case of O. aculeus only females were taken into the analysis

Results.
The daughter sporocysts of Microphallus pseudopygmaeus were located in the Onoba aculeus gonads. Microphallus piriformes sporocysts were located in the gonad - digestive gland complex of both Littorina species. This type of localization leads to parasitic castration in both sexes. Microscopy of the gonads showed the absence of active spermatozoids in infected males in contrast to uninfected ones. Furthermore, infected males of both Littorina species exhibited significantly reduced penis size (t22=3.56; P<0.05 - Littorina saxatilis and t38=8.26; P<<0.0001 - L. obtusata). The number of eggs and embrions in the reproductive systems of infected females of all three snail species varied from none to very few and was always less than in gonads of uninfected individuals.
The growth patterns of L. saxatilis, L. obtusata and O. aculeus are shown in Figures 4 A,B and 5. The snails' growth rate correlates negatively with the individual shell size at the beginning of the growth season. The smaller shell diameter at the moment of winter-grade line formation, the higher angle gain at the date of sampling.
Figure 4

Figure 5

Figure 4. Growth rate (D j ) of Littorina saxatilis (A) and L.. obtusata (B) as a function of shell diameter (d0). Figure 5. Growth rate (D j ) of Onoba aculeus as a function of shell diameter (d0).
Infestation with Microphallus piriformes had not induced any significant changes (t65=0.76; P>0.05) in growth rate of L. saxatilis (Figure 4A). Growth rate of L. obtusata infected with the same species significantly (t89=2.49; P<0.02) decreased in comparison with uninfected ones (Figure 4B). In contrast, O. aculeus infected with M. pseudopygmaeus displayed a significantly (t18=6.5; P< 0.0001) increased growth rate (Figure 5).

Discussion.
The very special way of parthenogenetic generations development is typical for the 'pygmaeus' microphallides. The specific feature is very early parasitic castration of the host due to sprouting of sporocyst' stolones directly to the snail gonad (Galaktionov, 1987). Daughter sporocysts of the 'pygmaeus' group are motionless and obtain all their nutrition from direct absorption through its tegumental surface. Thus, severe pathogenic pressure on the host during migration of sporocysts, cercariae and, moreover, rediae directly ingesting the host tissues is not characteristic of 'pygmaeus' parthenitae. These species are relatively low-pathogenic trematodes. Indeed, the infection with microphallides from the 'pygmaeus' group has no significant impact on the resistance of Littorina saxatilis to unfavourable environmental factors (Galaktionov, 1990). It was shown that infected and uninfected snails do not differ in resistance to heat stress and desiccation. Moreover, infected individuals are more resistant to subzero temperatures. Increased mortality of infected Littorina was only shown in some experiments on the resistance to low salinity. It is important to note that Galaktionov (1990) postulated low pathogenicity of 'pygmaeus' microphallides for L. saxatilis but not for L. obtusata. He proposed that these parasites have a significant role in selective mortality of last snail species in the field.
In this study relatively higher pathogenicity of Microphallus piriformes for L. obtusata is supported by the fact of lowered host growth in this host-parasite combination. It is interesting to note that one and the same trematode species can affect host growth rate differently in closely-related snail species. The different level of antagonism between M. piriformes and two Littorina species may be attributed to generally lower resistance of L. obtusata to unfavourable environmental factors (K.V. Galaktionov, personal communication).
It is clear that each host-parasite combination is governed by its own rules, associated with ecological and physiological peculiarities of the snail and trematode species. Nevertheless, within the wide range of ecological, life cyclic, morphological and biological diversity of trematodes in general, M. piriformes and M. pseudopygmaeus should be considered as a very similar species. It is enigmatic, however, that these trematodes cause several types of host growth response: 'dwarfism' in L. obtusata, 'normal' growth in L. saxatilis and 'gigantism' in O. aculeus.
Why is the growth response of Onoba and Littorina to parasitic castration by 'pygmaeus' microphallides so different? The problem may be dealt with within the context of two concepts. Both operate with the life history of snail host and state that parasitic castration of a host forms the basis of the phenomenon, but produce quite different conclusions.
The first concept, proposed by Minchella (1985), is based on the idea that variations in the host life history in response to parasite infestation should be a common feature of any co-adapting host-parasite systems. By combining the hypothesis with previous results, Minchella (1985) predicts gigantism in parasitized individuals of long-lived (perennial) snail species. Negative effects of parasitism on host reproduction in such snails are short-lived relative to the maximum life. In contrast, in short-lived (annual) host species gigantism should not be observed as a rule.
Whether or not the giant growth of infected snails may be considered as a 'co-adaptive' phenomenon we leave out of the scope of this paper, because of the discussion on this subject is fruitless for understanding physiological mechanisms of the phenomenon. The more important point is the evident disagreement between data obtained in our study and predictions of Minchella's concept. Onoba aculeus, L. saxatilis and L. obtusata in terms of this concept should be attributed to 'perennial' species (>1.5 y). Nevertheless, only Onoba demonstrates gigantic growth following the infestation and Littorina, that survives in the field much longer, displays either no growth alterations at all or even decreased growth rate.
The other concept, proposed by Sousa (1983), considers gigantism as a side, selectively neutral effect of parasitic castration and predicts it, in contrast to Minchella's (1985) hypothesis, in short-lived or semelparous snail hosts. Long-lived iteroparous species will rarely, if ever, exhibit enhanced growth following the infestation. As a summary, Sousa's concept considering energy equilibrium in a host-parasite system takes into account the following factors which influence the snail growth response to infestation. First, the annual amount of energy invested in host breeding which is one of the parameters of the specie's life history. Second, the individual investment in breeding that varies with the individual host age. And finally, costs for host reparation of damages induced by parasitism determined by antagonism between host and parasite.
It seems that growth response of Onoba and both Littorina to infestation is in accordance with Sousa's prediction: the shorter-lived snail species exhibit gigantism in contrast to longer-lived ones. However, Sousa as well as Minchella defines upper longevity limit for 'short-lived' snails as 1.5 y; therefore it is not possible to attribute O. aculeus to those. This species in the White Sea can overwinter three times and survive up to about three years. It is worth noting that only two another known species with enhanced growth under infestation in the field, Hydrobia ulvae and H. ventrosa have very similar longevity ranges (Gorbushin, 1997). So, we have to admit that neither Minchella's nor Sousa's hypothesises are able to predict pronounced differences in growth response of O. aculeus, L. saxatilis, L. obtusata, H. ulvae and H. ventrosa to infestation with trematode parthenitae.
Mouritsen & Jensen (1994) observed another contradiction between these hypotheses: both authors use H. ulvae as an example of gigantism among short-lived (Sousa, 1983) and long-lived (Minchella, 1985) species. Gorbushin (1997) emphasized that, indeed, modern knowledge on life history of most of gastropod species is not good enough for an adequate comparative analysis. In our opinion, the consideration of life history variations as a key element in understanding the phenomenon should be performed within much wider longevity range than has been done previously. Actually, about 10% of L. saxatilis population on Ryazhkov Island (the White Sea) survive up to seven years (Granovitch, 1992). This fact and the finding in Kruglaja Bay (the White Sea) of the L. littorea having 17 clearly pronounced winter-grade lines on it's shell (unpublished data) testify to a greater diversity of snail life-histories than both Minchella and Sousa announced.
Developing Sousa's standpoint, Gorbushin (1997) supposed that, due to parasitism being one of the environmental factors which influences the host's condition, costs for struggling with parasite (maintenance costs in terms of Sousa, 1983) should be relatively high in adults of longer-lived species, in contrast to shorter-lived ones investing extensively into reproduction and sacrificing the effectivity of reparative mechanisms. Hence, parthenitae of one and the same trematode species with wide specificity could be relatively more pathogenic for shorter-lived molluscs than for long-lived ones. Thus, despite the release of a large energy pool by parasitic castration in annual snail species, they do not show enhanced growth due to severe pathogenesis induced by parasite. Antagonism between the same parasite and a long-lived (>3-4 y) host is lower, however, parasitic castration due to relatively low reproduction effort does not release an energy pool large enough to provide additional growth. Gigantism as a trematode induced phenomenon would be expected in snail species having intermediate life history (longevity 2-3 y). The data presented in this paper is in accordance with the last hypothesis.
Other factors that are usually ignored when examining host-parasite interactions are elements of the physical and biotic environment where the interactions occur. The significance of environmental conditions in predetermination of snail growth response to trematode infestation was underestimated until the publication of Fernandez & Ecsh (1991) paper. The authors postulated that variations in available amount of energy to be shared between host and parasite should cause a different results in growth experiments using field and lab approach. Indeed, Gorbushin (1997) has shown that host-parasite interactions across different environments may have different outcomes. Growth rate of Hydrobia ulvae infected with Himasthla sp. (Gorbushin, 1997) and L. littorea infected with Renicola roscovita (Mouritsen et all, 1998) may be different in different populations and habitats respectively. Growth response of H.ventrosa to infestation with Bunocotyle progenetica may vary from normal growth to enhanced one with decreasing population density (Gorbushin, 1997). On the other hand, due to behavioural alterations induced by parasites, spatial segregation of infected snails in the field hypothetically may lead to more or less successful feeding. The case exemplifying this idea has been described by Granovitch (1992) for L. saxatilis infected with 'pygmaeus' microphallides. These parasites influence the behaviour of the snails, interfering with their normal tidal migrations.
Thus, in field gastropod populations trematode parthenitae may significantly alter the host growth. Whether or not this phenomenon leads to any consequences for the host population is a problem that requires special consideration in each host-parasite combination. Another enigmatic point is the physiological mechanism of energy relocation in snail-trematode system. Due to much noise of uncontrolled environmental factors in the field, further tending of the last problem should be performed in a lab using multifactor approach.
We are grateful to Dr Andrej Granovitch for introducing the authors to ecology and biology of the snail species studied and to Dr Alexandra Kharazova for editing the draft manuscript. Access to the field sites and laboratories was kindly allowed by the White Sea Biological Station ('Kartesh') of Zoological Institute of Russian Academy of Science (RAS). The investigation was supported financially by the Russian Foundation of Basic Research (RFBR) grant N 96-04-48-965 and by program 'Universities of Russia', grant N 3918

References
Fernandez, J. & Esch, G.W., 1991. Effect of parasitism on the growth rate of the pulmonate snail Helisoma anceps. Journal of Parasitology, 77, 937-944
Galaktionov, K.V., 1980. Four types of metacercarial species in molluscs Littorina saxatilis and L. obtusata from the Barents and White Sea. Vestnik Leningradskogo Universiteta, Series 3, 21-28. [In Russian with English summary].
Galaktionov, K.V., 1983. Description of species Microphallus pygmaeus (Levivnsen, 1881) nec. Odner, 1905 and M. piriformes (Odner,1905) nom. Nov. (Trematoda: Microphallidae). Vestnik Leningradskogo Universiteta, Series 15, 20-30. [In Russian with English summary].
Galaktionov, K.V., 1984. Microphallidae of 'pygmaeus' group. II Microphallus triangulatus sp. nov. (Trematoda: Microphallidae). Vestnik Leningradskogo Universiteta, Series 3, 5-11. [In Russian with English summary].
Galaktionov, K.V., 1987. Life-cycles of trematodes in intertidal biocenosises. In Life-cycles of parasites in biocenosises of nothern seas (ed. Yu. Polyansky), pp. 5-28. Apatity: Kola Scientific Center. [In Russian with English summary].
Galaktionov, K.V., 1990 The influence of the parasitizing of microphallide parthenitae (Trematoda) on the littoral mollusc Littorina saxatilis (Olivi) resistance to stress. In Morphology and ecology of parasites of marine animals (ed. A. Dobrovolsky), pp. 12-33. Apatity: Kola Scientific Center. [In Russian with English summary].
Galaktionov, K.V. & Dobrovolsky, A.A., 1987. Hermaphroditic generations of Trematodes. Leningrad: Nauka. [In Russian].
Gorbushin, A.M., 1993. Structure of winter growth interruption lines and mechanism of their formation on Hydrobia ulvae Pennant 1777 (Gastropoda, Prosobranchia) shell in the White Sea. Zoologichesky Zhurnal, 72, 29-34 [In Russian with English summary].
Gorbushin, A.M. 1997 Field evidence of trematode-induced gigantism in Hydrobia spp. (Gastropoda: Prosobranchia). Journal of Marine Biological Association of the United Kingdom, 77, 785-800.
Granovitch, A.I. 1992. The effect of trematode infection on the population structure of Littorina saxatilis (Olivi) in the White Sea. In: Proceedings of the Third International Symposium on Littorinid Biology (ed. J. Grahame et al.), pp. 255-263. London: The Malacological Society.
Huxham, M., Raffaelli, D. & Pike, A., 1995. The effect of larval trematodes on the growth and burrowing behaviour of Hydrobia ulvae (Gastropoda: Prosobranchia) in the Ythan estuary, north-east Scotland. Journal of Experimental Marine Biology and Ecology, 185, 1-17.
McClelland, G. & Bourns, T.K.R., 1969. Effect of Trichobilharzia ocellata on growth, reproduction and survival of Lymnaea stagnalis. Experimental Parasitoogy, 24, 137- 146.
Meuleman, E.A., 1972. Host-parasite interrelationships between the freshwater pulmonate Biomphalaria pfeifferi and the trematode Schistosoma mansoni. Netherlands Journal of Zoology, 22, 355-427.
Minchella, D.J., 1985. Host life history variation in response to parasitism. Parasitology, 90, 205-216.
Moose, J.W., 1963. Growth inhibition of young Oncomelania nosophora exposed to Schistosoma japonicum. Journal of Parasitology, 49, 151 - 152
Mouritsen, K.M. & Jensen, K.T., 1994. The enigma of gigantism: effect of larval trematodes on growth, fecundity, egestion and locomotion in Hydrobia ulvae (Pennant) (Gastropoda: Prosobranchia). Journal of Experimental Marine Biology and Ecology, 181, 53-66.
Mouritsen, K.M., Gorbushin, A.M. & Jensen, K.T., 1998. The effect of parasitic castration on growth rate of Littorina littorea: field evidence for the life history hypothesis and population dependent effect. Journal of Marine Biological Association of the United Kingdom, (in press).
Rothschild, A. & Rothschild, M. 1939. Some observations on the growth of Peringia ulvae (Pennant 1777) in the laboratory. Novites Zoologica., 41, 240-247.
Scarlato, O.A. ed., 1987. White Sea molluscs. Nauka, Leningrad [In Russian].
Sergievsky, S.O., Granovitch, A.I. & Mikhailova, N.A., 1991. Age structure of the populations of intertidal snails Littorina saxatilis and L.. obtusata in the White Sea. Proceedings of Zoological Institute of Academy of U.S.S.R. (Trudy Zoologicheskogo Instituta AN SSSR), 233, 79-126 [In Russian].
Sergievsky, S.O., Granovitch, A.I. & Sokolova, I.M., 1997. Long-term studies of Littorina obtusata and Littorina saxatilis populations in the White Sea. Oceanologica acta, 20, 1, 259-265.
Sluiters, J.F., Brussaard-Wust C.M. & Meuleman E.A., 1980. The relationship between miracidial dose, production of cercariae, and reproductive activity of the host in the combination Trichobilharzia ocellata and Lymnaea stagnalis. Zeitschrift fur.Parasitenkunde, 63, 13-26.
Sousa, W.P., 1983. Host life history and the effect of parasitic castration on growth a field study of Cerithidea californica Haldeman (Gastropoda: Prosobranchia) and its trematode parasites. Journal of Experimental Marine Biology and Ecology, 73, 273-296.
Sturrock, B.M. & Sturrock, R.F., 1970. Laboratory studies of the host-parasite relationship of Schistosoma mansoni and Biomphalaria glabrata from St. Lucia, West Indies. Annals of Tropical Medicine and Parasitology, 64, 357-363.
Wilson, R.A. & Denison J., 1980. The parasitic castration and gigantism of Lymnaea truncatula infected with the larval stages of Fasciola hepatica. Zeitschrift fur.Parasitenkunde, 61, 109-119.