Echinococcus: Biology and strain variation

ECHINOCUCCUS:

BIOLOGY AND STRAIN VARIATION

R. C. A. THOMPSON and A. .I. LYMBERY Division of Veterinary Biology, School of Veterinary Studies, Murdoch University, Murdoch, Western Australia 6150, Australia R. C. A. and LYMBERY A. J. 1990. Echinococcus: biology and strain variation. Znter~ti~~aiJournal for Pffr~itology 20: 457-470. Biology and strain variation in the causative agent of hydatid disease is reviewed with emphasis on developmental and genetic aspects. In vitro cultivation experiments have made a significant contribution to current knowledge of the developmental plasticity of Echinococcus. However, the mechanisms which regulate and determine developmental strategies in the parasite, as well as the characteristics, source and cytodifferentiation of germinal cells, are not understood. The nature, significance and origin of strain variation in Echinococcusare examined. Before we can fully appreciate the phenotypic consequences of genetic differentiation between populations, we need to know something about the genetic and environmen~l components of variation in traits such as development rate, host preference, host specificity, virulence and drug resistance. There is an urgent need for research on the developmental pathways by which genetic differences within and between strains of E. granulosusare translated to phenotypic differences in these traits. A~act-THaM~~N

INDEX KEY WORDS: Echinococcus;developmental biology; in vitro studies; strain variation; genetic variation; phenotypic variation; evolutionary biology.

INTRODUCTION taeniid in requiring two mammalian hosts for completion of its life cycle. However, compared to members of the genus Tueniu, Echinocuccus exhibits marked differences not only in morphology but also in its lower host specificity and much greater reproductive potential of the metacestode (Thompson, 1986). The basic life cycle patterns of the two major species, E. granulosus and E. multilocularis, are illustrated in Figs. 1 and 2. These may be considered to be natural cycles and in the case of E. granularus, is thought to also be ancestral (Rausch, 1986). However, the public health and economic significance of hydatid disease (echinococcosis/hydatidosis), as the most important of the cestode zoonoses, is directly attributable to human factors which have allowed interaction between natural (sylvatic) and domestic cycles (Thompson, 1988; Thompson & Allsopp, 1988) and have resulted, pa~ic~arly in the case of E. granulosus, in the widespread global perpetuation of Echinococcus in a variety of domestic, man-made life cycle patterns (Figs. 1 and 2). As a consequence, hydatid disease remains a problem of varying proportions on every continent-from a relatively localized problem with just one species, E. granulosus, in Australia to the widespread occurrence of both E. granulosus and E. rnult~~~~u~ar~s throughout mainland China (Rausch, 1986; Schwabe, 1986; Thompson & Lymbery, 1988; Thompson & Allsopp, 1988; Eckert, 1989; Schantz, in press; Schantz & Okelo, in press). Apart from human involvement, the public health and economic significance of hydatid disease is also Echinococcus is typically

attributable to two other major factors; (i) the lack of effective chemotherapeutic and prophylactic regimes and (ii) inherent flexibility of the parasite with respect to its development and genetic/evolutionary potential. It is this second factor on which we wish to focus in this review. DEVELOPMENTAL BIOLOGY Metacestode development

With the metacestode of Echinococcus, it is clear that we can only go so far in generalizing about larval development and that marked differences exist between species of ~ch~n~c~ccus as well as at the intraspecific level. It was the pioneering work of Dew (1922, 1925) in Australia that established the basic features of the endogenous development of unilocular hydatid cysts whereas it has taken longer to determine the histological features of the more complex, proliferating metacestode of E. mu~tilo~ularis (Rausch, 1954; Vogel, 1978). In fact, it was confirmed only recently that the infiltration of surrounding tissue in E. multilocularis infections was due to rod-like cellular protrusions consisting mainly of undifferentiated germinal cells which may detach and cause distant metastatic foci following their distribution via the circulatory system (Mehlhom, Eckert & Thompson, 1983; Eckert, Thompson & Mehihom, 1983). In vitro studies Although observations on natural and experimental infections have revealed details of sequential development in both intermediate and definitive hosts, it is primarily from in vitro studies that most information

457

458

R. C. A. THOMPSON and A. J. LYMBERY

Biology and strain variation

LION STRAIN

of Echinococcus

AUSTRALIAN SYLVATIC STRAIN?

PIG STRAIN?

CAMEL STRAIN?

BUFFALO STRAIN

KENYAN SHEEP STR

FIG 2. FIGS. 1 and 2. Life cycle patterns of Echinococcus granulosus and E. multilocularis showing presumed natural (sylvatic) cycles and some derived artificial cycles. For E. granulosus, forms perpetuated in different life cycle patterns may represent distinct strains, although the status of those in Fig. 2 requires further study.

R. C. A. THOMPSONand A. J. LYMBERY

460

and conceptual advances have been obtained concerning the seemingly unique developmental characteristics of Echinococcus. The difficulties associated with the in vivo maintenance of Echinococcus in the laboratory, the nature of the hosts involved and risks of human infection, combined with the amenability of its size, undoubtedly resulted in more emphasis being placed on the in vitro cultivation of Echinococcus than other taeniids. As a result of such work, the major developmental sequences have been duplicated in axenic culture and we can now maintain much of the life cycle in vitro (reviewed in Smyth & Davies, 1974a; Howell, 1986; Arme, 1987). In addition, such studies have proved particularly rewarding with respect to

FIG. 3. Secondary

revelations coccus.

of the developmental

potential

of Echino-

Dual potential The culmination of successful larval development in the intermediate host is the formation of protoscoleces. They consist of an invaginated scolex of which the overlying basal tegument has a protective mucopolysaccharide coating (Marchiondo & Andersen, 1983). Once fully developed, the protoscolex will remain dormant within the hydatid cyst until the integrity of the sterile (aseptic) cystic environment is altered. Physical or immunological damage to the cyst may result in death of protoscoleces following their

hydatid cysts (arrows) of Echinococcus granulosus in the peritoneal cavity of an experimentally infected rat.

461

Biology and strain variation of Echinococcus exposure to specific and/or non-specific components of the host’s humoral defence system or as a result of bacterial contamination. Cysts may also become ‘walledoff by an exuberant cellular response leading to starvation of the parasite. Alternatively, a cyst may rupture causing leakage of cyst contents and protoscoleces may themselves vesiculate and develop into new, secondary, hydatid cysts. The same phenomenon of secondary cyst production also occurs if protoscoleces are inoculated directly into the peritoneal cavity of a susceptible rodent (Fig. 3). If, however, protoscoleces are ingested by the correct species of definitive host, they will

evaginate and attach to the small intestinal mucosa in the Crypts of Lieberkiihn and develop into adult worms. However, even the adult worm can dedifferentiate under certain circumstances and develop in a cystic direction, as demonstrated when worms are exposed to unfavourable conditions in vitro (Fig. 4) @myth, 1969; Thompson, Deplazes & Eckert, in press). It is this dual development potential, and attempts to obtain strobilar development in vitro with E. granulosus and E. multilocularis, that have helped to explain the nature of the stimuli and conditions required to induce and support development.

4

FIG. 4. Vesiculating adult worm of Echinococcus granulosus in vitro. Worms were transferred to culture from an experimentally infected dog 35 days post-infection. As acidity of culture media decreased, worms vesiculated and became cystic (scale bar = 500 pm).

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R. C. A. THOMPSON and A. J. LYMBERY

Adult development Work on the common sheep strain of E. grunulosus

has shown that if protoscoleces are evaginated and exposed to diphasic culture conditions they will develop normally, into mature, segmented, adult worms (such worms fail to undergo fertilization for reasons not clearly understood and this barrier undoubtedly represents a major challenge for future research) (reviewed in Smyth & Davies, 1974a; Howell, 1986; At-me, 1987). Under monophasic culture conditions, although some growth occurs, worms fail to undergo either proglottization or segmentation. Diphasic conditions must include a nutritive substrate, for example coagulated serum as distinct from non-nutritive agar. The nature of the strobilization stimulus has been discussed in detail and is believed to involve nutritional and physical interactions (reviewed in Howell, 1986). It was assumed that such a stimulus would be a fundamental requirement for Echinococcus species, as it appears to be for some other taeniids such as Taenia serialis in vitro (Smyth, 1969). Intriguingly, however, this is not the case. It has been found that not all strains of E. granulosus will develop in vitro. In a series of comparative experiments which were to markedly alter our way of thinking about intraspecific variation in Echinococcus, Smyth and Davies (1974b) demonstrated that when exposed to exactly the same in vitro conditions that induce strobilar development in the sheep strain of E. granu~o,~u.~,the horse strain failed to undergo proglottization or segmentation. Evaginated worms of horse origin elongated slightly in vitro but failed to develop any further, although remaining alive, active and vermiform for many weeks. There is evidence to suggest that other strains of E. granulosus may also behave differently in vitro (Smyth, 1982). With E. multilocularis the situation with respect to sequential strobilar development in viva is very similar to that in E. granulosus, although development is much quicker (Thompson, 1986). However, under the same in vitro conditions as used for E. granulosus, E. multilo~ul~r~s responds erratically and in marked contrast to the relatively uniform developmental pattern achieved with E. gr~nulosus. fn vitro development of E. mult~lo~ularis is highly variable and although sexually mature forms can be produced from protoscoleces, segmentation is usually suppressed, thus demonstrating the independence of somatic and germinal differentiation (Smyth & Davies, 1975; Smyth, 1979; Smyth & Barrett, 1979; and reviewed in Howell, 1986; Arme, 1987). Recent studies suggest that this variability may have something to do with differences in preparative procedures between those used for E. granulosus and E. multilocularis due to the

different nature of the metacestode tissues (Thompson ef al., in press). However, this does not explain the int~guing observation that proglottization and segmentation of E. mult~lo~ulariscan be induced in vitro without a nutritive base, i.e. under monophasic conditions (Smyth & Davies, 1975; Smyth, 1979). In addition, we have found that E. multilocularis behaves differently to E. granulosus in that, although in vitro conditions may be suitable for the induction of proglottization and segmentation, further triggers appear to be necessary to sustain and stimulate maturation and subsequent proglottization (Thompson et al., in press). This is based on the observation that degeneration of in vitro cultured worms was related to the stage of development reached rather than the duration in culture. IE vitro studies of strobilar development thus demonstrate the complexity of developmental processes in Echinococcus, which appear to be dependent upon a diversity, and perhaps in some cases a series, of triggers which differ between species and strains. The ability of Echinococcus to develop into either cystic or adult forms in vitro and the fact that, at least with E. multilocularis, somatic and germinal developmental processes exhibit some degree of independence, suggest a very complicated process of cytodifferentiation and the possible existence of several primitive stem cell lines, as in other cestodes (Sulgostowska, 1972, 1974). In order to understand the developmental biology of Ech~~ococcus, it is impo~ant that we determine which lines of cells are responsible for initiating the separate processes of somatic and germinal differentiation in E. granulusus and E. multilocularis. However, studies at the light microscope and ultrastructural levels suggest that only one primitive cell type exists in both species (Sakamoto & Sugimura, 1970; Gustafsson. 1976). If there is only one such cell, it is obviously extremely sensitive to environmental conditions and further studies are required to characterize this cell, or cells, further. Germinal cells The isolation and in vitro cultivation of the germinal cells of E. granu~osus and E. multilocul~r~ would be of great benefit in determining their origin and characteristics. However, this has proved difficult and controversial because of the problems in isolating parasite cells to the exclusion of host cells (Fiori, Monaco, Scappaticci, Pugliese, Canu & Cappuccinelli, 1988; Howell & Matthaei, 1988). In addition, isolated parasite cells may he difficult to maintain for long periods in vitro (Pawlowski, Yap & Thompson, 1988). The ability to maintain Echinococcus lines would also be valuable for comparative studies on genomic

FIGS. 5 and 6. Histological sections through hydatid cysts of ~chjnocoee~ granulosusfrom a sheep (Fig. 5) and pig (Fig. 6). showing germinal (G), laminated fL) and adventitial (A) layers. Cellular proliferation adjacent to the Iaminated layers has resolved to form an adventitial layer in the sheep but not in the pig (scale bars = 100 pm).

Biology and strain variation

FIGS. 5 and 6.

of Echinococcus

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R. C. A. THOMPSON and A. J.

464

control mechanisms between the two species since, as emphasized by Howell (1986), species of Echinococcus provide unique models for studies concerned with genetic influences on development. The ability to cultivate cells of Echinococcus would also assist in confirming the origin of the laminated layer and its precise site of synthesis, about which there is still some controversy. Such studies may help to explain why the germinal cells behave differently in different host species and the factors responsible for initiating variable development. For example, in the normal intermediate rodent hosts, E. multilocularis develops rapidly and produces protoscoleces after which proliferation is curtailed, whereas in humans, proliferation may continue indefinitely with few if any protoscoleces produced (Rausch & Wilson, 1973). With E. granulosus we see similar contrasting development between hosts. For example, hydatid cysts of the common sheep strain appear very similar histologically in sheep, cattle and pigs, with apparently identical germinal and laminated layer formation (Figs. 5 and 6). However, whereas protoscolex production takes place readily in sheep, it is a rare occurrence in cattle and pigs (Thompson, Kumaratilake & Eckert, 1984; Thompson, Lymbery, Hobbs&Elliot, 1988). The reasons for this difference are not understood. It may be nutritional or related to the lack of adventitial layer formation in cattle and pigs since cellular infiltration adjacent to the cyst often does not resolve (Thompson, 1986). Immunological factors may thus provide the essential triggers which govern germinal cell proliferation and protoscolex production. Although some germinal cells initiate the formation of brood capsules and protoscoleces, a pool of uncommitted, undifferentiated germinal cells must remain in the germinal layer to initiate new cycles of asexual multiplication (Thompson, 1986). In addition, both protoscoleces and adult worms have a dual potential and therefore must retain some undifferentiated multipotential germinal cells. Asexual reproduction Unfortunately, the lack of information on the origin and cytodifferentiation of metacestode germinal cells represents a serious deficiency in our understanding of the biology of Echinococcus. The characteristic, asexual reproductive behaviour exhibited by species of Echinococcus represents the pinnacle of reproductive larval potential achieved by any taeniid. The asexual metacestode of Echinococcus has a virtually unlimited sequential generative capacity (Whitfield & Evans, 1983), and is of particular evolutionary and genetic significance and, as such, is central to theories which have attempted to explain the origins of strain variation in Echinococcus. STRAIN

VARIATION

A great many intraspecific variants, or strains, of Echinococcus have been described from different

LYMBERY

geographic areas or intermediate host species (Kumaratilake & Thompson, 1982; McManus & Smyth. 1986; Thompson & Lymbery, 1988) (Table 1). Smyth & Smyth (1964) proposed that two aspects of the reproductive behaviour of species of Echinococcus favour the production of different strains. Firstly, adults are presumed to be largely or exclusively selffertilizing, so that mutations in germ cells are likely to be transmitted in both heterozygous and homozygous form to offspring. Secondly, protoscoleces are produced asexually in the intermediate host, so that a large number of genetically identical individuals may arise from a single mutant offspring. This clone of mutant individuals may be recognized as a new strain. Another model of strain variation in Echinococcus was proposed by Rausch (1985). He argued that adults reproduce by cross-fertilization and that strains result from the differentiation of populations artificially isolated in different synanthropic cycles. The model proposed by Smyth & Smyth (1964) requires asexual reproduction of the metacestode to amplify rare mutations, while that proposed by Rausch (1985) does not. The fundamental point of departure of these models, however, is their contrasting assumptions about the breeding system of adult worms. Little empirical information is available on the predominant mode of fertilization in natural populations. Selfinsemination, but not cross-insemination, has been observed in E. granulosus, E. multilocularis and E. oligarthrus collected from naturally and experimentally infected definitive hosts (Smyth & Smyth, 1969; Kumaratilake, Thompson, Eckert & D’Alessandro, 1986). However, as discussed by Kumaratilake et al. (1986) Rausch (1985) and Thompson & Lymbery (1988) these observations do not provide evidence of the frequency of self-fertilization in nature. Lymbery, Hobbs & Thompson (1989) found that adult E. granulosus were aggregated, probably as a result of attraction between individual worms, in the intestine of experimentally infected dogs. Aggregative behaviour may function to enhance cross-fertilization between worms, but at least one other explanation, that it functions to enhance environmental quality (such as easier attachment or increased nutrient concentrations), is equally plausible. Electrophoretic analysis of the genetic structure of E. granulosus from domestic hosts in Australia suggested that both selfand cross-fertilization occur in natural populations (Lymbery & Thompson, 1988). Further studies of genetic variation within and between populations are likely to provide the most reliable estimates of the breeding system of species of Echinococcus. On present evidence, however, it seems probable that strain variation can be accounted for by aspects of the models presented by both Smyth & Smyth (1964) and Rausch (1985). Genetic andphenotypic dlferences Most strains of Echinococcus which have been described were separated initially on the basis of

465

Biology and strain variation of Echinococcus TABLE L-STRAINS OF Echinococcus

Strain*

Previous taxonomic designation

Known geographic dis~ibution

Definitive hosts

Intermediate hosts

Sheep, cattle, pigs macropods, MAN Sheep, cattle? MAN Macropods, cattle? MAN Cervids. MAN

E. grM&ws E.g. granulosus

Australian mainland, U.K., New Zealand, U.S.A. Tasmania Australian mainland, Asia?

Dog, dingo, fox Dog (fox) Dingo, dog

E.g. canadensis E.g. borealis

North America, Eurasia

Wolf, dog

5 Horse strain

E.g. equinus

6 Cattle straint

E. ortleppi E.g. ortleppi

7 African lion strain?

E. felidis E.g. felidis

U.K., Switzerland, Dog (fox?) Befgium, Italy, Syria, S. Africa, (New Zealand?) Switzerland, Belgium, Germany, Dog South Africa (Sri Lanka?) Several African countries Lion

1 Common sheep strain 2 Tasmanian sheep strain 3 Australian sylvatic strain t 4 Cervid strain (s)t

8 Kenyan sheep straint

Kenya

Dog, jackal

9 Camel straint

Africa, Middle East

Dog (fox?)

Russia, Eastern Europe Asia

Dog (fox?) Dog (fox?)

10 Pig straint 11 Buffalo strain?

Horses and other equines, cattle? MAN? Cattle, MAN (cervids?) Warthog, Bush pig (buffalo? wildebeest? zebra? impala? gazelle? giraffe? hippopotamus?) Sheep, goats, MAN(cattle? camel?) Camel (sheep? cattle? goat?) Pig (MAN?) Buffalo (cattle?)

E. rnU1~~~ European strain? Alaskan strain? North American strain t

Em. multilocularis Europe Alaska Em. sibiricensis

Central North America

Fox Dog Cat

Rodents, MAN

* Details in Kumaratilake &Thompson, 1982;Thompson, 1986;Thompson BrAllsopp, 1988; Thompson & Lymbery, 1988; Eckert & Thompson, 1988. t The status of these forms as distinct strains requires further work, particularly with respect to genetic characterization.

presume-d differences in intermediate host preference, usually inferred from epidemiological evidence. Subsequent characteri~tion has tradition~ly utilized morphology, but in some cases criteria such as

development in viva and in vitro, serotype, chemical composition, metabolism and more recently enzyme electrophoresis and DNA analysis have also been applied (Thompson & Lymbery, 1988). Techniques such as enzyme electrophoresis, restriction site analysis and DNA hybridization measure genetic variation on a finer scale than has previously been used to delimit strains. Lymbery & Thompson (1988) demonstrated substantial levels of genetic variation within the morphologically defined Australian mainland and Tasmanian sheep strains of E. gru~ulos~. Most of this variation was found between isolates from the same locality, and differences have even been found between protoscoleces from different cysts in the same individual host

(Lymbery & Thompson, 1989). These results contrast with electrophoretic and DNA analyses in Britain, which have found genetic differences between, but never within, the well-characterized sheep and horse strains (McManus & Smyth, 1979; McManus & Simpson 1985; McManus & Rishi, 1989). This may be indicative of real differences in the genetic heterogeneity of Australian and British strains but we believe that further analyses of British strains, using more enzyme loci or less conserved sequences of DNA as probes, may uncover levels of genetic variation similar to that found in Australian strains (Lymbery & Thompson, 1989). The discovery of genetic variation within strains previously defined on epidemiological and morphological grounds is not su~~sing. If adult E. gran~Zos~ reproduce by cross-fertili~tion, then mutation, migration and recombination will maintain variability within populations. Even if sexual reproduction is

466

R. C. A. THOMPSON and A. J. LYMBERY TABLE ~--DEVELOPMENTAL AND MORPHOLOGICALDIFFERENCES BETWEEN AUSTRALIAN MAMLAND AND TASMANIAN ~~E~TIcsTRA[N~~~~ Echinococcus~ranulosus AFTER 35 DAYSIN EXPERIMENTALLYINFEC:TEDloos

Mainland strain (eastern Australia) Percentage of worms with three or more segments* Percentage of worms with developing eggs in uterus* Total worm length (mm f S.E.M.)P Number of testes ( f S.E.M.)t Length of large hooks (pm f S.E.M.) t Length of small hooks (pm f S.E.M.) t

25 26 2.4 36.0 30.5 24.3

It f f f

Tasmanian strain

0.1 1.6 0.4 0.5

97 99 2.9 49.0 34.9 30.8

If f zt 4

0.1 1.8 0.4 0.7

* Data from Kumaratiiake. Thompson & Dunsmore. 1983. t Data from Kumaratiiake & Thompson, 1984.

entirely by self-fertilization, it is di~cult to estimate the extent to which this would reduce genetic diversity

within strains, as suggested by McManus & Smyth (1979) and Bryant & Flockhart (1986). Strains have traditionally been defined on epidemiological and morphological criteria. It is very likely that there is much underlying genetic variation within these strains which is detectable with biochemical or molecular techniques, but which does not produce phenotypic effects great enough to have been recognized as strain differences. This raises the question of what constitutes a strain. Thompson & Lymbery (1988) identified two conditions which should be satisfied before populations of ~c~inococc~s are regarded as different strains. Firstly, the populations should be genetically differentiated, and secondly they should differ in one or more characters of significance to the epidemiology and control of hydatid disease. Genetic differentiation implies a restriction in gene flow, and the potential for independent evolutionary change, between populations in different host cycles or geographic areas. There is little point, however, in designating all genetically differentiated populations as distinct strains. In our view. the term ‘strain’ is valuable not as an evolutionary unit or taxonomic category, but as a practical descriptor in the control and treatment of hydatid disease. For example, populations of E. gru~~~Qsus in domestic life cycles on the mainland of Australia and in Tasmania differ significantly in allelic frequencies, but at only two of 20 enzyme loci (Lymbery & Thompson. 1988). From these data, we calculated Nei’s unbiased coefficient of genetic distance (Nei, 1978) between the populations as 0.988, which is greater than values reported for local populations of most other organisms (Thorpe. 1982, 1983). Despite this close genetic similarity, extensive differences in development and morphology have been described between the populations (Table 2). From a practical viewpoint, the most important difference is in the prepatent period, with Tasmanian worms producing eggs approximately 7 days earlier than those from the mainland of Australia (Kumaratilake, Thompson & Dunsmore, 1983). We believe that this description is of enough epidemiological significance

to warrant the description of these two populations as distinct strains, despite their close overall genetic similarity. Development and strain variation

In recent years, there has been renewed interest in the integration of developmental biology and evolutionary theory (Bonner, 1982; Alberch, 1983; Stearns, 1983a). This interest stems from a recognition that there is an enormous gap in our knowledge of how patterns of gene activity are able to generate the complex phenotypes upon which natural selection operates. No-one doubts the importance of genomic regulatory mechanisms in controlling sequential gene activation, but there is abundant evidence that the dynamics of epigenetic* interactions impose their own constraints and directions on the developing organism (Alberch, 1982; Nijhout, Wray, Kremen & Teragawa, 1986; Green, 1987). At our present state of knowledge, phenotypes of complex, multicellular organisms could never be predicted from a total genomic sequence, even if we were able to attribute a particular structural or regulatory role to every region of the sequence. This is one reason (although not the only one) why genetic and phenotypic change between evolutionary lineages so often seem to be uncoupled. For example, it is possible that the Australian mainland and Tasmanian domestic strains of E. gra~fflos~s, although closely related genetically, differ in a way which directly or indirectly affects a critical rate-limiting step early in adult development. This could dramatically alter the rate of somatic and germinal differentiation and produce allometric changes in a great range of morphological characters. There is an additional level of complexity in the transition from genotype to phenotype. The structure of an organism is controlled not only by sequential gene activity and the internal dynamics of development, but also by external environmental influences. As discussed above, in E. granu~os~s environmental

* The term ‘epigenetic’ pertains to the causal mechanisms by which the genotype of an organism is expressed as a phenotype.

461

Biology and strain variation of Echinococcus TABLE

J-END

PRODLXI’S OF CARBOHYDRATE

nmol mg ’ dry wt/3 h f

METABOLISM UNDER AEROBIC AND ANAEROBIC CONDITIONS

s.E.M.) IN BRITISH SHEEP AND HORSE STRAINS OF

Anaerobic

Aerobic

Glycogen utilized Oxygen consumed Lactate Succinate Acetate Proprionate Pyruvate Malate Ethanol

Sheep strain

Horse strain

101.7 f 119.5 f 23.8 f 14.6 f 62.0 f 0 0.9 f 1.9 f 7.5 f

45.7 84.2 54.9 7.3 23.4 1.6 1.6 1.0 7.2

3.2 6.2 2.1 3.5 3.1 0.2 0.1 0.3

(EXPRESSED AS

Echinococcus granulosus*

f f f f f f f f f

12.1 6.0 5.1 0.5 1.5 0.1 0.4 0.1 0.8

Sheep strain

Horse strain

107.5 f _ 24.8 f 42.4 f 81.6 f 0 1.3 f 1.9 f 5.9 f

115.7 f 8.7

2.0 2.0 2.6 2.7 0.3 0.1 0.2

58.5 61.0 24.8 3.0 1.9 0.8 2.8

f f f f f f f

4.1 9.9 1.3 0.2 0.3 0.1 0.2

* Data from McManus & Smyth, 1978.

factors appear to control the differentiation of a protoscolex into either an adult tapeworm or a hydatid

cyst. These alternative developmental pathways are presumably activated by a switch at some threshold level of environmental stimulation (Smith-Gill, 1983). Similar switches may operate at many less fundamental stages of development, producing discrete phenotypic states which could be confused with fixed genetic differences of populations in different environments. For example, McManus & Smyth (1978,1982) found substantial differences in metabolic end products between protoscoleces of the British sheep and horse strains of E. granulosus (Table 3). Preliminary studies have suggested differences of similar magnitude between isolates from domestic and sylvatic intermediate hosts in Australia (Behm, Bryant & Thompson, cited in Bryant & Flockhart, 1986). These differences may indicate the use of different metabolic pathways by protoscoleces in different hosts, arising from either fixed genetic differences between strains or from developmental switches in response to environmental cues provided by the hosts. Environmental modification of the phenotype of an organism (phenotypic plasticity) does not always take the form of a switch between alternative pathways of development. Environmental factors may also influence rate constants along one pathway, producing a continuous phenotypic response, graded to the intensity of environmental stimulation (Smith-Gill, 1983). For example, in isolates of E. granulosus from sylvatic intermediate hosts in Australia, Hobbs, Lymbery & Thompson (in press) found continuous variation in certain rostellar hook characters. The number and size of hooks varied from that typical of protoscoleces in domestic intermediate hosts (sheep) to that characteristic of the previously described (Kumaratilake & Thompson, 1984) Australian sylvatic strain of the parasite (Fig. 7). One explanation of these results, which remains to be tested by appropriate crosstransmission experiments, is that the development of rostellar hook characters is affected by the host. This influence may be graded according to the time spent in

a particular

species of host, and be cumulative over generations. Phenotypic plasticity is not necessarily adaptive. The level of plasticity of a trait, however, is heritable and can therefore be moulded by natural selection (Via & Lande, 1985; Schlichting & Levin, 1986; Scheiner & Lyman, 1989). There is a common belief that adaptive plasticity, by uncoupling genotypes from short-term selection pressures, slows the rate at which traits evolve (Stearns, 1983b). Plasticity can also be viewed, however, as a directive force in evolution because it allows a population to survive environmental heterogeneity and thereby be exposed to new selection pressures. For example, assume that the metabolic and morphological characteristics of E. granulosus in sylvatic intermediate hosts in Australia are adaptive phenotypic responses, allowing parasites to survive either in the intermediate host itself or in the likely sylvatic definitive host. If an isolated sylvatic life cycle is maintained over many generations, these plastic responses could be genetically fixed as a result of the selection of random mutations in major genes (Baldwin effect; Simpson, 1953) or of the recombination of existing allelic variants of polygenes or modifiers (genetic assimilation; Waddington, 1957). The end

20 1 20

30

40 Number

50

60

3

of hook6

FIG. 7. Total number of rostellar hooks plotted against size of large hooks (pm) for isolates of Echinococcus granulosus from sheep (X) and macropod marsupials (0) in Australia. (Data courtesy of Russ Hobbs.)

R. C. A. THOMPSON and A. J. LYMBERY

468

result could then be a genetically differentiated population, adapted solely to a sylvatic life cycle. This scenario is hypothetical and we have no evidence for its occurrence. While genetic assimilation has been demonstrated in the laboratory (Waddington, 1956), Williams (1966) raised several objections to its importance as a major force in adaptive evolutionary change. CONCLUSIONS

In this review, we have concentrated on developmental biology and strain variation in Echinococcus, and have tried to emphasize how little we understand about many aspects of this medically and economically important parasite. These deficiencies in our knowledge all point to the need for future research to concentrate on the genetics of Echinococcus with respect to both development and evolutionary potential. The in-built capacity for Echinococcus to alter its developmental programme coupled with its ability to produce genetic variants, are of far-reaching significance for control. The practical importance of intraspecific variation in species of Echinococcus is that strains may differ in traits important to the diagnosis and treatment of hydatid disease as well as those which influence transmission patterns. If strains are genetically differentiated, then it is reasonable to assume that differences in these traits also have a substantial genetic component (Thompson & Lymbery, 1988). In many cases, the differences may be by-products of genetically fixed adaptive changes in developmental pathways. For example, differences in in vitro behaviour between the sheep and horse strains of E. granulosus in Britain surely reflect fundamental differences in developmental biology, which could have many phenotypic ramifications. A major feature of the attempted synthesis of developmental biology and evolutionary theory has been a concentration on the way development constrains evolution by biasing the production of variant phenotypes or by maintaining phenotypic flexibility (Maynard Smith, Burian, Kauffman, Alberch, Campbell, Goodwin, Lande, Raup & Wolpert, 1985). Constraints, however, are also opportunities and it is important to realize that the processes of development may provide direction to evolutionary change. To fully understand the significance of strain variation in species of Echinococcus, there is an urgent need to study the development of those phenotypic traits which are important to the life history of the organism and which affect the epidemiology and control of hydatid disease. This will require an integrated approach across the diverse disciplines of epidemiology, quantitative genetics, embryology, biochemistry and molecular biology. Acknowledgements-We Kumaratilake, Russ valuable contribution

would like to thank Lakshmi Hobbs and Kok Wei Yap for their to our research programme which has

been funded by the Australian Research Council, Clive and Vera Ramaciotti Foundation. Wool Research Trust, World Health Organization and Murdoch University. REFERENCES ALBERCHP. 1982. Developmental constraints in evolutionary processes. In: Evolution and Development (Edited by BONNERJ. T.). DD. 313-332. Springer. Berlin. ALBERCHP. 1983. Mapping genes tophenotypes. or the rules that generate form. Evolution 37: 861-863. ARME C. 1987. Cestoda. In: In vitro Methods for Parasite Cultivation (Edited by TAYLORA. E. R. & BAKERJ. R.), pp. 282-3 17. Academic Press, London. BONNERJ. T. 1982. Introduction. In: Evolution and Development (Edited by BONNERJ. T.), pp. 1-16. Springer, Berlin. BRYAN-~C. & FLOCKHART H. A. 1986. Biochemical strain variation in helminths. Advances in Parasitology25: 275-319. DEW H. R. 1922. Observations on the mode of development of brood capsules and scolices in the encysted stage of Taenia echinococcus. Medical Journal of Australia 2: 381384. DEW H. R. 1925. The histogenesis of the hydatid parasite (Taenia echinococcus) in the pig. Medical Journal of Australia I: 101-I IO. ECKERT J., THOMPSON R. C. A. & MEHLHORN H. 1983. Proliferation and metastases formation of larval Echinococcus multilocularis. I. Animal model, macroscopical and histological findings. Zeitschrift ftir Parasitenkunde 69: 737-748. ECKERTJ. & THOMPSONR. C. A. 1988. Echinococcus strains in Europe: a review. Tropical Medicine and Parasitology 39: 1-8. ECKERT J. 1989. New aspects of parasitic zoonoses. . Veterinary Parasitology 32: 37-55. FIORIP. L., MONACO G., SCAPPATICCI S., PUGLIESEE., CANU N. & CAPPUCCINELLIP. 1988. Establishment of cell cultures from hydatid cysts of Echinococcus granulosus. International Journalfor Parasitology 18: 297-305. GREEN P. B. 1987. Inheritance of pattern: analysis from phenotype to gene. American Zoologist 27: 657-673. G&A&& M.-K. S. 1976. Basic cell types in Echinococcus pranulosus (Cestoda. Cvclophyllidea). Acta Zoologica ‘j;ennical&‘l-16. _ _ _ HOBBS R. P., LYMBERY A. J. & THOMPSONR. C. A. (in press) Rostellar hook morphology of Echinococcus granulosus (Batsch, 1786) from natural and experimental Australian hosts. Parasitology. HOWELL M. J. 1986. Cultivation of Echinococcus species in vitro. In: The Biology of Echinococcus and Hydatid Disease (Edited by THOMPSONR. C. A.), pp. 143-163. Allen & Unwin, London. HOWELLM. J. & MA~THAEIK. 1988. In vitro culture ofhost or parasite cells? International Journal for Parasitology 18: 883-884. KUMARATILAKEL. M.&THOMPSON R. C. A. 1982. A review of the taxonomy and speciation of the genus Echinococcus Rudolphi 1801, Zeitschriffi’ir Parasitenkunde68: 121-146. KUMARATILAKEL. M.. THOMPSONR. C. A. & DUNSMOREJ. D. 1983. Comparative strobilar development of Echinococcus granulosus of sheep origin from different geographical areas of Australia in vivo and in vitro. International Journal for Parasito1og.v 13: 151-156. K~JMARATILAKEL. M. & THOMPSONR. C. A. 1984. Morphological characterization of Australian strains of Echinococcus granulosus. International Journal for Parasitology 14: 467417. I

.

.

Biology and strain variation of Echinococcus KUMARATILAKEL. M., THOMPSON R. C. A., ECKERTJ. & D’ALESSANDROA. 1986. Sperm transfer in Echinococcus

(Cestoda: Taeniidae). Zeitschriji fiir Parasitenkunde 72: 265-269.

LYMBERY A. J. & THOMPSON R. C. A. 1988. Electrophoretic analysis of genetic variation in Echinococcus gramdosus from domestic hosts in Australia. International Journalfor Parasitology 18: 803-S 11. LYMBERY A. J., HOBBSR. P. & THOMPSON R. C. A. 1989. The dispersion of Echinococcus granulosus in the intestine of dogs. Journal of Parasitology 75: 562-510. LYMBERY A. J. &THoM~~~NR. C. A. 1989. Geneticdifferences between cysts of Echinococcus granulosus from the same host. International Journalfor Parasitology 19:961-964. MARCHIOND~ A. A. & ANDERSEN F. L. 1983. Fine structure and freeze-etch study of the protoscolex tegument of Echinococcus multilocularis (Cestoda). Journal of Parasitology 69: 709-7 18.

469

RAUSCHR. L. 1985. Parasitology: retrospect and prospect. Journal of Parasitology 71: 139-151. RAUSCHR. L. 1986. Life-cycle patterns and geographical distribution of Echinococcus species. In: The Biology of Echinococcus and Hydarid Disease (Edited by THOMPSON R. C. A.), pp. 44-80. Allen & Unwin, London. SAKAMOTO T. & SUGIMURAM. 1970. Studies on echinococcosis. XXIII. Electron microscopical observations on histogenesis of larval Echinococcus multilocularis. Japanese Journal of Veterinary Research 18: 131-144. SCHANTZP. M. (in press) Echinococcosis/hydatidosis. In: Handbook

of Zoonoses,

Series

3, Parasitic Zoonoses

(Edited by STEELE J. H.). CRC Press, Boca Raton, Florida. SCHANTZ P. M. SCOKELOG. B. A. (in press) Echinococcosis/ hydatidosis. In: Tropical and Geographical Medicine (Edited by WARRENK. S. & MAHMOUD A. A. F.). McGraw-Hill, New York. SCHEINERS. M. & LYMANR. F. 1989. The genetics of phenotypic plasticity. I. Heritability. Journal of Evolutionary

MAYNARD SMITHJ., BURIANR., KAUFFMANS., ALBERCH P., CAMPBELL J., GOODWIN B., LANDER., RAUPD. & WOLPERT Biology 2: 9SlO7. L. 1985.Developmental constraints and evolution. Quarterly SCHLICHTING C. D. & LEVIND. A. 1986. Phenotypic plasticity: Review of Biology 60: 265-287. an evolving plant character. Biological Journal of the MCMANUSD. P. & SMUITTH J. D. 1978. Differences in the Linnean Society 29: 37-47. chemical composition and carbohydrate metabolism of SCHWABE C. W. 1986. Current status of hydatid disease: a Echinococcus granulosus (horse and sheep strains) and E. zoonosis of increasing importance. In: The Biology of multilocularis. Parasirology 77: 103-109. Echinococcus and Hydatid-Disease (Edited by THO&O~ MCMANUSD. P. & SMYTHJ. D. 1979. Isoelectric focusing of R. C. A.). vu. 81-l 13. Allen & Unwin. London. some enzymes from Echinococcus granulosus (horse and SIMPSON G. G. 1953. The Baldwin effect. &olurion 7: 110-l 17. sheep strains) and E. multilocularis. Transactions of the Royal SMITH-GILLS. J. 1983. Developmental plasticity: developSociety of Tropical Medicine and Hygiene 73: 259-265. mental conversion versus phenotypic modulation. American MCMANUSD. P. & SMYTHJ. D. 1982. Intermediary carboZoologist 23: 47-55. hydrate metabolism in protoscoleces of Echinococcus SMYTH J. D. & SMYTH M. M. 1964. Natural and experimental granulosus (horse and sheep strains) and E. mulrilocularis. hosts of Echinococcus granulosus and E. multilocularis, Parasitology 84: 35 l-366. with comments on the genetics of speciation in the genus MCMANUSD. P. & SIMPSON A. J. G. 1985. Identification of Echinococcus. Parasitology 54: 493-514. the Echinococcus (hydatid disease) organisms using cloned SMYTH J. D. 1969. Parasites as biological models. DNA markers. Molecular and Biochemical Parasitology Parasitology 59: 73-91. 17: 171-178. SMYTHJ. D. & SMY~HM. M. 1969. Self insemination in MCMANUSD. P. & SMYTH J. D. 1986. Hydatidosis: changing Echinococcus granulosus in vivo. Journal of Helminthology concepts in epidemiology and speciation. Parasitology 43: 383-388. Today 2: 163-168. SMYTHJ. D. & DAVIESZ. 1974a. In vitro culture of the MCMANUSD. P. & RISHIA. K. 1989. Genetic heterogeneity strobilar stage of Echinococcus granulosus (sheep strain): a within Echinococcusgranulosus isolates from different hosts review of basic problems and results. International Journal and geographical areas characterised with DNA probes. for Parasitology 4: 63 l-644. Parasitology 99: 17-29. SMYTH J. D. & DAVIESZ. 1974b. Occurrence of physiological MEHLHORNH., ECKERTJ. & THOMPSON R. C. A. 1983. strains of Echinococcusgranulosus demonstrated by in vitro Proliferation and metastases formation of larval Echinoculture of protoscoleces from sheep and horse hydatid coccus multilocularis. II. Ultrastructural investigations. cysts. International Journalfor Parasitology 4: 443445. Zeirschrifr ftir Parasitenkunde 69: 749-163. SMYTHJ. D. & DAVIESZ. 1975. In vitro suppression of NEI M. 1978. Estimation of average heterozygosity and segmentation in Echinococcus mulrilocularis with morphogenetic distance from a small number of individuals. logical transformation of protoscoleces into monozoic Genetics 89: 583-590. adults. Parasitology 71: 125-135. NIJHOUTH. F., WRAYG. A., KREMEN C. & TERACAWA C. K. SMYTHJ. D. 1979. Echinococcus granulosus and E. mulri1986. Ontogeny, phylogeny and evolution of form: an locularis: in vitro culture of the strobilar stages from algorithmic approach. Systematic Zoology 35: 445-457. protoscoleces. Angewandte Parasitologie 20: 137-147. PAWLOWSKI I. D., YAP K. W. & THOMPSON R. C. A. 1988. SMYTHJ. D. & BARRETI.N. J. 1979. Echinococcus multiObservations on the possible origin, formation and structure locularis: further observations on strobilar differentiation of calcareous corpuscles in taeniid cestodes. Parasitology in vitro. Revisra Zbtrica de Parasitologica 39: 39-53. Research 74: 293-296. SMYTHJ. D. 1982. Speciation in Echinococcus: biological and hUSCH R. L. 1954. Studies on the helminth fauna of Alaska. biochemical criteria. Revista Zberica de Parasitologica, XX. The histogenesis of the alveolar larva of Echinococcus Extra Volume: 25-34. species. Journal of Infectious Diseases 94: 178-186. STEARNS S. C. 1983a. Introduction to the symposium: the hUSCH R. L. & WILSONJ. F. 1973. Rearing of the adult inter-face of life-history evolution, whole-organism Echinococcus multilocularis Leuckart, 1863, from sterile ontogeny, and quantitative genetics. American Zoologisr larvae from man. American Journal of Tropical Medicine 23: 3-4. and Hygiene 22: 357-360. STEARNS S. C. 1983b. The evolution of life-history traits in

470

R. C. A. THOMPSON and A. J. LYMB~R’~

mosquitofish since their introduction to Hawaii in 1905: rates of evolution, heritabilities, and developmental plasticity. American Zo&gi.Tt U: 65-75. SIIL~~STOWZXAT. 1972. The development of organ systems in cestodes. I. A study of histology of ~~menolepis diminura (Rudolphi, 18 19) (Hymenolepididae). Acta Purusiiologica Polonica 20: 449462. SULGOSTOWSKA T. 1974. The development of organ systems in cestodes. II. Histogenesis of the reproductive system in Hynenolepis diminutn (Rudolphi, 18 19) (Hymenolepididae). Actu Parasitologica Polonica 22: 179-190. THQMPS~NR. C. A.. KUMARA~ILAKEL. M. & ECKERTJ. 1984. Observations on Echinococcusgrnnulosus of cattle origin in Switzerland. Inzernationul Journal .for Parasitology 14: 283-29 1. THOMPSON R. C. A. 1986. Biology and systematics of ~rhino~oc~~. In: The Eiolog~ o~Echinococcus and ~~d~fid Disease (Edited by THOMPSONR. C. A.). pp. 5-43. Allen & Unwin, London. THOMPSON R. C. A. 1988. Intraspecific variation and epidemiology. In: Parasites in Focus (Edited by MEHLHORN H.), pp. 391-411. Springer, Berlin. THOMPSON R. C. A. & ALLSOPP C. E. 1988. Hydatidosis: u Revienr of the Veterinary Aspects and Annotated Bibliography. Commonwealth Agricultural Bureaux, Wallingford, U.K. THOMPSONR. C. A. & LYMBERYA. J. 1988. The nature, extent and significance of variation within the genus Echinococcus. Advances in Parasito1og.v 27: 209-258. THOMPSONR. C. A., LYMBERYA. J., HOBBS R. P. & ELLIOTA.

D. 1988. Hydatid disease in urban areas of Western Australia: an unusual cycle involving western grey kangaroos (~arcropus .fuliginosu.~), feral pigs and domestic dogs. Austroliun Ve/erinar_v Journal 65: 188- 189. THOMPSONR. C. A., DEPLAZES P. & ECKERT J. (in press) Uniform strobilar development of Eehinococcus multilocuiaris in vitro from protoscolex to immature stages. Journal of’ Parasitolog?, THORPE J. P. 1982. The molecular clock hypothesis: biochemical evolution, genetic differentiation and systematics. Annual Review qfEcology and Systematics 13: 139-168. THORPE J. P. 1983. Enzyme variation, genetic distance and evolutionary divergence in relation to levels of taxonomic separation. In: Protein Polymorphism: Adaptive and Taxonomic Sign$cance (Edited by OXFORD G. S. & ROLLINSO~D.), pp. 13 1-I 52, Academic Press, London. VIA S. & LANDE R. 1985. Genoty~nvironment interaction and the evolution of phenotypic plasticity. ~v~Zuf~~~ 39: 505-522. VOGEL H. 1978. Wie wkhst der Alveolarechinokokkus? Tropenmedizin und Parasitologic 29: 1- 11. WADDINGTONC. H. 1956. Genetic assimilation ofthe hithorax phenotype. Evolution 10: l---13. WADDINGTONC. H. 19.57. The Strategy of the Genes. Allen & Unwin, London. WHITFIELD P. J. & EVANS N. A. 1983. Parthenogenesis and asexual multiplication among parasitic platyhelminths. Parasitology 86 (supplement): 121-160. WILLIAMS G. C. 1966. Adaptation and Natural Selection. Princeton University Press. Princeton. New Jersey.