Evolution of complex life cycles in trophically transmitted helminths. II. How do life-history stages adapt to their hosts?

We review how trophically transmitted helminths adapt to the special problems associated with successive hosts in complex cycles. In intermediate hosts, larvae typically show growth arrest at larval maturity (GALM). Theoretical models indicate that optimization of size at GALM requires larval mortality rate to increase with time between infection and GALM: low larval growth or paratenicity (no growth) arises from unfavourable growth and mortality rates in the intermediate host and low transmission rates to the definitive host. Reverse conditions favour high GALM size or continuous growth. Some support is found for these predictions. Intermediate host manipulation involves predation suppression (which decreases host vulnerability before the larva can establish in its next host) and predation enhancement (which increases host vulnerability after the larva can establish in its next host). Switches between suppression and enhancement suggest adaptive manipulation. Manipulation conflicts can occur between larvae of different ages/species a host individual. Larvae must usually develop to GALM before becoming infective to the next host, possibly due to trade-offs, e.g. between growth/survival in the present host and infection ability for the next host. In definitive hosts, if mortality rate is constant, optimal growth before switching to reproduction is set by the growth/morality rate ratio. Rarely, no growth occurs in definitive hosts, predicted (with empirical support) when larval size on infection exceeds growth/mortality rate. Tissue migration patterns and residence sites may be explained by variations in growth/mortality rates between host gut and soma, migration costs and benefits of releasing eggs in the gut.

Evolution of complex life cycles in trophically transmitted helminths. I. Host incorporation and trophic ascent.

Links between parasites and food webs are evolutionarily ancient but dynamic: life history theory provides insights into helminth complex life cycle origins. Most adult helminths benefit by sexual reproduction in vertebrates, often high up food chains, but direct infection is commonly constrained by a trophic vacuum between free-living propagules and definitive hosts. Intermediate hosts fill this vacuum, facilitating transmission to definitive hosts. The central question concerns why sexual reproduction, and sometimes even larval growth, is suppressed in intermediate hosts, favouring growth arrest at larval maturity in intermediate hosts and reproductive suppression until transmission to definitive hosts? Increased longevity and higher growth in definitive hosts can generate selection for larger parasite body size and higher fecundity at sexual maturity. Life cycle length is increased by two evolutionary mechanisms, upward and downward incorporation, allowing simple (one-host) cycles to become complex (multihost). In downward incorporation, an intermediate host is added below the definitive host: models suggest that downward incorporation probably evolves only after ecological or evolutionary perturbations create a trophic vacuum. In upward incorporation, a new definitive host is added above the original definitive host, which subsequently becomes an intermediate host, again maintained by the trophic vacuum: theory suggests that this is plausible even under constant ecological/evolutionary conditions. The final cycle is similar irrespective of its origin (upward or downward). Insights about host incorporation are best gained by linking comparative phylogenetic analyses (describing evolutionary history) with evolutionary models (examining selective forces). Ascent of host trophic levels and evolution of optimal host taxa ranges are discussed.

Why do larval helminths avoid the gut of intermediate hosts?

In complex life cycles, larval helminths typically migrate from the gut to exploit the tissues of their intermediate hosts. Yet the definitive host's gut is overwhelmingly the most favoured site for adult helminths to release eggs. Vertebrate nematodes with one-host cycles commonly migrate to a site in the host away from the gut before returning to the gut for reproduction; those with complex cycles occupy sites exclusively in the intermediate host's tissues or body spaces, and may or may not show tissue migration before (typically) returning to the gut in the definitive host. We develop models to explain the patterns of exploitation of different host sites, and in particular why larval helminths avoid the intermediate host's gut, and adult helminths favour it. Our models include the survival costs of migration between sites, and maximise fitness (=expected lifetime number of eggs produced by a given helminth propagule) in seeking the optimal strategy (host gut versus host tissue exploitation) under different growth, mortality, transmission and reproductive rates in the gut and tissues (i.e. sites away from the gut). We consider the relative merits of the gut and tissues, and conclude that (i) growth rates are likely to be higher in the tissues, (ii) mortality rates possibly higher in the gut (despite the immunological inertness of the gut lumen), and (iii) that there are very high benefits to egg release in the gut. The models show that these growth and mortality relativities would account for the common life history pattern of avoidance of the intermediate host's gut because the tissues offer a higher growth rate/mortality rate ratio (discounted by the costs of migration), and make a number of testable predictions. Though nematode larvae in paratenic hosts usually migrate to the tissues, unlike larvae in intermediates, they sometimes remain in the gut, which is predicted since in paratenics mortality rate and migration costs alone determine the site to be exploited.

To grow or not to grow? Intermediate and paratenic hosts as helminth life cycle strategies.

Larval helminths in intermediate hosts often stop growing long before their growth is limited by host resources, and do not grow at all in paratenic hosts. We develop our model [Ball, M.A., Parker, G.A., Chubb, J.C., 2008. The evolution of complex life cycles when parasite mortality is size- or time-dependent. J. Theor. Biol. 253, 202-214] for optimal growth arrest at larval maturity (GALM) in trophically transmitted helminths. This model assumes that on entering an intermediate host, larval death rate initially has both time- (or size-) dependent and time-constant components, the former increasing as the larva grows. At GALM, mortality changes to a new and constant rate in which the size-dependent component is proportional to that immediately before GALM. Mortality then remains constant until death or transmission to the definitive host. We analyse linear increasing and accelerating forms for time-dependent mortality to deduce why there is sometimes growth (intermediate hosts) and sometimes no growth (paratenic hosts). Calling i the intermediate or paratenic host, and j the definitive host, conditions favouring paratenicity are: (i) high values in host i for size at establishment, size-related mortality, expected intensity, (ii) low values in host i for size-independent mortality rate, potential growth rate, transmission rate to j, and ratio of death rate in j/growth rate in j. Opposite conditions favour growth in the (intermediate) host, either to GALM or until death without GALM. We offer circumstantial evidence from the literature supporting some of these predictions. In certain conditions, two of the three possible growth strategies (no growth; growth to an optimal size then growth arrest (GALM); unlimited growth until larval death) can exist as local optima. The effect of the discontinuity in death rate after GALM is complex and depends on mortality and growth parameters in the two hosts, and on the mortality functions before and after GALM.

The evolution of complex life cycles when parasite mortality is size- or time-dependent.

In complex cycles, helminth larvae in their intermediate hosts typically grow to a fixed size. We define this cessation of growth before transmission to the next host as growth arrest at larval maturity (GALM). Where the larval parasite controls its own growth in the intermediate host, in order that growth eventually arrests, some form of size- or time-dependent increase in its death rate must apply. In contrast, the switch from growth to sexual reproduction in the definitive host can be regulated by constant (time-independent) mortality as in standard life history theory. We here develop a step-wise model for the evolution of complex helminth life cycles through trophic transmission, based on the approach of Parker et al. [2003a. Evolution of complex life cycles in helminth parasites. Nature London 425, 480-484], but which includes size- or time-dependent increase in mortality rate. We assume that the growing larval parasite has two components to its death rate: (i) a constant, size- or time-independent component, and (ii) a component that increases with size or time in the intermediate host. When growth stops at larval maturity, there is a discontinuous change in mortality to a constant (time-independent) rate. This model generates the same optimal size for the parasite larva at GALM in the intermediate host whether the evolutionary approach to the complex life cycle is by adding a new host above the original definitive host (upward incorporation), or below the original definitive host (downward incorporation). We discuss some unexplored problems for cases where complex life cycles evolve through trophic transmission.

Schistocephalus cotti n. sp. (Cestoda: Pseudophyllidea) plerocercoids from bullheads Cottus gobio L. in an Arctic river in Finland, with a key to the plerocercoids of the Palaearctic species of the genus.

We compared plerocercoids of Schistocephalus Creplin, 1829 from Cottus gobio (n = 57) and Gasterosteus aculeatus f. semiarmatus (n = 45) from the River Utsjoki, Finland, taken only from single worm infections. Segment numbers in the two populations were distinct (G. aculeatus range 55-107, average 74 (SE 1.66), median 73; C. gobio range 122-189, average 146 (SE 1.78); median 144). The mean difference between populations, 71.47, t = 28.76 with 100 degrees of freedom, two-tailed p value <0.001, was considered extremely significant. Amplification of microsatellite loci that were originally designed for Schistocephalus from G. aculeatus was positive for all larvae from G. aculeatus (n = 20), whereas in no plerocercoids from C. gobio (n = 20) were any of the six microsatellites amplified, indicating that plerocercoids from G. aculeatus and C. gobio were two distinct genetic populations of Schistocephalus. The material from C. gobio is described as S. cotti n. sp. Plerocercoids of the Palaearctic species of Schistocephalus are identified as follows: S. nemachili Dubinina, 1959 with 228-235 or more segments, specific to Barbatula spp. (Balitoridae); S. pungitii Dubinina, 1959 with 62-92 (usually 70-80) segments, specific to Pungitius pungitius; S. solidus (Müller, 1776) in two forms, one in G. aculeatus f. leiurus and f. semiarmatus, with 48-100 (usually 65-75) segments, and the other in G. aculeatus f. trachurus, with 99-138 (usually 112-122) segments; and S. cotti n. sp. with 103-189 (usually 130-159) segments, probably specific to cottids. Nearctic Schistocephalus were not considered owing to the uncertain status of some North American records. Some other species of Schistocephalus of highly doubtful status were briefly noted. Cross-infection experiments and molecular studies are recommended to further elucidate the interrelationships between the various species of Schistocephalus.

Plasma etching and ashing: a technique for demonstrating internal structures of helminths using scanning electron microscopy.

Plasma etching and ashing for demonstrating the three-dimensional ultrastructure of the internal organs of helminths is described. Adult worms of the cestode Caryophyllaeides fennica were dehydrated through an ethanol series, critical point dried (Polaron E3000) and sputter coated with 60% gold-palladium (Polaron E5100) and glued to a standard scanning electron microscope (SEM) stub positioned as required for ashing. After initial SEM viewing of worm surfaces for orientation, stubs were placed individually in the reactor chamber of a PT7150 plasma etching and ashing machine. Worms were exposed to a radio frequency (RF) potential in a low pressure (0.2 mbar) oxygen atmosphere at room temperature. The oxidation process was controlled by varying the times of exposure to the RF potential between 2 to 30 min, depending on the depth of surface tissue to be removed to expose target organs or tissues. After each exposure the oxidized layer was blown from the surface with compressed air, the specimen sputter-coated, and viewed by SEM. The procedure was repeated as necessary, to progressively expose successive layers. Fine details of organs, cells within, and cell contents were revealed. Ashing has the advantage of providing three dimensional images of the arrangement of organs that are impossible to visualize by any other procedure, for example facilitating testes counts in cestodes. Both freshly-fixed and long-term stored helminths can be ashed. Ashing times to obtain the desired results were determined by trial so that some duplicate material was needed.

Optimal growth strategies of larval helminths in their intermediate hosts.

We consider optimal growth of larval stages in complex parasite life cycles where there is no constraint because of host immune responses. Our model predicts an individual's asymptotic size in its intermediate host, with and without competition from conspecific larvae. We match observed variations in larval growth patterns in pseudophyllid cestodes with theoretical predictions of our model. If survival of the host is vital for transmission, larvae should reduce asymptotic size as intensity increases, to avoid killing the host. The life history strategy (LHS) model predicts a size reduction <1/intensity, thus increasing the parasite burden on the host. We discuss whether body size of competing parasites is an evolved LHS or simply reflects resource constraints (RC) on growth fixed by the host, leading to a constant total burden with intensity. Growth under competition appears comparable with "the tragedy of the commons", much analysed in social sciences. Our LHS prediction suggests that evolution generates a solution that seems cooperative but is actually selfish.

Larval trypanorhynchs (Platyhelminthes: Eucestoda: Trypanorhyncha) from black-scabbard fish, Aphanopus carbo and oceanic horse mackerel, Trachurus picturatus in Madeira (Portugal).

Four different types of trypanorhynch postlarvae were found attached to the stomach mucosa, external stomach wall or free in the body cavity of two marine fishes from Madeira, namely blackscabbard fish, Aphanopus carbo and oceanic horse mackerel, Trachurus picturatus. Morphological features shown by SEM indicated that the postlarvae belonged to the species Tentacularia coryphaenae, Sphyriocephalus tergestinus, Nybelinia lingualis and possibly N. yamaguitii. Prevalence [mean intensity (range)] of T. coryphaenae, S. tergestinus and Nybelinia spp. in A. carbo (n = 135) was 12.6% [1.65 +/- 1.27(1-6)], 5.9% [1.57 +/- 0.79 (1-3)] and 2.2% [1.33 +/- 0.58 (1-2)] respectively. The prevalence of T. coryphaenae and S. tergestinus showed some seasonality, with a rise in prevalence of T. coryphaenae corresponding to a decrease in prevalence of S. tergestinus. However these differences were not significant. In T. picturatus (n = 304) only N. lingualis was found at a prevalence of 9.6%. Both S. tergestinus and N. lingualis were recovered only from the stomach mucosa or external stomach wall, while T. coryphaenae was observed either attached to the stomach mucosa or free in the visceral cavity of the fish. The paper presents the first scanning electron micrographs (SEM) of Sphyriocephalus tergestinus and a new geographical record of N. lingualis in T. picturatus.

Cystacanths of Bolbosoma vasculosum in the black scabbard fish Aphanopus carbo, oceanic horse mackerel Trachurus picturatus and common dolphin Delphinus delphis from Madeira, Portugal.

Cystacanths of the acanthocephalan, Bolbosoma vasculosum Rudolphi 1819, were found to be encapsulated in the connective tissues of the viscera of the black scabbard fish, Aphanopus carbo and oceanic horse mackerel, Trachurus picturatusfrom Madeira, Atlantic Ocean. Juvenile worms were obtained from the intestine of a stranded common dolphin, Delphinus delphis, also from Madeira. Cystacanths were 11-15 mm long, with a proboscis of 18-19 longitudinal rows, eight hooks per row, and two sets of trunk spines. Overall, the morphology and dimensions of the proboscis, neck and trunk corresponded to previous descriptions. Scanning electron microscopy of the proboscis structures and trunk spines is provided for the first time. The prevalence of B. vasculosum in A. carbo increased with fish length. There were no statistical differences in the prevalence and intensity of infection between seasons. The intensity of infection was similar for male and female fishes, but there were significant differences in relation to length, longer fishes having heavier infections. Aphanopus carbo from Madeira represents a new host record and a new geographic location for B. vasculosum.

Longer-term population dynamics of Gyrodactylus bullatarudis and G. turnbulli (Monogenea) on adult guppies (Poecilia reticulata) in 50-I experimental arenas.

Two suprapopulations of monogeneans, one each of Gyrodactylus bullatarudis and G. turnbulli, on two groups of ten experimentally infected adult guppies (Poecilia reticulata) were maintained separately in 50.1 aquaria and monitored over 210 days. The G. bullatarudis population had a pattern of initial growth, then a subsequent decline to extinction after 40 days. G. turnbulli, after initial population growth and decline, maintained low-intensity infections (0.33-3.3 parasites/host) on six hosts over 94 days and did not become extinct during the experiment. There were some differences between the host-site specificity of G. bullatarudis and G. turnbulli on adult P. reticulata as compared with previously observed infections on immature fish.

Use of protozoan communities for pollution monitoring.

A gradient of chronic organic pollution was identified in a small river in south-east England. The parasite fauna of the ubiquitous three-spined stickleback (Gasterosteus aculeatus L.) was studied at the extremes of the pollution gradient and trichodinid ciliates identified as a potential bioindicator. A simple technique was developed for the quantification of whole body-burdens of trichodinids on small fish. Three species of trichodinids were identified: Trichodina domerguei, T. tenuidens and Trichodinella epizootica at combined infestation intensities of < 14 to 137522/fish. Preliminary results are reported which may link the increased intensity of trichodinid infestation with increased concentration of sewage treatment works effluent.

Freeze fixation-dehydration as a method of preparation of Gyrodactylus (Monogenea) for SEM.

Freeze fixation-dehydration was used for the first time in the preparation of attached Gyrodactylus for SEM viewing. The technique provided instant immobilization of specimens before death and negligible shrinkage throughout the fixation-dehydration process. Comparisons of sample means of two linear measurements of attached opisthaptors showed 20% more shrinkage of Gyrodactylus fixed using 10% neutral buffered formalin than those which were freeze fixed. Freeze fixation-dehydration was excellent for the study of gross external morphology of attached Gyrodactylus. However, the freeze fixation-dehydration process may cause disruption of intracellular structural components making delicate tissues brittle and more prone to damage during subsequent manipulation.

Host response to initial and challenge infections, following treatment, of Gyrodactylus bullatarudis and G. turnbulli (Monogenea) on the guppy (Poecilia reticulata).

The host response of Poecilia reticulata Peters against two species of Gyrodactylus (G. bullatarudis Turnbull, 1956 and G. turnbulli Harris, 1986) was investigated by comparing the dynamics of infrapopulations arising from initial infections, challenge infections begun 1 day after formalin treatment of 3-day-old initial infections and challenge control infections. The primary host response of P. reticulata to Gyrodactylus was shown to be non-(Gyrodactylus)-species-specific. Observations of differences in host-site specificities of initial and challenge infection infrapopulations indicated that the host response is largely localised to areas of heavy infection. The exact nature of the response remains unknown.

Trichrome staining of Gyrodactylus sclerites and soft tissues following fixation in ammonium picrate-glycerin, including an improved rendition of the haptoral bars of G. turnbulli.

A simple technique using modified Mallory stain in the transferral of Gyrodactylus specimens from ammonium picrate-glycerin to a permanent mountant is described. Hamuli, their connecting bars and the penis sclerites are well defined by the technique as are muscles and tendons, cell nuclei, tegument and gland cells. As well as being useful in the study of general anatomy, the technique enhances the observation of the taxonomically important ventral and dorsal bars. In order to show this, improved illustrations of the dorsal and ventral bars of G. turnbulli are given along with explicit demonstrations of differences in morphology of the ventral bars of G. bullatarudis and G. rasini-two easily confused species.

[An attempt to devise a hypothesis of the seasonal maturation of helminths in the definitive host--the fish]. / Popytka sozdaniia gipotezy sezonnogo sozrevaniia gel'mintov v okonchatel'nom khoziaine--rybe.

The seasonal maturation of four species of helminths, Acanthocephalus clavula, A. lucii, Camallanus lacustris and Bunodera luciopercae, from the perch Perca fluviatilis is briefly described. It is noted that A. clavula, A. lucii and C. lacustris are found as mature worms in the intestine of the fish host throughout the year, whereas in Bunodera luciopercae there is a limited season of maturation. As a result of experimental studies it is suggested that B. luciopercae uses host gonadotrophin to initiate and stimulate the early phase of gametogenesis. The gonadotrophin may also activate the parasites' own endocrine system, which then takes over control of subsequent spermatogenesis and oogenesis. This formation is used for a hypothesis for seasonal maturation of helminths in the fish definitive host. It also provides a potential explanation for seasonal maturation of helminths of fish in polar and tropical zones of the world.