A field reciprocal transplant experiment reveals asymmetric costs of migration between lake and river ecotypes of three-spined sticklebacks (Gasterosteus aculeatus).

Theory of local adaptation predicts that nonadapted migrants will suffer increased costs compared to local residents. Ultimately this process can result in the reduction of gene flow and culminate in speciation. Here, we experimentally investigated the relative fitness of migrants in foreign habitats, focusing on diverging lake and river ecotypes of three-spined sticklebacks. A reciprocal transplant experiment performed in the field revealed asymmetric costs of migration: whereas mortality of river fish was increased under lake conditions, lake migrants suffered from reduced growth relative to river residents. Selection against migrants thus involved different traits in each habitat but generally contributed to bidirectional reduction in gene flow. Focusing particularly on the parasitic environments, migrant fish differed from resident fish in the parasite community they harboured. This pattern correlated with both cellular phenotypes of innate immunity as well as with allelic variation at the genes of the major histocompatibility complex. In addition to showing the costs of migration in three-spined sticklebacks, this study highlights the role of asymmetric selection particularly from parasitism in genotype sorting and in the emergence of local adaptation.

In vitro transition of Schistocephalus solidus (Cestoda) from coracidium to procercoid and from procercoid to plerocercoid.

With the present study, a culture system for successive life-cycle stages of the tapeworm Schistocephalus solidus was developed and this report documents for the first time, cultivation of the procercoid stage of S. solidus from eggs. Additionally we have transformed procercoids dissected from experimentally infected copepods and cultured procercoids into the early plerocercoid stage in vitro. Observations in the culture suggest that the coracidia can interact with their external environment and need no host specific stimuli, except for the components in the culture medium, for activation and hatching from the embryophore. Increasing the culture medium pH from 7.3 to 8.0 improved escape rates and frequencies of hook contractions, suggesting that the oncosphere may recognize and respond to environmental conditions along the host intestine. Procercoids in the culture did not stop growing indicating that conditions within the copepod may be important to limit growth and to induce transformation to plerocercoids. When procercoids are dissected from copepods and transferred to the culture, the outer tegument layers and cercomer starts to loosen. Comparison of the lectin staining of the loosened outer tegument layers and cercomer in procercoids dissected from copepods confirms that transitions of both, the oncosphere to procercoid and procercoid to plerocercoids, has taken place in the in vitro cultures.

MHC-based mate choice combines good genes and maintenance of MHC polymorphism.

Polymorphic genes of the major histocompatibility complex (MHC) are regarded as essential genes for individual fitness under conditions of natural and sexual selection. To test this hypothesis, we investigated the ultimate individual fitness trait--that of reproductive success. We used three-spined sticklebacks (Gasterosteus aculeatus) in seminatural enclosures, located in natural breeding areas where the experimental fish had been caught. During their reproductive period, fish were exposed continuously to their natural sympatric parasites. By genotyping almost 4000 eggs with nine microsatellites, we determined parenthood and inferred female mating decision. We found that with reference to their own MHC profile, female sticklebacks preferred to mate with males sharing an intermediate MHC diversity. In addition, males with a specific MHC haplotype were bigger and better at fighting a common parasite (Gyrodactylus sp.). This translated directly into Darwinian fitness since fish harbouring this specific MHC haplotype were more likely to be chosen and had a higher reproductive output. We conclude that females also based their mating decision on a specific MHC haplotype conferring resistance against a common parasite. This identifies and supports 'good genes'. We argue that such an interaction between host and parasite driving assortative mating is not only a prerequisite for negative frequency-dependent selection--a potential mechanism to explain the maintenance of MHC polymorphism, but also potentially speciation.

Disentangling the role of MHC-dependent 'good genes' and 'compatible genes' in mate-choice decisions of three-spined sticklebacks Gasterosteus aculeatus under semi-natural conditions.

To investigate and disentangle the role of major histocompatibility complex (MHC)-based 'good genes' and 'compatible genes' in mate choice, three-spined sticklebacks Gasterosteus aculeatus with specific MHC IIB genotypes were allowed to reproduce in an outdoor enclosure system. Here, fish were protected from predators but encountered their natural parasites. Mate choice for an intermediate genetic distance between parental MHC genotypes was observed, which would result in intermediate diversity in the offspring, but no mate choice based on good genes was found under the current semi-natural conditions. Investigation of immunological variables revealed that the less-specific innate immune system was more active in individuals with a genetically more divergent MHC allele repertoire. This suggests the need to compensate for an MHC-diminished T-cell repertoire and potentially explains the observed mate choice for intermediate MHC genetic distance. The present findings support a general pattern of mate choice for intermediate MHC diversity (i.e. compatible genes). In addition, the potentially dynamic role of MHC good genes in mate choice under different parasite pressures is discussed in the light of present and previous results.

Characterization of antisera raised against stickleback (Gasterosteus aculeatus) MHC class I and class II molecules.

The three-spined stickleback (Gasterosteus aculeatus) is an important model organism for investigations on the maintenance of polymorphism of the major histocompatibility complex (MHC) of vertebrates. Analysis of functional aspects of MHC diversity in stickleback would benefit from the availability of MHC specific reagents. Here we characterize antisera raised against recombinant fusion proteins of stickleback MHC class I alpha and class II alpha and beta. Western blot analysis using recombinant proteins confirmed the specificity of the antisera. In brain and muscle preparations, neither of the MHC types was detectable. High levels of each MHC receptor type were observed in gills and spleen and lower levels in head kidneys. In histological sections of gills, epithelial cells of primary and secondary lamellae stained positive with MHC class I antiserum, while single, scattered cells stained positive for MHC class II. In sections of spleen and head kidney, considerable numbers of cells positive for either MHC type were detected. Molecular weight shift in SDS-PAGE after deglycosylation of MHC class I alpha and class II beta confirmed the predicted glyco-protein character of the molecules. The majority of MHC II alpha was not glycosylated; only a small fraction of MHC II alpha was susceptible to deglycosylation. This suggests differential expression of the two stickleback MHC II alpha genes (Gaac-DAA, Gaac-DBA) only one of which (Gaac-DBA) has a site for N-linked glycosylation.

Individual MHC class I and MHC class IIB diversities are associated with male and female reproductive traits in the three-spined stickleback.

Genes of the major histocompatibility complex (MHC) are indispensable for pathogen defence in vertebrates. With wild-caught three-spined sticklebacks (Gasterosteus aculeatus) we conducted the first study to relate individual reproductive parameters to both MHC class I and II diversities. An optimal MHC class IIB diversity was found for male nest quality. However, male breeding colouration was most intense at a maximal MHC class I diversity. One MHC class I allele was associated with male redness. Similarly, one MHC class IIB allele was associated with continuous rather than early female reproduction, possibly extending the reproductive period. Both alleles occurred more frequently with increasing individual allele diversity. We suggest that if an allele is currently not part of the optimum, it had not been propagated by choosy females. The parasite against which this allele provides resistance is therefore unlikely to have been predominant the previous year - a step to negative frequency-dependent selection.

Infectivity of two nematode parasites, Camallanus lacustris and Anguillicola crassus, in a paratenic host, the three-spined stickleback Gasterosteus aculeatus.

Three-spined sticklebacks Gasterosteus aculeatus are frequent paratenic hosts of the nematode parasites Anguillicola crassus and Camallanus lacustris. As paratenic hosts, sticklebacks could spread infection by carrying high numbers of infective stages. In contrast, low infective ability of either parasite for the paratenic host could hinder the spread of infection. In the present study, G. aculeatus was, for the first time, infected under controlled laboratory conditions with defined doses of the parasites. Sticklebacks were exposed to 6, 12, 18 and 24 parasite larvae to determine the infective ability of the 2 nematode species. There were significantly higher infection rates for C. lacustris (18 to 49%) than for A. crassus (4 to 14%) at each exposure dose. In C. lacustris-infected sticklebacks, infection rates tended to be highest after exposure to 12 C. lacustris larvae and lowest after exposure to 24 parasites. In A. crassus-infected sticklebacks, no effect of parasite exposure dose on infection rates was observed. Immunity parameters such as respiratory burst activity and lymphocyte proliferation of head kidney leukocytes recorded 18 wk post exposure were not significantly affected by either parasite or exposure dose. Granulocyte:lymphocyte ratios were elevated only within the stickleback group showing the highest infection intensity of C. lacustris, i.e. to those exposed 18 parasites.

Genetic variation in MHC class II expression and interactions with MHC sequence polymorphism in three-spined sticklebacks.

Genes of the major histocompatibility complex (MHC) have been studied for several decades because of their pronounced allelic polymorphism. Structural allelic polymorphism is, however, not the only source of variability subjected to natural selection. Genetic variation may also exist in gene expression patterns. Here, we show that in a natural population of three-spined sticklebacks (Gasterosteus aculeatus) the expression of MHC class IIB genes was positively correlated with parasite load, which indicates increased immune activation of the MHC when infections are frequent. To experimentally study MHC expression, we used laboratory-bred sticklebacks that were exposed to three naturally occurring species of parasite. We found strong differences in MHC class IIB expression patterns among fish families, which were consistent over two generations, thus demonstrating a genetic component. The average number of MHC class IIB sequence variants within families was negatively correlated to the MHC expression level suggesting compensatory up-regulation in fish with a low (i.e. suboptimal) MHC sequence variability. The observed differences among families and the negative correlation with individual sequence diversity imply that MHC expression is evolutionary relevant for the onset and control of the immune response in natural populations.

Local differences in immunocompetence reflect resistance of sticklebacks against the eye fluke Diplostomum pseudospathaceum.

We investigated population differences in immunological adaptation of three-spined sticklebacks (Gasterosteus aculeatus) to one of their most abundant macroparasites, the eye fluke Diplostomum pseudospathaceum. We compared infection success in lab-bred fish of 2 populations in northern Germany, from a lake, where eye flukes are prevalent, and a river, where these parasites do not occur. In order to discriminate between protection through innate and acquired immunity, we exposed fish either only once or repeatedly. Lake fish were significantly less susceptible than river sticklebacks already after a single exposure, indicating that in sympatric hosts innate immunity plays the major role in the defence against this helminth infection. In both habitat types, previous exposures only marginally decreased infection rates within 12 weeks. Lake fish showed higher immunocompentence by means of respiratory burst activity and spleen size, regardless of the infection status. Furthermore, they were in a better energy status than river fish, as indicated by a higher hepatosomatic index and haematocrit value. Interestingly, F1 hybrid fish of both populations ranged between the pure habitat types in parasite susceptibility as well as in immunological and condition parameters. Our results suggest that sticklebacks from lakes are better adapted to cope with higher parasite abundance in this habitat.

How a complex life cycle can improve a parasite's sex life.

How complex life cycles of parasites are maintained is still a fascinating and unresolved topic. Complex life cycles using three host species, free-living stages, asexual and sexual reproduction are widespread in parasitic helminths. For such life cycles, we propose here that maintaining a second intermediate host in the life cycle can be advantageous for the individual parasite to increase the intermixture of different clones and therefore decrease the risk of matings between genetically identical individuals in the definitive host. Using microsatellite markers, we show that clone mixing occurs from the first to the second intermediate host in natural populations of the eye-fluke Diplostomum pseudospathaceum. Most individuals released by the first intermediate host belonged to one clone. In contrast, the second intermediate host was infected with a diverse array of mostly unique parasite genotypes. The proposed advantage of increased parasite clone intermixture may be a novel selection pressure favouring the maintenance of complex life cycles.

Modulation of granulocyte responses in three-spined sticklebacks Gasterosteus aculeatus infected with the tapeworm Schistocephalus solidus.

Leukocytes isolated from the head kidney and peripheral blood of 3-spined sticklebacks Gasterosteus aculeatus L. were analysed by means of flow cytometry during infection with the tapeworm Schistocephalus solidus (Müller, 1776). Although parasites increased their body weight continuously throughout the observation period (98 d), proportions of granulocytes increased in blood and head kidney only up to Day 63 post-infection (p.i.). Thereafter, declining proportions of granulocytes were observed in both organs. Thus the relative decrease in granulocyte number was not correlated to a decline in the parasitic load of the fish. To investigate a possible modulatory impact of S. solidus on granulocyte function, head kidney leukocytes were isolated at times before Day 63 p.i. and tested in vitro for their capacity to produce reactive oxygen species (ROS). Head kidney leukocytes from S. solidus-infected fish, analysed immediately after isolation (ex vivo, Day 40 p.i.), exhibited a higher ROS production when stimulated with phorbol myristate acetate (PMA), than leukocytes from naive, sham-treated control fish and fish that had resisted or cleared the infection (exposed but not infected). The latter showed an increased spontaneous ROS production that was not correlated to the numbers of granulocytes present in the head kidney isolates. In infected sticklebacks, spontaneous and PMA-induced ROS production was significantly correlated with numbers of granulocytes present in the head kidney isolates, suggesting that elevated ROS production was due to higher numbers of responding cells rather than an increased capacity of single cells. In vitro, after cultivation for 4 d in the presence of pokeweed mitogen (PWM) or extracts from S. solidus, head kidney leukocytes from control fish showed an increased ROS production and phagocytic activity compared with non-stimulated control cultures. In contrast, head kidney leukocytes from infected fish isolated on Days 48 and 44 p.i., failed to respond to S. solidus antigens in vitro. During S. solidus infection, granulocyte mobilisation resulted in elevated numbers of these cells in head kidneys, but the lack of an in vitro response to S. solidus antigens indicates an in vivo priming of granulocytes by the parasite. These observations may reflect the ability of S. solidus to impair the host's immune response once the parasite is developing in the body cavity of G. aculeatus.

Heredity of specific host-finding behaviour in Schistosoma mansoni miracidia.

Two strains of Schistosoma mansoni were used to investigate the hereditary basis of species-specific host recognition by analysing behavioural responses of miracidia to snail-conditioned water. An Egyptian strain of S. mansoni, capable of distinguishing its host snail Biomphalaria alexandrina from other snails was cycled repeatedly through Biomphalaria glabrata, the intermediate host of a Brazilian strain known to respond even to non-susceptible snails with high intensity. After 5 cycles in the non-natural host, miracidia of the Egyptian strain still retained their preference for the original host snail. In a second experiment, host-finding behaviour of hybrids between these two parasite strains was studied. In the F1 generation, hybrids of both parental combinations showed the same low degree of specificity as the pure-bred Brazilian strain. Approximately one quarter of F2 hybrids proved to be as discriminatory as the Egyptian strain, confirming dominant Mendelian inheritance of non-specificity in schistosome miracidial host-finding behaviour. Moreover, hybrids seem to have lost the ability to develop in B. alexandrina, possibly suggesting a link between host recognition and host compatibility. The heredity of this behavioural trait is of evolutionary and epidemiological significance, since a shift to low host-finding specificity might have been a prerequisite for S. mansoni to acquire new host snails after being introduced to South America by the slave trade.

Multiple parasites are driving major histocompatibility complex polymorphism in the wild.

Parasite mediated selection may result in arms races between host defence and parasite virulence. In particular, simultaneous infections from multiple parasite species should cause diversification (i.e. balancing selection) in resistance genes both at the population and the individual level. Here, we tested these ideas in highly polymorphic major histocompatibility complex (MHC) genes from three-spined sticklebacks (Gasterosteus aculeatus L.). In eight natural populations, parasite diversity (15 different species), and MHC class IIB diversity varied strongly between habitat types (lakes vs. rivers vs. estuaries) with lowest values in rivers. Partial correlation analysis revealed an influence of parasite diversity on MHC class IIB variation whereas general genetic diversity assessed at seven microsatellite loci was not significantly correlated with parasite diversity. Within individual fish, intermediate, rather than maximal allele numbers were associated with minimal parasite load, supporting theoretical models of self-reactive T-cell elimination. The optimal individual diversity matched those values female fish try to achieve in their offspring by mate choice. We thus present correlative evidence supporting the 'allele counting' strategy for optimizing the immunocompetence in stickleback offspring.

Rapid genetic divergence in postglacial populations of threespine stickleback (Gasterosteus aculeatus): the role of habitat type, drainage and geographical proximity.

The goal of this study was to evaluate the role of common ancestry, and of geographical or reproductive isolation, in genetic divergence in populations of threespine sticklebacks (Gasterosteus aculeatus). Using seven DNA microsatellite loci we compared the effects of habitat type, drainage system and geographical proximity on genetic distance among 16 populations situated in an area in Schleswig-Holstein (Germany) that became deglaciated approximately 12 000 years ago. Stickleback population structure correlated only weakly with drainage system, whereas the primary divergence was among habitat types. Phylogenetic analysis revealed that lake (n = 7) and river (n = 5) populations formed two distinct clades (Cavalli-Sforza's and Edwards' chord distance, 82-100% bootstrap support) at approximately equal genetic distances to a third clade, comprising putative estuarine (n = 4) ancestors. Allele frequencies in lake and river populations represented different subsets of the genetically more diverse estuarine populations. In nested amovas approximately twice the genetic variance was distributed among lake vs. river vs. estuarine populations as compared with the combined effects of drainage system and geographical distance. Limited gene flow between habitat types must have been established after postglacial colonization, suggesting ecological hybrid inferiority or behavioural mating barriers between ecotypes. Within estuarine and lake populations, population differentiation followed an isolation-by-distance model. Given the high observed heterozygosities within the 16 study populations (HO = 0.65-0.87), the mean divergence between lake and river population pairs (FST = 0.18 +/- 0.007) would be reached after 300-6000 generations in a stepwise mutation model, depending on the size of N(e). This demonstrates both the utility of hypervariable microsatellites for detecting recent population divergences and the danger of operating at temporal or spatial scales which are beyond their resolution.

Host-finding in Echinostoma caproni: miracidia and cercariae use different signals to identify the same snail species.

The snail host signals releasing host-finding responses in miracidia and cercariae of Echinostoma caproni were analysed by fractionation of snail-conditioned water (SCW). Cercariae responded non-specifically to organic and hydrophilic, low molecular weight components of SCW showing their typical turning response. Hydrolysis of peptides in SCW had no effect on cercarial responses. An artificial mixture of amino acids in concentrations determined from SCW as well as glycine alone in a concentration corresponding to the total concentration of amino acids in SCW showed nearly the same efficacy as SCW itself. Miracidia responded to a high molecular weight glycoprotein fraction, which could be isolated from SCW by ion-exchange and size-exclusion chromatography. In contrast to an Egyptian Schistosoma mansoni strain, the echinostome miracidia were not able to differentiate between different snail species. The results show for the first time that miracidia and cercariae of the same species may use different signals to identify the same snail host species. This indicates an independent evolution of host-finding mechanisms in the two parasite stages.

Snail host finding by Fasciola hepatica and Trichobilharzia ocellata: compound analysis of "miracidia-attracting glycoproteins".

The glycoconjugates from snail-conditioned water of Lymnaea truncatula and L. stagnalis which elicit typical host finding behavior in miracidia of Fasciola hepatica and Trichobilharzia ocellata were separated by anion-exchange chromatography and a two-step size-exclusion chromatography. We obtained fractions attractive for the parasites with MW of about 10(6) Da in both snail species. These fractions still contained species-specific information since miracidia responded only to molecules from their respective host snail. Analysis of the amino acid composition from the protein backbone revealed a similar composition in the effective fractions of both snails. Amounts of serine and threonine were higher than 30 mol %, which is typical for mucin-type glycoproteins. The carbohydrate moieties consisted mainly of galactose and fucose, but nine different other monosaccharides also were identified in smaller amounts. The heterogeneity of the molecules was also confirmed by the binding of six different lectins. Because of these characteristics, the effective molecules were termed "miracidia-attracting glycoproteins" (MAGs). MAGs may play an important role for parasite transmission, as they may increase the chance of an encounter between parasite and host and enable the miracidia to discriminate between their specific intermediate host and other unsuitable snail species.

Miracidial host-finding in Fasciola hepatica and Trichobilharzia ocellata is stimulated by species-specific glycoconjugates released from the host snails.

The miracidia of Fasciola hepatica and Trichobilharzia ocellata approach their host snails Lymnaea truncatula and L. stagnalis by increasing their rate of change of direction (RCD) in increasing gradients of snail-conditioned water (SCW), and they perform a turnback swimming in decreasing gradients. Both hostfinding responses in both species were induced by glycoconjugates with a molecular weight of > 30 kDa that were sensitive to hydrolysis with pronase E and oxidation with NaIO4. Alkaline cleavage revealed that they contained carbohydrates linked O-glycosidically via serine and N-acetylgalactosamine. Miracidia clearly preferred SCW from their specific host snail versus other sympatric snail species and did not respond to water conditioned with fish, tadpoles, or leeches. Differences in the chemical characteristics of SCW from the intermediate hosts L. truncatula and L. stagnalis could be shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotting, and subsequent carbohydrate detection. The first step of purification of the effective signaling SCW components from both snail species was achieved by ion-exchange chromatography.

Schistosoma mansoni miracidial host-finding: species specificity of an Egyptian strain.

The effect of snail-conditioned water (SCW) from Biomphalaria alexandrina, a pigmented and an albino strain of B. glabrata, and Lymnaea stagnalis on the host-finding behavior of miracidia of two Brazilian and one Egyptian strain of Schistosoma mansoni was studied. Miracidia of the Egyptian strain significantly preferred their suitable host B. alexandrina versus the other snail species with their behavior patterns of host location and their responses after contact with the host. However, miracidia of both Brazilian strains did not differentiate between SCW from three of the snail species; only the pigmented B. glabrata elicited weaker responses. The releasing cues of SCW for miracidial host-finding phases are macromolecular glycoconjugates. An analysis of SCW by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotting, and subsequent carbohydrate detection showed that the band patterns of glycoconjugates differed significantly among the four snail strains. Therefore, differing chemical characteristics of the signaling glycoconjugates could be the basis for the observed host specificity in miracidial host-finding.

Schistosoma mansoni and S. haematobium: miracidial host-finding behaviour is stimulated by macromolecules.

The miracidia of Schistosoma mansoni and S. haematobium approach their host snails by increasing their rate of change of direction (RCD) in increasing gradients of snail-conditioned water (SCW), and they perform a turnback response in decreasing gradients. After contact with the host "repeated investigation" is the typical host-specific response. Both species show no significant directed chemotactical orientation towards their snail hosts. All three host-finding responses (increased RCD, turnback response, and "repeated investigation") seem to be stimulated in both species by a similar component of SCW, a macromolecular glycoconjugate with a molecular weight > 30,000. The saccharide chains seem to be O-glycosidically linked via serine and N-acetylgalactosamine. The glycoconjugate is sensitive to lysozyme which may suggest that muramic acid as a gastropod-specific component is involved in the recognition process. Small molecular components of SCW, as well as magnesium chloride offered as pure chemical, may cause a moderate increase in the RCD. Therefore a minor contribution of these components to the host-finding response of schistosome miracidia cannot be excluded. That schistosome miracidia respond to complex macromolecules as host cues may indicate an adaptation to avoid interference of the host-finding with ubiquitous small molecular mud components and it might enable the miracidia to achieve a high degree of host-specificity in their host-finding.