Spore activation

Spore attachment

We used different combinations of hosts and parasite clones previously characterized to be resistant or susceptible to given Pasteuria clones [20]. We observed the fate of fluorescent spores of three parasite clones exposed to 14 D. magna host clones with the aim of identifying differences which correlate with the compatibility of a given host-parasite combination (Table 1). The parasites attach to the host oesophagus for all susceptible (compatible) host-parasite combinations, but they never do so for the resistant combinations (Table 1, Figures 1f and 2a). Thus, the result of this attachment-test was 100% consistent with the results of infection trials (Table 1). For susceptible combinations the host oesophagus was densely covered with spores forming a dense layer in the oesophagus, while there were no spores attached in resistant combinations. We never observed ambiguous cases (for example, only few spores attached). While spores in the mid and end gut moved with the flow of the food, those attached to the oesophagus were not disturbed by passing boluses of food, indicating that they strongly adhere. In resistant combinations spores were never seen attached to the oesophagus and all spores passed with the flow of the food through the gut (Figure 2b). Thus, spore attachment in the oesophagus was very specific to the D. magna and P. ramosa genotype and consistent with resistant/susceptibility status for each combination.

Influence of gender and culture conditions

Spore activation and resistance of other Daphniaspecies

Spore attachment is a key step in Daphnia-Pasteuriacoevolution

The Daphnia-Pasteuria system has become one of the prime examples of antagonistic coevolution. Host and parasites show strong genetic effects for resistance, virulence and infectivity; genotype × genotype interactions have been reported within and across populations and selection acts rapidly in natural populations [18, 19, 40, 41]. Our study suggests that the parasite-dependent [28] host population structure and the coevolution [29] described for this system are mainly driven by the properties of a unique step, the attachment step. First, this step revealed very strong host genotype by parasite genotype interactions (Table 1). Second, the attachment step is independent of the environmental conditions. Third, a recent study of D. magna - P. ramosa coevolution using resurrected host and parasite isolates from lake sediments showed a signal of fluctuating selection only for infectivity but not for parasite virulence [29]. Virulence (the parasite's effect on infected hosts) was also observed to evolve but at a slower rate [29]. The authors proposed that the difference between the evolution of virulence and infectivity resulted from different genes contributing to these traits. Here we give a mechanistic explanation for this finding. Infectivity depends on the attachment and, most likely, on the ligands present on the host and on the parasite. On the other hand, expression of virulence may depend on the host's immune response during the within-host proliferation step. It is likely that these processes are determined by different sets of genes.

The identification of the attachment step as the key step in the coevolutionary dynamics in this system will allow us to improve our understanding of the patterns of antagonistic coevolution. For example, evolutionary models studying the coevolution of the infectivity and the virulence steps [42] can fit our system in relation to the coevolution of the attachment and the proliferation steps. Those models typically characterize infection outcomes as binary (Yes/No), while empirical data suggest they are more quantitative [15–17]. We show that we can observe a binary outcome when individual steps of the infection process are considered. Furthermore, our method provides a fast and reliable way to test individuals and populations for their susceptibility to Pasteuria. Ongoing research in our group showed that up to 400 Daphnia individuals can be tested in a day (P Luijckx, in preparation). The assay we developed makes it possible to test for susceptibility without the potentially confounding effect of the within-host proliferation step in the infection trials.

From the environment to the host body cavity

The resting endospores of P. ramosa can remain dormant for decades under harsh environmental conditions [24, 29]. Before attachment to the host, the spores need to be activated (Figure 1d). The filter-feeding Daphnia capture particles, including parasites, from the water and transport them on a mucus-layered pathway from the phyllopods to the mouth. During this process, the parasite's exosporium opens by an unknown trigger, releasing the activated spore form within less than 10 min (Figure 1). Despite the fact that spore activation is a necessary step for the infection, this step is entirely unspecific with regard to Daphnia species and clone, host gender and the environmental conditions (Tables 1, 2, 3). The signal that triggers spore activation may be related to chemical substances in the mucus of the filtering apparatus, but other factors (for example, mechanical) cannot be excluded.

Once the activated spore enters the oesophagus, it will attach to the oesophagus wall if the host and the parasite genotype are compatible. There it presumably penetrates the gut wall and enters the host's body cavity. A similar attachment process on the cuticula is also known from P. penetrans but, in this case, the parasite seems to be able to attach to any area of the nematode's body surface [25]. It has been proposed that the lower part of P. nishizawae attaches to the host because this part is densely covered by microfibres [43]. In contrast, in P. ramosa it is the upper part of the peripheral fibres (Figure 1e) that are most densely covered with a layer of microfibres. These fibres may be involved in the attachment (Figure 1f).

An endospore adhesin epitope, situated on the exosporium of P. ramosa, has been identified and it has been suggested that it may be a ligand responsible for the recognition and the binding onto the host [44]. However, according to our results, it is unlikely that this epitope is involved in the attachment because the exosporium of P. ramosa is removed during the activation step. A later study, analysing surface proteins of P. ramosa spores by two-dimensional gel electrophoresis, proposed that a collagen-like protein may be responsible for the binding onto the host but might suffer the same problem of the previous study [45]. We propose that later studies on candidate proteins responsible for the specific attachment to the host in this system should investigate the spores once activated.

The development of Pasteuria, from the moment they attach to the oesophagus until the vegetative stage can be detected in the hemolymph (about 8 days at 20°C [46]), is unknown. Also, the penetration mechanism is poorly described. Sayre and Wergin [25] show a transmission electron micrograph of P. penetrans with a structure they call a 'germ tube' crossing the host epithelium. Our hypothesis is that the endospore makes a hole across the host epithelium and injects its cortex into the host. As one response of Daphnia to wounding is an increase of Phenoloxidase (PO) activity [47], one might expect the penetration process to trigger an immune response but this remains an open question. However, resolving the infection process will allow the study of the immune response during the proliferation step without the confounding effect of genetic variation in the attachment step.

Environment effects and the proliferation step

We found that environmental effects do not influence the activation and attachment step (Table 2). Excluding these steps, we suggest that the proliferation step is the one responsible for the reported sensitivity of the overall infection process for environment effects [32, 34]. The activation and the attachment steps seem independent of the host's immune system (defined as a system that is potentially able to kill parasites), while the proliferation step is likely to be governed by the host's immune system. The immune system may lead to variation between and within those Daphnia clones that allow Pasteuria attachment (and, thus, enable the parasite to enter the host), thereby contributing to local and temporal adaptation, maternal effects and induced resistance [29, 34, 48]. We suggest that future studies on host immunity should use only Pasteuria clones that can attach to a given clone of Daphnia so that all the variation observed is likely to originate from variation during the proliferation step. These factors highlight the importance of controlling the host and parasite genotypes and breaking down the infection process in order to understand the respective role of each step in host-parasite interactions.

Resolving the infection process leads to a better understanding of host-parasite interactions

Resolving the infection process in its sequential steps has been proposed in a number of theoretical models [10, 11] but experimental data are scarce. Our approach is transferable to other host-parasite systems and our results suggest that this can provide important new insights into host-parasite interactions and their evolution. Increasing the degree of the resolution of the infection processes highlights a universe of possibilities of the different levels at which host and parasites interact. The different steps might differ in how they are influenced by the environment. They might also differ in which sets of genes regulate them. As it is probably the case for our study system, different steps of the infection process might follow distinct evolutionary dynamics and be explained by different model (for example, balancing selection, directional selection) [10, 11]. However, because of the sequentiality of the steps, it is possible that the selection on one might depend on the selection on other steps. We propose that analysing infection as a succession of well characterized steps will help to reconcile the empirical data with predictions based on alternative coevolutionary models (for example, Red Queen and Selective Sweep models).

Spores of all P. ramosa clones tested, and which were isolated from natural D. magna populations, were activated by all D. magna clones as well as by six other Daphnia species (Table 3) and even a Cladoceran from a different genus, Simocephalus vetulus. Also, apart from the natural host, D. magna, D. dolichocephala, also became infected following attachment of the activated spores to the host oesophagus. This suggests that the triggers for spore activation and, to a lesser extent, for attachment are phylogenetically conserved. This may facilitate the host range evolution of the parasite. Indeed, despite its high specificity on the level of the host clone, P. ramosa infections have been reported in several species within the family Daphniidae [49]. It will be necessary to test more clones of different Daphnia species in order to determine their pattern of susceptibility and resistance to the parasite. Importantly, phylogenetically conserved steps of the infection process can be ruled out as major factors in coevolution, but are, perhaps, the most appropriate targets for vaccine and drug development. In fact, the genes involved in some infections steps have been worked out for some systems [50, 51] and can be of use in biomedicine for diseases control [52, 53].

Host and parasite

We used 14 isofemale lines (hereafter referred as clones) of D. magna and one clone each of six other Daphnia species (Tables 1 and 3). Unless otherwise stated, Daphnia clones were kept in standard medium (ADaM, [54] modified by using only 5% of the recommended Selenium dioxide concentration) at 20°C and fed with the chemostat cultured unicellular algae, Scenedesmus obliquus.

The parasites used were single genotypes of P. ramosa, C1, C14 and C19, characterized as clones in Luijckx et al. [20] and originated from D. magna populations in Moscow (Russia), Tvärminne (Finland) and Gaarzerfeld (Germany), respectively. Spore suspensions of Pasteuria were obtained by homogenizing infected D. magna in ADaM and quantifying spore density. The status of resistant or susceptible D. magna were defined previously [20]. The infection status of two further Finnish D. magna clones ('Kela-39-09' and 'Kela-18-10') exposed to Pasteuria clones were tested with the same protocol. All infections in these experiments were done with naïve individuals born to naïve mothers, kept under high food conditions. These conditions were applied because they are known to minimize the triggering of immune effect [34, 35, 55].

Fluorescence labelling of spores

Fluorescently labelled spores of P. ramosa were produced by homogenizing infected Daphnia in ADaM, followed by centrifugation at 10 000 g for 5 min at room temperature. The spore pellet was suspended in 0.5 mL of 0.1 M sodium bicarbonate (pH 9.1) containing 2.0 mg/mL of fluorescein-5(6)-isothiocyanate (F3651-100MG, Sigma-Aldrich, Miss, USA), a green fluorescent dye that stains proteins unspecifically [56]. Spores were incubated in the dark for 2 h at room temperature with occasional vortexing. The suspension was centrifuged at 10 000 g for 5 min and the supernatant removed. The spore pellet was suspended in distilled water and, again, subjected to centrifugation. This process was repeated until the supernatant was clear. Labelled spore suspensions can be stored at 4°C in the dark for several months.

The shape and location of the green labelled spores were examined in the transparent Daphnia using a microscope with fluorescent light (Leica DM 2500, at magnification 200 × and 400 ×) and filter cubes Leica B/G/R (bandpass filter excitation 420/30 nm; 495/15 nm; 570/20 nm - band pass filter suppression 465/20 nm; 530/30 nm; 640/40 nm). We increased the colour contrast by adding a red fluorescent dye to the medium in which the Daphnia were observed. This was done by preparing a solution of concentrated red dye (0.05 mL of DMSO with 0.0015 g of Tetramethylrhodamin-5-isothiocyanate; T0820-5MG by Sigma-Aldrich), which was homogenized in PBS to make the diluted dye (1 μL of concentrated solution with 10 mL of phosphate buffered saline). We added 1 μL of this solution to the Daphnia medium 10 min before observing the Daphnia. We obtained extended focus images using a camera Leica DFC 300FX and the program Leica Application Suite (Version 3.4.0, package 'Montage').

The separation of the different steps and their specificity

Adult Daphnia were put individually in 1 mL of medium in 24-well-plates and exposed for at least 1 h to around 17,000 labelled P. ramosa spores. Susceptible hosts exposed to labelled spores become infected, suggesting that the dye does little or no harm to the spores (data not shown).

Spore activation

Pilot trials revealed that the labelled spores remain in their typical spherical shape as long as they are not in contact with a host. Upon contact with the host phyllopods (swimming and respiratory appendages of branchiopod crustaceans), spores with a sombrero-like shape are observed (Figure 1c and 1d). We called this process 'spore activation'. We tested all combinations of 14 D. magna clones and three P. ramosa clones for spore activation (Table 1). The same was done for one clone each of six further Daphnia species but only in combination with one P. ramosa clone (Table 3). We used five replicates for each host-parasite combination [in total (14 × 3 × 5) + (6 × 1 × 5) = 190; details in Table 1].

Spore attachment

After exposure, Daphnia were placed on a microscopic slide and we examined the complete Daphnia body under a fluorescent microscope. The transparent body of Daphnia allowed us to determine in which body region activated fluorescent spores attach in the living animals. Once we determined the specific area, we tested resistant and susceptible Daphnia magna clones (five replicates of 14 clones, details in Table 1) for differences in attachment. The same was done with clones of other Daphnia species (five replicates of one clone per species; details in Table 3). In order to validate the assignment of individuals with apparently no spores attached to their oesophagus, we viewed the oesophagus of slightly squashed animals at 400 × magnification. For each experiment, the examiner was not informed whether the animals belonged to a susceptible or to a resistant clone. In order to confirm that the Daphnia ingested spores, the gut content was inspected for the presence of spores. All exposed animals had spores in the faeces. We call this procedure to test for spore attachment the 'attachment-test'.

Influence of gender and culture conditions

In order to see if the specificity pattern observed in the attachment-test was dependent on host sex or culture conditions, 10 host individuals of each sex were tested in each of six treatments. This was done with D. magna clones 'Kela-39-09' and 'Kela-18-10"'because these two Daphnia clones have the reverse pattern of infectivity to the two P. ramosa clones used and they are easily induced to produce male and female offspring in the laboratory. Daphnia were raised either at one of four temperatures (10°C, 15°C, 20°C, 25°C, with high food), two food levels (at 20°C, fed daily with 2.5 or 5 million algae) or two density levels (at 20°C, high food level, single Daphnia or Daphnia from crowded stock cultures; see Table 2). These conditions were chosen to represent various environments that are common in natural Daphnia populations. We did not employ a full factorial design, as our interest was not in establishing reaction norms but in testing for the influence of non-genetic conditions in general. Daphnia of both clones raised under these conditions were exposed to fluorescently labelled spores of P. ramosa C1 and C19. Given the very clear effects observed with the four combinations of hosts and parasites used and the range of conditions tested, we do not believe that other combinations would drastically change our results. However, we cannot exclude with certainty that some combinations might lead to a different result.

Resistance or susceptibility of other Daphniaspecies

One clone of each of six other Daphnia species (D. arenata, D. dolichocephala, D. galeata, D. barbata, D. similis and D. lumholtzi) were assayed for their propensity of oesophageal spore attachment using P. ramosa clone C19. For this assay, groups of five conspecific individuals were exposed to 200,000 P. ramosa spores of clone C19 in 20 mL medium. Four replicates per species were used. After 5 days we filled the jars to 100 mL and then changed the medium on a weekly basis. Animals were fed daily with 5 to 10 million algal cells per jar depending on the size of the Daphnia species. The infection status was investigated under a microscope with phase contrast (magnification 400 ×), at host death or 29 days after exposure.

Electron microscopy

In order to prepare Daphnia for transmission electron microscopy (TEM), infected individuals were fixed on ice in 4% glutaraldehyde buffer in Sorensen's phosphate buffer (0.1 M KH₂PO₄ and 0.1 M Na₂HPO₄) and kept in the dark for several hours. The animals were then rinsed five times on ice using the same buffer for a total of 5 min. Post-fixation was carried out with 1% OsO₄ in Sorensen's phosphate buffer on ice. After post-fixation, the Daphnia were again washed in Sorensen's phosphate buffer on ice, dehydrated in a graded acetone series, and finally embedded in the epoxydic resin EPON.

Transversal and sagittal sections were made through the oesophagus. Semi-thin sections (diamond knife, 0.7-1 μm) were cut in order to approach the right spot on the resin block using a RMC MT 6000-XL (RMC Inc, AZ, USA) ultramicrotome. In order to identify regions of interest for TEM, the tissue was stained using Richardson's dye [57] and examined under a light microscope. In order to see parasite structures using TEM, 5-8 ultrathin sections (diamond knife, 60 nm) were cut after every 10 semi-thin sections. The ultra-thin sections were mounted on Formvar-coated copper grids and stained with uranyl acetate and lead citrate to enhance the contrast. Ultrathin sections were analysed using a FEI Morgagni™ transmission electron microscope at 80 kV equipped with a digital camera.

For scanning electron microscopy (SEM), D. magna were fixed in 3% glutaraldehyde buffer in 0.1 M phosphate buffer for 2 h at 20°C. Samples were washed twice in distilled water for 5 - 10 s, dehydrated in graded ethanol series and critical point dried overnight (16 h). The specimens were coated with gold (20 nm) and viewed using a Philips XL 30 ESEM under high volume conditions from 5 to 15 kv.