INTRODUCTION
Along with the high importance of fish diseases caused by Myxozoa, studies of the life-cycles, parasite-host-relationships and developmental aspects of the phylum have resulted in a better understanding of life-history traits (Kent et al. Reference Kent, Andree, Bartholomew, El-Matbouli, Desser, Devlin, Feist, Hedrick, Hoffmann, Khattra, Hallett, Lester, Longshaw, Palenzeula, Siddall and Xiao2001). Most myxozoans probably exhibit a complex life-cycle, developing alternately in vertebrates (mostly teleosts) and invertebrates (mostly annelids). Non-motile, waterborne actinospores, shed by the aquatic invertebrate hosts, comprise the means of conveyance of the infective stages towards fish. Actinospores are assumed to be only viable for several days to few weeks (Markiw, Reference Markiw1992; Yokoyama et al. Reference Yokoyama, Ogawa and Wakabayashi1993; Xiao and Desser, Reference Xiao and Desser2000). Hence, special adaptations for rapid host recognition and attachment are essential for successful parasite transmission during the brief period of spore viability.
The discharge of their characteristic polar filaments anchors the actinospore to the fish surface. Gills, skin and the buccal cavity have been confirmed as portals of entry for M. cerebralis, while Thelohanellus hovorkai and Sphaerospora truttae prefer entry via the gills (Yokoyama and Urawa, Reference Yokoyama and Urawa1997; Holzer et al. Reference Holzer, Sommerville and Wootten2003). Tetracapsuloides bryosalmonae and Henneguya ictaluri enter via the skin, while the former was also reported to infect gills (Morris et al. Reference Morris, Adams and Richards2000) and the latter was additionally found in the intestine and the buccal cavity (Belem and Pote, Reference Belem and Pote2001; Longshaw et al. Reference Longshaw, Le Deuff, Harris and Feist2002). Consumption of infected oligochaetes may also aid infection in some species. Attachment to and penetration of hosts by actinospores, and subsequent migration of the amoeboid sporoplasm, should be considered as separate steps, which possibly require different host signals (Kallert et al. Reference Kallert, El-Matbouli and Haas2005a). Kallert et al. (Reference Kallert, El-Matbouli and Haas2005a) demonstrated that M. cerebralis actinospores become mechanically excitable after chemo-sensitization by host mucus prior to polar filament discharge. Such a combination of signals inhibits erroneous reactions upon contact with non-host aquatic organisms or any dead matter. However, no mechanoperceptive structure has yet been described in myozoans.
Many physiological aspects of this evolutionary successful parasite group remain unclear (Yokoyama, Reference Yokoyama2003) and a better understanding of host-invasion in Myxozoa requires information on their morphological and behavioural adaptations to parasitism and on the physiological requirements for host-recognition and host-attachment. Elucidation of these mechanisms is valuable for our understanding of the epidemiology of disease outbreaks caused by Myxozoa in fish populations. Moreover, artificial induction of spore responses may be an effective biological control approach to prevent great losses in hatchery and wild fish populations. Presenting preliminary findings with laboratory-cultured myxozoans, the present paper shows by which means myxozoan parasites ensure transmission success during the difficult task to recognize and invade fish hosts.
MATERIALS AND METHODS
Parasite cultivation
Myxospores were isolated by tissue homogenisation for 2–5 min at 11 500 rpm using an Ultra Turrax (IKA Labortechnik, Staufen, Germany). Tissue remnants were then removed by passing the homogenate through 100 μm mesh filters. The homogenate was then topped up to 500 ml with tap water with the suspension settling overnight at 4°C. All fish were kept at 16–18°C under a constant flow of well water and fed on conventional fish flakes. Oligochaete cultures were maintained in 5 litre plastic containers containing a 5–6 cm layer of autoclaved mud and coarsely grained sand (1:1 v/v) and were held at 12–15°C in the dark. Oligochaetes were fed weekly using a mixture of frozen Artemia, spray-dried Spirulina (MaBitech) and frozen lettuce. Autoclaved mud from the settling pond of a salmonid hatchery was added weekly. Only well water free of copper and chlorine was used for oligochaete cultures.
Henneguya nuesslini and host fish were obtained from a local hatchery (Bavaria, Germany). Myxospores were either obtained from muscle or pieces of the tail of adult, naturally infected brown trout. Cultivation of the parasite was conducted according to Kallert et al. (Reference Kallert, Eszterbauer, El-Matbouli, Erséus and Haas2005b). Four to seven-month-old (3–6 cm) parasite-free brook trout (Salvelinus fontinalis) and brown trout (Salmo trutta) were used for infection with actinospores from infected oligochaete cultures. Myxobolus parviformis actinospores originated from an established parasite culture described by Kallert et al. (Reference Kallert, Eszterbauer, Erséus, El-Matbouli and Haas2005c), using myxospores obtained from cysts isolated from gill filaments of laboratory-infected bream. For M. cerebralis cultivation, infected cultures of Tubifex tubifex were obtained from the long-term established laboratory cultures at the Institute of Zoology, Fish Biology and Fish Diseases of the University of Munich (Germany). Additionally, fresh oligochaetes (>200 000 individuals) from the salmonid hatchery were transferred to aerated plastic beakers (4 litre) and used for infection with M. cerebralis myxospores after a monitoring period (including control cultures without myxospore addition) as described by Kallert et al. (Reference Kallert, El-Matbouli and Haas2005a). Myxobolus pseudodispar originated from naturally infected oligochaetes from the salmonid hatchery. Actinospores from single oligochaetes were used for infection of laboratory-reared myxozoan-free roach (Rutilus rutilus) at a dose of ∼2000 actinospores per fish. The 18S rDNA sequence of this parasite isolate was submitted to GenBank (Accession no. EF466088) to enable future identification, as different isolates of spores morphologically identified as M. pseudodispar can show high variability (up to 4% difference) in these sequences. The isolate examined was most similar to M. pseudodispar ex Blicca bjoerkna (GenBank Accession no. AF466654).
Unless otherwise noted above, oligochaetes from a laboratory stock were infected by addition of ∼0·5–2×106 myxospores after incubating the freshly prepared oligochaete cultures for at least 1 week. Non-exposed control cultures did not produce notable amounts of actinospores. All species form Triactinomyxon-type actinospores (TAMs). They were harvested from oligochaete cultures by filtration using 20 μm nylon meshes or from single oligochaetes isolated in cell well plates according to the method of Yokoyama et al. (Reference Yokoyama, Ogawa and Wakabayashi1991).
Test substrates
The mucus substrates for experimental use were prepared as described previously (Kallert et al. Reference Kallert, Eszterbauer, Erséus, El-Matbouli and Haas2005c). Adult rainbow trout (Oncorhynchus mykiss) and carp (Cyprinus carpio) were obtained from a local distributor. Common bream (Abramis brama) and roach were caught in the river Aisch, Germany. Where necessary, mucus was concentrated by removing water from the solution by partial lyophilization in a SpeedVac, deionized water was used for dilution. Artificial incubation media (standard fresh water ‘SFW 100’ according to Meier-Brook (Reference Meier-Brook1978), containing 100 ppm Ca2+) were prepared by dissolving the respective salts in deionized water. In 1 volume of Ca2+-free water (7 mOsm/kg), the osmolality was restored according to the value of the Ca2+-containing mixture (12 mOsm/kg) by addition of NaCl. Osmolality was measured in triplicate using 150 μl samples by freezing-point-depression using an Osmometer Automatic (Knauer, Berlin), calibrated with deionized water and a 400 mOsm NaCl solution (12·687 g/kg). All osmolality values given refer to unbuffered substrates.
Polar filament discharge
Discharge rates
Polar filament discharge rates were determined as described by Kallert et al. (Reference Kallert, El-Matbouli and Haas2005a). Briefly, 9 μl of buffered test substrate were added to 21 μl of buffered spore suspension on slides and covered with a cover-slip (20×20 mm), followed by mechanical stimulation for 3 s at 50 Hz using a Mini Shaker Type 4810 (Brüel & Kjœr, Copenhagen). Buffered deionized water served as control in all experiments. All media containing parasites or test substrates were adjusted to pH 7·5 (5 mm sodium phosphate buffer). Spore suspensions were stored at 12°C and test substrates were kept on ice during use. Whenever possible, a blind protocol was used by coding the test substrates.
Discharge process
To visualize the process of polar filament discharge, video sequences were recorded using an Axiophot (Zeiss) and a video camera (Canovision EX1 8 mm, Canon). M. cerebralis actinospores were placed on a glass slide in 30–40 μl of water covered with a cover-slip and 4–12 μl of a 30% aqueous NH3 solution were added slowly from the side to induce filament firing. Still frames were taken as screenshots from the Video using Ulead Video Studio 7.0 SE software.
Ca2+-dependency
To investigate the role of Ca2+ in the surrounding medium on polar filament discharge, H. nuesslini actinospores were transferred from their original medium to SFW (‘Standard fresh water’ according to Meier-Brook, Reference Meier-Brook1978) with and without Ca2+. The original water of the actinospore suspension was replaced by transferring separate filtration units (10 ml plastic tubes with 20 μm mesh as bottom) in 5 ml cups with SFW for 30 min and repeated 4 times, changing the medium each time. Polar filament discharge rates were measured using carp mucus homogenate (1 mg/ml final concentration). Individual controls were included showing the discharge rates for actinospores incubated in each SFW mixture separately.
Discharge stimuli
The possible combination of chemical and mechanical stimuli for polar filament discharge in M. parviformis actinospores was tested as described by Kallert et al. (Reference Kallert, Eszterbauer, Erséus, El-Matbouli and Haas2005c). Briefly, immobilized single actinospores were exposed to a sequence of stimuli comprising 2·5 μl of control (buffered) water, mechanical stimulation (3-fold apical tipping), 2·5 μl of host mucus and repeated mechanical stimulation with 10 sec observation intervals between all steps. To test whether the actinospores were able to discharge at all, a saturated solution of urea was applied.
Sporoplasm release
To determine if actinospores had an intrinsic mechanism to cause their apical sutures to open upon polar filament discharge to enable sporoplasm release, H. nuesslini actinospores were microscopically observed after addition of rainbow trout mucus (1 mg/ml final concentration). The spore suspension (40 μl) was placed on a slide and plastilin spacers were used to ensure that pressure from the cover-slip did not affect sporoplasm release. Discharged and undischarged spores were observed for 15 min using a stereomicroscope. Sporoplasm release (emergence of at least 1/3 of the primary cell mass) with and without preceding polar filament discharge was recorded. The period from substrate mixing until sporoplasm release was recorded.
To confirm the cellular integrity of M. parviformis sporoplasms after emergence from the surrounding sheath, fluorescein-diacetate (FDA) staining was used as described by Yokoyama and Urawa (Reference Yokoyama and Urawa1997) and Markiw (Reference Markiw1992) on spore isolates incubated in carp mucus. Sporoplasm emission from M. pseudodispar actinospores was recorded using a digital camera (Moticam 2000, Motic) under a stereomicroscope, after a 1:3 (v/v) addition of carp mucus.
Statistical methods
Statistical analysis was performed using SPSS for Windows. Where necessary, data were arcsine square root transformed to obtain approximately normally distributed data; homogeneity was controlled using the Levene test. Mean values and standard errors were calculated from the transformed data and were retransformed thereafter. Differences between means were tested for statistical significance by a multivariate comparison procedure (Tukey HSD multiple t-test after one-way-ANOVA). Correlation between binominal variables was tested by cross-tabulation followed by calculation of Yates' continuity correction as well as the Phi-value and the contingency coefficient as symmetric measures. Mantel-Haenszel statistics (homogeneity of the odds ratio) was used to test for independence in response between the binary variables.
RESULTS
Polar filament discharge
Discharge process
Video analysis of polar filament discharge in M. cerebralis actinospores showed that complete extrusion requires less than 10 msec (Fig. 1A–C). Most notably, the polar filament first rapidly elongates, then contracts shortly after discharge to about half its length (Fig. 1D and E). This ‘dragging mechanism’ could be observed to pull the whole spore body about 35 μm along the slide by means of the sticky filament (Fig. 2A–C).
Polar filament discharge of Myxobolus cerebralis actinospores induced by addition of 30% ammonia (phase-contrast optics). (A) Actinospore apex with polar capsules (arrowhead). (B) The same spore having discharged one filament (arrowhead) 0·01 s later. (C) Discharge of a second filament 0·01 s later, both still elongated. (D) Different actinospore during polar filament discharge showing elongation of the first filament. (E) Both abridged discharged filaments of the same spore 10 s later. Counter included from the original video footage shows centiseconds (small scale) and sec (large scale).
Polar filament discharge of Myxobolus cerebralis actinospore induced by addition of 30% ammonia (bright-field optics, grid shows actinospore movement). (A) Actinospore prior to discharge. (B) The same spore with fully discharged filament. (C) The same spore with abridged filament less than 3 s later; note different location after movement along the slide. Counter included from the original video footage shows milliseconds (small scale) and sec (large scale).
Ca2+-dependency
H. nuesslini actinospores showed a significant discharge rate in SFW with Ca2+ (P<0·001 vs control), but discharged their polar filaments less frequently when the SFW was Ca2+-deficient (P<0·05, Table 1). When the osmolality of the Ca2+-deficient SFW (7 mOsm/kg) was adjusted to the value of Ca2+-containing water (12 mOsm/kg), the discharge rate was equal to that measured in Ca2+-containing water (P=0·57, Table 1). However, the responses in Ca2+-deficient water (with and without adjusted osmolality) were not significantly different from the respective controls (without mucus addition) (P=0·3 and 0·08 respectively). The responses to controls (without mucus) differed among the media (Table 1). Therefore, the discharge rates obtained in controls could be subtracted from the responses caused by mucus addition in the respective media for equalization. As a consequence, the discharge rate in Ca2+-containing water was significantly higher than that in Ca2+-deficient water with adjusted osmolality (P<0·05). The responses in both Ca2+-free media were not significantly different from each other (P=0·7).
Effect of Ca2+-ions on polar filament discharge of Henneguya nuesslini actinospores during response to carp mucus homogenate (1 mg/ml)
(The actinospores were transferred to artificial medium (‘Standard fresh water’ (SFW) according to Meier-Brook, Reference Meier-Brook1978). NaCl was used for osmolality equilibration of Ca2+-deficient water. Actinospores and substrates were mixed on a slide (21+9 μl) with application of vibrations (3 s, 50 Hz, Amplitude 0·5 mm). Letters indicate a significant difference (ab, P⩽0·001; ABCD, P⩽0·05; Tukey HSD). Total number of actinospores counted per substrate 286–398 (8 replicates).)
Discharge stimuli
Of 29 single M. parviformis actinospores, only 3 (10·3%) did react after the first mechanical stimulation, and 1 further specimen (3·8%) of the remaining reacted after mucus addition. None of the 25 remaining spores reacted after the ensuing mechanical stimulation within the observation period of 10 sec. General discharge ability of the actinospores was tested by addition of a saturated urea solution, showing that all spores had a functioning attachment apparatus.
Sporoplasm release
The period the amoeboid sporoplasm required to leave the valve structure after stimulation by mucus was determined for H. nuesslini actinospores. The majority of sporoplasms left the valves within 6 min, whereas most were emitted around 5 min (Fig. 3, with the mean time of emission around 5·4 min, s.d.=2·8). The observation of discharged and un-discharged H. nuesslini actinospores for 15 min after stimulation by mucus homogenate revealed that among actinospores with discharged polar filaments, 60·6% kept their sporoplasm inside the spore valve (109 individuals counted), while for non-discharged actinospores, 28·4% released their sporoplasms (95 individuals counted). In most undischarged actinospores in which the sporoplasm did not emerge, the amoeboid mass exhibited characteristic movements within the style of the spore (also observed in all other species) that were absent without mucus incubation. There was very weak correlation between the responses, as shown by the Phi-value of 0·114 and the contingency coefficient of 0·113 (with a significance of 0·104). Additionally, according to Mantel-Haenszel's chi-square analysis, there is no strong relationship between the two reactions (P=0·14). Therefore, the sporoplasm emission reaction is per se independent from polar filament discharge (no linear correlation) in H. nuesslini actinospores. Nevertheless, 39·4% more sporoplasms were released when polar filaments were discharged, but release was not an imperative consequence after filament discharge.
Duration until onset of the emission response of Henneguya nuesslini sporoplasms after stimulation with mucus in time categories representing the period (2 min per category) after stimulation in which the emission movement was observed (64 actinospores counted). A curve has been plotted showing Poisson-distribution.
Observing actinospores reacting to mucus, we could discern 2 different modes of sporoplasm emission in M. parviformis and M. pseudodispar. In one mode, the sporoplasm emerged as an amoeboid mass from the valve cells as typically seen on slides after incubation in fish mucus. Alternatively, a sheathed unit (corresponding to the ‘endospore’ described by Janiszewska, Reference Janiszewska1955) was emitted, out of which subsequently the sporoplasm emerged. The cellular integrity of the actively moving amoeboid primary cell could be confirmed by FDA-staining of M parviformis ‘endospores’ (Fig. 4). Unlike M. parviformis (see Kallert et al. Reference Kallert, Eszterbauer, Erséus, El-Matbouli and Haas2005c), the M. pseudodispar valve cell did not open in a splayed finger-like manner to release the sheath unit, and it was rather released from the valve cells as a ‘plug’ (Fig. 5).
Sporoplasm emission of Myxobolus parviformis sheath unit (‘endospore’) induced by addition of bream mucus after fluorecein-diacetate-staining. (A) Ball-like pseudopodium emerging first. (B) Apical opening and active emergence of the sporoplasm. (C) Fully emerged sporoplasm with sheath bag attached; note membrane staining of actively emerged amoeboid primary cell.
Expulsion of Myxobolus pseudodispar sheat unit (‘endospore’) induced by addition of roach mucus; note the plug-like apical architecture with no polar capsules left in the valve cells.
DISCUSSION
The experimental outcome along with a step-by-step analysis of morpho-physiological aspects and the reactions actuated by actinospores during host invasion provide valuable information on myxozoan transmission. An understanding of how actinospores recognize and enter the fish host is not only of importance concerning epidemiology of myxozoans, the information is useful also to researchers performing laboratory work with specimens of this phylum. For this study, we chose suitable species to investigate each of the mechanisms and tried to give comparative information about differences between certain species.
With respect to polar filament discharge, the observed speed of discharge and the length of the polar filament indicate that actinospores need close contact between their apical region and the host surface for successful attachment. In M. cerebralis actinospores, there is a putative chemically triggered thigmo-perceptive structure (Kallert et al. Reference Kallert, El-Matbouli and Haas2005a), suggested by the observation that the actinospores only discharged their filament when a chemical (mucus-derived) signal was present prior to physical contact. An intrinsic retraction mechanism was always observed immediately after discharge of the filament in several species. Its purpose could be to mediate ultimately close contact with the viscous fish surface or to pull the ‘endospore’-construct out of the valve envelope. The actinospores thereby have developed an excellent mechanism to pull their apical region tightly towards the host surface, enabling the sporoplasm to directly enter the epidermal layer. The artificially induced discharge without mechanical stimulation for visualization was only possible using ammonia as an effector for practical reasons. The use of ammonia instead of a natural trigger did not influence the characteristic course of the reaction. It remains unknown how the initial elongation and subsequent contraction of the polar filament actually occurs, but it is likely that either proteinaceous conformation changes upon hydration or the stored energy due to the well-known filament twist play a role.
Uspenskaya (Reference Uspenskaya1982) assumed Ca2+-ions to be involved in the polar filament discharge mechanism and suggested that it is an active process due to contractile proteins. The role of calcium as an effector or even the driving force behind the filament extrusion is also implicated in cnidarians: removal of calcium from the medium inhibited discharge of nematocysts in some species (Santoro and Salleo, Reference Santoro and Salleo1991; Kawaii et al. Reference Kawaii, Yamashita, Nakai and Fusetani1997; Russell and Watson, Reference Russell and Watson1995). Additionally, calcium plays a role in cell-signalling in cnidae-bearing complexes (Cannon and Wagner, Reference Cannon and Wagner2003). By Ca2+-replacement experiments, we showed that external Ca2+ was not a major effector for the discharge mechanism in H. nuesslini actinospores, but it appeared that the osmolality of the surrounding medium had to exceed a certain value to enable normal discharge rates. When the effect of osmolality differences was considered and the different responses in control media were subtracted from the discharge rates with mucus, Ca2+-ions indeed seemed to play a role. Discharge rates in Ca2+-containing water were doubled compared to those in Ca2+-deficient water at the same osmolality. However, in Ca2+-free medium with restored osmolality by NaCl-addition, filament discharge rates were higher than in media with Ca2+, indicating that eventually only an artificial effect of NaCl was observed. This may play a role in membrane conductivity or osmotic balance of the parasite primary cell and thus influence polar filament discharge. However, we assume that exterior Ca2+-ion-concentration does not play a significant role, considering that discharge was never fully abolished in Ca2+-deficient media. Whether actinospores are able to adjust to media with lower osmolality after prolonged incubation, should be further investigated.
In sporoplasm emission experiments, most H. nuesslini sporoplasms left their valve shell after about 5 min after little or no mechanical stimulation and with or without prior polar filament discharge – a result comparable with the data of Yokoyama et al. (Reference Yokoyama, Kim and Urawa2006). These authors further observed ‘slow’ and ‘fast’ reactions of 2 myxozoan species infecting different fish hosts. In Thelohanellus hovorkai actinospores, sporoplasms were released over a 30 min exposure time to host mucus, while Myxobolus arcticus actinospores reacted similarly to M. cerebralis in the current study. The aurantiactinomyxon type of T. hovorkai has a similar apical ultrastructure as M. parviformis triactinomyxon spores with rather counter-sunk polar capsules located below the apical valve cell margin (Kallert et al. Reference Kallert, Eszterbauer, Erséus, El-Matbouli and Haas2005c), in contrast to fast-reacting species such as M. cerebralis and H. nuesslini showing polar capsules with protruding tips. This may be an ecological adaptation in response to a need for rapid host recognition. Similarly, the low discharge rates and negative reactivity of M. parviformis actinospores in this work may be explained if this parasite requires an extended time to react, a feature which could reflect a specialization towards a certain host invasion route (e.g. via gills or oral uptake) or an environment that bears a higher risk of unintentional mechanical stimulation, for instance by large amounts of plankton. A more rigid apical structure may be an adaptation for prolonged viability, as these actinospores may be better protected or less sensitive than, for example, H. nuesslini actinospores. A difference in host preference is apparent between ‘quickly’ and ‘slowly’-reacting parasites; as the former infect stream inhabitants, while the latter seem to prefer bottom feeders in standing waters. However, M. pseudodispar is a fast-reacting species predominantly found in slow waters, which would represent an exception to the rule. However, the slow mode of sporoplasm emission observed by Yokoyama et al. (Reference Yokoyama, Kim and Urawa2006) may also be the result of increased mechanical sensitivity of the triactinomyxon-type actinospores, as no further mechanical stimulus was applied. This may have extended the period required for reaction of T. hovorkai specimens, as fewer polar filaments would have discharged. Surprisingly, M. parviformis actinospores did not elicit a similar reaction to a combination of chemical and mechanical stimulation compared to M. cerebralis. It is not known whether the incubation time of the chemical stimulus was too short for this species.
Although the experimental conditions of the sporoplasm emission experiments with H. nuesslini certainly do not completely mimic contact with a real host, we nevertheless demonstrated a suitable method to explore a putative built-in mechanism utilized by the parasite to escape from its covering transmission ‘vehicle’. Thus, it could be concluded, that the apical opening does not occur passively by an intrinsic mechanism, as many spores with discharged polar filaments did not open and sporoplasm release did not occur. Although polar filament discharge surely facilitates sporoplasm emission, we could show that it is not an imperative prerequisite. Enhanced movement of the sporoplasms and even a rearrangement of the polar capsules inside the valves were frequently observed during incubation of actinospores in fish mucus for several minutes. Thus, the sporoplams apparently were activated without being able to emerge. Hence, this suggests that even within an intact actinospore, the sporoplasm is able to recognize mucus components. It is possible that the sporoplasm may be capable of actively triggering polar filament discharge disposition and thereby affect their infectivity status.
A striking feature of many triactinomyxon-type actinospores is the ‘endospore’ structure which comprises a membrane-sheathed sporoplasm primary cell with polar capsules attached apically. Its purpose is probably the protection of the sporoplasm after attachment and prior to penetration of the host. El-Matbouli et al. (Reference El-Matbouli, Hoffmann, Schoel, McDowell and Hedrick1999) documented the ‘endospore’ structure by SEM on the fish host surface after exposure to M. cerebralis actinospores and described it as a “fibrous structure”, left after the sporoplasm had penetrated. It has also been observed in M. parviformis (Kallert et al. Reference Kallert, Eszterbauer, Erséus, El-Matbouli and Haas2005c) by light microscopy, and we observed it in the current study in M. pseudodispar and occasionally in M. cerebralis after activation by host mucus. The ‘endospore’ of M. pseudodispar often appears ball-like and is easily emitted by mechanical agitation. Its emission seems to be the result of host anchorage via the polar filament immediately after discharge and its characteristics (shape and mode of emission) differ between the species. As an example, M. cerebralis ‘endospores’ are rarely seen set free, whereas in M. pseudodispar this is very common after incubation in mucus and application of vibrations. Additionally, the amount of force needed for valve opening prior to sporoplasm release may be directly influenced by the sporoplasm itself, as we have shown the sporoplasm can recognize host cues even before polar filament discharge. In addition, sporoplasm emergence directly from the valvular apex directly would be rather difficult due to the forces actuated by water current on the whole valve shell, which is thus avoided in a very elegant manner. Morphological division of actinospores into ‘epi-’ and ‘endospore’, formerly suggested by Janiszewska (Reference Janiszewska1955), should be re-established, as it is not referred to in the current myxozoan taxonomy, although their presence and taxonomical impact are clearly visible. We suggest that the functional differences are taxonomically important and suggest them to be included among established parameters such as morphometrics and secondary cell count. Observations on the presence, composition, shape and function of the ‘endospore’ should be referred to in future actinospore descriptions and transmission studies.
Our findings with the myxozoan species in this study allowed us to compose a refined picture of the steps actinospores take during host invasion (Fig. 6). When in proximity to a host, the waterborne actinospores receive chemical cues from host mucus and become mechanically excitable (Fig. 6A). Upon apical contact, a not yet known mechanoreceptor induces fast discharge of the polar filament, which presumably depends on medium osmolality (Fig. 6B). Subsequent retraction of the filament pulls the apical region of the actinospore tightly into the mucous surface (Fig. 6C). Forces generated by a parachute-like mechanism caused by hydrodynamic forces on the floating appendices open the apical valve shell along their sutures (Fig. 6D) and the actinosporean shell is eventually discarded (Fig. 6E). For this purpose, actinospores possess an apical, species-specific suture lining along which the valves open. The sporoplasm (inside a sheath or ‘endospore’) is released being tightly attached (thereby physically and osmotically protected) to the host surface. The amoeboid mass leaves the membranous sheath (Fig. 6 (F)) and moves into the host (Fig. 6G) by penetration of the epidermal tissue (Fig. 6H) and finally follows a path towards its species-specific target cell type or organ. These mechanisms comprise an excellent set of adaptations for successful passive host recognition and invasion in an aquatic environment without energy-intensive active host-finding behaviour.
Schematical course of the events during host invasion by triactinomyxon-type actinospores. (A) Actinospore apical region encountering fish host surface, chemical sensitization. (B) Polar filament discharge after mechanical stimulus upon contact. (C) Adduction (arrow) of the actinospore apex for close contact to the host surface by means of filament constriction. (D) Opening of the apical valves among specific sutures by means of water current traction (arrow). (E) Release of the attached ‘endospore’, discarding of the valve shell. (F) Active emergence of the sporoplasm. (G) Fully emerged sporoplasm leaving the ‘endospore’ sheath on the host surface. (H) Penetration of the sporoplasm through the host integument and movement towards deeper layers.
We thank C. Loy for laboratory assistance and J. Borrelli for her help on the topic and culture maintenance. Additional thanks to the Department for Developmental Biology of the FAU Erlangen-Nürnberg and J. Hertel, B. Haberl, R. Rübsam and M. Kalbe for their help. This study was supported by the Deutsche Forschungsgemeinschaft.
