Transmission of Myxozoans to Vertebrate Hosts - Springer Link

Transmission of Myxozoans to Vertebrate Hosts

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Dennis M. Kallert, Daniel S. Grabner, Hiroshi Yokoyama, Mansour El-Matbouli, and Edit Eszterbauer

Abstract

This chapter reviews how waterborne myxozoan stages achieve transmission to vertebrate hosts, a process that depends on attachment to a host and subsequent invasion. Such transmission has largely been examined for actinospores that are produced by myxosporeans infecting annelid hosts, but we also consider malacosporean stages released from freshwater bryozoan hosts where possible. We review the functional morphology of spores and how this relates to the invasion process and consider cues that initiate the discharge of polar capsule filaments for attachment and sporoplasm activation. We summarize initial invasion steps and discuss factors that may influence transmission ranging from spore viability and infectivity to host cues. We also describe what is known about portals of entry to fish hosts and how non-specific host recognition may enable some myxosporeans to infect a broad range of hosts. Such non-specificity could promote diversification if speciation follows the acquisition of new hosts. We conclude by recommending experimental procedures to be adopted when handling and harvesting actinospores for experimental studies.

D.M. Kallert (&) Kallert and Loy GbR, Biological Studies and Scientific Services, Birkenweg 11, 91325 Adelsdorf, Germany e-mail: [email protected]

M. El-Matbouli Fish Medicine and Livestock Management, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria

D.S. Grabner Aquatic Ecology and Centre for Water and Environmental Research, University of Duisburg-Essen, Universitaetsstr. 5, 45141 Essen, Germany

E. Eszterbauer Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Hungary

H. Yokoyama Department of Aquatic Bioscience Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan

B. Okamura et al. (eds.), Myxozoan Evolution, Ecology and Development, DOI 10.1007/978-3-319-14753-6_13, © Springer International Publishing Switzerland 2015

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Keywords

Host recognition Actinospore Malacospore Sporoplasm process Polar capsule discharge Host cues Activation

13.1

Introduction

With the growing economic importance of fish diseases caused by myxozoans, research over recent decades has resulted in a greatly improved understanding of the life cycles and transmission of this enigmatic endoparasitic group. Despite reports of direct transmission in very few species (e.g. Enteromyxum spp.), all fully resolved myxozoan life cycles involve alternation between invertebrate and vertebrate hosts. Transmission to vertebrate hosts, predominantly fish, is achieved by highly specialized waterborne spores expelled from the invertebrate host. These immotile stages are adapted for dispersal, host recognition and attachment, the latter achieved by means of extrusible filaments from the characteristic polar capsules. The floating spores are relatively shortlived and, being immotile, rely on water currents to carry them to the vicinity of hosts prior to attachment and subsequent release of infectious sporoplasms. The actinospore transmission stages are generally released in large numbers and face obstacles such as predation, erroneous invasion attempts, and mechanical damage. They may also have to withstand physiological or osmotic stress prior to achieving transmission. The tremendous diversity of myxozoan transmission stage morphologies, host ranges, and habitats reflects the evolutionary need for efficient and quick host recognition and invasion mechanisms that have to be functional in a hostile environment by only a few specialized cell types. Knowledge on how transmission to vertebrate hosts is achieved is crucial for understanding the epidemiology of diseases caused by myxozoans. It is also fundamental for studying myxozoan life cycles, understanding host specificity and elucidating the molecular aspects of myxozoan development. This chapter provides an overview of how myxozoans manage to complete transmission to vertebrate hosts by examining

Invasion

attachment to hosts, how sporoplasms are activated for invasion, and what factors may influence transmission success. In particular, we review morphological and functional features of myxozoan spores, host recognition and specificity, invasion steps and the respective mechanisms. We conclude by reviewing experimental procedures and laboratory handling for investigations that involve actinospores.

13.2

Transmission Stages

13.2.1 Functional Morphology of Actinospores For most myxozoans, the vertebrate-infecting stage is not yet known. Nevertheless, a number of actinospore ‘types’ have been described (Lom and Dyková 2006) and these are typically triradiate with either three or six caudal processes that arise from the spore’s valve cells. The triactinomyxon type (Figs. 13.1 and 13.2) is the most common and possesses three valve cells (and associated caudal processes) and three polar capsules. Actinospore morphological features are highly variable, particularly the caudal processes, which are inflated osmotically when spores are released into water. These processes produce the characteristic final shape of the actinospore and lend buoyancy so the spore is able to drift passively in the water column. Variation in the length, shape, diameter, and symmetry of the caudal processes has been proposed to be adaptive for particular habitat conditions (Fig. 13.2), although very different types may be present in the same habitat (Xiao and Desser 1998b). Actinospores in standing waters have caudal processes whose collective large surface areas enhance buoyancy enabling them to remain in the water column for long periods. In contrast, neoactinomyxon-type actinospores

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Fig. 13.1 Schematic structure of a triactinomyxon actinospore (Kallert)

have very small caudal processes which may be advantageous for infecting bottom dwelling fish species such as cyprinids. The caudal processes

Fig. 13.2 Wet mount of a Henneguya nuesslini actinospore (triactinomyxon) (central image). See Fig. 12.1a for features. Scale bar = 100 µm Left insert Aurantiactinomyxon type spore from Branchiura sowerbyi, Scale bar = 20 µm Right insert malacosporean spore from Fredericella sultana. Scale bar = 10 µm (Kallert, Grabner, Székely)

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may also play a role in host attachment, although this requires experimental study. For instance, the very long appendices of certain aurantiactinomyxon-type spores may become entangled with gill filaments while very small caudal processes may decrease drag during attachment to fish living in fast flowing waters. Some actinospores form clusters (e.g. synactinomyxontype spores), with spores attached by the tips of their caudal processes to form cuboidal or spherically-shaped congregations of actinospores, with the apical (polar-capsule bearing) regions orientated outwards. Presumably, this is to increase the likelihood of transmitting a number of parasites infecting an individual fish host. Intraspecific variation in actinospore size and shape has been reported from the same host (Hallett et al. 2004). Whether this variation is functionally significant remains unknown.

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Another important actinospore feature is the number of secondary sporozooite cells enclosed within the primary sporoplasm cell. The function of sporozooites is to ultimately infect host tissues. Sporozooite numbers vary from two to over 60 (e.g. eight in Myxobolus pseudodispar and 64 in M. cerebralis). Some species may form less than their maximum number of sporozooites (Lom and Dyková 1992), which may be linked to host nutritional status or species. The polar capsules are located apical to the sporoplasm-harbouring style, often at the end of valve shell protrusions. Their conical plug or stopper often protrudes through the gaps where the surrounding valve cells do not overlap (Fig. 13.3). In the more compact spore types, such as the flowershaped aurantiactinomyxon, the polar capsules cluster centrally on one side, sometimes beneath a hump-like protrusion of the surrounding valve cells. The number of polar capsules and valve cells is usually the same and is an important species character. The capsulogenic cells are tightly joined to the valve cells through septal complexes or gap/tight junctions (see Chap. 8 for further discussion of cell junctions in Myxozoa) that are maintained throughout sporogenesis. An often overlooked structural feature of many actinospores is the endospore unit, which consists of a membrane-sheath that encloses the sporoplasm primary cell with the capsulogenic cells attached apically. Its purpose is most likely to maintain the integrity of the sporoplasm during host attachment after the valve cells have been shed. Scanning electron microscopic examination shows that the endospore of M. cerebralis is a fibrous structure that remains after the sporoplasm cells have penetrated the fish surface (El-Matbouli et al. 1999b). The endospore structure has also been observed in M. parviformis (Kallert et al. 2005a) and M. pseudodispar (Kallert et al. 2007).

13.2.2 Malacosporean Transmission Stages Vertebrate-infecting malacosporean spores (see Fig. 13.2) develop inside spherical sac-like or

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Fig. 13.3 Apical view of Myxobolus cerebralis actinospore in scanning electron microscopy. Note the protruding capsule tip stoppers (cones, arrowhead) through lip-like valve shell structures. Scale bar = 2 µm (Kallert)

motile worm-shaped stages, which are located in the body cavity of freshwater bryozoan hosts (Canning and Okamura 2004). The mechanism of spore-release from bryozoans is not yet clear but is likely to be via the vestibular pore from which bryozoan dormant stages (statoblasts) are released (Canning et al. 2002). Compared to most actinospores, the equivalent malacosporean spores released from bryozoans are small (19–20 µm), spherical and lack appendages. They possess four spherical polar capsules, four capsulogenic cells, eight valve cells (McGurk et al. 2005) (although recent confocal microscopy studies suggest there are more; Gruhl and Okamura, unpub. data) and two sporoplasms, each containing a secondary cell (Canning et al. 1996, 2000, 2007; McGurk et al. 2005). Ultrastructurally, specific types of sporoplasmosomes were detected in various developmental stages of malacosporeans, separate distinct from those observed in myxosporeans (Canning et al. 2000, 2002, 2007; Morris and Adams 2006a, 2007). Laboratory studies have indicated similar activation with polar capsule discharge and emission reactions comparable to those of myxosporean actinospores (Grabner and El-Matbouli 2010b; Grabner and Kallert, unpub. data).

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13.2.3 Actinospore Release Vegetative and sporogonic stages of actinosporeans are typically located intercellularly in the intestinal epithelium or in the coelom of annelid hosts (see Chap. 11 for discussion of different tissue tropisms in annelid hosts). Many actinospores are released through the digestive system, thus spore release may depend on feeding activity and may be influenced by temperature (El-Matbouli et al. 1999a). Seasonality in actinospore release varies among myxosporean species. Xiao and Desser (1998a) observed no apparent temporal fluctuations in spore emergence, whereas Yokoyama et al. (1993) reported that several actinospores in a goldfish pond emerged from oligochaetes between spring and summer when susceptible goldfish fry were present. The release of M. cultus actinospores from the oligochaete, Branchiura sowerbyi, was observed to peak around midnight implying actinospores emerge when fish are inactive at the bottom of ponds (Yokoyama et al. 1993).

13.3

Transmission Stage Longevity and Infectivity

Unlike myxospore stages whose hardened valves confer prolonged resistance and infectivity for months to years, infectivity of actinospores lasts for a period of days (Yokoyama et al. 1993; Xiao and Desser 1998b). For instance, at temperatures around 15 °C, M. cerebralis actinospores must invade their host in less than 60 h after release (Markiw 1992). Differences in estimated lifespans of actinospores (often referred to as viability or longevity) are likely to reflect different assay techniques. Ageing experiments have confirmed that periods of actinospore durability and reactivity differ among myxozoan species (Kallert and El-Matbouli 2008) (Fig. 13.4). The lowest ‘life-span’ was found for M. cerebralis actinospores. The viability of Henneguya nuesslini and M. pseudodispar actinospores was significantly longer upon storage, particularly at lower temperatures. Storage at lower temperatures yielded higher viability in all species.

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Reports of relatively long survivability of actinospores (up to two weeks) are presumably based on visible cellular breakdown (Yokoyama et al. 1993; Xiao and Desser 2000). The vital staining technique using fluorescein diacetate and propidium iodide (FDA/PI) yielded estimates of viability between 3 days and 4 weeks (Markiw 1992; Yokoyama et al. 1997; Özer and Wootten 2002; Wagner et al. 2003). Most of these studies on actinospore viability show a clear temperature-dependence while none of them address whether polar capsules are functional. Infectivity of actinospores over different time periods determined by infection experiments with susceptible fish hosts (El-Matbouli et al. 1999b) provide better inferences of the true life-span of actinospores in natural environments. Factors influencing infectivity besides durability include both a viable sporoplasm primary cell and functional polar capsule discharge (see below) for host attachment. Reactivity of actinospores may be influenced by several factors and certain isolates may not be reactive at all. For instance, defective polar capsules have been observed which are smaller than normal and show aberrant morphology (Kallert, unpub. data). It is unknown if spores with such nonfunctional polar capsules arise from development in unsuitable oligochaete hosts or deleterious mutations. It is possible that physical factors, such as osmotic or pH gradients, may influence spore development. Markiw (1992) observed dead sporoplasm cells but intact polar capsules within the same spores. In some cases actinospores with damaged processes, missing polar capsules or deformed or dull appearing sporoplasms remain fully infective (see Sect. 13.5.1). Due to their soft valve cells, bryozoan-borne malacospores are very short lived and lose infectivity in less than 24 h (de Kinkelin et al. 2002). Short life-span, small size and fragility of malacosporean actinospores make laboratory infection experiments difficult, as spores cannot be obtained efficiently by filtration of water. So far, the only useful methods for fish infection were either cohabitation of fish with infected bryozoans colonies (e.g. Feist et al. 2001; McGurk et al. 2006; Morris and Adams 2006b,

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Fig. 13.4 Percent viable actinospores (TAMs) of two myxozoan species determined by a FDA/PI-doublestaining (fluorescence microscopy) and b morphological characters (light microscopy) on the date of harvest (day 1) and after storage at 4 and 12 °C on two subsequent days (days 2 and 3). Total range of number of specimens per day/temperature a 30–42 (Myxobolus cerebralis), 65–86

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(Myxobolus pseudodispar). All combinations per species tested in 3 replicates b 135–157 (Myxobolus cerebralis), 87–137 (Myxobolus pseudodispar), all combinations per species tested in 6 replicates. SEM = standard error of the mean (from Kallert and El-Matbouli 2008, reprinted with permission from Folia Parasitologica)

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Most of our knowledge of the vertebrate invasion process of Myxozoa is based on studies of myxosporeans. Host encounter is likely to be transient so polar capsule discharge must be suitably timed to ensure a high likelihood of successful attachment. The apical region of actinospores must be in close proximity to the host surface (i.e. within the length of the polar filament). The combined sequence of actinospore invasion events is schematically shown in Fig. 13.5. After anchoring the spore via the polar filament to the

host surface, the apical valve shell sutures open possibly due to hydrodynamic forces (e.g. drag) acting on the extended caudal processes which are then discarded. The endospore unit remains on the host surface enabling sporoplasm penetration. Unfortunately, virtually nothing is known about the invasion of vertebrate hosts by malacosporean stages owing to difficulties in obtaining naturally released mature spores and their minute size. The activation response speed and excitability of invasion reactions are known to differ among species although polar capsule discharge itself is uniformly rapid. Kallert et al. (2005b) have shown that a sequential stimulation of chemical and mechanical host cues elicits polar capsule discharge in M. cerebralis. Yokoyama et al. (2006) observed slow and fast reactivity of two myxozoan species infecting different fish hosts: Thelohanellus hovorkai sporoplasms were released over a 30 min exposure time to host mucous, whilst M. arcticus actinospores reacted instantly. Exposure of single isolated M. parviformis actinospores to both host (bream) mucous and mechanical stimulation, as conducted with

Fig. 13.5 Inferred schematic course of events during host invasion by triactinomyxon-type actinospores. a Actinospore apical region encountering fish host surface and chemosensory stimulation. b Polar filament discharge after mechanical stimulus upon contact. c Rapid adduction (arrow) of the actinospore apex towards the host surface by means of filament constriction. d Opening of the apical valves along sutures and development of parachute-like form (arrow). e Endospore remains

attached while the valve shell is discarded. f Active emergence of the sporoplasm. g Fully emerged sporoplasm leaving the endospore sheath on host surface. h Penetration of the sporoplasm through the host integument and movement towards deeper layers. sc fish scales, ep fish epidermis, mu mucous layer, pc polar capsule, sp sporoplasm, sz sporozooites, va spore valves (modified from Kallert et al. 2007, reprinted with permission from Cambridge University Press)

2008; Grabner and El-Matbouli 2008, 2010a), exposure to homogenates of infected bryozoans colonies (Longshaw et al. 2002) or dissection of mature spore sacs to obtain defined numbers of spores (McGurk et al. 2005; Grabner and El-Matbouli 2010b). Fish have also been infected successfully by intraperitoneal injection of disrupted parasite material (Feist et al. 2001).

13.4

Invasion Process

13.4.1 Invasion Course and Speed

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M. cerebralis (Kallert et al. 2005b), elicited no reactions at all. The notably concealed polar capsule apertures of certain actinospores (e.g. those of M. parviformis) contrast with the more exposed polar capsules of other species (e.g. M. pseudodispar). The excitability threshold may relate to the relative degree of exposure of polar capsules. For instance, the polar capsules of the aurantiactinomyxon-type spore of T. hovorkai are counter-sunk below the apical valve cell margins. This morphological arrangement may result in the slow discharge onset of T. hovorkai actinospores in contrast to the fast discharge of actinospores in species such as M. cerebralis and H. nuesslini whose polar capsules have strikingly protruding tips. Since high flow rates may limit the time for actinospores to attach to their fish hosts (Hallett and Bartholomew 2008) the transmission of slow reacting species may be adversely affected by high water flow. The relative exposure timing for polar capsule discharge could be an adaptation to the speed of host recognition, based on different stimuli required in different habitats and hosts.

13.4.2 Attachment by Polar Capsule Discharge The feature that most strikingly connects myxozoans to their putative cnidarian ancestors is the intracellular nematocyst-like polar capsule produced within a capsulogenic cell. Its function is to anchor the actinospore to the host integument to enable host invasion. Polar filament discharge is irreversible and the associated polar capsule cell quickly disintegrates. Undischarged polar capsules are filled with an electron-dense mass surrounding the internal coiled filament. This electron dense mass is absent after discharge, which can thus be inferred by electron lucent polar capsules. The conical stopper on the capsule tip often bears ridges on its surface and points through the apical valve openings. Video analysis of polar filament discharge in M. cerebralis actinospores has shown that complete extrusion requires less than 10 ms (Kallert et al.

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2007). The polar filaments of M. cerebralis and H. nuesslini are sticky and rapidly elongate and then retract to about half their length in milliseconds after discharge (Kallert et al. 2007). The spore is thus moved by about 35 µm towards the host surface due to the adhesion of the filament. This intrinsic mechanism effects very close contact with the epithelial surface of the fish host. As a mechanical stimulus, a thigmotropic (touch sensitive) mechanism is assumed. The mechanism of perception is unknown and so is the cellular mechanism that triggers mechanical sensitivity. Presumably, the protein cone covering the capsule tip has a crucial role in triggering the reaction. Uspenskaya (1982) assumed that Ca2+ ions were involved in the polar filament discharge mechanism and suggested that it is an active process due to the action of contractile proteins. Discharge rates of actinospores in Ca2+-containing water were doubled compared to those in Ca2+-deficient water of the same osmolality (Kallert et al. 2007). Extreme pH values favour spontaneous discharge (Smith 2001).

13.4.3 Sporoplasm Emergence and Host Penetration Once the spore has attached to the host, the function of the sporoplasm is to disseminate the infective germ cells (sporozooites) into the host tissue (Fig. 13.6). This emission and penetration process is achieved by active movement via pseudopodia and amoeboid plasticity and is possibly facilitated by proteolytic activity (Adkison and Hedrick 2001). Sporoplasms of M. cerebralis can be found in superficial trout tissues only minutes after exposure (El-Matbouli et al. 1999b). Exposure of Henneguya ictaluri actinospores to the gills of channel catfish was observed to elicit sporoplasm protrusion and contraction, and pseudopodial amoeboid movement persisted for several hours until the sporoplasms were rounded up and detached from the valve cells of spores (Pote and Waterstrat 1993). Experimental observations have indicated that the apical spore opening does not occur passively

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by an intrinsic mechanism prior to sporoplasm emission, since activated and discharged actinospores, after fish mucous stimulation, do not automatically open (Kallert and El-Matbouli 2008). Enhanced movement of the sporoplasms and active rearrangements of the polar capsules have been frequently observed by the authors during incubation of actinospores with fish mucous homogenate in the absence of mechanical stimuli. This suggests that the sporoplasm primary cells can recognize host cues even inside the undischarged actinospore. Thus, although polar filament discharge facilitates sporoplasm emission, it is not vital for sporoplasm activation. Sporoplasms generally appear to be osmotically sensitive and can only be kept viable in suitable Ringer solutions. The primary cell of the sporoplasm usually degrades after host penetration to release the secondary cells that either become blood stages (e.g. some Sphaerospora spp.) or invade other tissues that are appropriate for subsequent development.

13.4.4 Portals of Entry and Initial Invasion Steps Portals of entry of actinospore sporoplasms have been demonstrated by fluorescent labelling Fig. 13.6 Apically emerging sporoplasm containing secondary sporozooites from Myxobolus parviformis actinospore. Scale bar = 20 µm (Kallert)

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(Yokoyama and Urawa 1997) and include gills, skin and the buccal cavity for M. cerebralis (Antonio et al. 1999) and gills for T. hovorkai and Sphaerospora truttae (Yokoyama and Urawa 1997; Holzer et al. 2003). In contrast, H. ictaluri infects via the intestine (Belem and Pote 2001). Consumption of infected oligochaetes may also serve as a route of fish infection by myxosporeans, although not by all species as exemplified by the transmission of M. pseudodispar (Székely et al. 2001). To date, the route and mechanism of malacosporean entry into fish hosts has only been studied for Tetracapsuloides bryosalmonae, although there is evidence that other malacosporeans also infect and develop spores in the kidney tubules of fish (Grabner and El-Matbouli 2010b; Bartošová-Sojková et al. 2014). There is no clear concensus on the route of malacosporean entry. Longshaw et al. (2002) detected stages of T. bryosalmonae by in situ hybridization (ISH) in skin epidermal mucous cells of rainbow trout one minute post exposure (p.e.) of fish to homogenized infected bryozoan colonies. T. bryosalmonae stages in gills were rarely encountered in their study. In contrast, attachment of T. bryosalmonae spores to the gill epithelium of rainbow trout was observed five minutes p.e. via ISH studies (Grabner and El-Matbouli 2010b). Initial penetrated stages

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were subsequently seen in the gills 30 min p.e., and no stages were detected attached to the outer surface or inner layers of the skin epithelium. Earlier ISH studies of both rainbow (Morris et al. 2000) and brown trout (Holzer et al. 2006) similarly concluded that the gills are the major route of entry of T. bryosalmonae into fish hosts. Initial events during host invasion have been studied most intensively in the myxosporean M. cerebralis. After sporoplasm penetration, intracellular multiplication within the first 60 min p.e. has been observed prior to intercellular migration in the epidermis and gill epithelium. This is followed by the disintegration of the syncytial primary cell encapsulating the secondary sporozooites, facilitating invasion of the host epidermal or gill epithelial cells (El-Matbouli et al. 1999b). Secondary cell proliferation is promoted by rapid synchronous mitosis (Daniels et al. 1976; El-Matbouli et al. 1995) resulting in production of cell-doublets via endogenous division of secondary cells into an enveloping cell and an inner cell. After entry into the host cell cytoplasm, celldoublets that survive traverse the host cell plasmalemma to enter the extracellular space. The presporogonic stages migrate deeper into subcutaneous layers concomitantly with continued proliferation of cell-doublets. In other myxozoans, such as T. bryosalmonae and Sphaerospora spp., presporogonic proliferation is observed in tissues other than those in which sporulation occurs and is known as extrasporogonic development. Amoeboid cells derived from malacosporean spores enter the fish host’s vascular system, where they accumulate and, after migration, undergo further development in target organs (Morris et al. 2000; Longshaw et al. 2002; Grabner and El-Matbouli 2010b). Unicellular extrasporogonic stages multiply in blood and proliferate in kidney interstitial tissue, where presporogonic stages can be found as cell doublets, consisting of a typical primary cell and a secondary cell. So-called S-T doublets migrate to the kidney tubule lumen, where the parasites transform into a pseudoplasmodium each developing into a single spore subsequently (Kent and Hedrick 1985, 1986; Morris and Adams 2008).

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Extrasporogonic developmental stages in blood are commonly found among the members of the genus Sphaerospora. Blood stages found in common carp (Cyprinus carpio) were designated by Molnár (1980) as “Csaba-parasites” (Cprotozoa) and by Lom et al. (1983) as “Unidentified Blood Organism” (UBO). These parasites were identified as the developmental stages of S. dykovae (syn S. renicola) (Csaba et al. 1984; Grupcheva et al. 1985) which was experimentally confirmed by Molnár (1984). Kovács-Gayer et al. (1982) and Körting (1982) described the occurrence of myxosporean developmental stages in the swimbladder of Sphaerosporainfected common carp. Such parasites, which seemed to represent intermediates of blood and renal stages were named “K protozoa or K stage” (in reference to the initials of Prof. Körting and Dr. Kovács-Gayer). Baska and Molnár (1988) characterized blood stages from six other cyprinid species, suggesting that other myxosporeans (most likely closely related Sphaerospora species) possess extrasporogonic blood stages. Extrasporogonic proliferation of S. dykovae begins with a small primary cell that contains a secondary cell. Remarkably, primary cells constantly rotate in the blood stream (Lom and Dyková 1992). The function of this movement has not been clarified, although it has been suggested this may be linked to host immune evasion (Hartigan et al. 2013).

13.4.5 Specific Versus Non-specific Host Recognition Whether actinospores are able to react specifically to susceptible host species has been a matter of considerable debate. T. hovorkai has been reported to react specifically to certain cyprinids (Yokoyama et al. 1997). In a later study T. hovorkai actinospores discharged only in response to the mucous of the susceptible host, whereas M. arcticus actinospores reacted to both non-susceptible and susceptible hosts (Yokoyama et al. 2006). El-Matbouli et al. (1999b) concluded, based on the number of spores that

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attached to host surfaces, that M. cerebralis actinospores distinguish carp from rainbow trout. The speed of sporoplasm release has been observed to vary in the presence of mucous from different fish species (Xiao and Desser 2000). Thus, besides differences in reactivity, specific reactions could never been shown conclusively. Other studies provide further evidence for a lack of host specificity during initial invasion steps. Actinospores of several myxosporeans have been shown to react to mucous of a variety of fish species. Field-collected actinospore isolates of different morphotypes reacted to mucous of salmon, trout, stickleback and bream by polar filament discharge and sporoplasm emission in studies by McGeorge et al. (1997) and Özer and Wootten (2002). Similarly, raabeia-type actinospores of M. cultus reacted to mucous from common carp, loach, rainbow trout, catfish, and Japanese eel, as well as to mucin from bovine submaxillary gland (Yokoyama et al. 1995a). Actinospores of H. nuesslini, M. parviformis, M. pseudodispar and M. cerebralis readily respond to mucous of roach, rainbow trout, bream and common carp (Kallert et al. 2005b, 2007, 2011; Kallert, unpub. data). More detailed studies have found no significant differences in the numbers of actively emerging M. cerebralis sporoplasms on epithelial surfaces of susceptible and resistant strains of rainbow trout and of common carp upon experimental exposure (Kallert et al. 2009). Stages of M. arcticus have been observed penetrating non-susceptible salmonids, although no further reproduction and development was reported (Yokoyama et al. 2006). The reduction in myxospore load in rainbow trout infected with M. cerebralis actinospores that were preincubated with common carp specimens, suggests that decoy fish species may lower spore abundance. Furthermore, a similar number of actinospores were observed to attach and penetrate gill tissue of the less susceptible Hofer strain rainbow trout (Kallert et al. 2009). The discovery of non-specific invasion behaviour of several actinospores from limnic European Myxosporea further indicates that host specificity in Myxosporea is presumably not a result of parasite choice (Kallert et al. 2011). In

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myxosporeans, the positive effect of specific recognition on transmission is presumably outbalanced by the benefits of a rapid response upon contact with any fish to increase the likelihood of a successful host invasion. The lack of specificity in discharge may be compensated for by the release of considerable numbers of transmission stages. Such non-specific host recognition may provide a means of acquiring of new host species, thus promoting diversification if speciation follows infection of novel hosts. Non-specificity of host recognition may therefore have contributed to the great radiation exhibited by the Myxosporea (see Chap. 3 for further discussion).

13.4.6 Host Cues For successful transmission to fish, accidental polar capsule discharge upon contact with other surfaces must be avoided. In actinospores, this is achieved by perception of chemical discharge triggers prior to activation of mechanical sensitivity. As is evident from the above discussion, numerous studies have shown reactivity of various actinospore samples to fish mucous substrates (Uspenskaya 1995; Yokoyama et al. 1995a, b, 2006; McGeorge et al. 1997; Xiao and Desser 2000; Özer and Wootten 2002; Kallert et al. 2005b, 2007, 2009, Kallert and El-Matbouli 2008). However, the most abundant teleost mucous components, carbohydrates and amino compounds, are unlikely to be involved owing to their ubiquitous distribution in aquatic environments. Yokoyama et al. (1995a) found that a low molecular (