Inosine-arginine salt as a promising agent for in vitro activation of waterborne fish-pathogenic myxozoan actinospores.

Mucus-derived nucleosides serve as key host cues for myxozoan actinospore fish host recognition, but to date their use for experimental actinospore activation in the laboratory or application in disease prevention has not progressed very far. One obstacle has been the low solubility of pure inosine and guanosine. To overcome this, we used inosine-arginine salt (ino-arg), which incorporates both high activation properties and high solubility. We tested its efficacy both in microassays directly observing reactions of actinospores of 2 distantly related myxozoan species, Myxobolus cerebralis and M. pseudodispar in comparison to inosine, as well as its actinospore-inactivation properties by premature polar capsule discharge in an infection experiment. Ino-arg was considerably more effective in eliciting polar capsule discharge and sporoplasm emission at much lower concentrations than pure inosine and, in contrast to the latter, remained dissolved in aqueous solution. Ino-arg exposure of M. pseudodispar actinospores resulted in polar capsule discharge and sporoplasm emission before host contact and subsequently in a lower infection rate in roach Rutilus rutilus.

Analysis of rainbow trout Oncorhynchus mykiss epidermal mucus and evaluation of semiochemical activity for polar filament discharge in Myxobolus cerebralis actinospores.

As myxozoan actinospores are stimulated by fish epidermal mucus to attach to their hosts via extrusion of filaments from specialized organelles, the polar capsules, mucus components were tested for discharge triggering activity on Myxobolus cerebralis actinospores. Using various methodological approaches, a selective exclusion of candidate substances based on experimental outcome is provided and the physiochemical traits of the putative agents are explored to create a basis for the isolation of the host recognition chemostimuli. Activity was detected in compounds that can be characterized as small molecular, amphiphilic to slightly hydrophobic organic substances. They were separable by chromatographic methods using reversed phase C18 supports. An active fraction was isolated by solid phase extraction comprising at least nine UV-detectable constituents as shown by thin-layer chromatography. By means of biochemical fractionation and analysis of host fish mucus, non-volatile inorganic electrolytes, all volatiles, free L-amino acids, glycoproteins, bound and free hexoses, sialic acids, glycans, proteins, urea, amines and inositols were shown not to trigger polar filament discharge. The results contribute to the identification of the attachment host cues and enable a more focused laboratory activation of myxozoan actinospores.

Differentially expressed parasite genes involved in host recognition and invasion of the triactinomyxon stage of Myxobolus cerebralis (Myxozoa).

The host recognition and invasion process of Myxobolus cerebralis actinospores (triactinomyxon, TAM) was studied on a genetic level. A small-scale in vitro assay was developed to activate a large number of TAMs simultaneously, and to monitor the host invasion in the absence of live fish. The transcriptomes of non-activated and in vitro-activated TAMs were compared by suppressive subtractive hybridization (SSH) to identify parasite genes involved in the host invasion process. Differential screening and a subsequent BLAST search revealed 15 of 452 SSH-library clones expressed differently in activated TAMs. None of the 15 transcripts obtained has previously been identified from M. cerebralis. Quantitative real-time PCR was used to examine the relative expression profile of 8 selected transcripts upon TAM activation and after penetration of the host. Four of these were found to be up-regulated in activated TAMs, while expression was relatively low in non-activated TAMs and in infected fish tissue, indicating that they are relevant genes during host recognition or subsequent host invasion of M. cerebralis TAMs.

Myxozoan transmission via actinospores: new insights into mechanisms and adaptations for host invasion.

Various mechanisms that enable and improve transmission success of myxozoan actinospore stages towards fish hosts are described, based upon a combination of experimental data and functional analysis of morphological characters. For this purpose, laboratory-reared actinospores of Myxobolus cerebralis, Myxobolus parviformis, Henneguya nuesslini and Myxobolus pseudodispar were employed to exemplarily investigate aspects of host attachment and invasion. The process of polar filament discharge of M. cerebralis actinospores was analysed, showing that full discharge occurs in less than 10 msec. Additionally, a mechanism that rapidly contracts the discharged filament after discharge is described for the first time. Its purpose is most likely to bring the actinospore apex rapidly into intimate contact with the surface of the host. Unlike M. cerebralis, M. parviformis actinospores did not discharge polar filaments after mechanical and chemical stimulation, suggesting a different mode of triggering. For H. nuesslini actinospores, experimental results indicated that polar filament discharge is independent of external calcium-ion concentration but is influenced by osmolality. After attachment of an actinospore and prior to penetration into the host, an ensheathed unit ('endospore'), containing the sporoplasm, was emitted from the valves in a manner which varied from species to species. Experimentally induced sporoplasm emission was time-dependent and was found to be independent of polar filament discharge in H. nuesslini. Remarkably, it could be concluded that the sporoplasm is able to recognize host-stimuli while still within the intact spore. An updated summary of the sequential course of events during host recognition and invasion by actinospores is given.

Polar filament discharge of Myxobolus cerebralis actinospores is triggered by combined non-specific mechanical and chemical cues.

This study presents initial evidence for the requirement of both chemical and mechanical stimuli to discharge polar capsules of Myxobolus cerebralis actinospores, the causative agent of salmonid whirling disease. The obligate need for combined discharge triggers was concluded from data obtained in a before/after experimental set-up carried out with individual locally immobilized actinospores. Homogenized rainbow trout mucus as chemostimulus and tangency of the apical region of the spores to achieve mechanical stimulation were applied subsequently. The actinospores showed discharged polar filaments exclusively when mucus substrate application was followed by touching the polar capsule-bearing region, but not when either stimulus was offered solely to the same individuals. We measured filament discharge rates to mucus preparations in a microscopic assay using supplementary vibration stimuli to ensure mechanical excitation. The actinospores responded similarly to different frequencies, which suggested a touch-sensitive recognition mechanism. Discharge specificity for salmonid mucus could not be confirmed, as mucus of common carp and bream could trigger similar filament expulsion rates. To a lesser extent homogenized frog epidermis and bovine submaxillary mucin could also stimulate the attachment reaction. In contrast, mucus of a pulmonate freshwater snail elicited no response.

The life cycle of Henneguya nuesslini Schuberg & Schroder, 1905 (Myxozoa) involves a triactinomyxon-type actinospore.

The life cycle of the histozoic myxozoan parasite Henneguya nuesslini was investigated in two salmonid host species. Naive brown trout, Salmo trutta, and brook trout, Salvelinus fontinalis, were experimentally infected in two trials by triactinomyxon type actinospores from naturally infected Tubifex tubifex. In exposed common carp, Cyprinus carpio, no myxospore production was detected. The parasite formed cysts with mature myxospores in the connective tissue of the fish 102 days post-exposure. The morphology of both actinosporean and myxosporean stages was described by light microscopy and a 1417-bp fragment of the 18S rDNA gene was sequenced. Sequence analysis confirmed the absolute congruence of the two developmental stages and assisted in determining species identity. Host range, tissue specificity and myxospore measurements provided sufficiently distinctive features to confirm species validity and were thus crucial for identification. The triactinomyxon spores had 16 secondary germ cells, unique dimensions, a very opaque sporoplasm matrix and three conspicuously protruding, pyriform polar capsules. This is the first record of a Henneguya sp. life cycle with a triactinomyxon-type actinospore, which suggests a close relationship with the Myxobolus group and a polyphyletic origin of the genus Henneguya.