The effects of Myxobolus cerebralis myxospore dose on triactinomyxon production and biology of Tubifex tubifex from two geographic regions

The aquatic oligochaete Tubifex tubifex is an obligate host of Myxobolus cerebralis, the causative agent of salmonid whirling disease. Tubifex tubifex can become infected by ingesting myxospores of M. cerebralis that have been released into sediments upon death and decomposition of infected salmonids. Infected worms release triactinomyxons into the water column that then infect salmonids. How the dose of myxospores ingested by T. tubifex influences parasite proliferation and the worm host are not well understood. Using replicated laboratory experiments, we examined how differing doses of myxospores (50, 500, 1,000 per worm) influenced triactinomyxon production and biomass, abundance, and individual weight of 2 geographically distinct populations of T. tubifex. Worm populations produced differing numbers of triactinomyxons, but, within a population, the production did not differ among myxospore doses. At the lowest myxospore dose, 1 worm population produced 45 times more triactinomyxons than myxospores received, whereas the other produced only 6 times more triactinomyxons than myxospores. Moreover, total T. tubifex biomass, abundance, and individual weight were lower among worms receiving myxospores than in myxospore-free controls. Thus, T. tubifex populations differ in ability to support the replication of M. cerebralis, and infection has measurable consequences on fitness of the worm host. These results suggest that variability in whirling disease severity observed in wild salmonid populations may partially be attributed to differences in T. tubifex populations.     

Prevalence of Myxobolus cerebralis infections among genetic lineages of Tubifex tubifex at three locations in the Madison River, Montana

Host biodiversity can impact disease risk and influence the transmission of parasitic disease. Stream sediment-dwelling worms, Tubifex tubifex (Clitellata: Oligochaeta), are the definitive host of the parasite Myxobolus cerebralis (Myxozoa: Myxosporea), which causes whirling disease in salmonid fishes. Genetic diversity of T. tubifex is correlated with host susceptibility to M. cerebralis , and mitochondrial Lineage III is generally shown to be more likely to be infected and produce the triactinomyxon (TAM) spores than other lineages. We determined the mitochondrial lineage, relative abundance, and prevalence of infection of T. tubifex collected at 3 sites in the Madison River, Montana, where previous study had shown variation in whirling disease prevalence and severity in caged trout fry. We also compared visual identification of TAMs released from cultured worms with a molecular genetic assay (diagnostic polymerase chain reaction [PCR]) for parasite detection of both infected and uninfected worms. We estimated that mitochondrial Lineage III was most abundant at the site previously shown to have high fish disease and was also most likely to be infected. The 2 techniques for detecting parasite infection did not always agree, and the likelihood of PCR (+) and spore (-) was not significantly different from PCR (-) and spore (+). Differences in the relative infection prevalence for these 2 lineages may explain the wide range of infection in natural streams.     

Persistent infection of Myxobolus cerebralis, the causative agent of salmonid whirling disease, in Tubifex tubifex

The objective of this study was to quantify and determine the periodicity in the release of the triactinomyxon (TAM) stage of Myxobolus cerebralis, the causative agent of salmonid whirling disease, by its aquatic oligochaete host Tubifex tubifex. For this, 24 individual T. tubifex (infected as a group at 15 C) were examined daily for the release of M. cerebralis TAMs, and the number of waterborne TAMs released by each worm was quantified. The duration of the infection in these worms was also monitored using a polymerase chain reaction (PCR) diagnostic test. TAMs were first released 74 days postexposure (PE) and continued to be released until 132 days PE. During this period, each worm released on average, 1.5 x 10(3) waterborne TAMs 12 times; however, no pattern or periodicity was noted. The results of the PCR diagnostic tests conducted at 5, 7, 9, and 15 mo PE were positive, and the persistent infection was confirmed at 606 days PE (approximately 20 mo) when the remaining worms began releasing TAMs again. Similar results were observed in naturally infected T. tubifex, indicating that these worms remain infected for the duration of their natural lifespan and are capable of shedding viable TAMs, in temporally separate periods. These findings open the possibility of a seasonal periodicity in TAM release by T. tubifex.     

Differential propagation of the metazoan parasite Myxobolus cerebralis by Limnodrilus hoffmeisteri, Ilyodrilus templetoni, and genetically distinct strains of Tubifex tubifex

Whirling disease, caused by the parasite Myxobolus cerebralis, has infected rainbow trout (Oncorhynchus mykiss) and other salmonid fish in the western United States, often with devastating results to native populations but without a discernible spatial pattern. The parasite develops in a complex 2-host system in which the aquatic oligochaete Tubifex tubifex is an obligate host. Because substantial differences in whirling disease severity in different areas of North America did not seem explainable by environmental factors or features of the parasite or its fish host, we sought to determine whether ecological or genetic variation within oligochaete host populations may be responsible. We found large differences in compatibility between the parasite and various laboratory strains of T. tubifex that were established from geographic regions with different whirling disease histories. Moreover, 2 closely related species of tubificids, Limnodrilus hoffmeisteri and Ilyodrilus templetoni, which occur naturally in mixed species assemblages with T. tubifex, were incompatible with M. cerebralis. Virulence of the parasite was directly correlated with the numbers of triactinomyxon spores that developed within each strain of T. tubifex. Thus, the level of virulence was directly related to the compatibility between the host strain and the parasite. Genetic analyses revealed relationships that were in agreement with the level of parasite production. Differences in compatibilities between oligochaetes and M. cerebralis may contribute to the spatial variance in the severity of the disease among salmonid populations.     

Ultraviolet irradiation inactivates the waterborne infective stages of Myxobolus cerebralis: a treatment for hatchery water supplies

The effects of ultraviolet (UV) irradiation on the viability of the waterborne triactinomyxon stages of Myxobolus cerebralis were evaluated by vital staining and the infectivity for juvenile rainbow trout Oncorhynchus mykiss. A dose of 1300 mWs cm-2 was required to inactivate 100% of the triactinomyxons held under a static collimated beam of UV as determined by vital staining. Juvenile rainbow trout were protected from infections with M. cerebralis when exposed to 14,000 or 1400 triactinomyxon spores per fish that had been treated with the collimating beam apparatus (1300 mWs cm-2). Among all fish receiving UV-treated triactinomyxons, none had clinical signs of whirling disease, or evidence of microscopic lesions or spores of M. cerebralis after 5 mo at water temperatures of 15 degrees C. In contrast, 100% of the fish receiving the higher dose of untreated triactinomyxons developed clinical signs of whirling disease and both microscopic signs of infection and spores were detected in all of the high and low dose trout receiving untreated triactinomyxon exposures. Two additional trials evaluated the Cryptosporidium Inactivation Device (CID) for its ability to treat flow-through 15 degrees C well water to which triactinomyxons were added over a 2 wk period. CID treatments of a cumulative dose exceeding 64,000 triactinomyxons per fish protected juvenile rainbow from infections with M. cerebralis. Rainbow trout controls receiving the same number of untreated triactinomyxons developed both microscopic lesions and cranial spore concentrations up to 10(4.6) per 1/2 head, although no signs of clinical whirling disease were observed. UV (126 mWs cm-2, collimated beam apparatus) was also effective in killing Flavobacterium psychrophilum, the agent causing salmonid bacterial coldwater disease, as demonstrated by the inability of bacterial cells to grow on artificial media following UV treatment.     

Effects of water temperature and substrate type on spore production and release in eastern Tubifex tubifex worms infected with Myxobolus cerebralis

Eastern Tubifex tubifex worms were exposed to Myxobolus cerebralis spores at 9, 13, 17, and 20 C in 1-L jars that contained sand, mud, or leaf litter as substrata. Beginning 60 days after exposure, water from each jar was filtered daily and examined for the presence of waterborne triactinomyxon spores (TAMs). On discovering a single TAM from an experimental jar, 48 T. tubifex worms from that jar were placed individually into 24-well plates. Spores released from individual infected T. tubifex worms were quantified to determine the first day of TAM release from infected worms, the infection rate, the total number of TAMs released per worm, and the duration of release. No TAMs were found in any of the jars incubated at 20 C or in uninfected, control worms at any temperature. The total number of TAMs released by infected worms in mud and sand was highest at 13 C compared with other temperatures. Infection rates among individual worms increased with temperature between 9 and 17 C. Higher temperatures (up to 17 C) induced earlier TAM releases among infected worms, and substratum did not influence this production parameter. The average duration of TAM release decreased as the temperature increased from 9 to 17 C, and there was a significant effect of substratum in the groups maintained at 13 and 17 C. In all temperature treatments between 9 and 17 C, the duration of release was least in the worms maintained in leaf litter, as was the total number of TAMs released during the experimental period and the median number of TAMs per production day.     

Prevalence and susceptibility of infection to Myxobolus cerebralis,and genetic differences among populations of Tubifex tubifex

The prevalence of infection and susceptibility of the aquatic oligochaete Tubifex tubifex to Myxobolus cerebralis, was examined in 2 studies on the upper Colorado River, Colorado, USA, where whirling disease occurs in wild trout populations. In the first study, the prevalence of infection ranged from 0.4 to 1.5%, as determined by counting the number of T. tubifex releasing triactinomyxons of M. cerebralis directly following their collection from the field. The susceptibility of those T. tubifex not releasing triactinomyxons was assessed by the number of these oligochaetes releasing triactinomyxons 3 mo following experimental exposures to spores of M. cerebralis. The prevalence of infection following experimental exposures of these T. tubifex ranged from 4.2 to 14.1%. In a second study, all T. tubifex collected at 2 different times directly from the 2 field sites in Colorado were exposed to spores of M. cerebralis. Individual oligochaetes representing those groups of T. tubifex releasing and those groups not releasing triactinomyxons at 3 mo were screened with molecular genetic markers. T. tubifex populations found at the 2 study sites consisted of 4 genetically distinct lineages that varied with respect to their susceptibility to experimental exposure to M. cerebralis. Lineages I and III contained the most oligochaetes susceptible to M. cerebralis and were the most prominent lineages at Windy Gap Reservoir, a site of high infectivity for wild rainbow trout on the upper Colorado River. In contrast, at the Breeze Bridge site which is below Windy Gap Reservoir and where M. cerebralis infections are less severe in wild trout, oligochaetes in lineages V and VI that are resistant to M. cerebralis were more prominent. These results suggest that certain habitats, such as Windy Gap Reservoir, are conducive to large and more homogenous populations of susceptible T. tubifex lineages that may serve as point sources of infection for M. cerebralis. Although not a direct objective of this study, there was no evidence of M. cerebralis infections among any oligochaetes other than those that would be classified as T. tubifex by standard morphological characteristics.     

Epizootiology of Myxobolus cerebralis, the causative agent of salmonid whirling disease in the Rock Creek drainage of west-central Montana

Whirling disease, caused by the myxozoan parasite Myxobolus cerebralis, remains a health threat to salmonid fish in the western United States. Although various aspects of this host-parasite system have been studied, investigations examining the overall epizootiology of whirling disease in an ecosystem are lacking. Therefore, in June 1998, studies were initiated in the Rock Creek watershed of west-central Montana and continued through 2003 to assess the intensity of infection in trout using sentinel cages stationed throughout the drainage. Additional studies determined the percentage of the annelid worm, Tubifex tubifex, releasing M. cerebralis at various localities in Rock Creek and whether there was a seasonal or daily periodicity in the release of the triactinomyxon stage of the parasite from T. tubifex. Lastly, habitat and water quality parameters, and the effects of habitat restoration on transmission of M. cerebralis, were assessed. Overall, the intensity of M. cerebralis infections in sentinel trout increased significantly throughout the drainage between June of 1998 and 2003, with the biggest jump occurring between 1998 and 1999. In addition, the range of M. cerebralis expanded considerably over the period of study. There was no strict correlation between habitat condition and the occurrence of the parasite; fish became heavily infected in optimal and marginal habitats. However, fish exposed at a locality that had the lowest habitat ranking consistently had the highest intensity of infection. The parasite has apparently caused a dramatic decline in rainbow trout densities, but the brown trout population numbers have increased, and the overall fish density remains high. Although a major habitat restoration project did not seem to have an effect on decreasing disease intensity, this was not surprising because the restored area was located just downstream from a "hotspot" of infected T. tubifex.     

Development of Myxobolus portucalensis Saraiva & Molnár, 1990 (Myxosporea: Myxobolidae) in the oligochaete Tubifex tubifex (Müller)

The extrapiscine development of Myxobolus portucalensis, a myxosporean parasite of the subcutaneous connective tissue of eel Anguilla anguilla L. was studied in the experimentally infected oligochaete Tubifex tubifex. After infecting parasite-free tubificids with mature spores of M. portucalensis, development of actinosporean stages was first observed with a light microscope 26 days after infection. Triactinospores of M. portucalensis emerged from the worms after 160 days of intra-oligochaete development. It was observed in histological sections that early pansporocysts were located in the gut epithelium of the experimental oligochaetes. Mature pansporocysts, each containing eight triactinospores, appeared 149 days after infection. After the rupture of pansporocysts, free actinospores were found in the lumen of the oligochaete gut. Released actinospores were floating in the water and showed a typical triactinomyxon form. Each triactinospore had three pyriform polar capsules, a sporoplasm with 32 secondary cells, a moderately long style and three slightly curved, sharply pointed caudal processes. The prevalence of infection in T. tubifex proved to be 52.5% (n = 40). No infection was found in Limnodrilus hoffmeisteri or in control Tubifex specimens     

Variability in triactinomyxon production from Tubifex tubifex populations from the same mitochondrial DNA lineage infected with Myxobolus cerebralis, the causative agent of whirling disease in salmonids

Myxobolus cerebralis, the causative agent of whirling disease, infects both salmonid fish and an aquatic oligochaete, Tubifex tubifex. Although M. cerebralis has been detected in river drainages throughout the United States, disease severity among wild fish populations has been highly variable. Tubifex tubifex populations have been genetically characterized using sequences from the 16S mitochondrial DNA (mtDNA) gene, the 18S ribosomal RNA gene, the internal transcribed spacer region 1 (ITS1), and randomly amplified polymorphic DNA (RAPD). Our earlier work indicated that large differences in compatibility between the parasite and populations of T. tubifex may play a substantial role in the distribution of whirling disease and resulting mortality in different watersheds. In the present study, we examined 4 laboratory populations of T. tubifex belonging to 16S mtDNA lineage III and 1 population belonging to 16S mtDNA lineage I for triactinomyxon (TAM) production after infection with M. cerebralis myxospores. All 4 16S mtDNA lineage III populations produced TAMs, but statistically significant differences in TAM production were observed. Most individuals in the 16S mtDNA lineage III-infected populations produced TAMs. The 16S mtDNA lineage I population produced few TAMs. Further genetic characterization of the 16S mtDNA lineage III populations with RAPD markers indicated that populations producing similar levels of TAMs had more genetic similarity.     

Interactions among two strains of Tubifex tubifex (Oligochaeta: Tubificidae) and Myxobolus cerebralis (Myxozoa)

Host-parasite interactions influence host population growth, host evolution and parasite success. We examined the interactions among Myxobolus cerebralis, the parasite that causes salmonid whirling disease, and resistant and susceptible strains of the oligochaete host Tubifex tubifex. Strains of T. tubifex with diverse genotypes often coexist in nature and have variable susceptibilities to M. cerebralis infection. Further, parasite proliferation differs by several orders of magnitude among T. tubifex strains. We examined total biomass produced by individual T. tubifex, including progeny production and adult growth, parasite proliferation and prevalence of infection using 2 strains of T. tubifex at 2 myxospore doses in a response-surface experimental design. Total biomass production per individual oligochaete and progeny biomass produced by an individual adult oligochaete were density-dependent for both resistant and susceptible individuals and the effects did not change with the addition of myxospores. However, both resistant and susceptible adults had highest growth when exposed to M. cerebralis. The presence of resistant oligochaetes in mixed cultures did not reduce the infection prevalence or parasite proliferation in susceptible individuals. In natural aquatic communities, resistant strains of T. tubifex may not reduce the effects of M. cerebralis on the salmonid host, particularly if sufficient numbers of susceptible T. tubifex are present.     

Detection of myxozoan parasites in oligochaetes imported as food for ornamental fish

To determine the potential for dissemination of myxozoan parasites by transfer of their alternate oligochaete hosts, shipments of tubificid worms obtained from an overseas commercial aquarium supplier were screened for actinospore stages of myxozoan parasites. At least 7 different triactinomyxon types were identified. The morphological characteristics of the actinospores recovered from these tubificids shared characteristics with triactinomyxons characterized in other surveys, particularly from eastern Europe. Analysis of the screened samples by polymerase chain reaction and comparison of morphological data indicated that these actinospores did not correspond to the triactinospore of Myxobolus cerebralis. Although identification of these triactinomyxon types was beyond the scope of this study, these data suggest that the unregulated import-export and exchange of live organisms for ornamental fish food may result in accidental introduction or dissemination of myxozoan parasites.     

The effect of cohabitation of Tubifex tubifex (Oligochaeta: Tubificidae) populations on infections to Myxobolus cerebralis (Myxozoa: Myxobolidae)

The competitive interactions between susceptible and resistant Tubifex tubifex (Oligochaeta: Tubificidae) exposed to Myxobolus cerebralis (Myxozoa: Myxobolidae) infections were investigated in two laboratory trials. Competition was assessed by the total parasite production over the course of the trials in mixed and pure cultures of M. cerebralis exposed worms, and by the genetic analyses of worms from the control and experimental groups at the beginning and end of the experiments. Mixed cultures of resistant and susceptible worms showed a 70% reduction in production of parasites released when compared with pure cultures of susceptible worms. In studies with laboratory and field-collected oligochaetes the mixed cultures at the end of the cohabitation experiments were dominated by resistant Tubifex from lineage V (HB strain) this strain of Tubifex has a competitive advantage over worms from other lineages. The results of this study suggest that certain species of Tubifex may be dead-end hosts to M. cerebralis by absorbing or inactivating the parasite and may also show greater survival compared to susceptible oligochaetes in certain whirling disease enzootic habitats.     

Tubifex tubifex from Alaska and their susceptibility to Myxobolus cerebralis

Although widespread throughout the continental United States, Myxobolus cerebralis, the myxozoan parasite that causes whirling disease in salmonids, has not been reported from the state of Alaska. As part of a risk assessment for the introduction and establishment of M. cerebralis into Alaska, the distribution of the invertebrate host Tubifex tubifex was surveyed, and its genetic composition and susceptibility to the parasite were determined. Many oligochaetes, but no T. tubifex, were collected from southeastern Alaska; however, 4 lineages of T. tubifex (I, III, IV, and VI) were identified from south-central Alaska. Lineage IV had not been previously described in North America, and its susceptibility to M. cerebralis was unknown. When lineage IV T. tubifex and 3 mixed lineage (I, III, IV, and VI) groups were exposed to M. cerebralis, only lineage III became infected under our experimental conditions. Infection occurred in this lineage even when it made up just 3% of the population. Implications of the presence of nonsusceptible lineages of T. tubifex on Alaskan salmonids would be significant in areas where these lineages dominate T. tubifex populations.     

The parasite that causes whirling disease, Myxobolus cerebralis, is genetically variable within and across spatial scales

Understanding the genetic structure of parasite populations on the natural landscape can reveal important aspects of disease ecology and epidemiology and can indicate parasite dispersal across the landscape. Myxobolus cerebralis (Myxozoa: Myxosporea), the causative agent of whirling disease in the definitive host Tubifex tubifex, is native to Eurasia and has spread to more than 25 states in the USA. The small amounts of data available to date suggest that M. cerebralis has little genetic variability. We examined the genetic variability of parasites infecting the definitive host T. tubifex in the Madison River, MT, and also from other parts of North America and Europe. We cloned and sequenced 18S ribosomal DNA and the internal transcribed spacer-1 (ITS-1) gene. Five oligochaetes were examined for 18S and five for ITS-1, only one individual was examined for both genes. We found two different 18S rRNA haplotypes of M. cerebralis from five worms and both intra- and interworm genetic variation for ITS-1, which showed 16 different haplotypes from among 20 clones. Comparison of our sequences with those from other studies revealed M. cerebralis from MT was similar to the parasite collected from Alaska, Oregon, California, and Virginia in the USA and from Munich, Germany, based on 18S, whereas parasite sequences from West Virginia were very different. Combined with the high haplotype diversity of ITS-1 and uniqueness of ITS-1 haplotypes, our results show that M. cerebralis is more variable than previously thought and raises the possibility of multiple introductions of the parasite into North America.     

Whirling disease: host specificity and interaction between the actinosporean stage of Myxobolus cerebralis and rainbow trout Oncorhynchus mykiss

Scanning electron microscopic studies were conducted on rainbow trout Oncorhynchus mykiss in the first 60 min after their exposure to the triactinomyxon spores of Myxobolus cerebralis. The results demonstrated that as early as 1 min post exposure the whole process, from the attachment of the triactinomyxon spores to the complete penetration of their sporoplasm germs, had occurred. The triactinomyxon spores sought out the secretory openings of mucous cells of the epidermis, the respiratory epithelium and the buccal cavity of trout and used them as portals of entry. Exposure experiments of the triactinomyxon spores of M. cerebralis to non-salmonid fish, such as goldfish Carassius auratus, carp Cyprinus carpio, nose Chondrostoma nasus, medaka Oryzias latipes, guppy Poecilia reticulata and also the amphibian tadpole Rana pipiens as well as to rainbow trout fry indicated a specificity for salmonids. Attempts to activate the triactinomyxon spores by exposure to mucus prepared from cyprinid and salmonid fish showed no significant differences from those conducted in tap water. The results suggest that the simultaneous presence of both mechano- and chemotactic stimuli was required for finding the salmonid fish host.     

The severity of whirling disease among wild trout corresponds to the differences in genetic composition of Tubifex tubifex populations in central Colorado

We analyzed the geographic distribution of Tubifex tubifex from various river drainages in central Colorado by genetic screening with specific mitochondrial 16S ribosomal DNA (mt 16S rDNA) markers. Four distinct mt 16S rDNA lineages are evident. The sites varied with respect to land- and water-use practices. All sites represented habitats presumed capable of supporting oligochaetes. At the locations where whirling disease has had the greatest impact on resident rainbow trout, T. tubifex, representing lineages I and III (genotypes known to be susceptible to Mxyobolus cerebralis), were most commonly found. In contrast, at sites less affected by whirling disease, T. tubifex of lineages V and VI that are more resistant to M. cerebralis infections were more abundant. The predominance of resistant T. tubifex worms (lineages V and VI) at low-impact sites supports the conclusion that when these genotypes are in greater abundance, the potential for more severe effects of whirling disease on wild rainbow trout populations may be diminished.     

Efficacy of passive sand filtration in reducing exposure of salmonids to the actinospore of Myxobolus cerebralis

The aquatic oligochaete Tubifex tubifex parasitized by Myxobolus cerebralis releases triactinomyxon (TAM) actinospores that can infect some species of salmonids and cause salmonid whirling disease. Silica sand was tested as a filtration medium for removal of TAMs from water containing the parasite. Laboratory tests indicated sand filtration removed > 99.99% of TAMs. In 2 different field tests, groups of 1 mo old rainbow trout Oncorhynchus mykiss were exposed for 2 wk to filtered and unfiltered water from a spring-fed pond enzootic for M. cerebralis. In November 2000, the exposure dose was estimated as between 3 and 5 TAMs fish(-1). During a March 2001 exposure, the estimated dose was between 286 and 404 TAMs fish(-1). Fish were held for 6 mo post exposure (p.e.) in laboratory aquaria for observation and evidence of clinical signs of whirling disease. We used 4 diagnostic techniques to assess the prevalence and severity of infection by M. cerebralis among fish exposed to filtered and unfiltered water. These included polymerase chain reaction (PCR) for genomic DNA of the parasite, histological evaluation for tissue damage, tissue digestion for quantification of cranial myxospores of the parasite, and total non-sampling mortality that occurred over 6 mo p.e. All diagnostic tests verified that the prevalence and severity of infection was significantly reduced among fish in treatment groups exposed to filtered water compared to those exposed to unfiltered water in both the low-dose and high-dose exposures.     

Alternation of Actinosporean and Myxosporean Phases in the Life Cycle of Zschokkella nova (Myxozoa)

ABSTRACT. Experimental evidence has been gathered to show that the life cycle of the myxozoan gallbladder parasite Zschokkella nova Klokacewa, 1914, which infects the fish Carassius carassius , has a complex life cycle with alternation of two hosts (fish and Oligochaeta) and two developmental phases (myxosporean and actinosporean). The gut epithelium of the oligochaete, Tubifex tubifex , exposed experimentally to Z. nova , obtained from C. carassius , became infected with organisms resembling Actinosporea. The spore structure and cube-like network of the interconnected spores is reminiscent of Siedleckiella silesica Janiszewska, 1952, although the spores are very different in size and number of sporoplasm nuclei. The life cycle of Z. nova resembles that of the whirling disease agent Myxosoma cerebralis described by Wolf and Markiw, which also alternates between fish and oligochaete hosts.