Variation and trade-offs in life history traits of the protist parasite Monocystis perplexa (Apicomplexa) in its earthworm host Amynthas agrestis.

The life history of a parasite describes its partitioning of assimilated resources into growth, reproduction, and transmission effort, and its precise timing of developmental events. The life cycle, in contrast, charts the sequence of morphological stages from feeding to the transmission forms. Phenotypic plasticity in life history traits can reveal how parasites confront variable environments within hosts. Within the protist phylum Apicomplexa major clades include the malaria parasites, coccidians, and most diverse, the gregarines (with likely millions of species). Studies on life history variation of gregarines are rare. Therefore, life history traits were examined for the gregarine Monocystis perplexa in its host, the invasive earthworm Amynthas agrestis at three sites in northern Vermont, United States of America. An important value of this system is the short life-span of the hosts, with only seven months from hatching to mass mortality; we were thus able to examine life history variation during the entire life cycle of both host and parasite. Earthworms were collected (N = 968 over 33 sample periods during one host season), then parasites of all life stages were counted, and sexual and transmission stages measured, for each earthworm. All traits varied substantially among individual earthworm hosts and across the sites. Across sites, timing of first appearance of infected earthworms, date when transmission stage (oocysts packed within gametocysts) appeared, date when number of both feeding (trophic) cells and gametocysts were at maximum, and date when 100% of earthworms were infected differed from 2-8 weeks, surprising variation for a short season available for parasite development. The maximal size of mating cells varied among hosts and across sites and this is reflected in the number of oocysts produced by the gametocyst. A negative trade-off was observed for the number of oocysts and their size. Several patterns were striking: (1) Prevalence reached 100% at all sites by mid season, only one to three weeks after parasites first appeared in the earthworms. (2) The number of parasites per host was large, reaching 300 × 103 cells in some hosts, and such high numbers were present even when parasites first appeared in the host. (3) At one site, few infected earthworms produced any oocysts. (4) The transmission rate to reach such high density of parasites in hosts needed to be very high for a microbe, from >0.33% to >34.3% across the three sites. Monocystis was one of the first protist parasites to have its life cycle described (early 19th century), but these results suggest the long-accepted life cycle of Monocystis could be incomplete, such that the parasites may be transmitted vertically (within the earthworm's eggs) as well as horizontally (leading to 100% prevalence) and merogony (asexual replication) could be present, not recognized for Monocystis, leading to high parasitemia even very early in the host's season.

APOLOCYSTIS BOSANQUETI N. SP. (APICOMPLEXA: EUGREGARINORIDA) FROM THE INVASIVE EARTHWORM AMYNTHAS AGRESTIS (ANNELIDA: MEGASCOLECIDAE), WITH SIGNIFICANCE FOR THE MONOPHYLY OF THE FAMILY MONOCYSTIDAE.

Apolocystis bosanqueti n. sp., a parasite of an important invasive earthworm in North America, Amynthas agrestis, is described from a site in northern Vermont. The earthworm host follows an annual life cycle in Vermont, so the entire life cycle of the parasite can be observed in 7 mo. In spring, the parasites were first seen in juvenile worms as paired gamonts (suggesting precocious association). These paired gamonts mature into gametocytes that form an opaque structure, with a thick gelatinous envelope (epicyst), that becomes full of zygotes. The resulting gametocyst becomes packed with ∼105 fusiform oocysts. The mature orbicular gametocysts are large (∼1 mm in diameter) and visible to the naked eye through the body wall of the host's anterior segments. The new species most resembles Apolocystis herculea described from many lumbricid earthworm species in Europe but differs from that parasite because Ap. herculea infects the intestinal wall in the posterior of the host rather than the anterior segments. A survey of 9 other earthworm species sympatric with Am. agrestis revealed that only Amynthas tokioensis, also an invasive species, was infected with Ap. bosanqueti, albeit much less commonly. Diagnosis for the family Monocystidae is problematic because cardinal characters are lacking, and the commonly cited character, a trophozoite with no anterior differentiation, is violated in most genera placed in the family. For the first time, a molecular phylogeny is presented that includes 3 genera of monocystids with diverse cell morphology (including the new species) and supports the monophyly of the family. The only morphological character that may be used to diagnose the Monocystidae is the morphology of oocysts, which are fusiform with extended terminal tips. A comparison of oocysts from 7 parasites recovered from local earthworms, including from 3 monocystid species in the phylogeny, confirms the utility of this diagnostic trait. The 2 hosts of the new species were most likely introduced from Japan, so the range of Apolocystis likely extends into East Asia.

Effect of substrate on the proliferation of Myxobolus cerebralis in the mitochondrial lineages of the Tubifex tubifex host.

The study goal was to examine the effects of sand and mud on the propagation of Myxobolus cerebralis, the whirling disease agent, in four mitochondrial 16S ribosomal DNA lineages (I, III, V, VI) of its oligochaete host, Tubifex tubifex (Tt). In all the lineage groups held continuously in either substrate (non-shifted) or transferred from sand to mud (shifted), substrate influenced parasite proliferation only in lineage III. Sporogenesis and release of triactinomyxon spores (TAMs) were more prevalent in lineage III Tt in mud compared to sand. Low-infection prevalence and lack of parasite development in lineage I is associated with the greater number of resistant worms and were not affected by substrate type. Substrate did not impact Tt from lineages V and VI that failed to develop any parasite stages in either substrate even after shifting from sand to mud. The relationship between the microbial community in the substrate and parasite proliferation in lineage III was described but not analyzed due to small sample size. Substrate-associated bacteria were hypothesized as essential dietary source for the oligochaete host feeding selectively on fine (mud)-microflora. Progeny was produced by all lineage groups shifted to mud with disparate survival profiles in lineage V and VI and high mortalities in lineage III. Our study demonstrates that substrate type can alter parasite proliferation in lineage III. Conversely, parasite development and infectivity were not altered in lineage V and VI that are refractory to the parasite nor among the more resistant phenotypes (I), regardless of substrate type.

Two new species of Bacillidium (Microsporidia) from coelomocytes of Branchiura sowerbyi (Oligochaeta: Naididae) in China.

Bacillidium spp. exclusively infect oligochaetes and these microsporidian pathogens are typically characterized by their rod-shaped spores. Seven Bacillidium spp. are presently reported from different organs of oligochaetes. Here, we describe two new Bacillidium species, Bacillidium sinensis n. sp. and Bacillidium branchilis n. sp., from coelomocytes of Branchiura sowerbyi. This is the first report of Bacillidium spp. in oligochaetes from China. Both species of Bacillidium elicit the formations of opaque xenoma-like lesions in coelomocytes of the host. A diplokaryotic nucleus occurs in all life stages of these two new Bacillidium species. Mature spores of B. sinensis are 15.9 ± 0.6 (14.7-17.1) µm long (average ± standard error, range, n = 50) and 2.5 ± 0.1 (2.3-2.7) µm wide in fresh preparations. A new type of exospore (sixteen-layered exospore) is discovered from B. sinensis n. sp. which is distinctly different from B. branchilis n. sp., and other Bacillidium spp. (double-layered exospore) reported previously. These two Bacillidium species are morphologically distinguished from each other and all Bacillidium spp. described previously in terms of hosts, infection sites, spore size, spore wall or polar filament thickness. BLASTn searches indicated that these two new microsporidian parasites are surprisingly most similar to Janacekia tainanus (94.76% for B. sinensis and 90. 2% for B. branchilis) isolated from the fat body of midge larva (Kiefferulus tainanus). Phylogenetic analysis demonstrates that the two novel taxons cluster with J. debaisieuxi, J. tainanus, and Bacillidium sp. within the Jirovecia-Bacillidium-Janacekia clade. Other available 18S rRNA gene sequences for microsporidia that infect oligochaetes include J. sinensis, B. vesiculoformis, Neoflabelliforma aurantiae, and Bacillidium sp., but these do not form a single cluster with B. sinensis and B. branchilis, but are instead dispersed through the clade. Based on the ultrastructural features and molecular characteristics, two new species within the genus Bacillidium, B. sinensis n. sp. and B. branchilis n. sp., are designated.

Stomatocystis goerresi, a new species of gregarine parasite (Apicomplexa, Monocystidae) from the invasive Japanese earthworm Amynthas tokioensis (Megascolecidae), with a description of the parasite's life cycle.

Stomatocystis goerresi sp. n., a gregarine (phylum Apicomplexa, Monocystidae) parasite of an important invasive earthworm in North America, Amynthas tokioensis (Beddard), is described. This is the second species placed into the genus, and details of its morphology and life cycle support Stomatocystis Bandyopadhyay, Mitra et Göçmen, 2006 as a valid taxon. The new species is described using standard nomenclature, measurements, shape descriptors, and photographs of living cells. The parasite was found only in A. tokioensis, and absent in sympatric earthworm species, suggesting it arrived when the earthworms were introduced from their origin from Japan. The species is distinctive from the type species in the genus, S. indica Bandyopadhyay, Mitra et Göçmen, 2006, in being substantially larger in all stages, found in only the host's seminal vesicles, and found in a different host species from East Asia. The distinctive trophozoites/gamonts develop a large funnel structure ringed with a collar of pronounced ridges, and the funnel appears even in the smallest cells. This funnel varies greatly in relative size (to the cell body) and shape, sometimes forming a large fan. The life cycle of S. goerresi is described including distinctive syzygy in which the funnels fuse and then produce a large cell with local centres of isogamete production (thus sex without gender). Gametes are large ( ~5 µm) spheres with complex tips. Oocyst production is large, > 1,000 per mature gametocyst. The genus Stomatocystis is placed into the Monocystidae, but the life cycle of the new species differs from those of other monocystid taxa, which may mean the Monocystidae are not monophyletic or life cycles are variable within the family. Prevalence of S. goerresi at the type locality was high (~ 90%). The parasites destroy the earthworm's organ of sperm self-storage thus eliminating the male function in the hermaphroditic host which may influence the ability of the earthworm to invade and be successful at new sites.

Field and Laboratory Observations on the Life History of Gordius terrestris (Phylum Nematomorpha), A Terrestrial Nematomorph.

To date, all free-living adult hairworms have been reported from aquatic habitats. However, in Oklahoma, a recently described gordiid, Gordius terrestris, is consistently encountered in terrestrial habitats. We found this gordiid species has a unique egg morphology, unlike that of any other hairworm species, with an outer shell separated by distinct space from a thick inner membrane surrounding the developing larva. Because of this unique egg morphology and the occurrence of free-living hairworms in terrestrial habitats, it was hypothesized that G. terrestris represents the first report of a hairworm species with a terrestrial life cycle. In this study, we observed thousands of free-living adult worms in terrestrial habitats such as wet lawns and underneath wet sod during the winter. We found evidence of worms mating in these terrestrial habitats, followed by female worms burrowing and ovipositing in the soil. In the laboratory, significantly more females burrowed in the soil than males, providing a plausible explanation for the extreme male-biased sex ratio observed for free-living worms found on wet lawns. Finally, we collected terrestrial earthworms infected with the cyst stage of this gordiid species in the field and confirmed those observations by infecting earthworms with G. terrestris larvae in the laboratory. Taken together, these observations strongly support the hypothesis that G. terrestris has a terrestrial life cycle.

A New Species of Monocystis (Apicomplexa: Gregarina: Monocystidae) from the Asian Invasive Earthworm Amynthas agrestis (Megascolecidae), with an Improved Standard for Monocystis Species Descriptions.

Monocystis perplexa n. sp., a parasite of an important invasive Japanese earthworm in North America, Amynthas agrestis, is described from a site in Vermont. An improved standard for Monocystis species descriptions is proposed including a standard nomenclature to reduce synonymies, a standard set of biometrics and shape descriptions for living cells, and a DNA genomic sequence for the 18S rRNA (∼1,700 base pairs). Comparing morphologies of Monocystis parasites in sympatric earthworm species indicates that M. perplexa is specific to A. agrestis in the study region. Also, polymerase chain reaction primers specific to M. perplexa amplified samples of A. agrestis earthworms taken from several sites in Japan. This suggests the parasite entered North America from Japan, the origin of the invasive Amynthas earthworm, and thus M. perplexa would be the first Monocystis described from the diverse Japanese Amynthas earthworms and the first from East Asia. Monocystis perplexa was found in every population of A. agrestis surveyed in Vermont, always reaching 100% prevalence by late summer (the host has an annual life cycle in Vermont). The 18S gene sequence differed from that of Monocystis agilis from the sympatric earthworm Lumbricus terrestris (the only other sequence available for Monocystis), and a genetic similarity tree places them closest among other gregarines. Many of the 95 described species of Monocystis are very similar in morphology (based on species descriptions), so the 18S gene can act as a barcode for Monocystis species and thus will help to eliminate both synonymies and reveal cryptic species.

In vitro and in vivo assays reveal that cations affect nematocyst discharge in Myxobolus cerebralis (Cnidaria: Myxozoa).

Myxozoans are parasitic, microscopic cnidarians that have retained the phylum-characteristic stinging capsules called nematocysts. Free-living cnidarians, like jellyfish and corals, utilize nematocysts for feeding and defence, with discharge powered by osmotic energy. Myxozoans use nematocysts to anchor to their fish hosts in the first step of infection, however, the discharge mechanism is poorly understood. We used Myxobolus cerebralis, a pathogenic myxozoan parasite of salmonid fishes, and developed two assays to explore the nature of its nematocyst discharge. Using parasite actinospores, the infectious stage to fish, we stimulated discharge of the nematocysts with rainbow trout mucus in vitro, in solutions enriched with chloride salts of Na+, K+, Ca2+ and Gd3+, and quantified discharge using microscopy. We then used quantitative polymerase chain reaction to evaluate the in vivo effects of these treatments, plus Mg2+ and the common aquaculture disinfectant KMnO4, on the ability of M. cerebralis actinospores to infect fish. We found that Mg2+ and Gd3+ reduced infection in vivo, whereas Na+ and K+ over-stimulated nematocyst discharge in vitro and reduced infection in vivo. These findings align with nematocyst discharge behaviour in free-living Cnidaria, and suggest phylum-wide commonalties, which could be exploited to develop novel approaches for controlling myxozoan diseases in aquaculture.

Involvement of sphaeractinomyxon in the life cycle of mugiliform-infecting Myxobolus (Cnidaria, Myxosporea) reveals high functionality of actinospore morphotype in promoting transmission.

Four new actinospore types belonging to the sphaeractinomyxon collective group (Cnidaria, Myxosporea) are described from the coelomic cavity of a marine Baltidrilus sp. (Oligochaeta, Naididae) inhabiting a northern Portuguese estuary. Host identification supports the usage of marine oligochaetes, namely of the family Naididae Ehrenberg, 1828, as definitive hosts for myxosporeans inhabiting estuarine/marine environments. The absence of mixed infections in the host specimens analysed is suggested to reflect the influence of host-, parasite- and environmental-related factors regulating myxosporean-annelid interactions. Molecular analyses matched the SSU rDNA sequences of three of the four new types with those of mugiliform-infecting Myxobolus spp., namely Myxobolus mugiliensis and a Myxobolus sp. from flathead grey mullet Mugil cephalus, and Myxobolus labrosus from thicklip grey mullet Chelon labrosus. These results directly link, for the first time, the sphaeractinomyxon collective group to a myxospore counterpart, further confirming their previously hypothesized specific involvement in the life cycle of myxobolids that infect mullets. Acknowledging this life cycle relationship, the functionality of the sphaeractinomyxon morphotype is suggested to have been decisive for the evolutionary hyperdiversification of the genus Myxobolus in mullets. Unlike other actinospore morphotypes, sphaeractinomyxon lack valvular processes, which implies a limited capability for buoyancy. Considering the benthic-feeding nature of mullets, this feature is most likely crucial in promoting successful transmission to the vertebrate host.

Morphological and molecular characterization of a new freshwater microsporidium, Jirovecia sinensis sp. n. (Microsporidia) infecting the coelomocytes of Branchiura sowerbyi (Oligochaeta: Naididae) in China.

We report a new microsporidium Jirovecia sinensis sp. n. from a freshwater oligochaete, Branchiura sowerbyi collected in Hongze city, Jiangsu province, East China. Numerous whitish hypertrophied coelomocytes of 0.33-0.59 mm in diameter indicated infection. Transmission electron microscopy observations revealed that all developmental stages were diplokaryotic. The earliest life stages observed were meronts that were in direct contact with host cytoplasm, accumulated peripherally in the hypertrophied coelomocytes and connected with host cytoplasm through many pinocytotic canals. Mature spores are rod-shaped with a blunt end, measuring 17.0 ± 0.1 (14.9-18.5) µm long and 2.0 ± 0.2 (1.7-2.2) µm wide. The most conspicuous character of the novel microsporidian parasite is the tail-like posterior prolongations, with a length of 29.6-40.8 µm. Mature spores have a manubrium with a diameter of 447-485 nm which consist of six density-discontinuous concentric circles. Spores possess a collar-shaped anchoring disk and a bipartite polarplast with an anterior lamellar region and a posterior tubular section. SSU rDNA-based phylogenetic analysis indicated with high support values that the new species clustered with two Bacillidium species (B. vesiculoformis and Bacillidium sp.) infecting the freshwater oligochaetes and Janacekia debaisieuxi infecting the insect Simulium maculatum. Based on the ultrastructural features and molecular characteristics, a new species in the genus Jirovecia, Jirovecia sinensis sp. n., is designated.

Characterisation of sphaeractinomyxon types (Cnidaria: Myxozoa) from marine and freshwater oligochaetes in a Portuguese estuary, with the demise of the endocapsa collective group.

Six types of sphaeractinomyxon are reported from the coelomic cavity of oligochaetes collected from the Minho River estuary in northern Portugal. Four new types are morphologically and molecularly described from freshwater species belonging to the genera Psammoryctides Hrabe and Potamothrix Vejdovský et Mrázek in the upper estuary, thus significantly increasing the number of known freshwater sphaeractinomyxon. In the lower estuary, sphaeractinomyxon types 8 and 10 of Rangel et al. (2016) are recorded infecting the marine oligochaete Tubificoides pseudogaster (Dahl). A single specimen of T. pseudogaster further displayed infection by one of the four new types found in the upper estuary, suggesting the involvement of sphaeractinomyxon in the life cycles of myxosporean species that infect migratory fish hosts. The acquisition of these second hosts is proposed to have allowed the myxosporean counterparts of sphaeractinomyxon to cross environmental barriers and conquer new habitats. Phylogenetic analyses of the SSU rRNA gene reveal the four new types clustering within the monophyletic clade of mugiliform-infecting myxobolids, strengthening the previously proposed involvement of the sphaeractinomyxon collective group in the life cycles of this specific group of myxosporeans. Endocapsa types also cluster within the latter clade, having actinospores that differ from those of sphaeractinomyxon only in the presence of valvular swellings that do not change when in contact with water. In this study, however, one type was found displaying actinospores with and without valvular swellings in the same oligochaete specimen. This overlap in actinospore morphology is given as grounds for the demise of the endocapsa collective group.

Delimitation of five astome ciliate species isolated from the digestive tube of three ecologically different groups of lumbricid earthworms, using the internal transcribed spacer region and the hypervariable D1/D2 region of the 28S rRNA gene.

BACKGROUND: Various ecological groups of earthworms very likely constitute sharply isolated niches that might permit speciation of their symbiotic ciliates, even though no distinct morphological features appear to be recognizable among ciliates originating from different host groups. The nuclear highly variable ITS1-5.8S-ITS2 region and the hypervariable D1/D2 region of the 28S rRNA gene have proven to be useful tools for the delimitation of species boundaries in closely related free-living ciliate taxa. In the present study, the power of these molecular markers as well as of the secondary structure of the ITS2 molecule were tested for the first time in order to discriminate the species of endosymbiotic ciliates that were isolated from the gastrointestinal tract of three ecologically different groups of lumbricid earthworms. RESULTS: Nineteen new ITS1-5.8S-ITS2 region and D1/D2-28S rRNA gene sequences were obtained from five astome species (Anoplophrya lumbrici, A. vulgaris, Metaradiophrya lumbrici, M. varians, and Subanoplophrya nodulata comb. n.), which were living in the digestive tube of three ecological groups of earthworms. Phylogenetic analyses of the rRNA locus and secondary structure analyses of the ITS2 molecule robustly resolved their phylogenetic relationships and supported the distinctness of all five species, although previous multivariate morphometric analyses were not able to separate congeners in the genera Anoplophrya and Metaradiophrya. The occurrence of all five taxa, as delimited by molecular analyses, was perfectly correlated with the ecological groups of their host earthworms. CONCLUSIONS: The present study indicates that morphology-based taxonomy of astome ciliates needs to be tested in the light of molecular and ecological data as well. The use of morphological identification alone is likely to miss species that are well delimited based on molecular markers and ecological traits and can lead to the underestimation of diversity and overestimation of host range. An integrative approach along with distinctly increased taxon sampling would be helpful to assess the consistency of the eco-evolutionary trend in astome ciliates.

Transcriptomic analysis of oligochaete immune responses to myxosporeans infection: Branchiura sowerbyi infected with Myxobolus cultus.

The Myxozoa are endoparasites characterized by a two-host life cycle that typically involves invertebrates and vertebrates as definitive and intermediate hosts, respectively. However, little is known about invertebrate-myxosporean interactions, particularly about patterns of host immune defense. We used RNA-sequencing to identify genes that are possibly involved in the immune responses of the oligochaete Branchiura sowerbyi naturally infected with Myxobolus cultus. De novo assembly of the B. sowerbyi transcriptome yielded 119,031 unigenes, with an average length of 896 bp and an N50 length of 1754 bp. Comparative transcriptome analysis revealed 4059 differentially expressed genes (DEGs) between M. cultus-infected and uninfected B. sowerbyi groups, including 3802 upregulated genes and 257 downregulated genes. Among the B. sowerbyi immune factors implicated in the responses to M. cultus infection, DEGs related to lectins, ubiquitin-mediated proteolysis, phagocytosis, oxidative-antioxidative responses, proteases, and protease inhibitors were upregulated. The expression of some immune-related molecules such as calmodulin, heat shock proteins, antimicrobial peptides, lysenin, and serum amyoid A protein were also significantly upregulated. The expression patterns of 14 immune-related DEGs identified by RNA-seq were validated by quantitative real-time polymerase chain reaction. This study is the first attempt to characterize the B. sowerbyi transcriptome and identify immune-related molecules possibly associated with M. cultus infection. It is also the first report of invertebrate host-myxosporean interactions at the transcriptomic level. Our results will facilitate the elucidation of adaptive evolution mechanisms of myxosporean parasites in the definitive host and the genetic basis for differences in resistance of invertebrate hosts of different genotypes to a myxosporean species.

Evolutionary Origin and Host Range of Plagiotoma lumbrici (Ciliophora, Hypotrichia), an Obligate Gut Symbiont of Lumbricid Earthworms.

Four common earthworm species, the anecic Lumbricus terrestris, the endogeic Octolasion tyrteum as well as the epigeic Eisenia fetida and Dendrobaena veneta, were examined for the presence of the microbial gut symbiont Plagiotoma lumbrici. The evolutionary origin of this endobiotic microbe was reconstructed, using the 18S rRNA gene, the ITS1-5.8S-ITS2 region, and the first two domains of the 28S rRNA gene. Plagiotoma lumbrici was exclusively detected in the anecic Lumbricus terrestris. Multigene analyses and the ITS2 secondary structure robustly determined the phylogenetic home of Plagiotoma lumbrici populations within the oxytrichid Dorsomarginalia (Spirotrichea: Hypotrichia) as a sister taxon of the free-living Hemiurosomoida longa. This indicates that earthworms obtained their gut endosymbiont by ingesting soil/leaf litter containing oxytrichine ciliates that became adapted to the intestinal tract of earthworms. Interestingly, according to the literature data, Plagiotoma lumbrici was detected in multiple anecic and some epigeic but never in endogeic earthworms. These observations suggest that Plagiotoma lumbrici might be adapted to certain gut conditions and the lifestyle of anecic Lumbricidae, such as Lumbricus, Aporrectodea, and Scherotheca, as well as of some co-occurring epigeic Lumbricus species.

PCR detection of Heterakis gallinarum in environmental samples.

Heterakis gallinarum is a widely distributed cecal nematode that parasitizes gallinaceous birds including chickens and turkeys. H. gallinarum infection poses a problem for the poultry industry as the nematode egg serves as a vector for the protozoan parasite, Histomonas meleagridis, the causative agent of histomonosis. The only means of detecting H. gallinarum in the environment is microscopic identification of the eggs in soil or feces; however, H. gallinarum eggs are often mistaken for those of Ascaridia galli. Three primer sets were designed from sequences cloned from the H. gallinarum genome to develop a diagnostic PCR. Each of these primer sets amplified a single product from H. gallinarum, but were unable to amplify DNA from H. meleagridis, Ascaridia galli, or Cestode sp. H. gallinarum DNA was amplified from Lumbricus sp. (earthworms) and Alphitobius diaperinus (darkling beetles), confirming that the earthworm acts as a paratenic host for H. gallinarum and suggesting that the darkling beetle may be a carrier for this nematode.

Molecular data infers the involvement of a marine aurantiactinomyxon in the life cycle of the myxosporean parasite Paramyxidium giardi (Cnidaria, Myxozoa).

An aurantiactinomyxon type is described from the marine naidid Tubificoides pseudogaster (Dahl, 1960), collected from the lower estuary of a Northern Portuguese River. This type constitutes the first of its collective group to be reported from Portugal, and only the fourth described from a marine oligochaete worldwide. Extensive morphological comparisons of new aurantiactinomyxon isolates to all known types without available molecular data are proposed to be unnecessary, given the artificiality of the usage of morphological criteria for actinosporean differentiation and the apparent strict host specificity of the group. Recognition of naidid oligochaetes as the hosts of choice for marine types of aurantiactinomyxon and other collective groups, suggests that the family Naididae played a preponderant role in the myxosporean colonization of estuarine communities. Molecular analyses of the type in study further infer its involvement in the life cycle of Paramyxidium giardi (Cépède, 1906) Freeman and Kristmundsson, 2018, a species that infects the kidney of European eel Anguilla anguilla (Linnaeus, 1758) and that has been reported globally, including from Portuguese waters. The low intraspecific difference registered in relation to Icelandic isolates of P. giardi (0.6%) is hypothesized to result from the emergence of genotypically different subspecies due to geographic isolation.

Thecamoeba quadrilineata (Amoebozoa, Lobosa) as a new member of amphizoic amoebae-first isolation from endozoic conditions.

A free-living soil amoeba Thecamoeba quadrilineata (Carter, 1856) Lepsi, 1960 (Amoebozoa: Thecamoebidae) was isolated from endozoic conditions for the first time. Presence of amoebae was detected after 4 days following inoculation of the gut of the earthworm Lumbricus terrestris on agar plate with Escherichia coli. On the basis of our isolation, we consider T. quadrilineata as further amphizoic amoeba species. This study enlarges the range of amphizoic tendency in members of the genus Thecamoeba and stresses the need for further research on the pathogenic potential of Thecamoeba species.

The potential role of the sphaeractinomyxon collective group (Cnidaria, Myxozoa) in the life cycle of mugiliform-infecting myxobolids, with the morphological and molecular description of three new types from the oligochaete Tubificoides insularis.

Three new types of sphaeractinomyxon (Cnidaria, Myxozoa) are described from the coelomic cavity of the marine oligochaete Tubificoides insularis, collected from the Alvor estuary, Algarve, Portugal. Another known type is also registered from this location and host: Sphaeractinomyxon type 10 of Rangel et al. (2016), which was originally described from the marine oligochaete Tubificoides pseudogaster in the Aveiro estuary, Portugal. Phylogenetic analysis revealed the case isolates and all other available SSU rRNA sequences of sphaeractinomyxon clustering within a clade containing Myxobolus spp. that infect mullets, thus suggesting that this collective group plays a role in the life cycle of mugiliform-infecting myxobolids. Also clustering within this clade were all types of tetraspora and endocapsa, calling into question the distinctiveness of these collective groups. Acknowledging a previous work showing that the pansporocysts of sphaeractinomyxon produce a variable number of actinospores, we suggest that the tetraspora collective group be deemed invalid and its types transferred to sphaeractinomyxon. In turn, endocapsa requires validation through the description of new types truly differentiating them from sphaeractinomyxon.

Elimination of Myxobolus cerebralis in Placer Creek, a Native Cutthroat Trout Stream in Colorado.

Placer Creek, a tributary of Sangre de Cristo Creek in Colorado's San Luis Valley, supported an allopatric core conservation population of native Rio Grande Cutthroat Trout Oncorhynchus clarkii virginalis during much of the 20th century. After the failure of gabion barriers in the late 1990s, Brook Trout Salvelinus fontinalis infected with Myxobolus cerebralis invaded from Sangre de Cristo Creek. By 2005, whirling disease (WD) and competition from Brook Trout reduced Rio Grande Cutthroat Trout numbers to less than 10% of the total trout population. New barriers were constructed in 2006 and the stream was treated with rotenone in 2007 and 2009 to eliminate all fish prior to the reintroduction of Rio Grande Cutthroat Trout. Results of WD research studies in Montana, California, and Colorado indicated it might be possible to break the life cycle of the parasite in some situations. Our management interventions included (1) reducing the fish population in the stream to zero for approximately 14 months, (2) introducing lineage V and VI Tubifex tubifex worms, which are not susceptible to M. cerebralis, and (3) eliminating a small off-channel pond that provided optimal habitat that sustained a localized high-density population of lineage III T. tubifex, the oligochaete host susceptible to M. cerebralis. Electrofishing during the fall of 2009 and spring of 2010 indicated the drainage was devoid of fish. Fry, juvenile, and adult Rio Grande Cutthroat Trout were stocked in September and October of 2010 and 2011. Approximately 975,000 lineage V and VI T. tubifex were introduced into Placer Creek between 2010 and 2012 as possible oligochaete competitors for the lineage III worms. The off-channel pond was filled in, and the surface was reseeded in April 2012. No evidence of M. cerebralis infection was detected among more than 280 Rio Grande Cutthroat Trout tested between July 2012 and July 2016, indicating the parasite had been eradicated from the Placer Creek basin upstream of the barriers.

Distribution and Prevalence of Myxobolus cerebralis in Postfire Areas of Plumas National Forest: Utility of Environmental DNA Sampling.

Myxobolus cerebralis is a myxozoan parasite and the etiological agent of whirling disease in salmonids. The parasite's life cycle involves waterborne spores and requires both a salmonid fish and the benthic freshwater oligochaete worm Tubifex tubifex (Oligochaeta: Tubificidae). Wildfires can lead to the erosion of fine sediments into stream channels and have been implicated as promoting environmental conditions that are suitable for the survival and success of T. tubifex, whose presence in turn can affect the prevalence of M. cerebralis. Analysis of environmental DNA (eDNA) has the potential to be a powerful tool for evaluating the presence of microorganisms, for which direct observation is impossible. We investigated the presence of M. cerebraliseDNA in river water and river sediment samples collected from areas affected by recent fire activity in Plumas National Forest, California. We compared eDNA loads in the environment to M. cerebralis infection in T. tubifex and sentinel-exposed Rainbow Trout Oncorhynchus mykiss and the presence of T. tubifex lineages in the same environment. For the latter, we developed a multiplex quantitative PCR assay for detection of T. tubifex lineages I, III, and V. Lineage IIIT. tubifex and M. cerebralis (eDNA as well as DNA extracted from fish and worm tissues) were detected only in samples obtained from areas affected by the Moonlight wildfire. The association between M. cerebralis infection in sentinel-exposed fish and eDNA detection in environmental samples only approached significance at a P-value of 0.056. However, given the difference in relative effort between the two sampling methods (host versus nonhost environment), our data suggest that eDNA sampling of water and substrate is a promising approach for surveillance of myxozoan fish parasites.