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.

The life cycle of Thelohanellus kitauei (Myxozoa: Myxosporea) infecting common carp (Cyprinus carpio) involves aurantiactinomyxon in Branchiura sowerbyi.

Thelohanellus kitauei is a freshwater myxosporean parasite causing intestinal giant cystic disease of common carp. To clarify the life cycle of T. kitauei, we investigated the oligochaete populations in China and Hungary. This study confirms two distinct aurantiactinomyxon morphotypes (Aurantiactinomyxon type 1 and Aurantiactinomyxon type 2) from Branchiura sowerbyi as developmental stages of the life cycle of T. kitauei. The morphological characteristics and DNA sequences of these two types are described here. Based on 18S rDNA sequence analysis, Aurantiactinomyxon type 1 (2048 bp) and Aurantiactinomyxon type 2 (2031 bp) share 99.2-99.4 %, 99.8-100 % similarity to the published sequences of T. kitauei, respectively. The 18S rDNA sequences of these two aurantiactinomyxon morphotypes share 99.4 % similarity, suggesting intraspecific variation within the taxon, possibly due to geographic origin. Phylogenetic analyses demonstrate the two aurantiactinomyxon types clustered with T. kitauei. Regardless, based on 18S rDNA synonymy, it is likely that Aurantiactinomyxon type 1 and 2 are conspecific with T. kitauei. This is the fourth elucidated two-host life cycle of Thelohanellus species and the first record of T. kitauei in Europe.

Small subunit ribosomal RNA sequence links the myxospore stage of Henneguya mississippiensis n. sp. from channel catfish Ictalurus punctatus to an actinospore released by the benthic oligochaete Dero digitata.

There are more than 200 species of Henneguya described from fish. Of these, only three life cycles have been determined, identifying the actinospore and myxospore stages from their respective hosts. Two of these life cycles involve the channel catfish (Ictalurus punctatus) and the freshwater oligochaete Dero digitata. Herein, we molecularly confirm the life cycle of a previously undescribed Henneguya sp. by matching 18S ribosomal RNA (rRNA) gene sequence of the myxospore stage from channel catfish with the previously described actinospore stage (Aurantiactinomyxon mississippiensis) from D. digitata. Gill tissue from naturally infected channel catfish contained pseudocysts restricted to the apical end of the primary lamellae. Myxospores were morphologically consistent with Henneguya spp. from ictalurid fishes in North America. The spores measured 48.8 ± 4.8 µm (range = 40.7-61.6 µm) in total spore length. The lanceolate spore body was 17.1 ± 1.0 µm (14.4-19.3 µm) in length and 5.0 ± 0.3 µm (4.5-5.5 µm) in width. The two polar capsules were 6.2 ± 0.4 µm (5.8-7.0 µm) long and 5.0 ± 0.3 µm (4.5-5.5 µm) wide. The polar capsule contained eight to nine coils in the polar filament. The two caudal processes were of equal length, measuring 31.0 ± 4.1 µm (22.9-40.6 µm). The 1980-bp 18S rRNA gene sequence obtained from two excised cysts shared 99.4% similarity (100% coverage) to the published sequence of A. mississippiensis, an actinospore previously described from D. digitata. The sequence similarity between the myxospore from channel catfish and actinospore from D. digitata suggests that they are conspecific, representing alternate life stages of Henneguya mississippiensis n. sp.

Effect of cadmium on the susceptibility of Tubifex tubifex to Myxobolus cerebralis (Myxozoa), the causative agent of whirling disease.

Environmental pollutants alter a wide range of host-parasite interactions in various ways. In some cases, pollution leads to a significant increase in parasite abundance, causing epidemics of parasitic diseases. In other cases, toxicants restrict the transmission success of parasites, resulting in reduction of their abundance. However, very little is known regarding whether and to what extent aquatic pollution affects myxozoan obligate parasites commonly found in fish. We investigated the effect of cadmium (Cd) on the aquatic oligochaete Tubifex tubifex infected with the myxozoan Myxobolus cerebralis. The oligochaetes were experimentally exposed to M. cerebralis myxospores and kept in various concentrations of Cd for 4 mo. Neither survival nor reproduction of the worms was affected by the metal, but infection prevalence and numbers of triactinosmyxon spores produced by individual worms were higher in the Cd-exposed group than the unexposed control. A comparative assay of a lethal Cd concentration (LC50) on infected and non-infected T. tubifex revealed that infected worms are more resistant to the acute toxicity of Cd, probably because uptake of Cd was reduced by the infection. These results suggest that the abundance of M. cerebralis likely increases in polluted waters and escalates the risk of whirling disease in the respective area.

Characterization of glycans in the developmental stages of Myxobolus cerebralis (Myxozoa), the causative agent of whirling disease.

Glycans and sugar-binding molecules (lectins) form an interactive recognition system, which may enable parasitic organisms to adhere to host cells and migrate into target tissues. The aim of the present study was to analyse surface-associated glycans in the developmental stages of Myxobolus cerebralis (Hofer), the causative agent of whirling disease. A panel of biotin-labelled plant lectins was used to detect a broad spectrum of glycan motifs with high specificity. Binding sites were detected histochemically in the tissue sections of infected rainbow trout, Oncorhynchus mykiss (Walbaum), and infected Tubifex tubifex (Müller), and were characterized by light, fluorescence and transmission electron microscopy. With mannose-specific lectins [Lens culinaris agglutinin, Pisum sativum agglutinin, Canavalia ensiformis agglutinin (LCA, PSA, CanA)] mannose-containing glycans were detected in all the developmental stages and host tissues. No binding sites for galactose-specific lectins were present in M. cerebralis spores but reactivity with host tissues occurred. Diversity in glycans was detected by N-acetyl-D-galactosamine-specific lectins in sporoplasm cells of M. cerebralis and triactinomyxon spores. In the group of lectins with monosaccharide-specificity for N-acetyl-D-glucosamine (GlcNAc), the reactivity of Datura stramonium agglutinin (DSA), Lycopersicon esculentum agglutinin (LEA) and Solanum tuberosum agglutinin (STA) was restricted to polar capsules whereas Griffonia simplicifolia agglutinin II (GSA II) also bound to sporoplasm cells of stages in the fish host but not in those present in infected T. tubifex. Moreover, Triticum vulgaris (wheat germ) agglutinin (WGA) and succinylated WGA indicated the presence of N-acetyl-D-glucosamine polymers in polar capsules. No specificity for spores was observed concerning 'bisected'N-glycans and no reactivity in parasitic stages was observed with the fucose-binding lectin Ulex europaeus agglutinin (UEA) I, Sambucus nigra agglutinin (SNA) (specific for alpha2,6-sialylated glycans) and Maackia amurensis agglutinin (MAAI) (specific for alpha2,3-sialylated glycans). Arachis hypogaea (peanut) agglutinin (PNA), Erythrina cristagalli agglutinin (ECA), GSA I, Sophora japonica agglutinin (SJA), Dolichos biflorus agglutinin (DBA) and GSA II detected reactive sites solely confined to the developmental stages of M. cerebralis and were not reactive in the fish host. These parasite-specific glycans may play a role in the adhesion process of the parasite to fish epidermis prior to infection, but may provide protection to the host by activating the complement system, or stimulating an adaptive immune response as putative antigens.

Aploparaksis kornyushini n. sp. (Cestoda: Hymenolepididae), a parasite of the woodcock Scolopax rusticola (L.), and its life-cycle.

Aploparaksis kornyushini n. sp. is described from a woodcock Scolopax rusticola L. from Lithuania, Russia (Tver' Region) and the Ukraine. Initially, one specimen of this tapeworm was described and figured by Kornyushin (1975) as A. scolopacis Yamaguti, 1935 together with another specimens belonging to the latter species. A. kornyushini n. sp. and A. scolopacis are morphologically very similar species. They can be distinguished by the slightly different length of the rostellar hooks and by the shape of the cirrus, which lacks basal bulbus in the new species. A. kornyushini can be readily distinguished from the remaining species of Aploparaksis Clerc, 1903 from woodcocks by the structure of its fully-developed embryophore, which has polar thickenings and two large or a few smaller lateral projection; this combination of characters is unknown for embryophores other Aploparaksis spp. (except for A. scolopacis). The life-cycle of A. kornyushini was studied under experimental conditions in Lithuania. The metacestodes were located under the chlorogogenous tissue of the intestine of Dendrobaena octaedra (Lumbricidae). The metacestode exhibits a pattern of postembryonal development typical for the cysticercoid modification termed an 'ovoid diplocyst'.

Development and reproduction of Stratiolaelaps scimitus (Acari: Laelapidae) with fungus gnat larvae (Diptera: Sciaridae), potworms (Oligochaeta: Enchytraeidae) or Sancassania aff. sphaerogaster (Acari: Acaridae) as the sole food source.

Stratiolaelaps (=Hypoaspis) miles (Berlese) (Acari: Mesostigmata: Laelapidae) is a polyphagous soil-dwelling predatory mite that is widely marketed for use in greenhouse production systems to manage populations of dark-winged fungus gnats, Bradysia spp. (Diptera: Sciaridae) and for supplemental control of thrips. The suggestion by Walter and Campbell (2003, Biol. Control 26: 253-269) that North American commercial cultures of S. miles may actually be S. scimitus was confirmed. The development and reproduction at 21-23 degrees C of S. scimitus provided ad libidum with one of three different prey--the fungus gnat Bradysia aff. coprophila (Lintner), potworms (Enchytraeidae), or Sancassania aff. sphaerogaster (Zachvatkin, 1937) (Acari: Astigmata: Acaridae)--were compared. Developmental duration of the egg and non-feeding larval stages were 2.47 and 1.11 days, respectively; mortalities were 8.3 and 5.5%. Stratiolaelaps scimitus failed to develop beyond the protonymphal stage when provided with S. aff. sphaerogaster alone, although some feeding was observed. Development and reproduction of S. scimitus was successful on both fungus gnat larvae and enchytraeids, with no influence of prey on protonymphal duration (4.70 days) and mortality (8.3%), or on deutonymphal duration (4.61 days) and mortality (6.1%). Adult female S. scimitus feeding on potworms, compared to those feeding on fungus gnat larvae, had a significantly shorter pre-oviposition period (2.69 vs. 4.59 days). However, diet did not influence other adult female developmental or reproductive characteristics (oviposition period, 18.6 days; post-oviposition period, 6.2 days; total adult longevity, 27.3 days; total number of eggs, 26.5). S. scimitus reared on potworms tended (p = 0.06) to have a higher intrinsic rate of increase, a higher finite rate of increase and a shorter doubling time (rm = 0.142 day(-1), lambda = 1.153, Dt = 4.85 days) than those reared on fungus gnat larvae (rm = 0.105 day(-1), lambda = 1.110, Dt = 6.58 days), but differences in net reproductive rate (R0) and generation time (G) were not significant.

Aploparaksis demshini n. sp. (Cestoda: Hymenolepididae), a parasite of the woodcock Scolopax rusticola Linnaeus, and its life-cycle.

Aploparaksis demshini n. sp. is described from a woodcock Scolopax rusticola L. from different parts of the Palaearctic (Lithuania, Karelia, the Urals, Primorskiy Kray). It differs from the most similar species A. belopolskajae Bondarenko, 1988, a parasite of snipes Gallinago spp., in the form and length of the rostellar hooks and the smaller cirrus, and from two other similar species, A. clavata Spasskaya, 1966 and A. schilleri Webster, 1955, by having an embryophore with polar thickenings and a spindle-shaped cirrus. The life-cycle of the parasite was studied under experimental conditions. The metacestodes were commonly located under the chlorogogenous tissue of the intestine of the earthworms Eisenia foetida(Savigny), Dendrobaena octaedra (Savigny) and E. nordenskioldi(Eisen), and in the wall of the intestine of the enchytraeid Briodrilus arcticus(Bell). The metacestodes exhibit a pattern of postembryonal development typical for the cysticercoid modification termed an 'ovoid diplocyst'.

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.

Evaluation of an innate immune reaction to parasites in earthworms.

Encapsulation is an essential process of the invertebrate immune system and includes the prophenoloxidase (proPO) cascade. We present an assay for evaluating this immune response, now newly adapted to earthworms. Coelomic fluid is withdrawn and coelomocytes are stained with l-Dopa. We studied assay repeatability and the correlation between number of PO-active cells and infection level of the parasitic protozoan Monocystis sp. in the earthworm Lumbricus terrestris. Our study showed high assay repeatability although the expected negative relationship between PO-active coelomocytes and parasite load was not observed; yet a suggestion toward a positive relationship was detected. This finding is contrary to previous assumptions that presume coelomocyte concentrations to be the independent variable determining parasite load.

Trypanosoma cruzi is lysed by coelomic cytolytic factor-1, an invertebrate analogue of tumor necrosis factor, and induces phenoloxidase activity in the coelomic fluid of Eisenia foetida foetida.

A cytolytic protein named Coelomic Cytolytic Factor-1 (CCF-1) was isolated from the coelomic fluid of the earthworm Eisenia foetida foetida. Despite the absence of any gene homology, CCF-1 showed functional analogy with the mammalian cytokine tumour necrosis factor (TNF), particularly based on similar lectin-like activity. Indeed, both CCF-1 and TNF recognise N,N'-diacetylchitobiose and exert lytic activity on African Trypanosoma brucei brucei. In this report, we show that South-American Trypanosoma cruzi trypomastigotes, but not epimastigotes, were lysed by earthworm coelomic fluid or purified CCF-1. However, T. cruzi was less susceptible to lysis than T. brucei brucei. This lytic effect of coelomic fluid and CCF-1 on T. cruzi trypomastigotes was partially inhibited in the presence of anti-CCF-1 monoclonal antibody, antibody neutralising the lectin-like activity of TNF or N,N'-diacetylchitobiose. In contrast, this lytic effect was completely inhibited when using T. b. brucei. In addition, T. cruzi components, upon recognition by CCF-1 in E. f. foetida coelomic fluid, triggered the prophenoloxidase cascade, an invertebrate defence mechanism. These results further extend the functional analogies of CCF-1 and TNF, suggesting that both molecules share a similar lectin-like activity that has been conserved as an innate recognition mechanism in invertebrates and vertebrates. They also establish a link between stercorarian (T. cruzi) and salivarian (T. brucei) trypanosomatids having divergent phylogenetic origins and patterns of evolution, but possessing closely related cell surface sugar moieties.

Electron microscopic study of a new microsporean Microsporidium epithelialis sp. n. infecting Tubifex sp. (Oligochaeta).

The cytology of a new microsporean parasite Microsporidium epithelialis sp. n. from the intestinal epithelial cells of the freshwater oligochaete Tubifex sp. (Tubificidae) is described. The microsporean occurred together with an actinosporean of the genus Triactinomyxon, which was found between the epithelial cells. The merogonic and sporogonic stages (mature spores included) of the microsporean parasite are monokaryotic. An individual sporophorous vesicle surrounds each spore. The fixed and stained spore has an average dimension of 1.9-2.5 x 0.9-1.2 microm. The spores are oval with a characteristic surface layer, showing ornamentation-like projections, which are in close contact to the exospore. A short polar filament forming three to four coils traverses the polaroplast with two lamellar layers. The ultrastructure and other characteristic features of this microsporean parasite are distinct from those of the microsporean species described so far from oligochaetes.

Envelope protein of parasitic wasp symbiont virus, polydnavirus, protects the wasp eggs from cellular immune reactions by the host insect.

Cotesia kariyai polydnavirus (CkPDV) virions are present in the oviducts of C. kariyai wasp and are injected with eggs into the hemocoel of the host armyworm Pseudaletia separata larvae during parasitization. Evidence that the presence of polydnavirus particles on the surface of the wasp eggs may be essential for prevention of cellular immune reactions by the host hemocytes was obtained by isolating an immunoevasive factor from CkPDV virions. The purified proteinaceous factor protects foreign materials from adhesion and encapsulation by hemocytes of the host P. separata larvae but not by those of common cutworm Spodoptera litura larvae which is an incompatible host for the C. kariyai wasp. Purification procedures consisted of extraction with ethanol/trifluoroacetic acid and reverse-phase high performance liquid chromatography. A factor with a molecular mass of approximately 50 kDa is demonstrated to be present on the envelope of CkPDV virion by immunoelectronmicroscopic observations. Furthermore, immunoreactive proteins are found in plasma of the armyworm larvae but not in the common cutworm larvae, indicating that only the natural host of C. kariyai wasp shares a similar epitope with CkPDV. The sequence of 23 amino acid residues at the amino terminus of the factor was determined to be Ile-Ser-Val-Glu-Asn-Val-Xaa-Thr-Thr-Gly-Ile-Phe-Leu-Asp-Ser-Gly-Glu-Xaa- Val- Pro-Tyr-Ala-Thr-Lys-Pro.

[Histopathologic study of the host-parasite relationship: the earthworm-, wild boar-metastrongyle model]. / Etude histo-pathologique de la relation hôtes-parasite: le modèle lombric, sanglier-métastrongles.

A histopathological study was conducted on two hosts of metastrongyles (Metastrongylus sp.): earthworms and wild boars (Sus scrofa Linnaeus, 1758). In the earthworm intermediate host there were rare granulomas outside the normal site occupied by larvae (blood sinuses of calciferous glands). Given the small number of these reactions, they could not constitute a limiting factor for parasitism in this host. However, the lungs of wild boars contained numerous lesions associated with the presence of these nematodes. Such lesions, often inflammatory, were part of an immunological defence mechanism which controls the extent of parasitism in wild boar.

The role of tubificid worms as an intermediate host in the life cycle of Myxobolus pavlovskii (Akhmerov, 1954).

Myxobolus pavlovskii from the gills of silver carp (Hypophthalmichthys molitrix) were used in attempts to transmit the infection under laboratory conditions. Spores of M. pavlovskii were placed in aquaria that contained sterilized sand and had been filled with tap water at 15-16 degrees C. Furthermore, oligochaetes (90% Tubifex tubifex) were added and examined by wet mounts and histology. Hexactinomyxon spores developed after about 3 months only in T. tubifex that had been exposed to M. pavlovskii. Myxosporea-free silver carp developed cysts in their gills that contained M. pavlovskii at 120 days after contact with Hexactinomyxon spores, proving that M. pavlovskii also needs an oligochaete intermediate host for its development.

The possible role of the soil fauna in the epizootiology of cysticercosis in cattle. I. Earthworms -- the biotic factor in a transmission of Taenia saginata eggs.

The role of earthworms in the transmission of Taenia saginata eggs was determined by inoculating 30 earthworms directly: lumbricus terrestris, Allolobophora caliginosa and Eisenia foetida with tapeworm eggs. The remaining earthworms (280 specimens) were examined by contaminating their medium indirectly with T. saginata eggs or segments. It has been concluded that the eggs of tapeworms may be transmitted by the digestive tracts of earthworms. It seems that in the natural environment earthworms may contribute to the horizontal and the vertical spreading eggs in soil and the same may play an important role in the epizootiology of cysticercosis in cattle.