Portals of entry and systemic localization of proliferative gill disease organisms in channel catfish Ictalurus punctatus

Immunized rabbit serum adsorbed in live catfish was used in indirect fluorescent antibody test (IFAT) to detect developmental life stages of Henneguya ictaluri n. sp. This myxozoan parasite is associated with proliferative gill disease in channel catfish Ictalurus punctatus (Rafinesque) in the USA. Specific pathogen free fingerlings were experimentally infected with the actinosporean stage of H. ictaluri and necropsied 24, 48, 72, and 96 h post-infection. At 24 h post-infection parasite stages were observed primarily in the gastric mucosa and submucosa but were also observed in the skin and buccal cavity. Ovoid organisms were detected in heart and blood vessels of the liver. From 48 to 72 h after exposure, fewer fluorescent organisms were located in all organs, with the exception of the gills, than were observed at 24 h. These organisms appeared to be degenerating except for those in the gills, which appeared to be multinucleated. By 96 h post-infection, the organisms could not be detected in fish tissues with the exception of the stages in the gills, which appeared to be a preferred site of development. Throughout the entire 96 h period of study, no stage of the organism was detected in the brain. Infected tissue sections treated with non-immune rabbit serum and non-infected tissue sections treated with immune rabbit sera all showed negative results by IFAT.     

Five New Species of Henneguya (Protozoa: Myxosporida) from Ictalurid Fishes*

SYNOPSIS. Four new species of Henneguya (myxosporidan) are described from Ictalurus punctatus Rafinesque (channel catfish). They are as follows: Henneguya postexilis sp. n. and Henneguya longicauda sp. n. from the gills; Henneguya adiposa sp. n. from the adipose fin; and Henneguya diversis sp. n. from the liver, kidney, connective tissue of muscles and fins, and tumor-like external growths. Henneguya pellis sp. n. is described from the dermis of Ictalurus furcatus (LeSueur) (blue catfish). The development stages of all described species are discussed. Henneguya exilis Kudo was found on the gills of one I. punctatus; notes on its spore characteristics are presented.     

Small Subunit Ribosomal RNA Sequences Unite Alternate Actinosporean and Myxosporean Stages of Myxobolus cerebralis the Causative Agent of Whirling Disease in Salmonid Fish

ABSTRACT. The alternating myxosporean and actinosporean stages of the myxozoan parasitc Myxobolus cerebralis (Hofer 1903) from its salmonid fish and aquatic oligochaete hosts, respectively, were compared for sequence homology of the small subunit (18S) ribosomal RNA genes. A 99.8% similarity between the sequences of these two stages was substantially greater than that of M. cerebralis compared to two other Myxobolus sp. from salmonid fish. Our results are the first molecular evidence confirming the alternating stages initially described by Wolf and Markiw [25] for the life cycle of M. cerebralis but found in two different taxonomic classes (Myxosporea and Actinosporea) are indeed forms of the same organism. Sequencing of rRNA genes of the actinosporean stage followed by development of specific primers for DNA amplification of the myxosporean stage, as in our study, should be applied to solve other myxozoan life cycles. Additionally, these approaches will in the future provide useful diagnostic reagents for the detection and study of this important group of fish pathogens.     

Comparative study of ultrastructure of interlamellar and intralamellar types of Henneguya exilis Kudo from channel catfish

The inter- and intralamellar types of Henneguya exilis Kudo (Myxosporida) infections from channel catfish are similar in spore structure and sporogenesis, but differ in the structure of their plasmodium wall and surface coat and in their relationship with the host cells. The 2 clinical types differ also in the sites of development and growth patterns of plasmodia within a gill filament. Interlamellar plasmodia are limited by 2 outer unit membranes which give rise to both single-and double-membraned pincytic canals. Intralamellar plasmodia are limited by a single outer unit membrane which gives rise to single-membraned pinocytic canals. Interlamellar plasmodia are covered by a fine granular coat of highly variable thicknesses; in some regions there is direct contact between the parasite and cells of the host. There is some evidence that host cell cytoplasm as well as interstitial material are taken in by interlamellar plasmodia. In contrast, intralamellar plasmodia are covered by a fine granular coat of almost uniform thickness, which prevents direct contact between the parasite and cells of the host; probably only interstitial material is taken by these plasmodia.     

放射孢子虫在中国的首次发现

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Henneguya polarislonga n. sp. (Cnidaria: Myxosporea) parasitizing Astyanax lacustris (Lütken, 1875) with an insight on its life cycle

Henneguya polarislonga n. sp. is described from the gills of Astyanax lacustris (Lütken, 1875) (Characiformes, Characidae) from streams of the Middle Paranapanema River, Upper Paraná River basin, in the São Paulo State, Brazil. The proposed new species is supported by a combination of morphological and molecular characterization (partial fragment of the SSU rDNA). Thirty specimens of A. lacustris were analyzed and myxospores were found in the gill lamellae of one specimen (Prevalence = 3.3%). Henneguya polarislonga n. sp. was morphometrically and genetically distinct from other Henneguya spp. previously described in Astyanax spp., mainly differing in the total length and caudal appendages length of the mature myxospores. Comparative analysis of the SSU rDNA sequences revealed that Seisactinomyxon-type actinospores previously reported parasitizing oligochaetes are developmental stages of the life cycle of Henneguya polarislonga n. sp. (similarity of 99.9%). This is the first time that an actinospore and a myxospore are correlated through molecular analysis in Brazil, contributing to the knowledge of the myxozoan ecology and biodiversity.     

Morphology and 18S rDNA of Henneguya gurlei (Myxosporea) from Ameiurus nebulosus (Siluriformes) in North Carolina

Henneguya gurlei was isolated from Ameiurus nebulosus captured in North Carolina and redescribed using critical morphological features and 18S small-subunit ribosomal RNA (SSU rDNA) gene sequence. Plasmodia are white, spherical, or subspherical, occur in clusters, measure up to 1.8 mm in length, and are located on the dorsal, pectoral, and anal fins. Histologically, plasmodia are located in the dermis and subdermally, and the larger cysts disrupt the melanocyte pigment layer. The spore body is lanceolate, 18.2 +/- 0.3 microm (range 15.7-20.3) in length, and 5.4 +/- 0.1 microm (range 3.8-6.1) in width in valvular view. The caudal appendages are 41.1 +/- 1.1 microm (range 34.0-49.7) in length. Polar capsules are pyriform and of unequal size. The longer polar capsule measures 6.2 +/- 0.1 microm (range 5.48-7.06), while the shorter is 5.7 +/- 0.1 microm (range 4.8-6.4) in length. Polar capsule width is 1.2 +/- 0.03 microm (range 1.0-1.54). The total length of the spore is 60.9 +/- 1.2 microm (range 48.7-68.5). Morphologically, this species is similar to other species of Henneguya that are known to infect ictalurids. Based on SSU rDNA sequences, this species is most closely related to H. exilis and H. ictaluri, which infect Ictalurus punctatus.     

Hungactinomyxon,a new actinosporean type and collective group(Myxozoa) from <Emphasis Type="Italic">Branchiura sowerbyi</Emphasis> Beddard (Oligochaeta)

Actinosporeans are characterised by great morphological diversity. As it has been proved that the class Actinosporea is a synonym of the Myxosporea, actinosporean genera have only been regarded only as actinosporean collective groups. While most actinosporeans are released individually by their oligochaete hosts, members of the synactinomyxon, siedleckiella and antonactinomyxon collective groups are released as eight connected structural elements. These actinosporean types are differentiated by the type of unit and junction of the caudal processes. On the basis of these characteristics, a new actinosporean type, constructed from eight echinactinomyxon units, is described as hungactinomyxon. Adjacent units are joined by two of their three processes and form two interconnected cubes, each containing four echinactinomyxons. Molecular biological studies also suggest that this new actinosporean type differs from other actinosporean types built up from eight structural elements for which 18S rDNA sequences are available in GenBank.     

Actinosporean infections in oligochaetes in a river system in southwest Ireland with descriptions of three new forms

Sediment samples were collected in April, June and August 1998 from a river in southwest Ireland. A total of 1,836 oligochaete specimens were collected from these sediments, and examined for actinosporean infections. Actinosporean-infected oligochaetes have been determined, symptoms and course of infection have been considered and the spores released from them have been described. Spore characteristics for each actinosporean were defined and the prevalence of infection in each oligochaete species was also determined. The overall prevalence of infection with actinosporeans within the oligochaete sample was approximately 2%. Seven types were distinguished, belonging to echinactinomyxon, aurantiactinomyxon and neoactinomyxum forms, from Tubifex ignotus, Lumbriculus variegatus and Limnodrilus hoffmeisteri, and compared with actinosporean types previously reported in the literature. Three of these forms have not been previously described, while the other 4 are morphologically and morphometrically similar to known forms. These actinosporeans may be stages in myxozoan life cycles.     

Postembryonic development, fecundity and food consumption of Dichroplus exilis (Orthoptera: Acrididae) under controlled conditions

Dichroplus exilis is a widely distributed species in Southern South America. Although there have been reports of D. exilis as an agricultural pest, some recent observations suggest that the damage attributed to D. elongatus may actually have been caused by D. exilis. This study was conducted to determine the postembryonic life cycle stages, fertility and food consumption of this species under controlled conditions (30 degrees C, 14L-10D, 40% RH). Individuals employed belong to the laboratory-hatched first generation (F1), from adults (n = 64, female = 28, male = 36) collected in natural grasslands near Rafaela, Santa Fe province in North-Eastern Argentina. Three cohorts of 16, 17 and 20 individuals were monitored independently in acetate tubes on a daily basis, until death of the last insect. Average fecundity was 381.84, 38.54 eggs per female. Egg-pod incubation time was 14.4, 1.08 days and six nymphal instars were recorded. Nymphal development time was 41.38, 0.71 days (I = 8.73, 0.20; II = 6.38, 0.24; III = 5.64, 0.33; IV = 7.15; 0.43; V=9.76, 0.54; IV = 7.85, 0.95). The recorded food consumption was 9.89, 1.08 (mg/ind/day) for nymphs IV, 18.04, 0.73 (mg/ind/day) for nymphs V-IV, 16.76, 1.06 (mg/ind/day) for pre-reproductive males, 28.09, 1.81 (mg/ind/day) for pre-reproductive females, 7.71,0.91 (mg/ind/day) for reproductive males and 13.06, 0.71 (mg/ind/day) for reproductive females, while the average adult food consumption, regardless of sex and reproductive status, was 16.41, 4.32 mg/day. Average food consumption of adult females was 17.47, 1.15 mg, and was significantly higher than that of males (10.83, 0.91mg). Data obtained in this study showed that D. exilis exhibits at least some of the biological attributes needed to configure an actual or potential agricultural pest, albeit not yet recognized as such. Field monitoring of grasshopper communities in areas where damage by D. exilis is suspected is envisaged in order to determine its possible status as a pest.     

Parasites of native and nonnative fishes of the Little Colorado River, Grand Canyon, Arizona

A 2-yr, seasonal, parasitological study of 1,435 fish, belonging to 4 species of native fishes and 7 species of nonnative fishes from the lower Little Colorado River (LCR) and tributary creeks, Grand Canyon, Arizona, yielded 17 species of parasites. These comprised 1 myxozoan (Henneguya exilis), 2 copepods (Ergasilus arthrosis and Lernaea cyprinacea), 1 acarine (Oribatida gen. sp.), 1 piscicolid leech (Myzobdella lugubris), 4 monogeneans (Gyrodactylus hoffmani, Gyrodactylus sp., Dactylogyrus extensus, and Ligictaluridus floridanus), 4 nematodes (Contracaecum sp., Eustrongylides sp., Rhabdochona sp., and Truttaedacnitis truttae), 3 cestodes (Bothriocephalus acheilognathi, Corallobothrium fimbriatum, and Megathylacoides giganteum), and 2 trematodes (Ornithodiplostomum sp. and Posthodiplostomum sp.). Rhabdochona sp. was the only adult parasite native to the LCR. Infection intensities of Ornithodiplostomum sp. and B. acheilognathi were positively correlated with length of the humpback chub Gila cypha. Adult helminths showed a high degree of host specificity, except B. acheilognathi, which was recovered from all fish species examined but was most abundant in cyprinids. Abundance of B. acheilognathi in the humpback chub was highest in the fall and lowest in the summer in both reaches of the LCR. There was no major taxonomic difference in parasite assemblages between the 2 different reaches of the river (LC1 and LC2). Parasite community diversity was very similar in humpback chub, regardless of sampling site or time. The parasite fauna of the LCR is numerically dominated by B. acheilognathi and metacercariae of Ornithodiplostomum sp. The richest and most diverse component community occurred in a nonnative species, the channel catfish Ictalurus punctatus, but infracommunity species richness was highest in a native host, humpback chub.     

Molecular characterization and ornithine-urea cycle genes expression in air-breathing magur catfish (Clarias magur) during exposure to high external ammonia

The air-breathing magur catfish (Clarias magur) is a potential ureogenic teleost because of its functional ornithine-urea cycle (OUC), unlike typical freshwater teleosts. The ability to convert ammonia waste to urea was a significant step towards land-based life forms from aquatic predecessors. Here we investigated the molecular characterization of some OUC genes and the molecular basis of stimulation of ureogenesis via the OUC in magur catfish. The deduced amino acid sequences from the complete cDNA coding sequences of ornithine transcarbamyolase, argininosuccinate synthase, and argininosuccinate lyase indicated that phylogenetically magur catfish is very close to other ureogenic catfishes. Ammonia exposure led to a significant induction of major OUC genes and the gene products in hepatic and in certain non-hepatic tissues of magur catfish. Hence, it is reasonable to assume that the induction of ureogenesis in magur catfish under hyper-ammonia stress is mediated through the activation of OUC genes as an adaptational strategy.     

Molecular relationships and phylogeny in a community of myxosporeans and actinosporeans based on their 18S rDNA sequences

The community of myxosporeans and actinosporeans inhabiting a typical Scottish highland stream and the outflow area of an adjacent salmon hatchery was analysed on the basis of their 18S rDNA sequences. Nine myxosporeans belonging to the genera Sphaerospora, Chloromyxum, Zschokkella, Myxidium, Hoferellus and Myxobilatus were identified from mature spores in different organs of the fish species present. Twelve actinosporean types belonging to the collective groups of neoactinomyxum, aurantiactinomyxon, raabeia, echinactinomyxon and synactinomyxon were found to be released from oligochaete worms collected from sediments. Twenty of the 21 sequences obtained from these myxozoans are new entries to the myxozoan database, and the genera Chloromyxum, Hoferellus and Myxobilatus were entered for the first time. Study of the molecular relationships between the different taxa and with other myxozoan sequences available showed that the myxosporeans inhabiting the urinary system clearly cluster together, independently of host species or spore morphology. However, the sequences of the two Sphaerospora species encountered show considerable differences from other members of this group and all other freshwater myxosporeans, and they were found to occupy an ancestral marine position. Three actinosporeans, i.e. Neoactinomyxum eiseniellae, Aurantiactinomyxon pavinsis and Raabeia 'type 3' were found to represent alternate life cycle stages of Chloromyxum sp., Chloromyxum truttae and Myxidium truttae, respectively (approximately 1400 identical base pairs each). Three other actinosporeans encountered (two echinactinomyxon and one raabeia type) showed over 92% sequence identity with myxosporeans from GenBank, whereas all other actinosporeans formed a closely related group devoid of any known myxosporeans.     

Cladistic analysis of myxozoan species with known alternating life-cycles

The phylogenetic relationships of 15 myxozoan taxa with known alternating life-cycles were investigated in order to provide insight into the puzzling matches between myxosporeans and actinosporeans of the myxozoan life-cycle data. Phylogenetic analyses were performed using two partitioned data-sets of life-cycle stages, myxosporean stage from fish hosts versus actinosporean stage from annelid hosts, and a combined data-set of myxosporean and actinosporean stages. A cnidarian parasite of fish, Polypodium hydriforme Ussov, 1885, was used as the outgroup. The supraspecific level grouping in the conventional classification of actinosporeans was not supported in the analysis of the partitioned data from the actinosporean phase, which yielded two equally parsimonious trees. Analysis of the partitioned data from the myxosporean phase provided 24 equally parsimonious trees and did not support the current classification of myxosporeans. The analyses of the partitioned data of myxozoans by life-cycle stage revealed a lack of taxonomic congruence between the two life-stage partitions. Two equally parsimonious trees were obtained from analysis of the combined data. The suborder Variisporina of the Myxozoa was not supported by the total evidence trees, while the monophyly of the species of Myxobolus Butschli, 1882 and of the Myxidiidae were supported. The cladograms from the combined data revealed that these myxozoan species formed four major monophyletic groups. Among them, two were supported by the partitioned data of the actinosporean phase. The phylogenetic signals and the better resolution reflected by the trees of combined data suggest that the phylogenetic total evidence approach should be employed in future studies of the systematics of myxozoans.     

Henneguya adiposa Minchew (Myxosporida) in the channel catfish: ultrastructure of the plasmodium wall and sporogenesis.

Wall ultrastructure and sporogenesis were studied in plasmodia of Henneguya adiposa Minchew which infects the channel catfish, Ictalurus punctatus (Rafinesque). Plasmodia were located among connective tissue bands of the adipose fin and were always separated from host fibrocytes by collagen fibers. The plasmodium wall consisted of a single unit membrane which was continuous with numerous pinocytic canals extending into the parasite's ectoplasm. The membrane was highly convoluted, producing an irregular parasite surface, and was covered by a fine granular coat of almost uniform thickness. Early sporogenic stages were located in a zone of cytoplasm rich in mitochondria, just interior to the zone of pinocytic canals. Later sporogenic stages, including mature spores, were concentrated in the center of the plasmodia. Sporogenesis began with the envelopment of one generative cell, the sporont, by a 2nd, nondividing, cell--the enveloping cell. The sporont and its progeny proceeded through a series of divisions until 10 cells were present within the enveloping cell. Once divisions were completed, the 10 cells became arranged into 2 indentical spore-producing units, each consisting of one binucleate sporoplasm and 2 capsulogenic cells, all surrounded by 2 valvogenic cells. Later stages of spore development indicated that capsulogenesis, valvogenesis and sporoplasm maturation occurred concimitantly.     

Henneguya adiposa Minchew (Myxosporida) in the Channel Catfish: Ultrastructure of the Plasmodium Wall and Sporogenesis*

Wall ultrastructure and sporogenesis were studied in plasmodia of Henneguya adiposa Minchew which infects the channel catfish, Ictalurus punctatus (Rafinesque). Plasmodia were located among connective tissue bands of the adipose fin and were always separated from host fibrocytes by collagen fibers. The plasmodium wall consisted of a single unit membrane which was continuous with numerous pinocytic canals extending into the parasite's ectoplasm. The membrane was highly convoluted, producing an irregular parasite surface, and was covered by a fine granular coat of almost uniform thickness. Early sporogenic stages were located in a zone of cytoplasm rich in mitochondria, just interior to the zone of pinocytic canals. Later sporogenic stages, including mature spores, were concentrated in the center of the plasmodia. Sporogenesis began with the envelopment of one generative cell, the sporont, by a 2nd, nondividing, cell—the enveloping cell. The sporont and its progeny proceeded through a series of divisions until 10 cells were present within the enveloping cell. Once divisions were completed, the 10 cells became arranged into 2 identical spore-producing units, each consisting of one binucleate sporoplasm and 2 capsulogenic cells, all surrounded by 2 valvogenic cells. Later stages of spore development indicated that capsulogenesis, valvogenesis and sporoplasm maturation occurred concomitantly.     

Histopathological changes in fish gills infected with myxosporidian parasites of the genus Henneguya

Tissue reaction to invasion by two Henneguya species was studied in gills of three species of fish. Henneguya psorospermica and Henneguya creplini develop in secondary lamellar arterioles in perch ( perca fluviatilis ) and ruff ( Acerina cernua). In pike ( Esox lucius), H. psorospermica develops in the artery. During the growth of the vegetative stage, the gill tissue may undergo local atrophy or hyperplasia of the epithelium and displacement and disfiguration of the lamellae. After the myxosporidian cyst, surrounded by a host tissue capsule has reached maturity and contains fully-formed spores, an inflammatory reaction may set in which reduces the cyst to granulomatous tissue. This inflammatory process is essentially of the same type as that observed in mammals. There is evidence that inflammatory responses are of regular occurrence in the final stages of most myxosporidian infections. Transfer from low to room temperature provokes a rapid elimination of the cysts by granulomatous inflammation.