Laboratory and Field Studies on Glugea stephani (Hagenmuller), a Microsporidan Parasite of Pleuronectid Flatfishes*

SYNOPSIS. The microsporidan Glugea stephani is a common parasite of juvenile English sole (Parophrys vetulus) in Yaquina Bay, Oregon. Field observations indicated that fish became infected only in the upper estuary where summer temperatures were above 15 C and the incidence of infection reached 79.8% in the late fall. Laboratory infections developed and parasite growth occurred only at or above 15 C. The parasite was successfully transmitted to juvenile English sole by brine shrimp (Artemia salina) and amphipod (Corophium spinicorne) vectors as well as by direct ingestion of spores by the host. Infections that resulted from ingestion of spore-carrying vectors were much heavier than those resulting from the direct ingestion of spores. The speckled sanddab (Citharichthys stigmaeus), a nonpleuronectid flatfish, and chum salmon (Oncorhynchus keta) were refractory to G. stephani infection in the laboratory. Results of this study suggest that G. stephani is potentially lethal to young pleuronectid flatfishes when heavy infections involve the entire intestine and reduce the capacity to absorb nutrients. Under these circumstances, starvation is probably the direct or indirect cause of death. The restriction of infection to fish that reach the upper estuary very likely mitigates the impact of G. stephani caused mortality on the entire English sole population on the Yaquina Bay nursery ground.     

Effects of low temperature on the development of the microsporidan Glugea stephani in English sole (Parophrys vetulus)

Glugea stephani requires temperatures above 15 C for development in juvenile pleuronectid flatfishes in Yaquina Bay, Oregon. The effect of low temperature (10 C) on the development of recently established parasites was tested experimentally in juvenile English sole (Parophrys vetulus). Low temperature arrested parasite development, but did not kill the protozoan which resumed development on return to 19-20 C after as long as 42 days at 10 C. No parasites detectable with the light microscope were found in fish examined after 70 days at 10 C. Although most juvenile English sole move permanently from the estuary to cooler ocean waters in fall and do not contribute to the continuation of the parasite life cycle, the cycle may be maintained by low numbers of English sole that overwinter in the estuary.     

Parasitology of the English Sole, Parophrys vetulus Girard in Oregon, U.S.A.

A total of 877 juvenile English sole ( Parophrys vetulus Girard) from the Yaquina Bay estuary and742 juvenileandadultsole from the Pacific Ocean off Oregon were examined forparasites. Fifteen species of parasites were found in juvenile English sole on the estuarine nursery ground. Differences in the prevalence and intensity of parasite infection between size classes of juvenile sole and between sole occupying the upper and lower estuary were determined. An additional 14 parasite species were found in offshore English sole, bringing the total observed in all fish to 29 species. Parasites acquired only in the estuary included the microsporidan Glugea stephani , the acanthocephalan Echinorhynchus lageniformis , and the nematode Philometra americana . Those acquired only in offshore areas included the trematodes Otodistomum veliporum and Zoogonus dextrocirrus , the leech Oceanobdella sp. and three species of copepods. An attempt to use parasite data to indicate the presence of distinct English sole stocks along the Oregon coast was inconclusive.     

Temporal changes in the prevalence of parasites in two Oregon estuary-dwelling fishes

The parasite faunas of juvenile English sole (Parophrys vetulus) in 1971-1972 and staghorn sculpin (Leptocottus armatus) in 1971 from Yaquina Bay, Oregon, were compared with faunas found in the same estuary in 1997-2000 (English sole) and 1999-2000 (staghorn sculpin). The 7 most commonly occurring parasites in 1971 were compared with the same species observed during the same month and sampling sites in 1997-2000. Multivariate community analysis of juvenile English sole parasites supported the suggestion that the 1971 parasite data were representative of the early-1970s time period. Four of the parasite species infecting English sole and 6 of those infecting staghorn sculpins had significantly lower prevalences in 1997-2000. Parasite species with significantly lower prevalences also had reduced intensity levels. One parasite (Glugea stephani) of English sole increased in prevalence in the 1997-2000 samples in association with the warm estuarine temperatures during the 1997 El Ni?o year. Although the causes for the changes in occurrence of other parasites were not determined, ecological changes in Yaquina Bay that may have influenced parasite ecology include apparent changes in the estuary ichthyofauna that occurred between the sampling periods. Such changes could be associated with increases in the number of California sea lions (Zalophus californianus) subsequent to establishment of the Marine Mammal Protection Act in 1972.     

Infection of plaice Pleuronectes platessa L. with Glugea (Nosema) stephani (Hagenmüller 1899) (Protozoa: Microsporidia) in a fish farm and under experimental conditions

One tank of plaice in the W.F.A. Fish Cultivation Unit at Hunterston, known to habour an infection with the microsporidian Glugea stephani , was sampled over a 2 year period and showed a mean level of infection of 49.4 % with an apparent trend towards a decrease. Experimental oral transmission was achieved, without the use of intermediate or carrier hosts, and the dependence of G. stephani on high temperatures was demonstrated. Most fish were able to tolerate the infection, but some mortalities in young fish were attributed to the parasite, particularly in the 2 months following the experimental infection. The parasite in the fish farm is believed to have been introduced with plaice stocks imported from more southern waters.     

The occurrence of Glugea stephani (Hagenmuller, 1899) in American winter flounder, Pseudopleuronects americanus (Walbaum) from the New York-New Jersey lower bay complex

American winter flounder, Pseudopleuronectes americanus , Collected in the New York-New Jersey bay complex, contained cysts in the intestinal tract which were subsequently identified as the microsporidan Glugea stephani . These fish represent 13 collections taken at approximately monthly intervals over a one and a half year period. The winter flounder varied in size from 8 to 33 cm and consequently represented a fish population of diverse age. During this study, a total of 1840 apparently healthy fish were dissected and examined for the presence of Glugea cysts , with an overall parasite infection rate of 6.63%. There was no correlation between size of the fish and percentage of infection. This percentage represents the mean carrying rate of this parasite in the fish population, since these are the fish that have survived the initial infection. This report extends the reported host range to the New York-New Jersey area. As a further aid in the characterization of G. stephani , we have included ultrastructural details of the spore morphology.     

Experimental horizontal transmission of Enterocytozoon salmonis to chinook salmon, Oncorhynchus tshawytscha.

Enterocytozoon salmonis was transmitted to chinook salmon Oncorhynchus tshawytscha by feeding tissues infected with the parasite and by cohabitation of noninfected fish with experimentally infected fish. Affected fish (dead and survivors) in both transmission trials had gross and microscopic signs of the disease and merogonic and sporogonic stages of the parasite. There were no morbidities or mortalities, or evidence of the parasite among control fish in either study. Results suggest that the parasite may be contracted by indirect contact among healthy and infected fish held in crowded ponds or net pens or by direct ingestion of spores found in the water.     

Experimental Horizontal Transmission of Enterocytozoon salmonis to Chinook Salmon, Oncorhynchus tshawytscha

Enterocytozoon salmonis was transmitted to chinook salmon Oncorhynchus tshawytscha by feeding tissues infected with the parasite and by cohabitation of noninfected fish with experimentally infected fish. Affected fish (dead and survivors) in both transmission trials had gross and microscopic signs of the disease and merogonic and sporogonic stages of the parasite. There were no morbidities or mortalities, or evidence of the parasite among control fish in either study. Results suggest that the parasite may be contracted by indirect contact among healthy and infected fish held in crowded ponds or net pens or by direct ingestion of spores found in the water.     

Life History and Electron Microscopy of a Haplosporidian,Nephridiophaga blattellae (Crawley) n. comb., in the Malphigian Tubules of the German Cockroach,Blattella germanica (L.)*

SYNOPSIS. Rearing of cockroaches without sporozoan parasites of the Malpighian tubules permitted the 1st experimental infections reported for these organisms as well as study of the intracellular early stages of infection. Infectivity tests show that the route of invasion is peroral rather than transovarian; that fresh spores obtained from either the Malpighian tubules or the hindgut are infective; that fresh spores held in aqueous suspension for 36 hr are not infective; that all ages and both sexes of cockroaches are susceptible, although older cockroaches are more readily infected experimentally; that a single peroral dose of a few hundred spores is sufficient to produce infection; that the earliest signs of progressive infection in the Malpighian tubules appear 15 days after ingestion of spores; and that the infection, spread mainly by schizogony, is well established by 23 days. Spores are acid-fast. The PAS-positive spore membrane is not dissolved by 20-min treatment with hot KOH, but tests for chitin were inconclusive. The life history of the parasite is described. “Encapsulation” of the parasite has been observed. Electron microscope studies, the first reported for the class Haplosporea, show similarities of the cytoplasmic structure of this haplosporidian with that of some members of the sporozoan class Telosporea, though no specialized structures were seen. Finding of intracellular stages together with failure of interspecific infection attempts suggest that the microorganism should be placed in the genus Nephridiophaga Ivanic, 1937 with the specific name blattellae Crawley, 1905.     

Effect of water temperature on the development, release and survival of the triactinomyxon stage of Myxobolus cerebralis in its oligochaete host.

The development of the triactinomyxon stage of Myxobolus cerebralis and release of mature spores from Tubifex tubifex were shown to be temperature dependent. In the present work, the effect of temperature over a range of 5-30 degrees C on the development and release of the triactinomyxon stages of M. cerebralis was studied. Infected T. tubifex stopped releasing triactinomyxon spores 4 days after transfer from 15 degrees C to 25 degrees C or 30 degrees C. Transmission electron microscopic examinations of the tubificids held at 25 degrees C and 30 degrees C for 3 days showed that all developmental stages degenerated and transformed to electron-dense clusters between the gut epithelial cells of T. tubifex. In contrast, tubificid worms held at 5 degrees C and 10 degrees C examined at the same time were heavily infected with many early developmental stages of triactinomyxon. At 15 degrees C, the optimal temperature for development, maturing and mature stages of the parasite were evident. Infected T. tubifex transferred from 15 degrees C to 20 degrees C stopped producing triactinomyxon spores after 15 days. However, 15 days at 20 degrees C was not sufficient to destroy all developmental stages of the parasite. When the tubificid worms were returned to 15 degrees C, the one-cell stages and the binucleate-cell stages resumed normal growth. It was also demonstrated that T. tubifex cured of infection by holding at 30 degrees C for 3 weeks and shifted to 15 degrees C could be re-infected with M. cerebralis spores. The waterborne triactinomyxon spores of M. cerebralis did not appear to be as short-lived as previously reported. More than 60% of experimentally produced waterborne triactinomyxon spores survived and maintained their infectivity for rainbow trout for 15 days at water temperatures up to 15 degrees C. In natural aquatic systems, the triactinomyxon spores may survive and keep their infectivity for periods even longer than 15 days.     

Effect, distribution, and prevalence of Glugea stephani (Microspora) in winter flounder (Pleuronectes americanus) living near two pulp and paper mills in Newfoundland

A study was conducted to determine the effects, geographical distribution, and prevalence of a microsporan parasite, Glugea stephani, in winter flounder (Pleuronectes americanus) in Newfoundland. Fish were captured by SCUBA divers in several coastal areas, including 2 embayments where pulp and paper mill effluent was discharged, as well as a number of pristine sites. Fish health was assessed by comparing histological profiles, condition factors (K), organosomatic indices and blood values between infected and uninfected samples. Multifocal xenomas of G. stephani were observed in several organs of fish taken near contaminated sites, whereas infected samples captured at a pristine site harbored the cysts only in the wall of the digestive tract. Proliferative inflammation, granuloma formation, and focal necrosis were associated with the infection primarily in the liver and kidney. Condition factors and blood values were lower and ovarian development inhibited or delayed in infected flounder. The multifocal infection occurred only in flounder in 2 embayments in western Newfoundland where pulp and paper mill effluent was discharged. Prevalence varied seasonally, with a peak in autumn and a low in spring. It is likely that the multifocal infection was associated with immunodepression after exposure to the contaminant.     

Food stoichiometry affects the outcome of Daphnia–parasite interaction

Phosphorus (P) is an essential nutrient for growth in consumers. P‐limitation and parasite infection comprise one of the most common stressor pairs consumers confront in nature. We conducted a life‐table study using a Daphnia–microsporidian parasite model, feeding uninfected or infected Daphnia with either P‐sufficient or P‐limited algae, and assessed the impact of the two stressors on life‐history traits of the host. Both infection and P‐limitation negatively affected some life‐history traits tested. However, under P‐limitation, infected animals had higher juvenile growth rate as compared with uninfected animals. All P‐limited individuals died before maturation, regardless of infection. The numbers of spore clusters of the microsporidian parasite did not differ in P‐limited or P‐sufficient hosts. P‐limitation, but not infection, decreased body phosphorus content and ingestion rates of Daphnia tested in separate experiments. As parasite spore production did not suffer even under extreme P‐limitation, our results suggest that parasite was less limited by P than the host. We discuss possible interpretations concerning the stoichiometrical demands of parasite and suggest that our results are explained by parasite‐driven changes in carbon (C) allocation of the hosts. We conclude that the impact of nutrient starvation and parasite infection on consumers depends not only on the stoichiometric demands of host but also those of the parasite.     

Validation of a quantitative PCR diagnostic method for detection of the microsporidian Ovipleistophora ovariae in the cyprinid fish Notemigonus crysoleucas

Microsporidian parasites are easily detected by light microscopy when infections are heavy and spores are present. However, early infections without spores, or light infections with low numbers of spores, are easily missed. This limitation has made it difficult to conduct investigations into microsporidian prevalence and transmission. In this study, we developed a quantitative TaqMan polymerase chain reaction assay to assess the presence of Ovipleistophora ovariae in the tissues of the cyprinid fish Notemigonus crysoleucas (golden shiner). The efficiency of the primer set was 100.8%, with a correlation coefficient of threshold position to copy number of 0.997 over 9 logs using a plasmid containing the cloned reaction product. No product was produced from other closely related microsporidian species (Nucleospora salmonis, Pseudoloma neurophila, Glugea stephani, Heterosporis sp., and O. mirandella). The coefficient of variation for replicate assays done on different days was 12.4%. The assay detects O. ovariae reliably at less than 10 genomic copies and 0.14 spores per reaction, but maximum sensitivity is only achieved when sonication is included as part of the DNA purification step. Using the assay, we found 4.44 x 10(1) to 7.91 x 10(6) copies microg(-1) host DNA in female golden shiners, with the spore density increasing during the spawning season. The parasite was also detected for the first time in the testes of male golden shiners at 2.60 x 10(1) to 8.62 x 102 copies microg(-1) host DNA.     

Spatial variability in digenean metacercariae infection of 0-group common sole Solea solea among nurseries along the French Atlantic coast

We sampled 0-group sole juveniles (N = 174) in September and October 2003 in 9 major nurseries located along the French Atlantic coast (English Channel and Bay of Biscay). 0-group sole were infected with 3 genera of digenean metacercariae, Timoniella spp. (Acanthostomidae), Prosorhynchus crucibulum and Prosorhynchus sp. A (Bucephalidae), Podocotyle sp. (Opecoelidae), and an unidentified species of Digenea. Parasite infection levels in the English Channel nurseries were lower, and the community composition was different from Bay of Biscay nurseries. We hypothesize that the difference between geographic areas was due to differences in first intermediate host communities and, to a lesser extent, because of lower temperatures in the English Channel compared to the Bay of Biscay. For the Bay of Biscay nurseries, mean total parasite abundance was strongly negatively correlated with mean annual river flow. This relationship may be the result of the more upstream location of 0-group sole nurseries in estuaries so that fish were further from local points of parasite transmission than those in embayed nurseries. Digenean metacercariae load may be influenced by 3 major local factors, i.e. abundance and proximity of the first intermediate hosts and cercariae dispersal capacities.     

Nosema pyrausta infection in Macrocentrus grandii, a braconid parasite of the European corn borer, Ostrinia nubilalis

Macrocentrus grandii which develop within Nosema pyrausta-infected larvae of the European corn borer, Ostrinia nubilalis, develop direct systemic infections from the ingestion of spores at the time of larval emergence from the host. Infections adversely affect pupal development and adult longevity. Infected females are unable to transmit the microsporidian to additional corn borer hosts. Pathogen development in the parasite host appears identical to its development in the corn borer host and mature spores show no morphological differences in size or shape when observed at the ultrastructural level. The prevalence of infection in natural parasite populations is 53.8% and closely parallels the 56.7% prevalence of infection in corn borer populations. Results suggest N. pyrausta may play a significant role in limiting M. grandii populations when levels of N. pyrausta in corn borers are high.     

Experimental infection of American winter flounder (Pseudopleuronectes americanus) with Glugea stephani (Microsporida)

Young-of-the-year Pseudopleuronectes americanus were captured in Sandy Hook Bay, New Jersey and maintained in aquaria at 16–19° C for 115 days in 1982 and 127 days in 1983. Half of them were experimentally exposed to Glugea stephani and the others were sham exposed. During the course of these experiments fish from both groups died as a result of G. stephani infection, demonstrating Glwgea -induced mortality in this species. Infections were found in both control and experimentally exposed fish. Mortality of Glwgea -infected fish in the exposed group was 63% while that in the controls was 28.5%.     

Vertical transmission of Nosema fumiferanae (Microsporidia: Nosematidae) and consequences for distribution, post-diapause emergence and dispersal of second-instar larvae of the spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae)

We examined vertical transmission of Nosema fumiferanae in the eastern spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae), and how it affects overwintering distribution and survival and spring emergence and dispersal of second-instar larvae in outbreak populations. Females containing 5.0 x 10(5) spores or more consistently produced 100% infected progeny. Transmission efficiency was still 50% at burdens as low as 0.2 x 10(5) spores per moth. Infection intensity in offspring increased with maternal spore load but became highly variable above 25 x 10(5) spores per female. Nosema multiplied in second instars for at least 1 month after they entered dormancy, regardless of temperature (2 degrees C versus 21 degrees C). Infection did not affect the distribution of overwintering larvae in a white spruce canopy. Dormancy survival between late-summer and the following spring was lower in families from infected females and was negatively correlated with larval infection intensity. Infection delayed larval emergence from hibernacula in the spring and resulted in delayed dispersal of emerged larvae, at least when parasite prevalence and infection intensities were high. Infected larvae were less successful in establishing feeding sites after dispersal. Our results underscore the potential of Nosema infection to negatively affect processes early in the budworm life cycle.     

Microsporidiosis in the wax moth Galleria mellonella (Lepidoptera: Pyralidae) caused by Vairimorpha ephestiae (Microsporidia: Burenellidae)

An experimental microsporidiosis of the wax moth caterpillars from laboratory population had been caused by oral infecting of early stages larvae and by intracavity injections of the spores of the microsporidian species Vairimorpha ephestiae. Peculiarities of microsporidiosis proceeding, manifestations of host defence reactions, and also an effect of the temperature of caterpillars cultivation and conditions of spores keeping on liability of the insects to the infection were studied. The effect of the microsporidia on the host organism was the early death or the delay of larvae development, but in several cases external manifestations of the effect of the parasite on the host were absent. The development of the parasites from the moment of infecting to the appearing of the mature spores congestions in the host organism proceeded 6 days. Microsporidia invaded insect fat body and caused its hypertrophy and disappearance of lipid granules. In the intestine and salivary glands microsporidia were not observed in the period from 6 to 16 day of the development. On the final stage of microsporidiosis the all contents of fatty tissue cells were replaced by spores of microsporidia. Under microscope only diplocaryotic spores of the Nozema type had been found in infected and died specimens, but not octospores. The spores threw out polar tubes under the change of pH in incubating solution from neutral to alkaline. The effects of microsporidiosis on the wax moth haemolymph were the increased rate of prohaemocytes, appearing of multinuclear free-circulating cells at 6 day after infection, and suppression of the reaction of haemolymph melanization with the mass sporogenesis of the parasite. The characteristic symptom of the wax moth microsporidiosis had been revealed, accumulation of black points and small spots of irregular form under cuticle ("reaction of attretization"). Increase of the temperature of insect cultivation up to 32 degrees C during 3 days after infection contributed to the full deliverance of the insects from the infection in first and second generations. It can be considered as a method of treatment of wax moth laboratory colonies from microsporidiosis. Oral infection of III and IV stage caterpillars by the spores being kept during 3-6 months under 4 degrees C in form of water suspension caused the death of 63.0-61.5 and 91% of caterpillars being cultivated under 25 and 21 degrees C respectively. Under the temperature of cultivation equal 30 degrees C the mortality did not differ from the control sample (8-10%). The spores extracted from dried bodies of caterpillars lost their vitality. It was demonstrated by the test on infectious ability in vivo and by acridine orange staining. This host-parasite system appears to be perspective in investigations of resistance mechanisms in insects and immunosuppressive features of entomopathogen microsporidia.     

Seasonal prevalence of the microsporidan, Glugea stephani (Hagenmuller), in winter flounder, Pseudopleuronectes americanus (Walbaum), from the New York-New Jersey Lower Bay Complex

During a 32 month period 26 monthly collections of winter flounder were conducted from various locations in the New York-New Jersey Lower Bay Complex. A total of 3125 flounder were captured and examined for the presence of the microsporidan, Glugea stephani. Of the total number of fish collected, 260 (8.32%) were infected with the protozoan. At least one G. stephani infected flounder was captured and identified each month, which indicates that the infection is present on a year-round basis. The monthly infection prevalence ranged from 0.63 to 25%. Increased parasitism corresponded with elevated water temperatures. Fish size was not a statistically significant factor for infection.     

Dosage effect study of the microsporidian Octosporea muscaedomesticae in the adult black blowfly Phormia regina,with reference to the reproductive cycle of the pathogen

The quantitative pathogenicity of the microsporidian Octosporea muscaedomesticae in adult Phormia regina was studied. Dosage levels ranging from 10² to 10⁶ spores per fly were administered to five and six groups of newly emerged, starved adult flies in two trials. Rates of mortality and infection were determined. A direct relationship between number of spores ingested and subsequent infection rate was found in a 4-day trial while no such relationship was found in an 18-day trial, using the same source of inoculum and host flies from the same colony. The lack of a direct relationship between spore dose and rate of infection in the 18-day trial is explained on the basis of the short spore-to-spore development time of the parasite. New generations of spores formed within the host tissues obscure the results in relation to the spore dose initially administered. An appreciable number of spores in the inoculum is needed to initiate frank infection. The ID₅₀ (median infective dose) was 4.4 × 10⁴ spores per fly after 4 days.