Molecular identification and prevalence of plerocercoid larvae (Cestoda: Diphyllobothriidae) in some commercial fish species from Peru.

Diphyllobothriosis, a fish-borne zoonosis in South America, is mainly caused by the Pacific broad tapeworm Adenocephalus pacificus Nybelin, 1931, a parasite of considerable concern in fishery resources due to its impact on public health. A new diphyllobothrid, Diphyllobothrium sprakeri Hernández-Orts et al. Parasites Vectors 14:219, 2021, was recently described from sea lions from the Pacific Coast, but marine fish acting as intermediate hosts are unknown. The objective of this study was to confirm the presence of plerocercoid larvae of Diphyllobothriidae Lühe, 1910 (Cestoda: Diphyllobothriidea) in nine fish species of commercial importance in Peru. Of a total of 6999 fish (5861 Engraulis ringens, 853 Sciaena deliciosa, 6 Sciaena callaensis, 171 Scomber japonicus, 40 Trachurus murphyi, 40 Ariopsis seemanni, 18 Merluccius peruanus, 5 Sarda chiliensis, and 5 Coryphaena hippurus), 183 were infected with plerocercoid larvae, representing a total prevalence of 2.61% and a mean intensity of 3.2. Based on mtDNA cox1 sequences of 43 plerocercoids, a phylogenetic analysis revealed that 41 belong to A. pacificus and two to D. sprakeri. These findings are first molecular data for D. sprakeri larvae, and the infections of E. ringens and T. murphyi by plerocercoid larvae represent the first records of intermediate/paratenic hosts for this species. Hence, the findings of the current study enhance our understanding of the presence of diphyllobothriid species in commercial fish from the Southeastern Pacific Ocean and their potential impact on seafood safety for local human populations.

Population genetic structure of diphyllobothriid tapeworms (Cestoda: Diphyllobothriidea) parasitising fish in the Baikal Rift Zone.

Tapeworms of the genus Dibothriocephalus are widely distributed throughout the world, and some are agents of human diphyllobothriasis, one of the most important fish-borne zoonoses caused by a cestode parasite. Until now, the population genetic structure of diphyllobothriid tapeworms in the Baikal Rift Zone (BRZ) has remained unexplored. The major aim of this study was to analyse the population genetic structure of D. dendriticus and D. ditremus parasitising fish in the BRZ based on internal transcribed spacer 1 (ITS1) and mitochondrial gene cytochrome oxidase subunit I (cox1) sequences. We found that both species had complex population genetic structures. Each species formed 2 clades (D. dendriticus: Clade 1 & 2; D. ditremus Clade A & B) that differed in genetic diversity. D. dendriticus haplotypes in Clade 1 formed a star-like sub-network with a main haplotype, whereas the haplotypes in Clade 2 formed a diffuse network. We assumed that the complex population genetic structure of D. dendriticus was a consequence of populations evolving under different palaeoecological conditions during the Last Glacial Maximum. In contrast to D. dendriticus, both clades in the D. ditremus samples formed a diffuse network. Our findings revealed hypothetical pathways in the formation of the population genetic structure of diphyllobothriids in the BRZ. On one hand, isolation by distance played an important role; on the other hand, lake recolonisation from refugia and a genetic bottleneck after the end of the Last Glacial Maximum had a possible influence.

Effects of fish species composition on Diphyllobothrium spp. infections in brown trout - is three-spined stickleback a key species?

Subarctic populations of brown trout (Salmo trutta) are often heavily infected with cestodes of the genus Diphyllobothrium, assumedly because of their piscivorous behaviour. This study explores possible associations between availability of fish prey and Diphyllobothrium spp. infections in lacustrine trout populations. Trout in (i) allopatry (group T); (ii) sympatry with Arctic charr (Salvelinus alpinus) (group TC); and (iii) sympatry with charr and three-spined stickleback (Gasterosteus aculeatus) (group TCS) were contrasted. Mean abundance and intensity of Diphyllobothrium spp. were higher in group TCS compared to groups TC and T. Prevalence, however, was similarly higher in groups TCS and TC compared to group T. Zero-altered negative binomial modelling identified the lowest probability of infection in group T and similar probabilities of infection in groups TC and TCS, whereas the highest intensity was predicted in group TCS. The most infected trout were from the group co-occurring with stickleback (TCS), possibly due to a higher availability of fish prey. In conclusion, our study demonstrates elevated Diphyllobothrium spp. infections in lacustrine trout populations where fish prey are available and suggests that highly available and easily caught stickleback prey may play a key role in the transmission of Diphyllobothrium spp. parasite larvae.

Diphyllobothrium sp. in Canis familiaris from the subtropical area of Argentina (Puerto Iguazú, Misiones).

This paper reports the first finding of Diphyllobothrium sp. eggs in Canis familiaris (domestic dog) from Puerto Iguazú, a subtropical city of Misiones province, Argentina. In 2013, two positive cases of Diphyllobothrium sp. eggs were detected during an annual parasitological survey of dogs. Dog feces were collected in vials containing 10% formalin and processed using Telemann's sedimentation and Sheather's flotation techniques. The two cases were detected in rural areas of the municipality. Since Misiones is not a part of the endemic area of diphyllobothriasis and given the fact that it is located in the three-border area of Argentina, Brazil and Paraguay, we consider this finding of great importance to public health. We stress the need for updating the current knowledge about the life cycle of these parasites considering the range of intermediate and definitive hosts, their zoonotic potential, and the epidemiological situation in non-endemic areas.

[Cytomorphological and biochemical characteristics of the whitefish, Baikal omul Coregonus migratorius, infected by plerocercoids of Diphyllobothrium dendriticum (Cestoda: Pseudophyllidae)].

The cytomorphological and biochemical composition of the blood has been studied for the deep-dwelling (bottom) morphotype of the Baikal omul infected by plerocercoids of Diphyllobothrium dendriticum, during the fish spawning migration. A decrease in hemoglobin synthesis and an increase in the proliferative activity of erythroid cells have been registered. The changes in the leukocyte population composition, immunoglobulin, and total protein concentrations evidenced a disorder of blood cell proliferation and differentiation and suppression of the immune response in the infected fish. The changes in the humoral and cell factors of homeostasis in Coregonus migratorius infected by D. dendriticum during the spawning period remain within the limits of the adaptive possibilities of the species.

[The specific features of Diphyllobothrium latum circulation in the natural foci of the Kostroma region].

Three hundred pikes in the Kostroma section of the Gorky Reservoir, in the Kostroma Overflow, and in the Galich and Chukloma Lakes were examined for infection with Diphyllobothrium latum larvae. In the Kostroma section of the Gorky Reservoir and in the Kostroma Overflow, diphyllobothriasis was recorded in 66.7% (12/18) and 14.7% (11/75) of the pikes, respectively. The high intensity of the epidemic process in diphyllobothriasis was confirmed in the Kostroma Region. The number of notified cases was noted to tend to increase: 35 and 173 cases in 1994 and 1999, respectively.

Diphyllobothrium stemmacephalum (Cestoda: Diphyllobothriidae) found from a harbor porpoise in northern Japan, with comments on a geographical gap with human infection cases in southern Japan.

Even though the cetacean tapeworm Diphyllobothrium stemmacephalum occurs in both cold and warm waters, human infections and final host occurrences have been confined to temperate areas in and near Japan. We recently obtained a strobila of this cestode that was excreted from a harbor porpoise accidentally caught offshore of Hokkaido of northern Japan. Genetic analysis of 28S rDNA and cox1 genes confirmed that the cestode was D. stemmacephalum. Our finding sets the northernmost record of D. stemmacephalum in the western Pacific, suggesting that the risk of human infections by this parasite in northern Japan deserves further attention.

Comparative efficacy of candling and glass plate compression for detection of diphyllobothriosis in rainbow trout (Oncorhynchus mykiss) musculature.

The efficiency of the direct candling technique on fillets (candling 1) was compared with examination of cuts 4 mm thick or less (candling 2) and glass plate compression for the detection of plerocercoids of Diphyllobothrium spp. in muscles of rainbow trout, Oncorhynchus mykiss. Application of the three procedures gave the following results (percentage of infected fish/percentage of isolated plerocercoids): candling 1: 40.9/22, candling 2: 29.5/18.8, glass plate compression: 29.5/59.2, and combination of candling 1 and 2: 70.5/40.8. The combination of the three techniques yielded 100% sensitivity: 44 infected fish were detected of 77 trout examined. When different regions of the musculature were compared using the three techniques, a high density of plerocercoids and the highest percentage of infection (90.9%; 40 infected trout) were detected in the ventral musculature. Candling 1, candling 2 and glass plate compression on the ventral musculature gave the following case numbers and percentages, respectively, for the total of 44 cases: 9 (20.5%), 9 (20.5%), and 22 (50%).

Marine fish imported from Argentina as source of human diphyllobothriosis in Europe? Ecological evidence from dolphins.

Diphyllobothriosis caused by the Pacific broad tapeworm Adenocephalus pacificus (syn. Diphyllobothrium pacificum) is an emerging parasitic disease reported also from non-endemic areas, including Europe (Spain). The origin of these human cases is unknown but should be related to fresh marine fish imported from endemic areas. In this study, we molecularly confirmed common dolphins Delphinus delphis off Argentina as euparatenic transit hosts of A. pacificus. Preliminary analysis of their stomach content, together with data from previous studies from the Southwest Atlantic, showed that common dolphins feed almost exclusively on schooling Argentine hake Merluccius hubbsi and Argentine anchovy Engraulis anchoita. Therefore, we suggest that Argentine hake and Argentine anchovy may represent the intermediate hosts of A. pacificus in the Southwest Atlantic, but also in Europe to where M. hubbsi is imported on ice (unfrozen).

Diphyllobothrium sprakeri n. sp. (Cestoda: Diphyllobothriidae): a hidden broad tapeworm from sea lions off North and South America.

BACKGROUND: The systematic of several marine diphyllobothriid tapeworms of pinnipeds has been revised in recent years. However, 20 species of Diphyllobothrium from phocids and otariids are still recognized as incertae sedis. We describe a new species of Diphyllobothrium from the intestine of California sea lions Zalophus californianus (Lesson) (type-host) and South American sea lions Otaria flavescens (Shaw). METHODS: Zalophus californianus from the Pacific coast of the USA and O. flavescens from Peru and Argentina were screened for parasites. Partial fragments of the large ribosomal subunit gene (lsrDNA) and the cytochrome c oxidase subunit 1 (cox1) mitochondrial gene were amplified for 22 isolates. Properly fixed material from California sea lions was examined using light and scanning electron microscopy. RESULTS: A total of four lsrDNA and 21 cox1 sequences were generated and aligned with published sequences of other diphyllobothriid taxa. Based on cox1 sequences, four diphyllobothriid tapeworms from O. flavescens in Peru were found to be conspecific with Adenocephalus pacificus Nybelin, 1931. The other newly generated sequences fall into a well-supported clade with sequences of a putative new species previously identified as Diphyllobothrium sp. 1. from Z. californianus and O. flavescens. A new species, Diphyllobothrium sprakeri n. sp., is proposed for tapeworms of this clade. CONCLUSIONS: Diphyllobothrium sprakeri n. sp. is the first diphyllobothriid species described from Z. californianus from the Pacific coast of North America, but O. flavescens from Argentina, Chile and Peru was confirmed as an additional host. The present study molecularly confirmed the first coinfection of two diphyllobothriid species in sea lions from the Southern Hemisphere.

Detection and identification of Diphyllobothrium nihonkaiense plerocercoids from wild Pacific salmon (Oncorhynchus spp.) in Japan.

We investigated the risk of diphyllobothriasis from ingestion of wild Pacific salmon in Japan by surveying Diphyllobothrium plerocercoids in 182 salmon samples obtained from Japan. The plerocercoids were not detected in chum salmon (Oncorhynchus keta) (0/26), called Akizake in Japan, caught between September and November. However, the detection rate of plerocercoids in chum salmon, called Tokishirazu in Japan, caught between early April and June, was 51.1% (24/47) with an average of two plerocercoid larvae per fish. The detection rates of cherry salmon (Oncorhynchus masou) and pink salmon (Oncorhynchus gorbuscha) were 12.2% (10/82) and 18.5% (5/27), respectively, and the average number of plerocercoids per fish was 0.45 (37 larvae/82 fishes) and 0.22 larvae (6 larvae/27 fishes), respectively. Plerocercoids isolated from O. keta and O. masou were identified as Diphyllobothrium nihonkaiense on the basis of molecular analysis of the cox1 and nad3 genes. Moreover, four tapeworms (three from O. keta and one from O. masou) were obtained by infecting golden hamsters with plerocercoids. The morphological features of these tapeworms were similar to those of D. nihonkaiense isolated from humans. Therefore, we think that O. keta and not O. masou is the most important source of plerocercoid infections in Japan.

[Compared parasitic infection of Ligula intestinalis (Cestoda: Diphyllobothridae) in Cyprinidae species: Rutilus rubilio and Scardinius erythrophthalmus in two dam reserves in Tunisia]. / Infestation compaéee de Ligula intestinalis (Cestoda: Diphyllobothridae) chez deux poissons Cyprinidés : Rutilus rubilio et Scardinius erythrophthalmus dans deux retenues de barrages en Tunisie.

Monitoring quantitative parameters of parasitism by ligula intestinalis (Cestoda: Diphyllobothridae) was performed by examining 516 fish belonging to two introduced freshwater species: Rutilus rubilio (350 individuals) and Scardinius erythrophthalmus (166 individuals). These fish were collected in two dam reserves in Tunisia, Sidi Salem and Nebhana. The analysis of the aquatic bird's composition in these two reserves revealed the existence of piscivorous bird species that were previously reported as final host of Ligula. Monitoring the bird's composition highlighted higher relative abundance and frequency in Sidi Salem than in Nebhana dam reserve. The analyses of the prevalence, mean intensity and abundance of the parasite revealed the most important values in roach, Rutilus rubilio which seems to be the preferential second intermediate host of the parasite Ligula intestinalis in these environments. Comparative analysis of parasitism in both explored sites suggests that Ligula intestinalis presents two different strategies of infestation. In Sidi Salem reserve, which is larger than Nebhana and containing on important and diversified piscivorous species, the parasite infects a maximum of host individuals with low parasite mean intensity values. However, at Nebhana, which is a smaller reserve, the parasite infects fewer individuals than Sidi Salem but with higher mean intensity. The highest prevalence values were recorded in large size classes of roach species in Sidi Salem reserve.

[Morphofunctional characteristics of thymus and immunological parameters in golden hamster under experimental invasion with the cestode Diphyllobothrium dendriticum].

Morphofunctional changes induced by the tapeworm in the thymus and blood of its definitive host, by an example of the experimental model "Diphyllobothrium dendriticum--Mesocricetus auratus", have been investigated. The morphofunctional changes in the thymus and immunological transformations in the organism of infested hamster are evidences of rather high immunogenic effect of the D. dendriticum antigens on the definitive host at the period of active growth of the parasites.

Endohelminths of European Perch (Perca fluviatilis) from Selected Localities in Poland with an Emphasis on Search of the Broad Fish Tapeworm Dibothriocephalus latus.

INTRODUCTION: A piscivorous fish European perch (Perca fluviatilis) is present in all types of lakes and brackish waters in Poland. Previous ichthyoparasitological surveys revealed broad spectrum of endohelminths in perch from different aquatic environments. Among them, detection of Diphyllobothrium sp. and Dibothriocephalus latus (syn. Diphyllobothrium latum) in the Pomeranian Bay is of particular interest, since D. latus is one of the causative agents of diphyllobothriasis, fish-borne parasitic zoonosis. Besides, D. latus eggs were previously detected in coprological samples of otter, wolf and lynx from the Bialowieza Primeval Forest. AIM: To conduct parasitological examinations of European perch from the Pomeranian Bay in order to detect the spectrum of its endohelminths and to provide a pilot study on helminths of perch from different water bodies in the Bialowieza Primeval Forest. Due to zoonotic character of D. latus, we have focused our attention to this tapeworm. RESULTS: The larvae of tapeworm Triaenophorus nodulosus and thorny-headed worm Acanthocephalus lucii were detected in perch from the Pomeranian Bay. In perch from different localities in the Bialowieza Primeval Forest, T. nodulosus, A. lucii and tapeworm Proteocephalus percae were detected. D. latus plerocercoids were found neither in musculature nor in peritoneal cavity and other internal organs of any of the fish examined from both studied localities in Poland. CONCLUSION: Future screening implementing morphological and molecular markers is needed in order to understand the current distribution of D. latus in Europe.

Gastrointestinal parasites in captive and free-living wild birds in Goiania Zoo / Parasitas gastrointestinais em aves silvestres em cativeiro e de vida livre no Zoológico de Goiânia

Helminths are endoparasites that infect a variety of bird species. Endoparasite infections can cause severe diseases, including kill captive avian hosts and represents a problem to maintenance and conservation. The eggs and larval forms of these parasites are usually eliminated in the host feces. The main interest of this study is to report the occurrence of eggs and oocysts in feces from captive wild birds in Goiânia Zoo and free-living birds in its surroundings. The fecal samples were subjected to parasitological examination to identify the presence of helminths and to classify their eggs based on morphological characteristics. Eggs of nematode parasites (Positive/N) were identified as Ascaridia spp. in Brotogeris chiriri (2/2), Dromaius novaehollandiae (1/1) and Rhea americana (2/2); Ascarididae in Pavo cristatus nigripensis (1/1); Capillaria spp. in Ara chloropterus (1/1) and Penelope jacucaca (1/1); Capillaria plagiaticia in Anodorhynchus hyacinthinus (1/2) and Ara spp. (2/2); Capillaria venusta in Rhamphastus tucanus (1/2); and unidentified nematode eggs from Amazona amazonica (2/2). Eimeria spp. oocyst was founded in feces from A. hyacinthinus (1/2). Considering free-living birds, Diphyllobothrium spp. eggs were founded in Ardea alba (2/2) and Nycticorax nycticorax (2/3). Co-infection of Eustrongylides spp. was founded in A. alba (1/2). This is the first occurrence of: Ascaridia spp. parasitizing B. chiriri; and C. venusta parasitizing R. tucanus. In conclusion, the helminth eggs found in the abovementioned host bird species are consistent with those reported in the literature, and treatment and control protocols were based on their identification.

Helmintos são endoparasitas que infectam uma variedade de espécies de aves. As infecções por endoparasitas podem causar doenças graves, podendo matar aves em cativeiro e representam um problema de manutenção e conservação. Os ovos e as formas larvais desses parasitas são geralmente eliminados nas fezes do hospedeiro. O principal objetivo deste estudo é relatar a ocorrência de ovos e oocistos em fezes de aves silvestres em cativeiro no Zoológico de Goiânia e em aves de vida livre em seu entorno. As amostras fecais foram submetidas a exame parasitológico para identificação da presença de helmintos e classificação dos ovos com base nas características morfológicas. Ovos de nematóides (Positivo/N) foram identificados como Ascaridia spp. em Brotogeris chiriri (2/2), Dromaius novaehollandiae (1/1) e Rhea americana (2/2); Ascarididae em Pavo cristatus nigripensis (1/1); Capillaria spp. em Ara chloropterus (1/1) e Penelope jacucaca (1/1); Capillaria plagiaticia em Anodorhynchus hyacinthinus (1/2) e Ara spp. (2/2); Capillaria venusta em Rhamphastus tucanus (1/2); e ovos de nematóide não identificado de Amazona amazonica (2/2). Oocistos de Eimeria spp. foram encontrados nas fezes de A. hyacinthinus (1/2). Considerando pássaros de vida livre, ovos de Diphyllobothrium spp. foram encontrados em Ardea alba (2/2) e Nycticorax nycticorax (2/3). Coinfecção de Eustrongylides spp. foi encontradada em A. alba (1/2). Esta é a primeira ocorrência de: Ascaridia spp. parasitando B. chiriri; e C. venusta parasitando R. tucanus. Em conclusão, os ovos de helmintos encontrados nas espécies de aves hospedeiras acima mencionadas são consistentes com os relatados na literatura, e os protocolos de tratamento e controle foram baseados em sua identificação.

[Prevalence of Diphyllobothrium latum, L., 1758 infestation in Perca fluviatilis from Lake Leman]. / Prévalence de l'infestation par Diphyllobothrium latum, L., 1758 chez les Perches (Perca fluvatilas) du lac Léman.

Diphyllobothriasis is contracted by consuming raw or undercooked freshwater fish and is still present on the shores of lake Leman. The aim of this study was to evaluate the prevalence of Diphyllobothrium latum plerocercoid larvae in Perca fluviatilis from this lake. Four to 10% of perch fillets examined in November 2003, February 2004, April 2004 and January 2005, were infested with D. latum larvae. The identification of the larvae was confirmed by PCR and sequencing of the 18S rDNA.

High morphological plasticity and global geographical distribution of the Pacific broad tapeworm Adenocephalus pacificus (syn. Diphyllobothrium pacificum): molecular and morphological survey.

The most important causative agent of human diphyllobothriosis in South America, Diphyllobothrium pacificum, is transferred to the original genus Adenocephalus Nybelin, 1931; revised and redescribed on the basis of the evaluation of an extensive material collected mainly from northern fur seal, Callorhinus ursinus, from St. Paul Island, Alaska. Detailed analysis of morphological and morphometrical data shows a high variability in most of the characteristics traditionally used in diagnosis of diphyllobothriid tapeworms. Phylogenetic analyses based on newly characterised sequences of mitochondrial cytochrome c oxidase subunit 1 and nuclear large subunit ribosomal RNA genes consistently reveal Adenocephalus pacificus as a sister lineage to the clade formed of the remaining Diphyllobothrium species and other genera (Digramma, Diplogonoporus, Ligula). Despite the generally similar morphology, A. pacificus can be differentiated from the closely related taxa in the presence of transverse papilla-like tegumental protuberances distributed anteriorly, separated by narrow semicircular grooves on the ventral surface of proglottids between their anterior margin and the anterior edge of the male gonopore, and relatively small eggs. A. pacificus displays a relatively low host specificity (found in 9 of 16 otariids, and in accidental hosts such as man, dog and jackal, the latter representing a new host) and a uniquely wide geographical distribution on both hemispheres. In addition, suitability of morphological criteria used in diagnostics of diphyllobothriid cestodes is discussed.

The influence of changing prey availability on the prevalence of Diphyllobothrium in river otters from Yellowstone National Park.

Parasite prevalence in predatory mammals is influenced by numerous factors including diet, sex, season, and habitat. We examined the effect of such factors on the prevalence of gastrointestinal helminths in North American river otters ( Lontra canadensis ) in Yellowstone Lake, Yellowstone National Park. Otters in this ecosystem have recently experienced a decline in their main prey, Yellowstone cutthroat trout ( Oncorhynchus clarkii bouvieri), and have, in turn, increased consumption of alternative foods. Helminths were found in 13.2% of otter fecal samples. The dominant parasite was a Diphyllobothrium sp. ( Diphyllobothrium ditremum or Diphyllobothrium dendriticum ), a cestode acquired from cutthroat trout. Truttaedacnitis truttae and Contracaecum spp. nematodes were incidental parasites in otter feces, and acanthocephalan eggs were found in 1 sample. The prevalence of trout remains and a Diphyllobothrium sp. in otter feces was higher during the cutthroat trout spawning season. A Diphyllobothrium sp. was more prevalent in the feces of female otters. There was no relationship between annual declines in the frequency of trout in otter feces and prevalence of parasites. Helminth prevalence and species richness in Yellowstone otters was low and likely reflects low predator densities and few intermediate hosts. This is the first study reporting the helminth fauna of river otters in the Greater Yellowstone Area and confirms the otter as a definitive host for Diphyllobothrium sp. in this region.

Leucocyte migration in rainbow trout (Oncorhynchus mykiss [Walbaum]): optimization of migration conditions and responses to host and pathogen (Diphyllobothrium dendriticum [Nitzsch]) derived chemoattractants.

A rainbow trout leucocyte-derived chemoattractant(s) was prepared and tested as a stimulant of leucocyte migration. It was used to optimize an in vitro leucocyte migration assay using a 48-well micro chemotaxis chamber. This assay has subsequently been used to test the chemoattractant activity of antigen extracts from the tegument of Diphyllobothrium dendriticum plerocercoids and conditioned medium obtained after in vitro maintenance of live plerocercoids. Leucocytes were found to have an increased directional motility (chemotactic response) to the host-derived chemoattractant(s) but a random increased motility (chemokinetic response) following stimulation/contact with parasite-derived antigens.

Infection of brown trout with Diphyllobothrium dendriticum procercoids.

The aims of this experimental study were to develop a practical method of controlling the number of Diphyllobothrium dendriticum procercoids delivered to a fish host; to examine the effect of different procercoid doses (3, 7 and 15) on the plerocercoid level in fish; and to examine the potential mortality caused by plerocercoids. The experiment was terminated after 2 months. The prevalence of infection at the lowest dose level, 17.2%, was statistically significantly lower than at medium and high levels, 63.3% and 56.7%, respectively. The mean intensity increased slightly along with the dosage. The contribution of D. dendriticum to the death of a few fish could not be proved. It is concluded that the present method is practical for use in larger experiments as well.