Cucumispora dikerogammari n. gen. (Fungi: Microsporidia) infecting the invasive amphipod Dikerogammarus villosus: a potential emerging disease in European rivers.

Dikerogammarus villosus is an invasive amphipod that recently colonized the main rivers of Central and Western Europe. Two frequent microsporidian parasites were previously detected in this species, but their taxonomic status was unclear. Here we present ultrastructural and molecular data indicating that these two parasites are in fact a single microsporidian species. This parasite shares numerous characteristics of Nosema spp. It forms elongate spores (cucumiform), developing in direct contact with host cell cytoplasm; all developmental stages are diplokaryotic and the life cycle is monomorphic with disporoblastic sporogony. Initially this parasite was described as Nosema dikerogammari Ovcharenko and Kurandina 1987. However, phylogenetic analysis based on the complete sequence of SSU rDNA places the parasite outside the genus Nosema and it is therefore ascribed to a new genus Cucumispora. The key features characteristic to this genus are: presence of a very well-developed, umbrella-shape anchoring disk covering the anterior part of polaroplast; arrangement of isofilar polar filament into 6-8 coils convoluted with different angles, voluminous diplokaryon, thin spore wall and relatively small posterior vacuole containing posterosome. The parasite infects most host tissues but mainly muscles. It showed high rates of horizontal trophic transmission and lower rates of vertical transmission.

First record of a Kabatana sp. microsporidium infecting fish in the Atlantic Ocean.

Two-spotted goby Gobiusculus flavescens from the Swedish Gullmarsfjord regularly present subcutaneous creamy-white patches in the body musculature, associated with Kabatana sp. infection. Analysis of the 16S rRNA gene of the microsporidium showed 98.54% homology with Kabatana newberryi infecting a marine goby from California, indicating that the Swedish microsporidium is either a different strain of K. newberryi or a closely related species. This represents the first record of a Kabatana species in the Atlantic Ocean. The genetic similarity of the 2 microsporidia was paralleled by close infection phenotypes. Infected muscle fibres were swollen compared to adjacent non-infected fibres, and mature spore masses were found throughout the skeletal musculature. No xenoma formation was detected. Since G. flavescens is an established model species in behavioural ecology, the host-parasite system is ideally suited for testing how microsporidian infections affect host behaviour and fitness.

Association with host mitochondrial haplotypes suggests that feminizing microsporidia lack horizontal transmission.

The amphipod crustacean Gammarus duebeni hosts two feminizing microsporidian parasites, Nosema granulosis and Microsporidium sp. Samples of G. duebeni were collected from three sites on the Scottish island of Great Cumbrae and screened for microsporidia using polymerase chain reaction. Associations between the prevalence of the two feminizing parasites and haplotypes of the host mitochondrial gene cytochrome oxidase I (COI) were investigated. The prevalence of both parasites varied significantly among the host's COI haplotypes, suggesting that horizontal transmission is rare or absent in the life cycles of the feminizing microsporidia and that all transmission must therefore be vertical. Life cycles in which all transmission is vertical are common among bacterial parasites but have never before been demonstrated in Eukaryotic parasites.

Infection of Gammarus duebeni populations by two vertically transmitted microsporidia; parasite detection and discrimination by PCR-RFLP.

We screened a population of the brackish water crustacean Gammarus duebeni from the Isle of Cumbrae for the presence of vertically transmitted microsporidia. We compared 2 screening techniques; light microscopy and PCR-based detection using generic 16S rDNA microsporidian primers. Fifty percent of females from this population tested positive for vertically transmitted microsporidia. The PCR screen was 100% efficient in comparison with existing LM based screening. In addition, the PCR screen produced bands of 2 sizes suggesting that more than 1 species of microsporidian was present. Sequencing revealed 2 distinct species of vertically transmitted microsporidia; 33% of females were infected with the feminizer Nosema granulosis and 17% were infected with a new species which we provisionally designate Microsporidium sp. On the basis of sequence information, we developed a discriminatory PCR-RFLP test based on MspI and HaeIII digests. This screen allows rapid detection and discrimination of vertically transmitted microsporidia in natural field populations. We applied the PCR-RFLP screen to a second G. duebeni population from the Isle of Man. This population also hosted these 2 parasite species. In total 45% of females harboured N. granulosis and 10% harboured Microsporidium sp. No dual-infected individuals were found in either population. The occurrence of 2 vertically transmitted parasites within a population has implications for our understanding of parasite-host relationships in the field and we discuss factors affecting the dynamics of parasite-parasite competition and coexistence.

Two species of feminizing microsporidian parasite coexist in populations of Gammarus duebeni.

The amphipod crustacean Gammarus duebeni hosts two species of vertically transmitted microsporidian parasites, Nosema granulosis and Microsporidium sp. A. Here it is demonstrated that these co-occurring parasite species both cause infected females to produce female-biased broods. A survey of European G. duebeni populations demonstrates that these two parasites co-occur in six of 10 populations. These findings contrast with the theoretical prediction that two vertically transmitted feminizing parasites should not coexist in a panmictic population of susceptible hosts at equilibrium. Possible explanations for the co-occurrence of the two feminizing microsporidia in G. duebeni include the recent invasion of a new parasite, horizontal transmission of one or both parasites and the spread of alleles for resistance to the dominant parasite in host populations.

Establishment of the new genus Paranosema based on the ultrastructure and molecular phylogeny of the type species Paranosema grylli Gen. Nov., Comb. Nov. (Sokolova, Selezniov, Dolgikh, Issi 1994), from the cricket Gryllus bimaculatus Deg.

The ultrastructure of the microsporidian parasite Nosema grylli, which parasitizes primarily fat body cells and haemocytes of the cricket Gryllus bimaculatus (Orthoptera, Gryllidae) is described. All observed stages (meront, meront/sporont transitional stage ("second meront"), sporont, sporoblast, and spore) are found in direct contact with the host cell cytoplasm. Nuclei are diplokaryotic during almost all stages of the life cycle, but a brief stage with one nucleus containing an abundance of electron-dense material is observed during a "second merogony." Sporogony is disporous. Mature spores are ovocylindrical in shape and measure 4.5+/-0.16micromx2.2+/-0.07 microm (n=10) on fresh smears and 3.3+/-0.06 micromx1.4+/-0.07 microm (n=10) on ultrathin sections. Spores contain 15-18 coils of an isofilar polar filament arranged in one or two layers. Comparative phylogenetic analysis using rDNA shows N. grylli to be closely related to another orthopteran microsporidian, Nosema locustae, and to Nosema whitei from the confused flour beetle, Tribolium confusum. Antonospora scoticae, a parasite of the communal bee Andrena scotica, is a sister taxon to these three Nosema species. The sequence divergence and morphological traits clearly separate this group of "Nosema" parasites from the "true" Nosema clade containing Nosema bombycis. We therefore propose to change the generic name of N. grylli and its close relative N. locustae to Paranosema n. comb. We leave N. whitei in former status until more data on fine morphology of the species are obtained.