Mode of action of Pseudomonas fluorescens strain CL145A, a lethal control agent of dreissenid mussels (Bivalvia: Dreissenidae).

Pseudomonas fluorescens strain CL145A (Pf-CL145A) has demonstrated promise as an efficacious and selective agent for the control of macrofouling Dreissena spp. mussels. Herein, we report trials to investigate the mode of action of this biocontrol agent against Dreissena polymorpha, the zebra mussel. Exposure to dead Pf-CL145A cells achieved the same temporal pattern and percentage mussel mortality as did live cells, thereby excluding infection as the possible lethal mode of action. Histological analysis revealed pathologies consistent with the cause of death being intoxicating natural products associated with Pf-CL145A cells. Irrespective of whether the mussels were exposed to live or dead Pf-CL145A cells, examination of tissues from histological sections revealed that: (1) at the end of the 24-h treatment period there was massive hemocyte infiltration into the lumina of both the digestive gland and stomach; and (2) mussel deaths occurred following lysis and necrosis of the digestive gland and sloughing of stomach epithelium. These trials provide strong evidence that the lethal mode of action of Pf-CL145A is intoxication.

Pseudomonas fluorescens strain CL145A - a biopesticide for the control of zebra and quagga mussels (Bivalvia: Dreissenidae).

Zebra mussels (Dreissena polymorpha) and quagga mussels (Dreissena rostriformis bugensis) are the "poster children" of high-impact aquatic invasive species. In an effort to develop an effective and environmentally acceptable method to control their fouling of raw-water conduits, we have investigated the potential use of bacteria and their natural metabolic products as selective biological control agents. An outcome of this effort was the discovery of Pseudomonas fluorescens strain CL145A - an environmental isolate that kills these dreissenid mussels by intoxication (i.e., not infection). In the present paper, we use molecular methods to reconfirm that CL145A is a strain of the species P. fluorescens, and provide a phylogenetic analysis of the strain in relation to other Pseudomonas spp. We also provide evidence that the natural product lethal to dreissenids is associated with the cell wall of P. fluorescens CL145A, is a heat-labile secondary metabolite, and has degradable toxicity within 24 h when applied to water. CL145A appears to be an unusual strain of P. fluorescens since it was the only one among the ten strains tested to cause high mussel mortality. Pipe trials conducted under once-through conditions indicated: (1) P. fluorescens CL145A cells were efficacious against both zebra and quagga mussels, with high mortalities achieved against both species, and (2) as long as the total quantity of bacterial cells applied during the entire treatment period was the same, similar mussel mortality could be achieved in treatments lasting 1.5-12.0 h, with longer treatment durations achieving lower mortalities. The efficacy data presented herein, in combination with prior demonstration of its low risk of non-target impact, indicate that P. fluorescens CL145A cells have significant promise as an effective and environmentally safe control agent against these invasive mussels.

Interactions between parasitism and biological responses in zebra mussels (Dreissena polymorpha): Importance in ecotoxicological studies.

Given that virtually all organisms are hosts for parasites, the investigation of the combined effects of contamination and parasitism is important in the framework of aquatic bioindication procedures. To assess the impact of such multistresses at the host cellular level, we sampled parasitized zebra mussel (Dreissena polymorpha) populations from two sites in northeast France that presented different levels of contamination. Experimental groups were formed based on parasite species and host gender and tested by histochemistry and automated image analysis for biological responses, such as structural changes of the lysosomal system and neutral lipid accumulation. Infected organisms displayed smaller and more numerous lysosomes compared with uninfected congeners, and infection further elevated the effect of the chemical contamination on this biomarker. In contrast, co-infection of females with selected parasites did produce inverse results, i.e. a more developed lysosomal system and neutral lipid depletion. Our data, therefore, suggest that parasitism in zebra mussels represents a potential confounding factor in ecotoxicological studies and must be taken into account in environmental risk assessment studies.

Ophryoglena hemophaga n. sp. (Ciliophora: Ophryoglenidae): a parasite of the digestive gland of zebra mussels Dreissena polymorpha.

Ophryoglena hemophaga n. sp. is described from a freshwater Dreissena polymorpha population in the Rhine delta of the Netherlands. This is the first ophryoglenine species (order Hymenostomatida, suborder Ophryoglenina) recorded as a molluscan parasite. As is typical of ciliates in the suborder Ophryoglenina, O. hemophaga exhibits a polymorphic life history with cystment and reproduction by palintomy. Trophonts were observed within digestive gland lumina, and zebra mussel hemocytes were present in some of their digestive vacuoles. The presence of a single, longitudinal tract of multiple contractile vacuoles represents its most unique feature and distinguishes it from all other described Ophryoglena spp. The number of somatic kineties of O. hemophaga (range 100 to 124) is also distinguishing as it is one of the lowest for [corrected] an Ophryoglena sp. Other characteristics of this species include: ovoid to elongate trophonts 96 to 288 microm in length, with an elongate macronucleus 41 to 65 microm in length; tomonts 50 to 150 microm in diameter producing a clear mucous cyst envelope, whose thickness is approximately half of the tomont diameter; elongated theronts 96 to 131 microm in length which emerge after 1 to 3 cell divisions taking 36 to 48 h at 20 +/- 3 degrees C. Protomonts and theronts are, respectively, negatively and positively phototactic--characteristics that likely aid in maintenance of infection in zebra mussel populations.

Comparison of rDNA sequences from colchicine treated and untreated sporocysts of Phyllodistomum folium and Bucephalus polymorphus (Digenea).

The most frequently used antimitotic agent in cytogenetic studies is colchicine. We investigated whether the initial treatment of trematodes for karyological analysis with colchicine would have mutagenic or degradational effect on rDNA sequences. Dreissena polymorpha is the intermediate host of Phyllodistomum folium and Bucephalus polymorphus, and the sporocyst stage of these trematode species develop, respectively, in the gills and gonads of this mussel. Sporocysts of P. folium and B. polymorphus were obtained from D. polymorpha collected from waterbodies in Belarus and in Lithuania. 5.8S and 28S rDNA genes, ITS1 and ITS2 of P folium and B. polymorphus were sequenced and compared, and no nucleotide sequence differences between colchicine treated and untreated trematodes were found. Based on these results, we conclude that colchicine treatment for 3-5 h has no mutagenic or degradational effect on rDNA sequences. During the course of this investigation, two genetically different P. folium samples were noted in Belarus.

Histological analysis of trematodes in Dreissena polymorpha: their location, pathogenicity, and distinguishing morphological characteristics.

Four families of trematodes were observed in histological sections during a 1992-1997 investigation of the parasites of zebra mussels Dreissena polymorpha. These included Aspidogastridae, i.e., Aspidogaster, Echinostomatidae, Bucephalidae, i.e., Bucephalus polymorphus, and Gorgoderidae, i.e., Phyllodistomumfolium. This article describes the precise location of these trematodes in the tissues of D. polymorpha, provides graphic evidence of their effect on the organs they inhabit, and highlights the distinguishing morphological characteristics. Evidence of defense reaction of host to trematode infection, i.e., encapsulation of Aspidogaster and nacrezation of B. polymorphus, is also presented and is the first such report for zebra mussels. The histological photomicrographs included represent the first comprehensive series published on trematode infection of zebra mussels. These images, in conjunction with the morphological descriptions presented, should assist researchers in identifying the 4 major trematode taxa that they are likely to encounter in the tissue sections of zebra mussels.

Aquastella gen. nov.: a new genus of saprolegniaceous oomycete rotifer parasites related to Aphanomyces, with unique sporangial outgrowths.

The oomycete genus Aquastella is described to accommodate two new species of parasites of rotifers observed in Brooktrout Lake, New York State, USA. Three rotifer species--Keratella taurocephala, Polyarthra vulgaris, and Ploesoma truncatum--were infected, and this is the first report of oomycete infection in these species. Aquastella attenuata was specific to K. taurocephala and Aquastella acicularis was specific to P. vulgaris and P. truncatum. The occurrence of infections correlated with peak host population densities and rotifers were infected in the upper layers of the water column. Sequencing of 18S rRNA and phylogenetic analysis of both species placed them within the order Saprolegniales, in a clade closely related to Aphanomyces. The Aquastella species were morphologically distinct from other rotifer parasites as the developing sporangia penetrated out through the host body following its death to produce unique tapered outgrowths. Aquastella attenuata produced long, narrow, tapering, finger-like outgrowths, whilst A. acicularis produced shorter, spike-like outgrowths. We hypothesize that the outgrowths serve to deter predation and slow descent in the water column. Spore cleavage was intrasporangial with spore release through exit tubes. Aquastella attenuata produced primary zoospores, whereas A. acicularis released spherical primary aplanospores, more typical of other genera in the Aphanomyces clade.

Occurrence of zebra mussel parasites: modelling according to contamination in France and the USA.

Parasites can be reliable tool in assessing the effects of ecosystem disturbances. However, they can respond in different ways and any changes in assemblages are not easily predictable. Descriptive modelling could be a first step since providing information on the relative importance of a pollutant on parasite occurrence. We chose the zebra mussel, as test organism and twelve sites in France and the United States. Contaminants had not the same impact on microparasite occurrence. Metals enhanced the infection, except zinc associated only with higher prevalence of the commensal ciliate Conchophthirus acuminatus. We should note that Rickettsiales-like organism infection is higher at higher Ni and Cr concentrations. Models indicated also that the most polluted sites were also those with higher rates of co-infections. Therefore, the continuous contamination of freshwater ecosystems implies a significant risk promoting the development of parasites that may affect bivalve populations and other species belonging to their life-cycle.

Zebra mussel (Dreissena polymorpha) parasites: potentially useful bioindicators of freshwater quality?

In environmental quality bioassessment studies, analysis of host-parasite interactions may well be a valuable alternative to classical macroinvertebrate sampling approaches. Herein, we investigated whether zebra mussel (Dreissena polymorpha) parasites could be useful biomonitoring tools. Mussel populations were sampled twice at two sites in northeastern France representing different levels of contamination and were characterized for parasite infection following standard histological methods. Our results indicated that sites of different environmental quality (i.e. chemical contamination) exhibited different parasite communities characterized by different trematode species and parasite associations. An additional significant finding was the positive correlation established between the prevalence of Rickettsiales-like organisms and metal contamination. Multivariate analyses were valuable in examining parasite communities.

Seasonal dynamics of endosymbiotic ciliates and nematodes in Dreissena polymorpha.

We report the results of a two-year study in the Svisloch River (Minsk, Belarus) on the dynamics of infection in Dreissena polymorpha by nematodes and three ciliate species Conchophthirus acuminatus, Ophryoglena sp., and Ancistrumina limnica. Although these endosymbionts were present in most of the samples, their prevalence and infection intensity differed significantly. C. acuminatus and A. limnica infection intensities in both years of the study had a maximum in summer and were positively correlated with water temperature. In contrast, Ophryoglena sp. and nematode infection intensities were considerably lower in summer versus winter and were negatively correlated with temperature. In the first long-term study to monitor the size and reproductive rate of C. acuminatus, we found that mean length was negatively correlated with temperature and that temperature was positively correlated with asexual reproduction, with a peak of cell division in April as water temperatures increased.

Field and laboratory studies of Ophryoglena sp. (Ciliata: Ophryoglenidae) infection in zebra mussels, Dreissena polymorpha (Bivalvia: Dreissenidae).

This study, conducted in the Dnieper-Bug Canal in Belarus, is the first to monitor the seasonal (June-November) dynamics of infection with the parasitic ciliate Ophryoglena sp. in a zebra mussel (Dreissena polymorpha) population. Mean population prevalence and intensity of infection varied, respectively, from 11 to 62% and from 0.9 to 24.1 ciliates/mussel. Mean prevalence was highly correlated with mussel length in mussels <20 mm (R(2)=0.97) and was lower in larger mussels. Mean infection intensity in mussels 1-25 mm long was similarly correlated with their size (R=0.98), reached a maximum in the 20-25 mm size-class, and then sharply decreased, thus providing evidence, albeit limited, that high intensity of infection might be lethal. Transinfection of zebra mussels by Ophryoglena sp. was achieved in the laboratory-a first for a protozoan parasite of D. polymorpha; from an initial complete lack of infection, mean prevalence and intensity rose, respectively, to 86.7% and 8.3 ciliates/mussel.