Distribution of predatory bacteria that attack chromatiaceae in a sulfurous lake

Predatory bacteria that attack Chromatiaceae (purple sulfur bacteria) recovered from Lake Cisó (a mostly anaerobic holomictic lake) have been studied over two annual cycles. During the mixing period the lake was completely anaerobic; both predator and prey populations were found along the water column, and even at the surface. Throughout the stratification period maximum Chromatiaceae occurred between a depth of 1 and 3 m depth. The maximum numbers of predators and prey (Chromatiaceae) also occurred in this range.A collapse took place in the lake during the second annual cycle in 1986. It brought about changes in the physicochemical parameters of the lake, thus altering the population dynamics. Nevertheless, during both cycles the number of predatory bacteria was maximum immediately below the depth at which the maximum number of prey bacteria occurred. Send offprint requests to: N. Gaju.     

Increased Diversity of Predacious <Emphasis Type="Italic">Bdellovibrio</Emphasis>-Like Organisms (BLOs) as a Function of Eutrophication in Kumaon Lakes of India

Predation by Bdellovibrio-like organisms (BLOs) results in bacterial community succession in aquatic ecosystems. The effects of nutrient loading on the distribution and phylogeny of BLOs remain largely unknown. To this end, we present our findings on BLO diversity from four north-Indian lakes that are variable in their trophic status; Nainital is eutrophic, both, Bhimtal and Naukuchiatal are mesotrophic and Sattal remains oligotrophic, respectively. Initially, total heterotrophic bacteria and BLOs were quantified by most probable number (MPN) analyses using Pseudomonas putida and Escherichia coli as prey bacteria. Total bacterial numbers were at least two-logs higher in the eutrophic lake samples compared with oligotrophic lake. Similarly, BLO numbers were approximately 39-fold higher using Pseudomonas sp., which is likely the preferred prey within these lakes. Conversely, significant differences were not observed between mesotrophic and oligotrophic BLO numbers when E. coli was used as the prey. PCR-RFLP of small subunit rDNA (SSU rDNA) of BLOs, followed by cloning, sequencing, and taxonomic categorization revealed distinct differences such that, eutrophic lake consisted of higher BLO diversity compared with mesotrophic and oligotrophic lake, most likely due to both, higher numbers and availability of a diverse population of prey bacteria resulting from nutrient loading in this ecosystem.     

Isolation and characterization of some moderately halophilic bacteria with lipase activity

Lipases are an important class of enzymes which catalyze the hydrolysis of long chain triglycerides and constitute the most prominent group of biocatalysts for biotechnological applications. There are a number of lipases, produced by some halophilic microorganisms. In this study, some lipase producing bacteria from the Maharla salt lake located in south of Iran were isolated. All isolates were screened for true lipase activity on plates containing olive oil. The lipase activity was measured using titrimetric methods. Among thirty three isolates, thirteen strains demonstrating orange zone around colonies under UV light, were selected for identification using the molecular methods and some morphological characteristics. The bacterium Bacillus vallismortis BCCS 007 with 3.41 ± 0.14 U/mL lipase activity was selected as the highest lipase producing isolate. This is the first report of isolation and molecular identification of lipase producing bacteria from the Maharla lake.     

National surveillance of <Emphasis Type="Italic">Salmonella enterica</Emphasis> in food-producing animals in Japan

A total of 518 fecal samples collected from 183 apparently healthy cattle, 180 pigs and 155 broilers throughout Japan in 1999 were examined to determine the prevalence and antimicrobial susceptibility of Salmonella. The isolation rates were 36.1% in broilers, 2.8% in pigs and 0.5% in cattle. S. enterica Infantis was the most frequent isolate, found in 22.6% of broiler fecal samples. Higher resistance rates were observed against oxytetracycline (82.0%), dihydrostreptomycin (77.9%), kanamycin (41.0%) and trimethoprim (35.2%). Resistance rates to ampicillin, ceftiofur, bicozamycin, chloramphenicol and nalidixic acid were <10%. CTX-M-2 β-lactamase producing S. enterica Senftenberg was found in the isolates obtained from one broiler fecal sample. This is the first report of cephalosporin-resistant Salmonella directly isolated from food animal in Japan.     

Isolation and characterisation of bacteria from the haloalkaline Lake Elmenteita, Kenya

Culture-independent studies show that soda lake environments harbour diverse groups of bacteria and archaea. In this study different enrichment and isolation media were used in an attempt to isolate novel groups of bacteria from Lake Elmenteita. Different media were prepared using filter-sterilised water from the lake. The isolates recovered were purified on tryptic soy agar supplemented with 1% sodium carbonate and 4% sodium chloride. Phylogenetic analysis of 181 partial 16S rRNA gene sequences with excellent quality showed that the majority of the isolates were affiliated to the class Gammaproteobacteria and to the genus Bacillus. Isolates from the genus Halomonas and Bacillus constituted 37 and 31% of the total sequenced isolates, respectively. Other groups recovered were related to Marinospirillum, Idiomarina, Vibrio, Enterococcus, Alkalimonas, Alkalibacterium, Amphibacillus, Marinilactibacillus and the actinobacteria Nocardiopsis and Streptomyces. Fifty-one different genera were represented with 31 and 15 cultures scoring with their nearest neighbour similarities below 98 and 97%, respectively. Some novel taxa were identified which had not been isolated previously from the soda environment. The results show that the use of different media with varying compositions can help retrieve novel bacterial diversity from the soda lake environment.     

Biostimulation of Estuarine Microbiota on Substrate Coated Agar Slides: A Novel Approach to Study Diversity of Autochthonous Bdellovibrio- and Like Organisms

Characterization of Bdellovibrio- and like organisms (BALOs) from environmental samples involves growing them in the presence of Gram-negative prey bacteria and isolation of BALO plaques. This labor-intensive enrichment and isolation procedure may impede the detection and phylogenetic characterization of uncultivable BALOs. In this article, we describe a simple slide biofilm assay to improve detection and characterization of BALO microbiota. Agar spiked with biostimulants such as yeast extract (YE), casamino acids (CA), or concentrated cells of Vibrio parahaemolyticus P5 (most widely used prey bacteria for isolation of halophilic BALOs) was plated onto buffed glass slides and exposed to water samples collected from Apalachicola Bay, Florida. After incubating for a week, diversity of the biofilm bacterial community was studied by culture-dependent and culture-independent molecular methods. The results revealed that most probable numbers (MPNs) of BALOs and total culturable bacteria recovered from YE agar slide were significantly higher than the numbers on CA- or P5-spiked agar slides. Polymerase chain reaction–restriction fragment length polymorphism followed by 16S rDNA sequencing of clones from different biostimulants resulted in identification of a plethora of Gram-negative bacteria predominantly from the alpha, gamma, delta-proteobacteria, and the Cytophaga–Flavobacterium–Bacteroides group. Corresponding to the higher biomass on the YE agar slide, the BALO clone library from YE was most diverse, consisting of Bacteriovorax spp. and a novel clade representing Peredibacter spp. Microbiota from all three biostimulated biofilms were exclusively Gram-negative, and each bacterial guild represented potential prey for BALOs. We propose the use of this simple yet novel slide biofilm assay to study oligotrophic aquatic bacterial diversity which could also potentially be utilized to isolate marine bacteria with novel traits.     

Predator/Prey Interaction between <Emphasis Type="Italic">Pfiesteria piscicida</Emphasis> and <Emphasis Type="Italic">Rhodomonas</Emphasis> Mediated by a Marine Alpha Proteobacterium

The dinoflagellate Pfiesteria piscicida coexists with bacteria in aquatic environments and as such, may interact with them at the physiological level. This study was designed to investigate the influence of bacteria, present in a clonal culture of Pfiesteria piscicida, on the predator/prey relationship of this dinoflagellate with the alga Rhodomonas. A series of replenishment experiments with bacteria isolated from P. piscicida clonal culture and the bacteria-free P. piscicida derived from the same culture were carried out. In the presence of bacteria, the number of P. piscicida increased significantly when incubated with alga Rhodomonas. This enhanced growth was almost entirely due to the increased consumption rate of Rhodomonas by P. piscicida since in bacteria-free (axenic) cultures Rhodomonas were consumed at significantly reduced rates relative to cultures with bacteria. Subsequent replenishment experiments with individual bacterial isolates showed that a single isolate was responsible for the increased predation rate of P. piscicida. The presence or absence of this specific bacterium determined the outcome of the interaction between P. piscicida and Rhodomonas. Partial sequence analysis of the 16S rDNA of this isolate indicated that it was a novel marine alpha proteobacterium with sequence similarities to a Roseobacter sp. and a bacterium recently isolated from a toxic dinoflagellate Alexandrium sp.     

Response of Bdellovibrio and Like Organisms (BALOs) to the Migration of Naturally Occurring Bacteria to Chemoattractants

A dual culture-based and non–culture-based approach was applied to characterize predator bacterial groups in surface water samples collected from Apalachicola Bay, Florida. Chemotaxis drop assays were performed on concentrated samples in an effort to isolate predator bacteria by their chemotactic ability. Yeast extract (YE) and casamino acids (CA) proved to be strong chemoattractants and resulted in three visibly distinct bands; however, dextrose, succinate, pyruvate, and concentrated cells of Vibrio parahaemolyticus P5 as prey did not elicit any response. The three distinct bands from YE and CA were separately collected to identify the chemotactic microbial assemblages. Plaque-forming unit assays from different chemotaxis bands with P5 as prey indicated 5- (CA) to 10-fold (YE) higher numbers of predator bacteria in the outermost chemotactic bands. Polymerase chain reaction–restriction fragment length polymorphism and 16S rDNA sequencing of clones from different chemotaxis bands resulted in identification of Pseudoalteromonas spp., Marinomonas spp., and Vibrio spp., with their numbers inversely proportional to the numbers of predators—i.e., Bdellovibrio spp. and Bacteriovorax spp—in the chemotaxis bands. This study indicates that predatorial bacteria potentially respond to high densities of microbial biomass in aquatic ecosystems and that chemotaxis drop assay may be an alternate culture-independent method to characterize predatorial bacterial guilds from the environment.     

Long‐term impacts of invasive species on a native top predator in a large lake system

1. Declining abundances of forage fish and the introduction and establishment of non‐indigenous species have the potential to substantially alter resource and habitat exploitation by top predators in large lakes. 2. We measured stable isotopes of carbon (δ¹³C) and nitrogen (δ¹⁵N) in field‐collected and archived samples of Lake Ontario lake trout (Salvelinus namaycush) and five species of prey fish and compared current trophic relationships of this top predator with historical samples. 3. Relationships between δ¹⁵N and lake trout age were temporally consistent throughout Lake Ontario and confirmed the role of lake trout as a top predator in this food web. However, δ¹³C values for age classes of lake trout collected in 2008 ranged from 1.0 to 3.9‰ higher than those reported for the population sampled in 1992. 4. Isotope mixing models predicted that these changes in resource assimilation were owing to the replacement of rainbow smelt (Osmerus mordax) by round goby (Neogobius melanostomus) in lake trout diet and increased reliance on carbon resources derived from nearshore production. This contrasts with the historical situation in Lake Ontario where δ¹³C values of the lake trout population were dominated by a reliance on offshore carbon production. 5. These results indicate a reduced capacity of the Lake Ontario offshore food web to support the energetic requirements of lake trout and that this top predator has become increasingly reliant on prey resources that are derived from nearshore carbon pathways.     

Isolation and characterization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains from different grain habitats in Turkey

Bacillus thuringiensis (Bt) is a gram-positive, spore-forming bacterium and it produces insecticidal crystal (cry) proteins during sporulation. Because the genetic diversity and toxic potential of Bt strains differ from region to region, strains have been collected and characterized all over the world. The aim of this study is to isolate Bt strains in grain-related habitats in Turkey and to characterize them on the basis of crystal morphology, cry gene content, and chromosomal and plasmid DNA profiles. Four approaches were taken analysis with phase contrast (PC) microscopy, polymerase chain reaction (PCR), pulsed field gel electrophoresis (PFGE) and plasmid isolation. Ninety-six samples were collected from Central Anatolia and the Aegean region. Bt was isolated from 61 of 96 samples (63.5) and 500 Bt-like colonies were obtained. One hundred and sixty three of the colonies were identified as Bt based on cry protein formation using PC microscopy. Among the examined colonies, the overall proportion identified (as Bt index) was 0.33. We found that 103 isolates were positive for the five different cry genes (cry1, cry2, cry3, cry4 and cry9) examined with PCR. In addition, plasmid profiling of 37 cry gene-positive isolates indicated that the 15 kb plasmid band was present in all isolates; however, 11 of 37 isolates had more than one plasmid band at different sizes. Finally, chromosomal DNA profiling by PFGE gave rise to different DNA patterns for isolates containing the same cry gene which suggests a high level of diversity among the Bt strains isolated.     

Characterization of Pasteurellaceae‐like bacteria isolated from clinically affected psittacine birds

Aims: The aim of the present investigation was to identify and characterize Pasteurella‐like isolates obtained from clinically affected psittacine birds. Methods and Results: A total of 37 isolates from psittacine birds tentatively classified with the family Pasteurellaceae were characterized phenotypically. The genetic relationship was investigated by sequencing of partial rpoB and 16S rRNA genes for selected isolates. The results obtained were compared with the data from 16 reference strains. Nine isolates were identified as Gallibacterium spp., 16 as Volucribacter spp. or Volucribacter‐like, while 11 isolates were classified as taxon 44 of Bisgaard. A single isolate was identified as Pasteurella multocida. Conclusions: Characterization of Pasteurellaceae by traditional methods is often inconclusive because of inconsistent reactions and phenotypic diversity. For the same reason, genotyping is essential to allow proper classification as demonstrated in the present study. Significance and Impact of the Study: Limited information exists on the isolation and significance of Pasteurellaceae associated with clinically affected psittacine birds showing signs of digestive and/or respiratory disorders. The present investigations demonstrated that these organisms are widely distributed among clinically affected birds, but isolation of these taxa cannot be unambiguously correlated with the symptoms observed.     

Use of mycelia as paths for the isolation of contaminant‐degrading bacteria from soil

Mycelia of fungi and soil oomycetes have recently been found to act as effective paths boosting bacterial mobility and bioaccessibility of contaminants in vadose environments. In this study, we demonstrate that mycelia can be used for targeted separation and isolation of contaminant‐degrading bacteria from soil. In a ‘proof of concept’ study we developed a novel approach to isolate bacteria from contaminated soil using mycelia of the soil oomycete Pythium ultimum as translocation networks for bacteria and the polycyclic aromatic hydrocarbon naphthalene (NAPH) as selective carbon source. NAPH‐degrading bacterial isolates were affiliated with the genera Xanthomonas, Rhodococcus and Pseudomonas. Except for Rhodococcus the NAPH‐degrading isolates exhibited significant motility as observed in standard swarming and swimming motility assays. All steps of the isolation procedures were followed by cultivation‐independent terminal 16S rRNA gene terminal fragment length polymorphism (T‐RFLP) analysis. Interestingly, a high similarity (63%) between both the cultivable NAPH‐degrading migrant and the cultivable parent soil bacterial community profiles was observed. This suggests that mycelial networks generally confer mobility to native, contaminant‐degrading soil bacteria. Targeted, mycelia‐based dispersal hence may have high potential for the isolation of bacteria with biotechnologically useful properties.     

Higher Isolation of NDM-1 Producing Acinetobacter baumannii from the Sewage of the Hospitals in Beijing

Multidrug resistant microbes present in the environment are a potential public health risk. In this study, we investigate the presence of New Delhi metallo-β-lactamase 1 (NDM-1) producing bacteria in the 99 water samples in Beijing City, including river water, treated drinking water, raw water samples from the pools and sewage from 4 comprehensive hospitals. For the bla _NDM-1 positive isolate, antimicrobial susceptibility testing was further analyzed, and Pulsed Field Gel Electrophoresis (PFGE) was performed to determine the genetic relationship among the NDM-1 producing isolates from sewage and human, as well as the clinical strains without NDM-1. The results indicate that there was a higher isolation of NDM-1 producing Acinetobacter baumannii from the sewage of the hospitals, while no NDM-1 producing isolates were recovered from samples obtained from the river, drinking, or fishpond water. Surprisingly, these isolates were markedly different from the clinical isolates in drug resistance and pulsed field gel electrophoresis profiles, suggesting different evolutionary relationships. Our results showed that the hospital sewage may be one of the diffusion reservoirs of NDM-1 producing bacteria.     

Application of machine learning to identify predators of stocked fish in Lake Ontario: using acoustic telemetry predation tags to inform management

Understanding predator–prey interactions and food web dynamics is important for ecosystem-based management in aquatic environments, as they experience increasing rates of human-induced changes, such as the addition and removal of fishes. To quantify the post-stocking survival and predation of a prey fish in Lake Ontario, 48 bloater Coregonus hoyi were tagged with acoustic telemetry predation tags and were tracked on an array of 105 acoustic receivers from November 2018 to June 2019. Putative predators of tagged bloater were identified by comparing movement patterns of six species of salmonids (i.e., predators) in Lake Ontario with the post-predated movements of bloater (i.e., prey) using a random forests algorithm, a type of supervised machine learning. A total of 25 bloater (53% of all detected) were consumed by predators on average (± S.D. ) 3.1 ± 2.1 days after release. Post-predation detections of predators occurred for an average (± S.D. ) of 78.9 ± 76.9 days, providing sufficient detection data to classify movement patterns. Tagged lake trout Salvelinus namaycush provided the most reliable classification from behavioural predictor variables (89% success rate) and was identified as the main consumer of bloater (consumed 50%). Movement networks between predicted and tagged lake trout were significantly correlated over a 6 month period, supporting the classification of lake trout as a common bloater predator. This study demonstrated the ability of supervised learning techniques to provide greater insight into the fate of stocked fishes and predator–prey dynamics, and this technique is widely applicable to inform future stocking and other management efforts.     

Isolation and characterization of bacteriophages infecting <Emphasis Type="Italic">Xanthomonas arboricola</Emphasis> pv. <Emphasis Type="Italic">juglandis</Emphasis>, the causal agent of walnut blight disease

Walnut orchards suffer from a blight caused by the bacteria Xanthomonas arboricola pv. juglandis. These bacteria can be infected by viral bacteriophages and this study was carried out to isolate and characterize bacteriophages from walnut orchards located throughout the South Island of New Zealand. Twenty six X. arboricola phages were isolated from three hundred and twenty six samples of plant material representing phyllosphere and rhizosphere ecosystems. The phage isolates were characterized by host-range, plaque and particle morphology, restriction digest and phylogenetic analysis and stability under various storage conditions. From capsid and tail dimensions the bacteriophages were considered to belong to the double-stranded DNA families Podoviridae and Siphoviridae. Of the twenty six bacteriophages, sixteen belonged to Podoviridae and were found both in the phyllosphere and rhizosphere. In contrast, Siphoviridae were present only in the rhizosphere isolates. Phage genome sizes ranged from 38.0 to 52.0 kb from a Hind III restriction digestion and had in common a 400 kb fragment that was identical at the DNA level. Despite the similar restriction patterns, maximum parsimony bootstrap analysis showed that the phage were members of different groups. Finally, we hypothesise that these phage might have use in a biocontrol strategy and therefore storage stability and efficacy was tested. Titres declined more than 50% over a 12-months storage period. Deep-freezing temperatures (−34°C) increased while chloroform decreased the stability.     

Isolation of Bdellovibrio sp. from Wastewater and Their Potential Application in Control of Salmonella paratyphi in Water

The problem of the increasing resistance of bacteria to conventional antibiotics gives the bacteria Bdellovibrio a great utility as a potential alternative source of antibiotics. Therefore, the preliminary goal of the present study was isolation and identification of antibiotic-resistant bacteria used as prey organisms for isolated Bdellovibrio sp., by xylose lysine desoxycholate (XLD) agar from different types of water in the Taif area, Saudi Arabia, and also to investigate water quality. Four antibiotic-resistant isolates of Salmonella sp. which were susceptible to Bdellovibrio were identified by morphological, biochemical and 16S rRNA characterization as Salmonella paratyphi. Seventeen strains of Bdellovibrio sp. were isolated from sewage wastewater using isolated S. paratyphi as prey bacteria by a double-layer plate. Only one of them causing a large plaque after 48 h of incubation at 37°C was designated Bdellovibrio AOA12. The shape of Bdellovibrio was confirmed by morphological characterization and electron microscopy. Bdellovibrio could lyse four antibiotic-resistant Gram-negative bacterial strains of Salmonella paratyphi but could not lyse Gram-positive bacteria such as Staphylococcus aureus and Bacillus cereus. The kinetic lysis of the Bdellovibrio as predator to four isolates of antibiotic-resistant Salmonella paratyphi as prey organisms was demonstrated. The results suggest that it may be possible to utilize Bdellovibrio to control antibiotic-resistant S. paratyphi in water.     

Atypical swimbladders of lake charr, Salvelinus namaycush, from Great Slave Lake, Northwest Territories, Canada

Swimbladder walls of lake charr, Salvelinus namaycush, from Great Slave Lake (GSL), Northwest Territories, Canada, were unusually thick for the species. The thinnest sections of the GSL bladders (mean = 2.44mm, range = 1.1–4.4mm) were significantly thicker (P = 0.001) than lake charr swimbladders collected from two small Province of Ontario lakes (means = 0.65 and 0.92mm), whose populations were assumed to be representative of the species. Variance in wall thickness was also greater in GSL lake charr than in charr from two small lakes (P < 0.02). Within individuals, some of the GSL bladder walls were markedly irregular in thickness, but whether these anomalies exist in situ or were artifacts of preservation remains uncertain. The bulk of the tissue in the thickest sections of the GSL swimbladders was in the tunica serosa (outer layer). The extent of the modification of the GSL swimbladders is extraordinary for northern fishes in postglacial lakes.     

Antimicrobial Activities and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry of <Emphasis Type="Italic">Bacillus</Emphasis> Isolates from the Marine Sponge <Emphasis Type="Italic">Aplysina aerophoba</Emphasis>

The aim of this study was to isolate bacteria that are resistant to the strong antimicrobial metabolites characteristic of Aplysina aerophoba. For this purpose, bacterial isolation was performed on agar plates to which sponge tissue extract had been added. Following screening for antifungal and antimicrobial activities, 5 strains were chosen for more detailed analyses. 16S ribosomal DNA sequencing revealed that all isolates belonged to the genus Bacillus, specifically B. subtilis and B. pumilus. Using a combination of matrix-assisted laser desorption/ ionization mass spectrometry typing of whole cells and antimicrobial bioassays against selected reference strains, the bioactive metabolites were identified as lipopeptides.     

Susceptibility of Caenorhabditis elegans to Burkholderia Infection Depends on Prior Diet and Secreted Bacterial Attractants

The nematode Caenorhabditis elegans may be killed by certain pathogenic bacteria and thus is a model organism for studying interactions between bacteria and animal hosts. However, growing nematodes on prey bacteria may influence their susceptibility to potential pathogens. A method of axenic nematode culture was developed to isolate and quantify interactions between C. elegans and potentially pathogenic strains of the Burkholderia cepacia complex. Studying these dynamics in liquid solution rather than on agar surfaces minimized nematode avoidance behavior and resolved more differences among isolates. Most isolates of B. cenocepacia, B. ambifaria and B. cepacia caused 60–80% mortality of nematodes after 7 days, whereas isolates of B. multivorans caused less mortality (<25%) and supported nematode reproduction. However, some B. cenocepacia isolates recovered from chronic infections were much less virulent (5–28% mortality). As predicted, prior diet altered the outcome of interactions between nematodes and bacteria. When given the choice between Burkholderia and E. coli as prey on agar, axenically raised nematodes initially preferred most lethal Burkholderia isolates to E. coli as a food source, but this was not the case for nematodes fed E. coli, which avoided toxic Burkholderia. This food preference was associated with the cell-free supernatant and thus secreted compounds likely mediated bacterial-nematode interactions. This model, which isolates interactions between bacteria and nematodes from the effects of prior feeding, demonstrates that bacteria can influence nematode behavior and their susceptibility to pathogens.     

Fleeing towards death – leech‐induced behavioural defences increase freshwater snail susceptibility to predatory fish

Prey species are often exposed to multiple predators, which presents several difficulties to prey species. This is especially true when the response to one predator influences the prey’s susceptibility to other predators. Predator‐induced defences have evolved in a wide range of prey species, and experiments involving predators with different hunting strategies allow researchers to evaluate how prey respond to multiple threats. Freshwater snails are known to respond to a variety of predators with both morphological and behavioural defences. Here we studied how freshwater snails Radix balthica responded behaviourally to fish and leech predators, both separately and together. Our aim was to explore whether conflicting predator‐induced responses existed and, if so, what effect they had on snail survival when both predatory fish and leeches were present. We found that although R. balthica increased refuge use when exposed to predatory fish, they decreased refuge use when exposed to predatory leeches. When both predators were present, snails showed a stronger response towards leech than fish and responded by leaving the refuge. This response made the snails more susceptible to fish predation, which increased snail mortality when exposed to both fish and leech compared to fish only. We show that predators that have a relatively low predation rate can substantially increase mortality rates by indirect effects. By forcing snails out of refuges such as rock and macrophyte habitats, leeches can indirectly increase predation from molluscivorous fish and may thus affect snail densities.