Non-target trials with Pseudomonas fluorescens strain CL145A, a lethal control agent of dreissenid mussels (Bivalvia: Dreissenidae)

In an effort to develop an efficacious and environmentally safe method for managing zebra mussels (Dreissena polymorpha) and quaggamussels (Dreissena rostriformis bugensis), we initiated a research project investigating the potential use of bacteria and their naturalmetabolic products as biocontrol agents. This project resulted in the discovery of an environmental isolate lethal to dreissenid mussels,Pseudomonas fluorescens strain CL145A (Pf-CL145A). In previous published reports we have demonstrated that: 1) Pf-CL145A’s mode ofaction is intoxication (not infection); 2) natural product within ingested bacterial cells lyse digestive tract epithelial cells leading to dreisseniddeath; and 3) high dreissenid kill rates (>90%) are achievable following treatment with Pf-CL145A cells, irrespective of whether thebacterial cells are dead or alive. Investigating the environmental safety of Pf-CL145A was also a key element in our research efforts, andherein, we report the results of non-target trials demonstrating Pf-CL145A’s high specificity to dreissenids. These acute toxicity trials weretypically single-dose, short-term (24-72 h) exposures to Pf-CL145A cells under aerated conditions at concentrations highly lethal todreissenids (100 or 200 mg/L). These trials produced no evidence of mortality among the ciliate Colpidium colpoda, the cladoceran Daphniamagna, three fish species (Pimephales promelas, Salmo trutta, and Lepomis macrochirus), and seven bivalve species (Mytilus edulis,Pyganodon grandis, Pyganodon cataracta, Lasmigona compressa, Strophitus undulatus, Lampsilis radiata, and Elliptio complanata). Lowmortality (3-27%) was recorded in the amphipod Hyalella azteca, but additional trials suggested that most, if not all, of the mortality couldbe attributed to some other unidentified factor (e.g., possibly particle load or a water quality issue) rather than Pf-CL145A’s dreissenidkillingnatural product. In terms of potential environmental safety, the results of these invertebrate and vertebrate non-target trials areencouraging, but it would be unrealistic to think that dreissenids are the only aquatic organisms sensitive to Pf-CL145A’s dreissenid-killingnatural product. Additional testing is needed to better define Pf-CL145A’s margin of safety by identifying the sensitivity of other susceptibleorganisms. The results of these non-target safety trials – in combination with equally promising mussel control efficacy data – have now ledto Pf-CL145A’s commercialization under the product name Zequanox®, with dead cells as the product’s active ingredient. The commercialavailability of only dead-cell Zequanox formulations will eliminate the risk of any possible non-target infection by Pf-CL145A, furtherreducing environmental concerns. During the non-target project reported herein, the limited quantities of Pf-CL145A cells that we were ableto culture severely restricted the number and size of our trials. In contrast, the availability of Zequanox will now greatly expand theopportunities for non-target testing. The trials reported herein – exposing non-target organisms under aerated conditions to unformulated,laboratory-cultured cells – clearly point to Pf-CL145A’s potential for high host specificity, but non-target trials with Zequanox – using Pf-CL145A cells cultured, killed, and formulated using industrial-scale protocols – will be even more important as they will define the nontargetsafety limits of the actual commercial products under a wide range of environmental conditions.