Fate of Bacillus thuringiensis subsp. israelensis in the Field: Evidence for Spore Recycling and Differential Persistence of Toxins in Leaf Litter

Bacillus thuringiensis subsp. israelensis is a bioinsecticide increasingly used worldwide for mosquito control. Despite its apparent low level of persistence in the field due to the rapid loss of its insecticidal activity, an increasing number of studies suggested that the recycling of B. thuringiensis subsp. israelensis can occur under specific, unknown conditions. Decaying leaf litters sampled in mosquito breeding sites in the French Rhône-Alpes region several months after a treatment were shown to exhibit a high level of larval toxicity and contained large amounts of spores. In the present article, we show that the high concentration of toxins found in these litters is consistent with spore recycling in the field, which gave rise to the production of new crystal toxins. Furthermore, in these toxic leaf litter samples, Cry4Aa and Cry4Ba toxins became the major toxins instead of Cyt1Aa in the commercial mixture. In a microcosm experiment performed in the laboratory, we also demonstrated that the toxins, when added in their crystal form to nontoxic leaf litter, exhibited patterns of differential persistence consistent with the proportions of toxins observed in the field-collected toxic leaf litter samples (Cry4 > Cry11 > Cyt). These results give strong evidence that B. thuringiensis subsp. israelensis recycled in specific breeding sites containing leaf litters, and one would be justified in asking whether mosquitoes can become resistant when exposed to field-persistent B. thuringiensis subsp. israelensis for several generations.     

Germination, Growth, and Sporulation of Bacillus thuringiensis subsp. israelensis in Excreted Food Vacuoles of the Protozoan Tetrahymena pyriformis

Spores of Bacillus thuringiensis subsp. israelensis and their toxic crystals are bioencapsulated in the protozoan Tetrahymena pyriformis, in which the toxin remains stable. Each T. pyriformis cell concentrates the spores and crystals in its food vacuoles, thus delivering them to mosquito larvae, which rapidly die. Vacuoles containing undigested material are later excreted from the cells. The fate of spores and toxin inside the food vacuoles was determined at various times after excretion by phase-contrast and electron microscopy as well as by viable-cell counting. Excreted food vacuoles gradually aggregated, and vegetative growth of B. thuringiensis subsp. israelensis was observed after 7 h as filaments that stemmed from the aggregates. The outgrown cells sporulated between 27 and 42 h. The spore multiplication values in this system are low compared to those obtained in carcasses of B. thuringiensis subsp. israelensis-killed larvae and pupae, but this bioencapsulation represents a new possible mode of B. thuringiensis subsp. israelensis recycling in nontarget organisms.     

Comparative Sensitivity to UV-B Radiation of Two Bacillus thuringiensis Subspecies and Other Bacillus sp.

Susceptibility of Bacillus thuringiensis spores and toxins to the UV-B range (280–330 nm) of the solar spectrum reaching Earth's surface may be responsible for its inactivation and low persistence in nature. Spores of the mosquito larvicidal B. thuringiensis subsp. israelensis were significantly more resistant to UV-B than spores of the lepidopteran-active subsp. kurstaki. Spores of subsp. israelensis were as resistant to UV-B as spores of B. subtilis and more resistant than spores of the closely related B. cereus and another mosquito larvicidal species B. sphaericus. Sensitivity of B. thuringiensis subsp. israelensis spores to UV-B radiation depended upon their culture age; 24-h cultures, approaching maximal larvicidal activity, were still sensitive. Maximal resistance to UV-B was achieved only at 48 h. Received: 13 December 2000/Accepted: 19 January 2001     

Protection from UV-B Damage of Mosquito Larvicidal Toxins from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. <Emphasis Type="Italic">israelensis</Emphasis> Expressed in <Emphasis Type="Italic">Anabaena</Emphasis> PCC 7120

A transgenic strain of the nitrogen-fixing filamentous cyanobacterium Anabaena PCC 7120 protected expressed δ-endotoxin proteins of Bacillus thuringiensis subsp. israelensis from damage inflicted by UV-B, a sunlight component that penetrates Earth's ozone layer. This organism, which serves as a food source to mosquito larvae and could multiply in their breeding sites, may solve the environment-imposed limitations of B. thuringiensis subsp. israelensis as a mosquito biological control agent. Received: 20 November 2001 / Accepted: 31 December 2001     

Marginal cross-resistance to mosquitocidal Bacillus thuringiensis strains in Cry11A-resistant larvae: presence of Cry11A-like toxins in these strains

Culex quinquefasciatus mosquito larvae resistant to the Cry11A toxin showed marginal cross-resistance to the multiple toxin crystals from B. thuringiensis subsp. israelensis and also to toxin crystals from three other mosquitocidal strains, i.e. B. thuringiensis subsp. fukuokaensis, subsp. jegathesan, and subsp. kyushuensis. Cross-resistance patterns of the Cry11A-resistant larvae to mosquitocidal strains of B. thuringiensis together with the immunological screening using antisera raised against Cry11A indicated the presence of Cry11A-like toxins in these strains and could be used as a screening tool for the identification of novel toxins. The Cry11A-resistant larvae had significantly less resistance to the Cry11B toxin from B. thuringiensis subsp. jegathesan. The occurrence of cytolytic toxins in all of these mosquitocidal strains partially explains the marginal cross-resistance observed with multiple toxin crystals since each of these crystals also contains cytolytic toxins.     

Properties and applied use of the mosquitocidal bacterium,Bacillus sphaericus

Strains of Bacillus sphaericus exhibit varying levels of virulence against mosquito larvae. The most potent strain, B. sphaericus 2362, which is the active ingredient in the commercial product VectoLex^®, together with another well-known larvicide Bacillus thuringiensis subsp. israelensis, is used to control vector and nuisance mosquito larvae in many regions of the world. Although not all strains of B. sphaericus are mosquitocidal, lethal strains produce one or two combinations of three different types of toxins. These are (1) the binary toxin (Bin) composed of two proteins of 42 kDa (BinA) and 51 kDa (BinB), which are synthesized during sporulation and co-crystallize, (2) the soluble mosquitocidal toxins (Mtx1, Mtx2 and Mtx3) produced during vegetative growth, and (3) the two-component crystal toxin (Cry48Aa1/Cry49Aa1). Non-mosquitocidal toxins are also produced by certain strains of B. sphaericus, for example sphaericolysin, a novel insecticidal protein toxic to cockroaches. Larvicides based on B. sphaericus-based have the advantage of longer persistence in treated habitats compared to B. thuringiensis subsp. israelensis. However, resistance is a much greater threat, and has already emerged at significant levels in field populations in China and Thailand treated with B. sphaericus. This likely occurred because toxicity depends principally on Bin rather than various combinations of crystal (Cry) and cytolytic (Cyt) toxins present in B. thuringiensis subsp. israelensis. Here we review both the general characteristics of B. sphaericus, particularly as they relate to larvicidal isolates, and strategies or considerations for engineering more potent strains of this bacterium that contain built-in mechanisms that delay or overcome resistance to Bin in natural mosquito populations.     

Cyt1A of Bacillus thuringiensis Delays Evolution of Resistance to Cry11A in the Mosquito Culex quinquefasciatus

Insecticides based on Bacillus thuringiensis subsp. israelensis have been used for mosquito and blackfly control for more than 20 years, yet no resistance to this bacterium has been reported. Moreover, in contrast to B. thuringiensis subspecies toxic to coleopteran or lepidopteran larvae, only low levels of resistance to B. thuringiensis subsp. israelensis have been obtained in laboratory experiments where mosquito larvae were placed under heavy selection pressure for more than 30 generations. Selection of Culex quinquefasciatus with mutants of B. thuringiensis subsp. israelensis that contained different combinations of its Cry proteins and Cyt1Aa suggested that the latter protein delayed resistance. This hypothesis, however, has not been tested experimentally. Here we report experiments in which separate C. quinquefasciatus populations were selected for 20 generations to recombinant strains of B. thuringiensis that produced either Cyt1Aa, Cry11Aa, or a 1:3 mixture of these strains. At the end of selection, the resistance ratio was 1,237 in the Cry11Aa-selected population and 242 in the Cyt1Aa-selected population. The resistance ratio, however, was only 8 in the population selected with the 1:3 ratio of Cyt1Aa and Cry11Aa strains. When the resistant mosquito strain developed by selection to the Cyt1Aa-Cry11Aa combination was assayed against Cry11Aa after 48 generations, resistance to this protein was 9.3-fold. This indicates that in the presence of Cyt1Aa, resistance to Cry11Aa evolved, but at a much lower rate than when Cyt1Aa was absent. These results indicate that Cyt1Aa is the principal factor responsible for delaying the evolution and expression of resistance to mosquitocidal Cry proteins.     

Synergism between Bacillus thuringiensis Spores and Toxins against Resistant and Susceptible Diamondback Moths (Plutella xylostella)

We studied the effects of combinations of Bacillus thuringiensis spores and toxins on the mortality of diamondback moth (Plutella xylostella) larvae in leaf residue bioassays. Spores of B. thuringiensis subsp. kurstaki increased the toxicity of crystals of B. thuringiensis subsp. kurstaki to both resistant and susceptible larvae. For B. thuringiensis subsp. kurstaki, resistance ratios were 1,200 for a spore-crystal mixture and 56,000 for crystals without spores. Treatment of a spore-crystal formulation of B. thuringiensis subsp. kurstaki with the antibiotic streptomycin to inhibit spore germination reduced toxicity to resistant larvae but not to susceptible larvae. In contrast, analogous experiments with B. thuringiensis subsp. aizawai revealed no significant effects of adding spores to crystals or of treating a spore-crystal formulation with streptomycin. Synergism occurred between Cry2A and B. thuringiensis subsp. kurstaki spores against susceptible larvae and between Cry1C and B. thuringiensis subsp. aizawai spores against resistant and susceptible larvae. The results show that B. thuringiensis toxins combined with spores can be toxic even though the toxins and spores have little or no independent toxicity. Results reported here and previously suggest that, for diamondback moth larvae, the extent of synergism between spores and toxins of B. thuringiensis depends on the strain of insect, the type of spore, the set of toxins, the presence of other materials such as formulation ingredients, and the concentrations of spores and toxins.     

Isolation of a Bacillus thuringiensis strain (serotype 8a:8b) highly and selectively toxic against mosquito larvae

An isolate of Bacillus thuringiensis designated as PG-14 obtained from the Philippines was highly toxic to the mosquitoes Aedes aegypti and Culex molestus but nontoxic to the silkworm, Bombyx mori, and adults of a daphnid. The degree of toxicity to mosquito larvae was the same as that of the reference strain of Bacillus thuringiensis subsp. israelensis (serotype 14). Parasporal inclusion produced by the isolate PG-14 was spherical or irregular in shape and morphologically similar to that produced by the reference strain of subsp. israelensis. The H antigenic structure of the isolate was identical to that of the reference strain of B. thuringiensis subsp. morrisoni (serotype 8a:8b). Differences were shown in the O antigenic structures and in the production of lecithinase. Thermostable exotoxin was not produced by the isolate PG-14. The results indicate the isolation of a B. thuringiensis strain which shows the same toxicity as that of subsp. israelensis.     

Variable Cross-Resistance to Cry11B from Bacillus thuringiensis subsp. jegathesan in Culex quinquefasciatus (Diptera: Culicidae) Resistant to Single or Multiple Toxins of Bacillus thuringienisis subsp. israelensis

A novel mosquitocidal bacterium, Bacillus thuringiensis subsp. jegathesan, and one of its toxins, Cry11B, in a recombinant B. thuringiensis strain were evaluated for cross-resistance with strains of the mosquito Culex quinquefasciatus that are resistant to single and multiple toxins of Bacillus thuringiensis subsp. israelensis. The levels of cross-resistance (resistance ratios [RR]) at concentrations which caused 95% mortality (LC₉₅) between B. thuringiensis subsp. jegathesan and the different B. thuringiensis subsp. israelensis-resistant mosquito strains were low, ranging from 2.3 to 5.1. However, the levels of cross-resistance to Cry11B were much higher and were directly related to the complexity of the B. thuringiensis subsp. israelensis Cry toxin mixtures used to select the resistant mosquito strains. The LC₉₅ RR obtained with the mosquito strains were as follows: 53.1 against Cq4D, which was resistant to Cry11A; 80.7 against Cq4AB, which was resistant to Cry4A plus Cry4B; and 347 against Cq4ABD, which was resistant to Cry4A plus Cry4B plus Cry11A. Combining Cyt1A with Cry11B at a 1:3 ratio had little effect on suppressing Cry11A resistance in Cq4D but resulted in synergism factors of 4.8 and 11.2 against strains Cq4AB and Cq4ABD, respectively; this procedure eliminated cross-resistance in the former mosquito strain and reduced it markedly in the latter strain. The high levels of activity of B. thuringiensis subsp. jegathesan and B. thuringiensis subsp. israelensis, both of which contain a complex mixture of Cry and Cyt proteins, against Cry4- and Cry11-resistant mosquitoes suggest that novel bacterial strains with multiple Cry and Cyt proteins may be useful in managing resistance to bacterial insecticides in mosquito populations.     

Ingestion and Adsorption of Bacillus thuringiensis subsp. israelensis by Gammarus lacustris in the Laboratory

Several groups of Gammarus lacustris adults were exposed to solutions containing 0.5 and 5.0 mg of Bacillus thuringiensis subsp. israelensis per liter for 1- or 24-h periods by using traditional static bioassay exposure procedures. During a postexposure holding period, fecal pellets were removed and plated on tryptic soy agar to determine B. thuringiensis subsp. israelensis spore content. The experiments verified that traditional exposure procedures assure ingestion of B. thuringiensis subsp. israelensis spores and provided a mean dose estimate of 1,948 spores ingested per test animal with a 95% confidence interval ranging from 891 to 4,296 (1-h exposure, 5.0 mg/liter). It was also found that dose level is highly dependent upon both exposure duration and concentration and that relatively short exposures can result in a relatively long-term retention of spores postexposure (≥30 days). Body burden experiments established that large numbers of spores adsorb to the bodies of test animals during exposure and may in part explain the long-term retention of spores in the test system postexposure. These results imply that in field applications of microbial control agents, toxicologically unaffected but exposed organisms might transport the agent to untreated sites, expanding the effective treatment area and the number of organisms exposed.     

Long lasting persistence of Bacillus thuringiensis Subsp. israelensis (Bti) in mosquito natural habitats

Background

The detrimental effects of chemical insecticides on the environment and human health have lead to the call for biological alternatives. Today, one of the most promising solutions is the use of spray formulations based on Bacillus thuringiensis subsp. israelensis (Bti) in insect control programs. As a result, the amounts of Bti spread in the environment are expected to increase worldwide, whilst the common belief that commercial Bti is easily cleared from the ecosystem has not yet been clearly established.

Methodology/Main Findings

In this study, we aimed to determine the nature and origin of the high toxicity toward mosquito larvae found in decaying leaf litter collected in several natural mosquito breeding sites in the Rhône-Alpes region. From the toxic fraction of the leaf litter, we isolated B. cereus-like bacteria that were further characterized as B. thuringiensis subsp. israelensis using PCR amplification of specific toxin genes. Immunological analysis of these Bti strains showed that they belong to the H14 group. We finally used amplified length polymorphism (AFLP) markers to show that the strains isolated from the leaf litter were closely related to those present in the commercial insecticide used for field application, and differed from natural worldwide genotypes.

Conclusions/Significance

Our results raise the issue of the persistence, potential proliferation and environmental accumulation of human-spread Bti in natural mosquito habitats. Such Bti environmental persistence may lengthen the exposure time of insects to this bio-insecticide, thereby increasing the risk of resistance acquisition in target insects, and of a negative impact on non-target insects.     

The dual-activity insecticidal protein,Cry2Aa,does not enhance the mosquitocidal activity of Bacillus thuringiensis subsp. israelensis

Cry2Aa, one of the major insecticidal proteins produced by Bacillus thuringiensis subsp. kurstaki HD1, is known to be active against both lepidopteran and dipteran larvae. In order to determine whether Cry2Aa could enhance or synergize the mosquitocidal activity of B. thuringiensis subsp. israelensis, we constructed a plasmid vector that harbored the cry2Aa operon and transformed crystalliferous and acrystalliferous strains of this bacterium. The wild-type B. thuringiensis subsp. israelensis, a recombinant B. thuringiensis subsp. israelensis producing Cry2A along with its native major mosquitocidal proteins, and a recombinant B. thuringiensis subsp. israelensis producing Cry2Aa alone were tested against three major mosquito species — Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus. Our results demonstrated that Cry2Aa does not synergize or enhance the mosquitocidal activity of B. thuringiensis subsp. israelensis against these important vectors of disease.     

Ingested particles reduce susceptibility of insect larvae to Bacillus thuringiensis

Susceptibility to Bacillus thuringiensis of mosquito and lepidopteran larvae is affected by feeding behaviour and nutritional value of the available food. Reduced mortality is attributed to feeding inhibition and dilution of the pathogen in the presence of nutritional and inert particles, which limit the amount of ingested toxin. These reasons are, however, not sufficient to explain the data presented here. Values of LC₅₀ (the concentration that kills 50% of exposed population) of B. thuringiensis subsp. israelensis (Berliner) against Aedes aegypti (L.) larvae and of B. thuringiensis subsp. kenyae (Berliner) against Spodoptera littoralis (Boisduval) larvae were about 20–217 and 2.3–44‐fold higher, respectively, in the presence of nutritional or biologically inert (non‐nutritional) particles than without. The number of B. thuringiensis spores in carcasses of B. thuringiensis ‐killed A. aegypti and S. littoralis larvae were between 1.9 and 5.6‐fold and between 8.5 and 12‐fold higher, respectively, in the presence of particles than without. In all cases, non‐nutritional particles better protected the exposed larvae than nutritious particles. We propose that another basic mechanism exists, that ingested particles protect midgut epithelial cells by covering their surface and thus preventing availability of the toxin to the gut receptors. Understanding the defence mechanisms of insects against B. thuringiensis toxicity may lead to improved pest management methods.     

Evaluation of four fresh-water unicellular cyanobacteria as potential hosts for mosquitocidal toxins

Summary For biocontrol of mosquitoes, mosquitocidal toxin genes from Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus have been cloned into a number of cyanobacteria. However, little is known about the persistence of such recombinant cyanobacteria in mosquito larval habitats. Four fresh water unicellular cyanobacteria, Synechococcus PCC6301, PCC7425, PCC7942 and Synechocystis PCC 6803, were evaluated under laboratory conditions related to mosquito breeding environments. Results indicated that Synechococcus PCC6301 was potentially the most suitable organism for use in the natural mosquito habitat as it could tolerate a wide range of temperatures, salinities, and biological and chemical insecticides. Moreover, strain PCC6301 could be ingested and digested by Culex quinquefasciatus larvae and could support the development of larvae to full insect maturity.     

Transfer of plasmids pBC 16 and pC 194 into Bacillus thuringiensis subsp. israelensis

A lysozyme sensitive strain of B. thuringiensis (strain O 016) was isolated and shown to be effectively transformed with plasmids pC 194 and pHV 33 using the protoplast transformation technique. The plasmid pC 194 from one successful transformant, strain O 016–194, was subsequently transferred to B. thuringiensis subsp. israelensis by a “conjugation-like” process. The plasmid pBC 16 from B. cereus could also be transferred to B. thuringiensis subsp. israelensis with high frequency using the conjugation-like process. Further, both plasmids, pC 194 and pBC 16, were transferred between strains of B. thuringiensis subsp. israelensis to yield transcipient strains that harbored and expressed properties of both plasmids. This work constitutes effective gene transfer system in B. thuringiensis subsp. israelensis.     

Fate of Bacillus thuringiensis subsp. israelensis under Simulated Field Conditions

The fate of Bacillus thuringiensis subsp. israelensis in a natural aquatic habitat was studied in a model system by using laboratory-simulated field waters and a mutant of the bacterium resistant to three antibiotics. Contact with mud of a sporal culture of the mutant resulted in an immediate disappearance of the larvicidal activity but had no influence on viability. The cessation of toxicity was caused by bacterial adsorption on soil particles, since 99.8% of the bacteria was found in the mud fraction within 45 min, with concurrent disappearance from the supernatant. When the mud was stirred, the bacteria could be redetected. The viability count of the mud suspension remained practically constant for at least 22 days, indicating that the spores were still fully viable but were incapable of germinating and multiplying in the mud under our experimental conditions. Approximately 8% of the colony forming ability of the bacteria could be separated from the mud by vigorous mixing followed by immediate filtration. The filtrated spores retained their toxicity, killing 90% of the larval populations even after 22 days incubation in the soil. The inactivation of the toxic activity of B. thuringiensis subsp. israelensis in the mud was therefore a reversible process and was probably due to masking of the bacteria, thus making the bacteria and their toxin inaccessible to the larvae. In the simulated field waters without mud, we observed only a very slow inhibition of the larvicidal activity. In contrast to the activity in the mud suspension, this activity could not be restored.     

Larvicidal activity of Bacillus thuringiensis subsp. israelensis, serovar H14 in Aedes aegypti: Histopathological studies

Bacillus thuringiensis subsp. israelensis, serovar H14, when applied as a primary commercial powder, caused the rapid death of Aedes aegypti larvae. Mortality started 6 min after application of 4 μg/ml of the pathogen and reached a maximum 27 min later. When the LC₅₀ (10 ng/ml) was applied, mortality began after 37 min and reached a maximum 120 min later. Histopathological changes in B. thuringiensis israelensis-treated larvae could be observed only in the midgut and caeca. In B. thuringiensis israelensis-treated “dead larvae”, the epithelial layer is disorganized, most of the cells have disappeared and the peritrophic membrane is broken. The epithelium in the B. thuringiensis israelensis-treated “living larvae” still maintains its monolayer structure, but with marked cellular hypertrophy and vacuolized cytoplasm. Also, the “brush border” is thinner and disrupted. Based on the fact that mortality of A. aegypti is a quick process, and because the histopathological changes caused by B. thuringiensis israelensis are similar to those found in lepidopterous larvae treated with pure δ-endotoxin of other B. thuringiensis variants, it is suggested that larvicidal activity of B. thuringiensis israelensis in A. aegypti is due to its δ-endotoxin.     

Production of Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus strain 1593 on media using a byproduct from a monosodium glutamate factory

Two newly developed media, H4 and H7, were found to be highly suitable for culturing Bacillus thuringiensis subsp. israelensis and B. sphaericus, respectively. These media contained 0.05% K₂HPO₄ and 4% HDL (H4 medium) or 0.05% K₂HPO₄ and 7% HDL (H7 medium); HDL is the by-product from a monosodium glutamate factory. Tests to compare endospore formation and toxicity values of B. thuringiensis subsp. israelensis in H4 medium and nutrient broth supplemented with salts and glucose (NBSG) medium were carried out in a 3-liter fermentor. The viable cell count and LC₅₀ value of B. thuringiensis subsp. israelensis in H4 medium at 48 hr were 2.5 × 10⁸ cells/ml and 10^?7.2 (dilution), respectively, while those in NBSG medium were 1.6 × 10⁸ cells/ml and 10^?6.5, respectively. In the case of B. sphaericus grown in H7 medium, the number of cells and LC₅₀ value were found to be 1.4 × 10⁹ cells/ml and 10^?7.8, respectively. B. sphaericus grown in nutrient broth supplemented with salt and yeast extract (NBSY) were found to produce 6.4 × 10⁸ cells/ml and an LC₅₀ value of 10^?6.8. The toxicity of B. thuringiensis subsp. israelensis was tested against Aedes aegypti larvae, while that of B. sphaericus was tested against Culex quinquefasciatus. The cost of 10 liters of medium for production of B. thuringiensis subsp. israelensis and in B. sphaericus and H4 and H7 was $0.02 and $0.03, respectively. The cost of these newly developed media was much less than that of NBSG medium ($7.05 per 10 liters) for cultivation of B. thuringiensis subsp. israelensis and NBSY medium ($11.67 per 10 liters) for cultivation of B. sphaericus.     

Interactions between Bacillus thuringiensis subsp. israelensis and Fathead Minnows, Pimephales promelas Rafinesque, under Laboratory Conditions

Interactions between Bacillus thuringiensis subsp. israelensis and fathead minnows, Pimephales promelas, were studied in laboratory exposures to two commercial formulations, Vectobac-G and Mosquito Attack. Mortality among fatheads exposed to 2.0 × 10⁶ to 6.5 × 10⁶ CFU/ml with both formulations was attributed to severe dissolved oxygen depletion due to formulation ingredients rather than to direct toxicity from the parasporal crystal. No adverse effects were observed at 6.4 × 10⁵ CFU/ml and below. Fathead minnows rapidly accumulated high numbers of spores with 1 h of exposure to 2.2 × 10⁵ CFU of Mosquito Attack per ml, producing whole-body counts of 4.0 × 10⁶ CFU per fish. Comparison of counts on gastrointestinal tract samples and whole-body samples and high numbers of spores in feces indicated that ingestion was the major route of exposure. B. thuringiensis subsp. israelensis spore counts decreased rapidly after transfer of fish to clean water, with a drop of over 3 orders of magnitude in 1 day. Spores were rarely detected in fish after 8 days but were detectable in feces for over 2 weeks. These findings suggest that fish could influence the dissemination of B. thuringiensis subsp. israelensis, and possibly other microbial agents, in the aquatic environment.