Quantitative polymerase chain reaction (QPCR) methods for beach monitoring by estimating abundance of Enterococcus spp. in recreational waters use internal, positive controls which address only the amplification of target DNA. In this study two internal, positive controls were developed to control for both amplification and cell lysis in assays measuring abundance of vegetative Gram-positive bacteria.Controls were constructed using Streptococcus gordonii DL-1, a naturally transformable, Gram-positive bacterium. Unique target sequences were provided by chromosomal insertion of a genetically modified, green fluorescent protein gene fragment. Results suggest that their use for control of lysis and amplification may be of significant value.The use of these controls and the establishment of data quality objectives to determine the tolerable level of decision error should ensure that environmental decisions based on QPCR data are technically and scientifically sound.QPCR measurements related to cell abundance may vary between samples as thick-walled Gram-positive bacteria are inherently difficult to lyse and substances present in recreational waters may inhibit amplification. As QPCR methods are considered for beach monitoring, it is essential to demonstrate that the data obtained accurately reflects the abundance of the bacterial indicator.

Sources of Enterococcus faecalis isolates from Pensacola Beach, FL. were identified using a library-based approach by applying the statistical method of average similarity to single and composite data sets generated from separate analyses. Data sets included antibiotic resistance analysis (ARA), rep-fingerprints, and fatty acid methyl ester (FAME) profiles. Use of a composite data set composed of ARA and rep-fingerprints, added to the confidence of the identifications. The addition of FAME data to composite data sets did not add to the confidence of identifications. Source identification was performed to better understand risk associated with higher densities of enterococci found in swash zone interstitial water (SZIW) as compared to adjacent bathing water on Pensacola Beach, FL. The "swash zone" is that area of the beach continually washed over by waves. As the potential sources of enterococci were limited in this environment, only two library units, sea gull and human, were constructed. Identification of the beach isolates using a composite data set indicated a sea gull origin. The clonality of the beach isolates suggested that the beach environment selects certain subspecies of E. faecalis.

A shading experiment was conducted over a growing season to measure the effects of light reduction on Vallisneria americana in Perdido Bay on the Florida–Alabama border and to determine the response of heterotrophic bacteria in the rhizosphere. Plants subjected to 92% light reduction showed the most pronounced effects in chlorophyll a concentration, above- and below-ground biomass, and leaf dimensions. These results further suggested that the V. americana life cycle, as exhibited in temperate waters, was impaired. Heterotrophic bacteria were enumerated and identified (i) from the roots and sediments of fully illuminated plants and from unvegetated sediments at three intervals and (ii) from the roots of plants that have been subjected to 92% light reduction for 3 months. Up to two orders of magnitude greater numbers of bacteria were enumerated from root samples than sediment samples on a dry weight basis. Bacteria enumerated from the roots of plants subjected to light reduction (1.3±1.1×108 CFU g−1) were significantly higher than numbers of bacteria enumerated from the roots of fully illuminated plants (4.8±1.8×107 g−1 in the summer) or sediment samples (1.4±0.03×106 g−1). This suggests the roots of seagrasses stressed by light reduction provided more nutrients for bacterial growth. Higher percentages of Gram-negative bacteria were isolated from roots (up to 85% in the fall) than sediments (0–15%). Examination of isolates for traits characteristic of rhizosphere bacteria (siderophore production, formation of the phytohormone indole-3-acetic acid, and antifungal activity) did not show a clear distinction between root-associated and sediment isolates. Taxonomic identifications of root-associated bacteria based on MIDI analysis of fatty acid methyl esters were consistent with bacteria known to be associated with other plants or found at oxic–anoxic interfaces. In addition, the bacterial identifications showed most species were associated with only roots or only sediments. These results support studies suggesting seagrass rhizospheres harbor distinct bacterial communities.

Ingestion of bacteria by oysters Crassostrea virginica and bactericidal activity of oyster hemocytes were studied using 4 environmental isolates (shellfish) and 3 clinical isolates (fecal) of Vibrio parahaemolyticus. Clinical isolates (2030, 2062, 2107) were obtained from the feces of patients with gastroenteritis who became ill during the 1998 food poisoning outbreak traced to consumption of raw oysters from Galveston Bay, Texas. This outbreak was the first reported occurrence in the United States of the virulent serotype O3:K6. Environmental isolates were from oysters (1094, 1100), crab (1163) and sardines (ATCC 17802). All isolates possessed the thermolabile direct hemolysin (tlh) gene, whereas only the clinical isolates possessed the thermostable direct hemolysin (tdh) gene, a vir- ulence determinant. On average, environmental isolates were more susceptible than clinical isolates to killing by oyster hemocytes, as determined by an in vitro dye reduction assay. Isolate 2062 was the most susceptible of the clinical isolates; it lacked identifiable capsular material present in the other clinical isolates and displayed the most diffuse colony morphology on nutrient agar plates. When oys- ters were exposed in vivo to mixtures of a clinical (2030) and an environmental (1163) isolate, more clinical than environmental isolates were found in the tissues and hemolymph.

A tetrazolium dye reduction assay was used to study factors governing the killing of bacteria by oyster hemocytes, In vitro tests were performed on bacterial strains by using hemocytes from oysters collected from the same location in winter and summer. Vibrio parahaemolyticus strains, altered in motility or colonial morphology (opaque and translucent), and Listeria monocytogenes mutants lacking catalase, superoxide dismutase, hemolysin, and phospholipase activities were examined in winter and summer. Vibrio vulnificus strains, opaque and translucent (with and without capsules), were examined only in summer. Among V. parahaemolyticus and L. monocytogenes, significantly (P < 0.05) higher levels of killing by hemocytes were observed in summer than in winter. L. monocytogenes was more resistant than V. parahaemolyticus or V. vulnificus to the bactericidal activity of hemocytes. In winter, both translucent strains of V. parahaemolyticus showed significantly (P < 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. In summer, only one of the V. parahaemolyticus translucent strains showed significantly (P < 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. No significant differences (P > 0.05) in killing by hemocytes were observed between opaque (encapsulated) and translucent (nonencapsulated) pairs of V. vulnificus. Activities of 19 hydrolytic enzymes were measured in oyster hemolymph collected in winter and summer. Only one enzyme, esterase (C4), showed a seasonal difference in activity (higher in winter than in summer). These results suggest that differences existed between bacterial genera in their ability to evade killing by oyster hemocytes, that a trait(s) associated with the opaque phenotype may have enabled V. parahaemolyticus to evade killing by the oyster's cellular defense, and that bactericidal activity of hemocytes was greater in summer than in winter.

Renewed interest in the use of Metarhizium anisopliae and its toxins for insect control prompted the following safety assessment. A neutral extract (methylene chloride, pH 7.2), derived from M. anisopliae cultures, was evaluated for toxicity and mutagenicity using aquatic animal bioassays and the Ames test. The average LC50 of the neutral extract obtained in static, acute 96-h tests conducted with ≤24-h-old Mysidopsis bahia was 2.41 mg L−1. By partially purifying destruxins from the neutral extract, it was shown that destruxins alone were not responsible for the observed toxicity in mysids. The neutral extract was fetotoxic to developing grass shrimp, Palaemonetes pugio, and frog, Xenopus laevis, embryos; the LC50 values were 52 and 32 mg L−1, respectively. Eye spot abnormalities were observed in shrimp and frog embryos exposed to the neutral extract. In extract-exposed frog embryos, moderate to severe cranial, facial, and gut malformations were also observed. The neutral extract was toxic to juvenile mosquito fish, Gambusia affinis, at an LC50 value of 141 mg L−1. Adult female G. affinis surviving a 24-h exposure to 200 μg ml−1 of the neutral extract produced healthy broods. After 3 months, no mortalities or adverse effects were observed in adult G. affinis fed a diet partially composed of a freeze-dried M. anisopliae culture. The neutral extract did not show mutagenicity in the Ames test using strains TA98 and TA100 with and without metabolic activation by rat liver S9. Significant (p  ≤ 0.05) mortalities were obtained when embryos of grass shrimp and inland silverside fish, Menidia beryllina, were exposed to the same lot of M. anisopliae conidiospores. Exposure of frog embryos to M. anisopliae conidiospores did not cause significant (p > 0.05) mortalities or malformations.

Experiments were performed in which developing embryos of the grass shrimp,Palaemonetes pugio,were exposed to conidiospores of the insect pathogenic fungus,Metarhizium anisopliae.Responses were variable, with significant (P≤0.05) adverse effects observed in five of six experiments conducted. Dead embryos and larvae with visible growth ofM. anisopliaewere observed in all experiments. Growth ofM. anisopliaewas occasionally observed on embryos and larvae prior to death. Delayed hatch was also observed. In one of the initial experiments, an increase inN-acetyl-β-d-glucosaminidase, EC 3.2.1.30 (NAGase), activity accompanied by an increase in virulence toward shrimp embryos was observed. Additional experiments in which conidiospores were produced on homogenized caterpillars suggested a positive correlation between virulence ofM. anisopliaetoP. pugioembryos and activity of spore-associated NAGase. Under these laboratory conditionsM. anisopliaewas an invasive pathogen of grass shrimp embryos, and the growth substrates on which their spores develop can influence the severity of effects on these nontarget arthropods.

A short-term (48 h) chemical toxicity test using larvae of the coot clam Mulinia lateralis was modified to evaluate potential toxicity and pathogenicity of microbial pest control agents. M. lateralis larvae, at the straight-hinged stage of development, were exposed to various microbial pest control agents including: a mosquito larvacide, Bacillus thuringiensis var. israelensis (Bti); a molluscicidal strain of Bacillus alvei; a viral pathogen of the gypsy moth Lymantria dispar nuclear polyhedrosis virus (LdNPV); and a broad host-range fungal insect pathogen, Metarhizium anisopliae. Mortalities significantly higher than heat-killed controls were obtained with Bti at a 10(-4) dilution of a commercial preparation, and with LdNPV at an occlusion body density of 1 x 10(6) ml(-1). Sodium dodecyl sulfate (SDS) and the water-soluble fraction of No. 2 fuel oil (WSFoil) were also tested to provide a measure of comparison, sensitivity and precision. SDS, toxic at an LC(50) of 6.3 mg l(-1), had a mean coefficient of variation of 23 %. The clam larval toxicity test was very sensitive to WSFoil; exposures resulted in an LC(50) Of <10% v/v. Because of its precision, sensitivity and simplicity, the hi, lateralis larval test has the potential to be useful for assessing adverse effects that microbial pest control agents may have on nontarget bivalves.

Developing embryos of the inland silverside fish, Menidia beryllina, and grass shrimp, Palaemonetes pugio, were exposed to conidiospores of the fungal weed control agent, Colletotrichum gloeosporioides f. sp. aeschynomene and the entomopathogen, Metarhizium anisopliae. Only Metarhizium anisopliae caused significant (p≤0.05) mortalities in the exposed embryos. Colletotrichum gloeosporioides did, however, cause fatal infections in adults when conidia were injected into the peritoneum of fish or the hemocoel of shrimp.

The fate of Bacillus sphaericus spores in the aquatic environment was investigated by suspending spores in dialysis bags in fresh and seawater. Spore viability was lost more rapidly in seawater. Neither B. sphaericus nor B. thuringiensis israelensis (B.t.i.) spores mixed with pond sediment appeared to attach to the sediment. However, rapid decrease in B.t.i. toxicity suggested attachment of parasporal bodies to sediment. B. sphaericus toxin settled more slowly and less completely. B. sphaericus spores fed to larvae of four aquatic invertebrates were mostly eliminated from the animal gut in less than one week. An exception was the cranefly (Tipula abdominalis) where spores persisted in the posterior gut for up to five weeks.

Developing embryos of the inland silverside fish Menidia beryllina were exposed to conidiospores of the entomopathogenic fungus Metarhizium anisopliae. Several adverse effects were observed in both embryos and newly hatched larvae. These included transitory effects on the heart resulting in decreased cardiac output or circulation velocity, rupture of the chorion, fungal growth on the mandibles of larvae, focal vertebral abnormalities in larvae and teratogenic expressions in embryos and larvae. An ordinal ranking system was used to enumerate responses to conidiospores. This ranking system allowed significance to be determined by nonparametric analysis of variance. Responses were highly variable with significant (p less than or equal to 0.05) adverse effects observed in 5 of the 6 experiments conducted. Heat-killed spores failed to cause significant adverse effects indicating that viable spores were required for the adverse effects.

Developing embryos of the inland silverside fish, Menidia beryllina, were exposed to conidiospores of the insect pathogenic fungus, Beauveria bassiana, that possessed activity against the migratory grasshopper, Melanoplus sanguinipes. Various adverse effects were observed in Menidia beryllina embryos and larvae. They included rupture of the chorion, embryo death, developmental defects (vertebral abnormalities) in the embryo or hatched larvae, and fungal infections on the mandibles of larvae. Although there was little evidence of a definitive dose-response trend based on densities of viable conidiospores, statistically significant (p≤0.01) responses were observed in tests in which conidiospore densities were as low as 7.1×103/ml and as high as 1.3×106/ml. Viable spores were required for adverse effects to occur; heat-killed spores failed to cause significant adverse effects.

Embryos, larvae and adult grass shrimp Palaemonetes pug10 were exposed to spores of the insect-control fungus Beauvena basslana Conidiospores attached to embryos held by gravid females and remained wlth the egg mass for at least 6 d In the first experiment where individual developing embryos contained in test tubes were exposed to conidiospores of B basslana. 2 of the 75 embryos became infected and died, and a third was abnormal at hatch In Expt 2 a repeat experiment, no adverse effects were observed In subsequent expenments, attempts were made to increase the probability of infection or abnormal development for embryos exposed to B basslana Strategies for these attempts included lowering the salinity, adding a carbon source for the fungus to the test water, or rendenng embryos free of potentially protective symbiotic bacteria with an antibacter~al agent All attempts were unsuccessful in promoting infection or abnormal development B bassiana did not cause any adverse effects duling shnmp larval development In adult shrimp lethal infections only occurred in specimens injected with eithei conldiospores or blastospores of B bassjana

Beauveria bassiana spores and metabolites were evaluated for toxicity and pathogenicity to Mysidopsis bahia. Static acute 96-h tests were conducted with ⩽24-h-old M. bahia using either conidiospores; the mycotoxin, beauvericin, or a nonpolar extract of the mycelia. Conidiospore densities of ⩾1.5×106/ml caused high mortalities. These mortalities were attributed to a high particulate density since heat-killed controls also proved lethal. Beauvericin, a cyclic depsipeptide produced by some strains of B. bassiana, was toxic at an LC50 of 0.56 mg/L. The toxicity of beauvericin persisted in sterile seawater for at least 3, but not, 8 weeks. A nonpolar extract of mycelia from B. bassiana, which contained approximately 1% beauvericin by weight, was toxic at an LC50 of 84.2 mg/L. In contrast, a nonpolar extract of mycelia from the fungal weed pathogen, Colletotrichum gloeosporioides f. sp. aeschynomene, was not toxic when tested up to 70.4 mg/L.

A fully enclosed test system was developed both to assess potential adverse effects of microbial pest control agents on nontarget aquatic invertebrates and to monitor their fate and survival. Eastern oysters Crassostrea virginica were exposed to various microbial pest control agents including the vegetative bacterium Pseudomonas fluorescens, bacterial spores of Bacillus sphaericus, and fungal spores of Colletotrichum gloeosporioides f. sp. aeschynomene. After an exposure of 3 d for the fungus, 14 d f or the bacterial spores, and 7 d f or the vegetative bacterial cells, half the oysters were placed into a fully enclosed 60 1 aquarium equipped with a recirculating water system which passed water through an ultraviolet-irradiation sterilizer at a rate of 11 min-1. The remaining oysters were placed into wire cages maintained in Santa Rosa Sound, Pensacola, Florida, USA. Plate counts, performed on homogenized oyster tissues, revealed that all of the microbial pest control agents were found in oysters after exposure. Oysters depurated each microorganism at a different rate. None of the agents colonized the oysters. For all microbes tested, rates of clearance from the oysters in the enclosed UV light depuration system were similar to rates of clearance from the oysters in Santa Rosa Sound. Histological examination of oyster tissues detected P fluorescens and B. sphaericus in the gut and C. gloeosporioides in the gill. Spore germination was not observed and no apparent signs of infectivity or pathogenicity were detected.

Bacillus sphaericus spores were suspended in bottles of filtered (0.45 microns) freshwater and seawater under various conditions of temperature, pH and salinity. Heat resistant culturable counts (spores) slowly decreased with time. Spores suspended in dialysis bags submerged in a freshwater pond or in flowing seawater underwent a more rapid drop in heat resistant spore counts than did spores held in bottles. Thus, laboratory studies may overestimate spore longevity in the environment. Spore settling rate was related to the nature of particulate material in the water column. Paraspores (or perhaps spores and toxin) of B. thuringiensis serovar israelensis (B.t.i.) had a greater tendency to adhere to and settle with suspended sediment and fine particulates than did paraspores of B. sphaericus. These observations may at least partially explain the greater persistence of B. sphaericus larvicidal activity in field tests than that of B.t.i..

A chemical toxicity and teratogenicity test was adapted to assess potential adverse effects of a microbial pest control agent on a nontarget fish. Developing embryos of the inland silverside, Menidia beryllina, were exposed to conidiospores of the insect-pathogenic fungus Beauveria bassiana. Embryo rupture and death were observed. Embryo rupture did not always result in death, nor was death always associated with embryo rupture. Adherence of spores to the chorion, followed by germination and penetration by the germ tube, probably caused the embryos to rupture. Statistically significant (P less than or equal to 0.05) responses were observed in tests in which conidiospore concentrations were greater than or equal to 8.3 x 10(4) or less than or equal to 1.5 x 10(6)/ml. Conidiospores treated with a dispersant (biological detergent) showed significantly less binding (P less than or equal to 0.01) to embryos than did untreated spores. Both detergent-treated and heat-killed spores failed to cause significant adverse effects.

Plasmid pSI30 was constructed to increase the sensitivity of detection of a genetically engineered micro‐organism (GEM) and its recombinant DNA in environmental samples. This broad host‐range, mobilizable plasmid contained chlorocatechol (clc) degradative genes, antibiotic resistance genes (ampicillin and kanamycin) and a fragment of eukaryotic DNA. The clc genes encode enzymes that convert 3‐chlorocatechol to maleylacetic acid permitting the host, Pseudomonas putida RC‐4, to grow on 3‐chlorobenzoate. This catabolic phenotype was exploited using enrichment procedures to detect RC‐4(pSI30) cells, freeliving in the water column or when irreversibly bound to surfaces. The eukaryotic DNA sequence provided a unique target allowing positive identification by DNA:DNA hybridization. Using the eukaryotic DNA sequence as a probe, no transfer of the plasmid to indigenous bacteria was detected. Persistence of RC‐4(pSI30) and its ability to multiply upon addition of 3‐chlorobenzoate were demonstrated 78 days after its addition to natural freshwater. In flow‐through microcosms RC‐4(pSI30), undetectable as free‐living cells, was found by enrichment as irreversibly bound sessile forms. These experiments revealed the stability of pSI30 and its utility in a ‘combination’ detection system for tracking the survival of a GEM and its DNA in environmental samples.

Elimination of Bacillus sphaericus spores ingested by midge larvae, snails, and oysters was most rapid among midge larvae. Spores remained in oysters up to 21 days and in snails up to 49 days. Viable spores were recovered in snail and oyster feces for these same periods. There was no indication of actively growing B. sphaericus in the animals. Passage through oyster gut detoxified the B. sphaericus mosquito larval toxin, but there was a 33% retention of toxicity following snail gut passage. Midge larvae reared to adults in spore-containing water carried spores in/on the adult body. This suggests that these animals could carry the bacteria to sites beyond the application area.