A set of DNA markers was developed that successfully identifies Bacillus thuringiensis ssp. kurst... more A set of DNA markers was developed that successfully identifies Bacillus thuringiensis ssp. kurstaki (Btk) when screened against other Bacillus species and subspecies. These subspecies-specific primer sets allowed detection and characterization of Btk within an environmental background that contained many Bacillus species. Because Btk is used as an active ingredient in many commercial formulations, yet is not naturally widely distributed in North America or Europe, these markers will prove useful in investigations on the environmental persistence and ecological fate of Btk.
Significant but subtle differentiation was detected for both microsatellite DNA and mitochondrial... more Significant but subtle differentiation was detected for both microsatellite DNA and mitochondrial DNA among four populations of American shad Alosa sapidissima. The data indicate that straying among rivers is sufficient to permit only marginal population ...
Page 1. 175 Coastal Management, 28:175–185, 2000 Copyright ã 2000 Taylor & Francis 0892-0753/... more Page 1. 175 Coastal Management, 28:175–185, 2000 Copyright ã 2000 Taylor & Francis 0892-0753/00 $12.00 + .00 The Cost of Nutrient Reduction: A Case Study of Chesapeake Bay ARTHUR J. BUTT Chesapeake Scientific Investigations Foundation, Inc. ...
Background: Spittle bugs and sharpshooters are well-known xylem sap-feeding insects and vectors o... more Background: Spittle bugs and sharpshooters are well-known xylem sap-feeding insects and vectors of the phytopathogenic bacterium Xylella fastidiosa (Wells), a causal agent of Pierce's disease of grapevines and other crop diseases. Specialized feeding on nutrient-deficient xylem sap is relatively rare among insect herbivores, and only limited genomic and transcriptomic information has been generated for xylem-sap feeders. To develop a more comprehensive understanding of biochemical adaptations and symbiotic relationships that support survival on a nutritionally austere dietary source, transcriptome assemblies for three sharpshooter species and one spittlebug species were produced. Findings: Trinity-based de novo transcriptome assemblies were generated for all four xylem-sap feeders using raw sequencing data originating from whole-insect preps. Total transcripts for each species ranged from 91 384 for Cuerna arida to 106 998 for Homalodisca liturata with transcript totals for Graphocephala atropunctata and the spittlebug Clastoptera arizonana falling in between. The percentage of transcripts comprising complete open reading frames ranged from 60% for H. liturata to 82% for C. arizonana. Bench-marking universal single-copy orthologs analyses for each dataset indicated quality assemblies and a high degree of completeness for all four species. Conclusions: These four transcriptomes represent a significant expansion of data for insect herbivores that feed exclusively on xylem sap, a nutritionally deficient dietary source relative to other plant tissues and fluids. Comparison of transcriptome data with insect herbivores that utilize other dietary sources may illuminate fundamental differences in the biochemistry of dietary specialization.
In a laboratory test, total luminescen ce spectro scopy was used to detect and measure the in viv... more In a laboratory test, total luminescen ce spectro scopy was used to detect and measure the in vivo presence of a biohazard surrogate, endospores of Bacillus m egaterium, within the parasitoid wasp C. congregata (Say) (H ymenoptera:Braconidae). Upon em ergence, exposed wasps were allowed to feed on ve different concentrations of endospores suspended in 5 mL of honey solution. After 12 h insects were chilled at 280 8 C for 5 min to permit capture. Aqueous suspensions were prepared by homogenizing the wasps in 3 m L of deionized water. The total excitation-emission matrix (EEM) was measured for each suspension by using an SLM Series 2 lumines-cence spectrometer. For wasps exposed to spore concentrations of 3.0 3 10 2 to 3.0 3 10 6 colony forming units (CFU)/mL, two intensity maxima were observed. The emission for tryptophan was identi ed at excitation (Ex) 300 nm and em ission (Em) 350 nm. A second emission that resulted from other biological materials including ni-troheterocyclic com pounds and endospores occurred at Ex 350 nm and Em 420 nm. Changes in the ratio of intensity between the tryp-tophan and second emission were found to be related to endospore material present in the suspensions at original feeding concentrations of 15 000 to 15 million spores. Intensity ratios for the positive samples equaled 0.9, while the intensity ratios for the control equaled 1.9. One notable difference recorded for the emission spectra was an apparent, but minor, red shift of approximately 5 nm of the second emission when compared against a signature library of pure cultures. Diagnostic information such as this should contribute to methods for the detection and potential identi cation of biohazard materials with the use of photoluminescence.
Quantifying bacterial abundance and biomass is fundamental to many microbiological studies. Direc... more Quantifying bacterial abundance and biomass is fundamental to many microbiological studies. Directly counting via epifluorescence microscopy has become the method of choice, especially for environmental samples, and conventional techniques require filtration of cells onto black polycarbonate membrane filters. We investigated the utility of instead capturing stained bacterial suspensions on bioadhesive slides, performing tests using pure cultures of bacteria, mixtures of cultured bacteria, and environmental samples from five habitat types. When compared to the standard filtration and flow cytometric approaches, bioadhesive slides were found to be an accurate and precise platform for rapid enumeration of bacteria. Total bacterial counts made using the three methods were positively correlated for acridine orange and Live/Dead® (L/D) staining (0.81 ≤r≤0.95, all p≤0.002). All platforms had similar precision, though counts obtained using bioadhesive slides were significantly higher than those made with polycarbonate filters and flow cytometry. The specific bioadhesive slides we used resulted in substantial cell mortality for certain pure cultures and river water samples, limiting their use for L/D determination. Cell enumeration using bioadhesive slides is particularly effective because it is highly precise at a wide range of cell concentrations, allows observation of cells that are not readily discernible on filters, reduces the number of steps and processing materials associated with sample analysis, and increases throughput.
Vesicular monoamine transporter 1 (VMAT-1) mRNA and protein were examined (1) to determine whethe... more Vesicular monoamine transporter 1 (VMAT-1) mRNA and protein were examined (1) to determine whether adult mouse brain expresses full-length VMAT-1 mRNA that can be translated to functional transporter protein and (2) to compare immunoreactive VMAT-1 proteins in brain and adrenal. VMAT-1 mRNA was detected in mouse brain with RT-PCR. Th e cDNA was sequenced, cloned into an expression vector, transfected into COS-1 cells, and cell protein was assayed for VMAT-1 activity. Immunoreactive proteins were examined on western blots probed with four diff erent anti-bodies to VMAT-1. Sequencing confi rmed identity of the entire coding sequences of VMAT-1 cDNA from mouse medulla oblongata/pons and adrenal to a Gen-Bank reference sequence. Transfection of the brain cDNA into COS-1 cells resulted in transporter activity that was blocked by the VMAT inhibitor reserpine
We tested the hypotheses that foraging insects can acquire human DNA from the environment and tha... more We tested the hypotheses that foraging insects can acquire human DNA from the environment and that insect-delivered human DNA is of sufficient quantity and quality to permit standard forensic analyses. Houseflies, German cockroaches, and camel crickets were exposed to dusty surfaces and then assayed for human mitochondrial and nuclear loci by conventional and qPCR, and multiplex STR amplification. Over two experiments, 100% of insect groups and 94% of dust controls tested positive for human DNA. Of 177 individuals, 33–67% tested positive and 13 yielded quantifiable human DNA (mean = 0.022 € 0.006 ng; mean dust control = 2.448 € 0.960 ng); four had at least one positive allele call for one or more locus; eight others showed multiple peaks at some loci. Results imply that application to routine forensic casework is limited given current detection methodology yet demonstrate the potential use of insects as environmental samplers for human DNA. Insects were first used in modern forensic investigations in the late 19th century and are now used routinely in homicide and other medico-legal investigations (1–3). Most recently, casework and simulated studies based on short tandem repeat (STR) analysis of DNA extracted from the gut contents of larval blow flies have demonstrated that blow flies can provide molecular evidence for the identification of both victims and criminals (4–6). Owing to the advances in molecular genotyping and the application of methods for low copy number DNA forensic analysis, the routine use of insects in casework is likely to expand (7). The employment of free-roaming arthropods as environmental samplers for the surveillance of biothreats has been the focus of our research for several years. Insects passively acquire or ingest multiple organic and inorganic materials while foraging in the environment, and these materials can be easily harvested for analyses using a variety of detection methodologies (8). In particular, scavenging insect species are adept at locating organic materials and may be useful for collecting environmental samples when stealth is indicated. Further, depending on the material of interest and the detection methodology employed, foraging insects may provide more informative samples than traditional sampling methods. This study investigates the potential use of three common insect species, the common housefly, the German cockroach, and a camel cricket, as collectors of human DNA that may be relevant to the surveillance of humans and human activity in enclosed structures. Two of these species, the housefly and German cockroach, are synanthropic, i.e., highly adapted to living in close association with humans and human dwellings, and are also cosmopolitan, i.e., the same species occurs throughout the world. Recently, Toothman et al. (9) reported that 97% of 36 environmental swab samples collected from dusty surfaces from multiple locations within a large academic building tested positive for human mitochondrial DNA (mtDNA) or human nuclear DNA. Samples yielded human DNA at levels (0.2–1.1 pg per cm 2) high enough to permit detection using standard forensic techniques, and 61% of samples contained human DNA of adequate quality to permit STR analysis with interpretable results. No obvious trends in human DNA quantity or quality were detected with respect to human traffic level and visual dust levels. Results of this study provide control data for interpreting results of the study reported herein; both studies were conducted concurrently, and samples were taken from the same locations and at the same time. We hypothesized that insects could acquire human DNA from dust while foraging in an indoor environment and, further, that insect-delivered human DNA would be of sufficient quantity and adequate quality to permit standard forensic analyses. Two manipu-lative experiments were performed to test the effect of insect species and sampling location on recovery of human DNA from insect foragers. The effects of human traffic level and apparent dust level on recovery of insect-borne human DNA were also examined to determine whether resulting samples could provide information about the relative number of past or present human inhabitants. Human DNA recovered from insects was subjected to qualitative and quantitative analyses using conventional and quantitative polymerase chain reaction (qPCR) methods for nuclear and
We observed ulcerative lesions on live Atlantic menhaden, Brevoortia tyrannus, during ichthyofaun... more We observed ulcerative lesions on live Atlantic menhaden, Brevoortia tyrannus, during ichthyofaunal sampling in the tidal James River in October 1999 (near Jamestown, VA, USA). Other synoptically collected fishes exhibited no signs of lesions or pre-ulcerative tissues. Live fish were classified as unremarkable (no dermal anomalies), pre-ulcerative (integument intact with boil-like swelling), and ulcerative (severe focal lesions). Specimens were analyzed for bacteria, fungi, and pathogenic protozoans including amphizoic amoe-bae, Pfiesteria piscicida, and Kudoa sp. No Pfiesteria were detected in any tissue specimen. All B. tyrannus examined, including tissues from unremarkable fish, tested positive for presence of the known fish parasite Kudoa. Only ulcerative lesions were also colonized by bacteria, fungi, and amphizoic amoebae. The absence of bacteria, fungi, and protozoans from unremarkable and pre-ulcerative fish suggests that association of other potential pathogens with B. tyrannus ulcers was due to secondary colonization following lesion formation as a result of Kudoa infection.
This study was undertaken to develop a quantitative polymerase chain reaction assay that would im... more This study was undertaken to develop a quantitative polymerase chain reaction assay that would improve the utility of PCR for detecting Haplosporidium nelsoni (MSX), a serious parasite of the eastern oyster Crassostrea virginica. A competitive PCR sequence was generated from the H. nelsoni small subunit ribosomal DNA fragment, originally described by Stokes and colleagues, that was amplified by the same PCR primers and had similar amplification performance. Assays performed using competitor dilutions ranging from 0.05 to 500 pg/µl DNA were used to test oyster samples designated using histological techniques as having " light " or " heavy " MSX infections. Visual diagnoses were confirmed equally well with three methods: densitometry of ethidium-bromide-stained agarose, densitometry of SYBRGreen-stained polyacrylamide gels, and analysis by GeneScan 3.0 of fluorescent products detected in ultrathin gels. Oysters diagnosed as negative for MSX tested as negative or light by PCR. Oysters with light MSX infections generally had less than 5 pg/µl infectious DNA. Oysters with heavy infections generally corresponded to 5 pg/µl or greater competitor dilutions.
From 24 mating sets, 6300 ¢ngerling of yellow perch (Perca £avescens) were stocked into one pond ... more From 24 mating sets, 6300 ¢ngerling of yellow perch (Perca £avescens) were stocked into one pond and equal numbers of progeny from six representative sets out of the 24 were stocked into each of two other ponds. After communal rearing for 21 months, total length and body weight were assessed for n 5 300 ¢sh in each of the three ponds and molecular pedigrees were performed for each sampled individual to assign the progeny back to the original parents. The overall average number of alleles per locus was A 516.4 and observed and expected heterozygosities were H o 5 0.88 and H e 5 0.77 respectively. The mean weight of random samples and the top 10% fast-growing ¢sh from the pond with all the sets was sig-ni¢cantly greater than those from either of the two replicate ponds with six crosses. For the two replicate ponds, no signi¢cant di¡erences were found in family rankings and assignment of the top10% fast-growing ¢sh, indicating that families with superior growth performance in one pond also exhibited the same superior growth performance in the replicate pond. However, there were no signi¢cant correlations detected in family mean weights of the top 10% ¢sh between any two of the three ponds.
A standard flow cytometric protocol was developed for estimating triploid induction in batches o... more A standard flow cytometric protocol was developed for estimating triploid induction in batches of larval fish. Polyploid induction treatments are not guaranteed to be 100% efficient, thus the ability to quantify the proportion of triploid larvae generated by a particular treatment helps managers to stock high-percentage spawns and researchers to select treatments for efficient triploid induction. At 3 d posthatch, individual Grass Carp Ctenopharyngodon idella were mechanically dissociated into single-cell suspensions; nuclear DNA was stained with propidium iodide then analyzed by flow cytometry. Following ploidy identification of individuals, aliquots of diploid and triploid cell suspensions were mixed to generate 15 levels (0–100%) of known triploidy (n = 10). Using either 20 or 50 larvae per level, the observed triploid percentages were lower than the known, actual values. Using nonlinear regression analyses, quadratic equations solved for triploid propor- tions in mixed samples and corresponding estimation reference plots allowed for predicting triploidy. Thus, an accurate prediction of the proportion of triploids in a spawn can be made by following a standard larval processing and analysis protocol with either 20 or 50 larvae from a single spawn, coupled with applying the quadratic equations or reference plots to observed flow cytometry results. Due to the universality of triploid DNA content being 1.5 times the diploid level and because triploid fish consist of fewer cells than diploids, this method should be applicable to other produced triploid fish species, and it may be adapted for use with bivalves or other species where batch analysis is appropriate.
A set of DNA markers was developed that successfully identifies Bacillus thuringiensis ssp. kurst... more A set of DNA markers was developed that successfully identifies Bacillus thuringiensis ssp. kurstaki (Btk) when screened against other Bacillus species and subspecies. These subspecies-specific primer sets allowed detection and characterization of Btk within an environmental background that contained many Bacillus species. Because Btk is used as an active ingredient in many commercial formulations, yet is not naturally widely distributed in North America or Europe, these markers will prove useful in investigations on the environmental persistence and ecological fate of Btk.
Significant but subtle differentiation was detected for both microsatellite DNA and mitochondrial... more Significant but subtle differentiation was detected for both microsatellite DNA and mitochondrial DNA among four populations of American shad Alosa sapidissima. The data indicate that straying among rivers is sufficient to permit only marginal population ...
Page 1. 175 Coastal Management, 28:175–185, 2000 Copyright ã 2000 Taylor & Francis 0892-0753/... more Page 1. 175 Coastal Management, 28:175–185, 2000 Copyright ã 2000 Taylor & Francis 0892-0753/00 $12.00 + .00 The Cost of Nutrient Reduction: A Case Study of Chesapeake Bay ARTHUR J. BUTT Chesapeake Scientific Investigations Foundation, Inc. ...
Background: Spittle bugs and sharpshooters are well-known xylem sap-feeding insects and vectors o... more Background: Spittle bugs and sharpshooters are well-known xylem sap-feeding insects and vectors of the phytopathogenic bacterium Xylella fastidiosa (Wells), a causal agent of Pierce's disease of grapevines and other crop diseases. Specialized feeding on nutrient-deficient xylem sap is relatively rare among insect herbivores, and only limited genomic and transcriptomic information has been generated for xylem-sap feeders. To develop a more comprehensive understanding of biochemical adaptations and symbiotic relationships that support survival on a nutritionally austere dietary source, transcriptome assemblies for three sharpshooter species and one spittlebug species were produced. Findings: Trinity-based de novo transcriptome assemblies were generated for all four xylem-sap feeders using raw sequencing data originating from whole-insect preps. Total transcripts for each species ranged from 91 384 for Cuerna arida to 106 998 for Homalodisca liturata with transcript totals for Graphocephala atropunctata and the spittlebug Clastoptera arizonana falling in between. The percentage of transcripts comprising complete open reading frames ranged from 60% for H. liturata to 82% for C. arizonana. Bench-marking universal single-copy orthologs analyses for each dataset indicated quality assemblies and a high degree of completeness for all four species. Conclusions: These four transcriptomes represent a significant expansion of data for insect herbivores that feed exclusively on xylem sap, a nutritionally deficient dietary source relative to other plant tissues and fluids. Comparison of transcriptome data with insect herbivores that utilize other dietary sources may illuminate fundamental differences in the biochemistry of dietary specialization.
In a laboratory test, total luminescen ce spectro scopy was used to detect and measure the in viv... more In a laboratory test, total luminescen ce spectro scopy was used to detect and measure the in vivo presence of a biohazard surrogate, endospores of Bacillus m egaterium, within the parasitoid wasp C. congregata (Say) (H ymenoptera:Braconidae). Upon em ergence, exposed wasps were allowed to feed on ve different concentrations of endospores suspended in 5 mL of honey solution. After 12 h insects were chilled at 280 8 C for 5 min to permit capture. Aqueous suspensions were prepared by homogenizing the wasps in 3 m L of deionized water. The total excitation-emission matrix (EEM) was measured for each suspension by using an SLM Series 2 lumines-cence spectrometer. For wasps exposed to spore concentrations of 3.0 3 10 2 to 3.0 3 10 6 colony forming units (CFU)/mL, two intensity maxima were observed. The emission for tryptophan was identi ed at excitation (Ex) 300 nm and em ission (Em) 350 nm. A second emission that resulted from other biological materials including ni-troheterocyclic com pounds and endospores occurred at Ex 350 nm and Em 420 nm. Changes in the ratio of intensity between the tryp-tophan and second emission were found to be related to endospore material present in the suspensions at original feeding concentrations of 15 000 to 15 million spores. Intensity ratios for the positive samples equaled 0.9, while the intensity ratios for the control equaled 1.9. One notable difference recorded for the emission spectra was an apparent, but minor, red shift of approximately 5 nm of the second emission when compared against a signature library of pure cultures. Diagnostic information such as this should contribute to methods for the detection and potential identi cation of biohazard materials with the use of photoluminescence.
Quantifying bacterial abundance and biomass is fundamental to many microbiological studies. Direc... more Quantifying bacterial abundance and biomass is fundamental to many microbiological studies. Directly counting via epifluorescence microscopy has become the method of choice, especially for environmental samples, and conventional techniques require filtration of cells onto black polycarbonate membrane filters. We investigated the utility of instead capturing stained bacterial suspensions on bioadhesive slides, performing tests using pure cultures of bacteria, mixtures of cultured bacteria, and environmental samples from five habitat types. When compared to the standard filtration and flow cytometric approaches, bioadhesive slides were found to be an accurate and precise platform for rapid enumeration of bacteria. Total bacterial counts made using the three methods were positively correlated for acridine orange and Live/Dead® (L/D) staining (0.81 ≤r≤0.95, all p≤0.002). All platforms had similar precision, though counts obtained using bioadhesive slides were significantly higher than those made with polycarbonate filters and flow cytometry. The specific bioadhesive slides we used resulted in substantial cell mortality for certain pure cultures and river water samples, limiting their use for L/D determination. Cell enumeration using bioadhesive slides is particularly effective because it is highly precise at a wide range of cell concentrations, allows observation of cells that are not readily discernible on filters, reduces the number of steps and processing materials associated with sample analysis, and increases throughput.
Vesicular monoamine transporter 1 (VMAT-1) mRNA and protein were examined (1) to determine whethe... more Vesicular monoamine transporter 1 (VMAT-1) mRNA and protein were examined (1) to determine whether adult mouse brain expresses full-length VMAT-1 mRNA that can be translated to functional transporter protein and (2) to compare immunoreactive VMAT-1 proteins in brain and adrenal. VMAT-1 mRNA was detected in mouse brain with RT-PCR. Th e cDNA was sequenced, cloned into an expression vector, transfected into COS-1 cells, and cell protein was assayed for VMAT-1 activity. Immunoreactive proteins were examined on western blots probed with four diff erent anti-bodies to VMAT-1. Sequencing confi rmed identity of the entire coding sequences of VMAT-1 cDNA from mouse medulla oblongata/pons and adrenal to a Gen-Bank reference sequence. Transfection of the brain cDNA into COS-1 cells resulted in transporter activity that was blocked by the VMAT inhibitor reserpine
We tested the hypotheses that foraging insects can acquire human DNA from the environment and tha... more We tested the hypotheses that foraging insects can acquire human DNA from the environment and that insect-delivered human DNA is of sufficient quantity and quality to permit standard forensic analyses. Houseflies, German cockroaches, and camel crickets were exposed to dusty surfaces and then assayed for human mitochondrial and nuclear loci by conventional and qPCR, and multiplex STR amplification. Over two experiments, 100% of insect groups and 94% of dust controls tested positive for human DNA. Of 177 individuals, 33–67% tested positive and 13 yielded quantifiable human DNA (mean = 0.022 € 0.006 ng; mean dust control = 2.448 € 0.960 ng); four had at least one positive allele call for one or more locus; eight others showed multiple peaks at some loci. Results imply that application to routine forensic casework is limited given current detection methodology yet demonstrate the potential use of insects as environmental samplers for human DNA. Insects were first used in modern forensic investigations in the late 19th century and are now used routinely in homicide and other medico-legal investigations (1–3). Most recently, casework and simulated studies based on short tandem repeat (STR) analysis of DNA extracted from the gut contents of larval blow flies have demonstrated that blow flies can provide molecular evidence for the identification of both victims and criminals (4–6). Owing to the advances in molecular genotyping and the application of methods for low copy number DNA forensic analysis, the routine use of insects in casework is likely to expand (7). The employment of free-roaming arthropods as environmental samplers for the surveillance of biothreats has been the focus of our research for several years. Insects passively acquire or ingest multiple organic and inorganic materials while foraging in the environment, and these materials can be easily harvested for analyses using a variety of detection methodologies (8). In particular, scavenging insect species are adept at locating organic materials and may be useful for collecting environmental samples when stealth is indicated. Further, depending on the material of interest and the detection methodology employed, foraging insects may provide more informative samples than traditional sampling methods. This study investigates the potential use of three common insect species, the common housefly, the German cockroach, and a camel cricket, as collectors of human DNA that may be relevant to the surveillance of humans and human activity in enclosed structures. Two of these species, the housefly and German cockroach, are synanthropic, i.e., highly adapted to living in close association with humans and human dwellings, and are also cosmopolitan, i.e., the same species occurs throughout the world. Recently, Toothman et al. (9) reported that 97% of 36 environmental swab samples collected from dusty surfaces from multiple locations within a large academic building tested positive for human mitochondrial DNA (mtDNA) or human nuclear DNA. Samples yielded human DNA at levels (0.2–1.1 pg per cm 2) high enough to permit detection using standard forensic techniques, and 61% of samples contained human DNA of adequate quality to permit STR analysis with interpretable results. No obvious trends in human DNA quantity or quality were detected with respect to human traffic level and visual dust levels. Results of this study provide control data for interpreting results of the study reported herein; both studies were conducted concurrently, and samples were taken from the same locations and at the same time. We hypothesized that insects could acquire human DNA from dust while foraging in an indoor environment and, further, that insect-delivered human DNA would be of sufficient quantity and adequate quality to permit standard forensic analyses. Two manipu-lative experiments were performed to test the effect of insect species and sampling location on recovery of human DNA from insect foragers. The effects of human traffic level and apparent dust level on recovery of insect-borne human DNA were also examined to determine whether resulting samples could provide information about the relative number of past or present human inhabitants. Human DNA recovered from insects was subjected to qualitative and quantitative analyses using conventional and quantitative polymerase chain reaction (qPCR) methods for nuclear and
We observed ulcerative lesions on live Atlantic menhaden, Brevoortia tyrannus, during ichthyofaun... more We observed ulcerative lesions on live Atlantic menhaden, Brevoortia tyrannus, during ichthyofaunal sampling in the tidal James River in October 1999 (near Jamestown, VA, USA). Other synoptically collected fishes exhibited no signs of lesions or pre-ulcerative tissues. Live fish were classified as unremarkable (no dermal anomalies), pre-ulcerative (integument intact with boil-like swelling), and ulcerative (severe focal lesions). Specimens were analyzed for bacteria, fungi, and pathogenic protozoans including amphizoic amoe-bae, Pfiesteria piscicida, and Kudoa sp. No Pfiesteria were detected in any tissue specimen. All B. tyrannus examined, including tissues from unremarkable fish, tested positive for presence of the known fish parasite Kudoa. Only ulcerative lesions were also colonized by bacteria, fungi, and amphizoic amoebae. The absence of bacteria, fungi, and protozoans from unremarkable and pre-ulcerative fish suggests that association of other potential pathogens with B. tyrannus ulcers was due to secondary colonization following lesion formation as a result of Kudoa infection.
This study was undertaken to develop a quantitative polymerase chain reaction assay that would im... more This study was undertaken to develop a quantitative polymerase chain reaction assay that would improve the utility of PCR for detecting Haplosporidium nelsoni (MSX), a serious parasite of the eastern oyster Crassostrea virginica. A competitive PCR sequence was generated from the H. nelsoni small subunit ribosomal DNA fragment, originally described by Stokes and colleagues, that was amplified by the same PCR primers and had similar amplification performance. Assays performed using competitor dilutions ranging from 0.05 to 500 pg/µl DNA were used to test oyster samples designated using histological techniques as having " light " or " heavy " MSX infections. Visual diagnoses were confirmed equally well with three methods: densitometry of ethidium-bromide-stained agarose, densitometry of SYBRGreen-stained polyacrylamide gels, and analysis by GeneScan 3.0 of fluorescent products detected in ultrathin gels. Oysters diagnosed as negative for MSX tested as negative or light by PCR. Oysters with light MSX infections generally had less than 5 pg/µl infectious DNA. Oysters with heavy infections generally corresponded to 5 pg/µl or greater competitor dilutions.
From 24 mating sets, 6300 ¢ngerling of yellow perch (Perca £avescens) were stocked into one pond ... more From 24 mating sets, 6300 ¢ngerling of yellow perch (Perca £avescens) were stocked into one pond and equal numbers of progeny from six representative sets out of the 24 were stocked into each of two other ponds. After communal rearing for 21 months, total length and body weight were assessed for n 5 300 ¢sh in each of the three ponds and molecular pedigrees were performed for each sampled individual to assign the progeny back to the original parents. The overall average number of alleles per locus was A 516.4 and observed and expected heterozygosities were H o 5 0.88 and H e 5 0.77 respectively. The mean weight of random samples and the top 10% fast-growing ¢sh from the pond with all the sets was sig-ni¢cantly greater than those from either of the two replicate ponds with six crosses. For the two replicate ponds, no signi¢cant di¡erences were found in family rankings and assignment of the top10% fast-growing ¢sh, indicating that families with superior growth performance in one pond also exhibited the same superior growth performance in the replicate pond. However, there were no signi¢cant correlations detected in family mean weights of the top 10% ¢sh between any two of the three ponds.
A standard flow cytometric protocol was developed for estimating triploid induction in batches o... more A standard flow cytometric protocol was developed for estimating triploid induction in batches of larval fish. Polyploid induction treatments are not guaranteed to be 100% efficient, thus the ability to quantify the proportion of triploid larvae generated by a particular treatment helps managers to stock high-percentage spawns and researchers to select treatments for efficient triploid induction. At 3 d posthatch, individual Grass Carp Ctenopharyngodon idella were mechanically dissociated into single-cell suspensions; nuclear DNA was stained with propidium iodide then analyzed by flow cytometry. Following ploidy identification of individuals, aliquots of diploid and triploid cell suspensions were mixed to generate 15 levels (0–100%) of known triploidy (n = 10). Using either 20 or 50 larvae per level, the observed triploid percentages were lower than the known, actual values. Using nonlinear regression analyses, quadratic equations solved for triploid propor- tions in mixed samples and corresponding estimation reference plots allowed for predicting triploidy. Thus, an accurate prediction of the proportion of triploids in a spawn can be made by following a standard larval processing and analysis protocol with either 20 or 50 larvae from a single spawn, coupled with applying the quadratic equations or reference plots to observed flow cytometry results. Due to the universality of triploid DNA content being 1.5 times the diploid level and because triploid fish consist of fewer cells than diploids, this method should be applicable to other produced triploid fish species, and it may be adapted for use with bivalves or other species where batch analysis is appropriate.
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Papers by Bonnie L Brown