Kathleen M. Yeater

Near-isogenic cotton germplasm lines that differ in fiber-bundle strength have temporal differences in fiber gene expression patterns as revealed by comparative high-throughput profiling

Gene expression profiles of developing cotton (Gossypium hirsutum L.) fibers from two near-isogenic lines (NILs) that differ in fiber-bundle strength, short-fiber content, and in fewer than two genetic loci were compared using an oligonucleotide microarray. Fiber gene expression was compared at five time points spanning fiber elongation and secondary cell wall (SCW) biosynthesis. Fiber samples were collected from field plots in a randomized, complete block design, with three spatially distinct biological replications for each NIL at each time point. Microarray hybridizations were performed in a loop experimental design that allowed comparisons of fiber gene expression profiles as a function of time between the two NILs. Overall, developmental expression patterns revealed by the microarray experiment agreed with previously reported cotton fiber gene expression patterns for specific genes. Additionally, genes expressed coordinately with the onset of SCW biosynthesis in cotton fiber correlated with gene expression patterns of other SCW-producing plant tissues. Functional classification and enrichment analysis of differentially expressed genes between the two NILs revealed that genes associated with SCW biosynthesis were significantly up-regulated in fibers of the high-fiber quality line at the transition stage of cotton fiber development. For independent corroboration of the microarray results, 15 genes were selected for quantitative reverse transcription PCR analysis of fiber gene expression. These analyses, conducted over multiple field years, confirmed the temporal difference in fiber gene expression between the two NILs. We hypothesize that the loci conferring temporal differences in fiber gene expression between the NILs are important regulatory sequences that offer the potential for more targeted manipulation of cotton fiber quality.

A combined functional and structural genomics approach identified an EST-SSR marker with complete linkage to the Ligon lintless-2 genetic locus in cotton (Gossypium hirsutum L.)

BackgroundCotton fiber length is an important quality attribute to the textile industry and longer fibers can be more efficiently spun into yarns to produce superior fabrics. There is typically a negative correlation between yield and fiber quality traits such as length. An understanding of the regulatory mechanisms controlling fiber length can potentially provide a valuable tool for cotton breeders to improve fiber length while maintaining high yields. The cotton (Gossypium hirsutum L.) fiber mutation Ligon lintless-2 is controlled by a single dominant gene (Li2) that results in significantly shorter fibers than a wild-type. In a near-isogenic state with a wild-type cotton line, Li2 is a model system with which to study fiber elongation.ResultsTwo near-isogenic lines of Ligon lintless-2 (Li2) cotton, one mutant and one wild-type, were developed through five generations of backcrosses (BC5). An F2 population was developed from a cross between the two Li2 near-isogenic lines and used to develop a linkage map of the Li2 locus on chromosome 18. Five simple sequence repeat (SSR) markers were closely mapped around the Li2 locus region with two of the markers flanking the Li2 locus at 0.87 and 0.52 centimorgan. No apparent differences in fiber initiation and early fiber elongation were observed between the mutant ovules and the wild-type ones. Gene expression profiling using microarrays suggested roles of reactive oxygen species (ROS) homeostasis and cytokinin regulation in the Li2 mutant phenotype. Microarray gene expression data led to successful identification of an EST-SSR marker (NAU3991) that displayed complete linkage to the Li2 locus.ConclusionsIn the field of cotton genomics, we report the first successful conversion of gene expression data into an SSR marker that is associated with a genomic region harboring a gene responsible for a fiber trait. The EST-derived SSR marker NAU3991 displayed complete linkage to the Li2 locus on chromosome 18 and resided in a gene with similarity to a putative plectin-related protein. The complete linkage suggests that this expressed sequence may be the Li2 gene.

Rhipicephalus (Boophilus) microplus aquaporin as an effective vaccine antigen to protect against cattle tick infestations

BackgroundVaccination as a control method against the cattle tick, Rhipicephalus (Boophilus) microplus has been practiced since the introduction of two products in the mid-1990s. There is a need for a vaccine that could provide effective control of R. microplus in a more consistent fashion than existing products. During our transcriptome studies of R. microplus, several gene coding regions were discovered to encode proteins with significant amino acid similarity to aquaporins.MethodsA cDNA encoding an aquaporin from the cattle tick, Rhipicephalus microplus, was isolated from transcriptomic studies conducted on gut tissues dissected from fully engorged adult female R. microplus.ResultsBioinformatic analysis indicates this aquaporin, designated RmAQP1, shows greatest amino acid similarity to the human aquaporin 7 family. Members of this family of water-conducting channels can also facilitate the transport of glycerol in addition to water. The efficacy of this aquaporin as an antigen against the cattle tick was explored in cattle vaccine trials conducted in Brazil. A cDNA encoding a significant portion of RmAQP1 was expressed as a recombinant protein in Pichia pastoris, purified under native conditions using a polyhistidine C-terminus tag and nickel affinity chromatography, emulsified with Montanide adjuvant, and cattle vaccinated intramuscularly. The recombinant protein provided 75% and 68% efficacy in two cattle pen trials conducted in Campo Grande, Brazil on groups of 6 one year old Holstein calves.ConclusionThe effectiveness of this vaccine in reducing the numbers of adult female ticks shows this aquaporin antigen holds promise as an active ingredient in cattle vaccines targeted against infestations of R. microplus.

Exploring the use of an anti-tick vaccine as a tool for the integrated eradication of the cattle fever tick, Rhipicephalus (Boophilus) annulatus

Bovine babesiosis, also known as cattle fever, is a tick-borne protozoal disease foreign to the United States. It was eradicated by eliminating the vector species, Rhipicephalus (Boophilus) annulatus and Rhipicephalus (Boophilus) microplus, through the efforts of the Cattle Fever Tick Eradication Program (CFTEP), with the exception of a permanent quarantine zone (PQZ) in south Texas along the border with Mexico. Keeping the U.S. free of cattle fever ticks in a sustainable manner is a critical national agricultural biosecurity issue. The efficacy of a Bm86-based anti-tick vaccine commercialized outside of the U.S. was evaluated against a strain of R. annulatus originated from an outbreak in Texas. Vaccination controlled 99.9 and 91.4% of the ticks 8 weeks and 5.5 months after the initial vaccination, respectively. Computer modeling of habitat suitability within the PQZ typically at risk of re-infestation with R. annulatus from Mexico predicted that at a level of control greater than 40%, eradication would be maintained indefinitely. Efficacy and computer modeling data indicate that the integration of vaccination using a Bm86-based anti-tick vaccine with standard eradication practices within the northwestern half of the PQZ could incentivize producers to maintain cattle on pasture thereby avoiding the need to vacate infested premises. Implementing this epidemiologically proactive strategy offers the opportunity to prevent R. annulatus outbreaks in the U.S., which would represent a significant shift in the way the CFTEP operates.

The Li2 Mutation Results in Reduced Subgenome Expression Bias in Elongating Fibers of Allotetraploid Cotton (Gossypium hirsutum L.)

Next generation sequencing (RNA-seq) technology was used to evaluate the effects of the Ligon lintless-2 (Li2) short fiber mutation on transcriptomes of both subgenomes of allotetraploid cotton (Gossypium hirsutum L.) as compared to its near-isogenic wild type. Sequencing was performed on 4 libraries from developing fibers of Li2 mutant and wild type near-isogenic lines at the peak of elongation followed by mapping and PolyCat categorization of RNA-seq data to the reference D5 genome (G. raimondii) for homeologous gene expression analysis. The majority of homeologous genes, 83.6% according to the reference genome, were expressed during fiber elongation. Our results revealed: 1) approximately two times more genes were induced in the AT subgenome comparing to the DT subgenome in wild type and mutant fiber; 2) the subgenome expression bias was significantly reduced in the Li2 fiber transcriptome; 3) Li2 had a significantly greater effect on the DT than on the AT subgenome. Transcriptional regulators and cell wall homeologous genes significantly affected by the Li2 mutation were reviewed in detail. This is the first report to explore the effects of a single mutation on homeologous gene expression in allotetraploid cotton. These results provide deeper insights into the evolution of allotetraploid cotton gene expression and cotton fiber development.

Abundance and Accumulation of Escherichia coli and Salmonella Typhimurium Procured by Male and Female House Flies (Diptera: Muscidae) Exposed to Cattle Manure

Abstract House flies, Musca domestica L., develop within and feed upon microbe-rich substrates such as manure, acquiring and potentially disseminating pathogenic bacteria. Because adult female flies frequent manure due to oviposition or nutrition requirements, we hypothesized females would consume more manure than males even in the presence of additional food sources (e.g. sugar), resulting in measurable differences in bacterial load between sexes. House fly acquisition of bacteria from manure inoculated with GFP-expressing E. coli or Salmonella sp. was examined for both sexes over 24 h in assays where 1) inoculated manure was the only food source and 2) both inoculated manure and sugar water were provided. We conducted assays with mated male and female flies separately to determine sex-specific effects on bacterial acquisition. Over 24 h, bacterial abundance increased in manure inoculated with S. Typhimurium, but not E. coli. In flies, bacterial abundance increased within sex only in S. Typhimurium assays. Overall, female flies harbored more bacteria than males; however, differences in abundance were only significant at early time points. In the E. coli manure–sugar assays, male and female colony-forming units (CFU) abundance differed at 4 and 12 h, while CFU abundance differed at 4 and 12 h in all S. Typhimurium assays. Fly digestive tract observations from manure–sugar assays supported these initial differences especially at 4 h where females contained manure and fly food, while males contained only sugar water. Identifying sex-specific effects on house fly acquisition and carriage of bacteria from manure facilitates risk assessment of pathogen transmission on farms.

Poultry litter and the environment: Physiochemical properties of litter and soil during successive flock rotations and after remote site deposition.

The U.S. broiler meat market has grown over the past 16 years and destinations for U.S. broiler meat exports expanded to over 150 countries. This market opportunity has spurred a corresponding increase in industrialized poultry production, which due to the confined space in which high numbers of animals are housed, risks accumulating nutrients and pollutants. The purpose of this research was to determine the level of pollutants within poultry litter and the underlying soil within a production facility; and to explore the impact of spent litter deposition into the environment. The study follows a production facility for the first 2.5 years of production. It monitors the effects of successive flocks and management practices on 15 physiochemical parameters: Ca, Cu, electrical conductivity, Fe, K, Mg, Mn, moisture, Na, NO3(-)/N, organic matter, P, pH, S, and Zn. Litter samples were collected in-house, after clean-outs and during stockpiling. The soil before house placement, after the clean-outs and following litter stockpiling was monitored. Management practices markedly altered the physiochemical profiles of the litter in-house. A canonical discriminant analysis was used to describe the relationship between the parameters and sampling times. The litter profiles grouped into five clusters corresponding to time and management practices. The soil in-house exhibited mean increases in all physiochemical parameters (2-297 fold) except Fe, Mg, %M, and pH. The spent litter was followed after deposition onto a field for use as fertilizer. After 20 weeks, the soil beneath the litter exhibited increases in EC, Cu, K, Na, NO3(-)/N, %OM, P, S and Zn; while %M decreased. Understanding the impacts of industrialized poultry farms on the environment is vital as the cumulative ecological impact of this land usage could be substantial if not properly managed to reduce the risk of potential pollutant infiltration into the environment.

Efficacy of Novaluron as a Feed-Through for Control of Immature Horn Flies, House Flies, and Stable Flies (Diptera: Muscidae) Developing in Cow Manure

ABSTRACT Two rates (0.4 mg/kg body weight/d and 0.6 mg/kg body weight/d) of a daily feed-through formulation of novaluron (Novaluron 0.67% active ingredient Cattle Mix), a newer benzoylphenyl urea insecticide, were evaluated for efficacy in controlling the larval stage of horn flies, Haematobia irritans (L.), house flies, Musca domestica L., and stable flies, Stomoxys calcitrans (L.), developing in cow manure. Both rates of feed-through novaluron, delivered consecutively for 10 d, reduced adult emergence of all three species when compared with the untreated control. The presence of deformed puparia indicated that novaluron had an insect growth regulator effect on the developing fly larvae. Both of the feed-through rates evaluated resulted in 100% reduction of adult stable fly emergence after the second day of feed-through treatment. The level of control efficacy observed against these three fly species make this feed-through formulation a candidate for use in an integrated livestock pest management program, particularly in confined cattle production situations where a feed-through product could be easily administered.

The effect of induced yellowing on the physicochemical properties of specialty rice

BACKGROUND Postharvest yellowing (PHY) of rice kernels can be a major problem in the rice industry. This is especially true with high-valued specialty rice, because profit loss will be greater. The objective of this work was to determine whether a significant change occurs in the physicochemical properties (apparent amylose and protein concentrations, viscosity profile and gelatinisation temperature) as a result of induced PHY. RESULTS In this study, four specialty rices (Basmati, Jasmine, Arborio and Sushi) were yellowed using a laboratory method. PHY increased apparent amylose concentration. It also significantly increased onset and peak gelatinisation temperatures. However, peak, breakdown and setback Rapid ViscoAnalyzer viscosities were decreased by PHY. Trough viscosity for Basmati and Jasmine decreased, whereas it increased for Arborio. Moisture and protein concentrations were unchanged by the yellowing process. Attempts to rehydrate the kernels after induced PHY caused them to fracture, thus making them unsuitable for their intended purpose. CONCLUSION This study indicates that rice that has been subjected to PHY shows a reduction not only in appearance but also in cooking and processing quality, decreasing its value. However, the changes differed for each rice type, with Jasmine being affected the least.

Effects of Bacterial Dose and Fly Sex on Persistence and Excretion of Salmonella enterica serovar Typhimurium From Adult House Flies (Musca domestica L.; Diptera: Muscidae)

Abstract Salmonella Typhimurium (Le Minor and Popoff 1987; Enterobacteriales: Enterobacteriaceae) is a pathogen that causes gastroenteritis in humans and can be harbored by house flies. Factors influencing excretion of S. Typhimurium from infected flies have not been elucidated but are essential for assessing transmission potential. We determined the persistence and excretion of a green fluorescent protein (GFP) expressing strain of S.Typhimurium from house flies. Individual male and female flies were fed either sterile Luria-Bertani (LB) broth (controls) or cultures of “high” (~105 colony forming units [CFU]) or “low” (~104 CFU) doses of bacteria (treatments). Bacterial persistence was determined over 16 h by culturing whole-fly homogenate. Both sex and dose affected persistence between 6 and 12 h postingestion. In a separate experiment, fly excretion events were monitored during this time interval and excreta droplets were individually cultured for bacteria. Female flies had more excretion events than males across treatments. We observed interactions of fly sex and bacterial abundance (dose), both on the proportion of Salmonella-positive droplets and the CFU shed per droplet (CFU/droplet). In the low-dose treatment, males excreted a greater proportion of positive droplets than females. In the high-dose treatment, males excreted more CFU/droplet than females. High-dose male flies excreted more CFU/droplet than low-dose males, but low-dose females excreted more CFU/droplet than high-dose females. Irrespective of sex, low-dose flies excreted a greater dose-adjusted CFU (CFU droplet/CFU fed) than high-dose flies. This study demonstrates that both bacterial abundance and fly sex may influence excretion of bacteria from flies, and should be considered when assessing the risk of house fly transmission of pathogens.