Germplasm of Ozark chinquapin (Castanea ozarkensis Ashe) can be cryopreserved by dormant winter buds.

Ozark chinquapin (Castanea ozarkensis Ashe) is a forest tree, endemic to the Ozark Mountain region in Eastern United States. Its nutritious nuts were consumed by Native Americans, European settlers, livestock, and wild animals and its wood was an important rot-resistant construction material. Once a significant tree in regional forest communities, the species was nearly eradicated by a chestnut blight caused by Cryphonectria parasitca (Murill) Barr fungus. Some individuals have survived as sprouts from adventitious root buds, but they rarely reach reproductive maturity. While some in situ restoration efforts are underway, the development of a viable ex situ germplasm preservation method is critical to the conservation of this important food-bearing species. Our experiment aimed to develop a cryopreservation method for C. ozarkensis dormant winter buds subjected to eight experimental treatments before desiccation, slow cooling, and storage in liquid nitrogen vapor. The highest post cryogenic viability was 91.2 % for dormant buds pretreated with 0.3 M sucrose for 16 h followed by 0.75 M sucrose for 3 h; this treatment is suggested for cryopreservation of dormant winter buds of Ozark chinquapin germplasm.

Assessment of Potential Anti-Methanogenic and Antimicrobial Activity of Ethyl Nitroacetate, α-Lipoic Acid, Taurine and L-Cysteinesulfinic Acid In Vitro.

Livestock producers need new technologies to maintain the optimal health and well-being of their animals while minimizing the risks of propagating and disseminating pathogenic and antimicrobial-resistant bacteria to humans or other animals. Where possible, these interventions should contribute to the efficiency and profitability of animal production to avoid passing costs on to consumers. In this study, we examined the potential of nitroethane, 3-nitro-1-propionate, ethyl nitroacetate, taurine and L-cysteinesulfinic acid to modulate rumen methane production, a digestive inefficiency that results in the loss of up to 12% of the host's dietary energy intake and a major contributor of methane as a greenhouse gas to the atmosphere. The potential for these compounds to inhibit the foodborne pathogens, Escherichia coli O157:H7 and Salmonella Typhimurium DT104, was also tested. The results from the present study revealed that anaerobically grown O157:H7 and DT104 treated with the methanogenic inhibitor, ethyl nitroacetate, at concentrations of 3 and 9 mM had decreased (p < 0.05) mean specific growth rates of O157:H7 (by 22 to 36%) and of DT104 (by 16 to 26%) when compared to controls (0.823 and 0.886 h-1, respectively). The growth rates of O157:H7 and DT104 were decreased (p < 0.05) from controls by 31 to 73% and by 41 to 78% by α-lipoic acid, which we also found to inhibit in vitro rumen methanogenesis up to 66% (p < 0.05). Ethyl nitroacetate was mainly bacteriostatic, whereas 9 mM α-lipoic acid decreased (p < 0.05) maximal optical densities (measured at 600 nm) of O157:H7 and DT104 by 25 and 42% compared to controls (0.448 and 0.451, respectively). In the present study, the other oxidized nitro and organosulfur compounds were neither antimicrobial nor anti-methanogenic.

Effect of a maternal high-fat diet with vegetable substitution on fetal brain transcriptome.

Maternal dietary conditions play a major role in fetal growth and brain development. The primary aim of this study was to determine the effects of 5% of energy substitution by vegetables in a maternal dietary fat on placental and fetal weight and on fetal brain gene expression. Two-month-old female C57BL/6 mice were fed 16% (normal-fat, NF), 45% fat (HF), or HF substituted with vegetables (5% energy, HF+VS) diets for 12 weeks. Dams were then bred with NF diet-fed male mice. Placenta and fetal weights were measured at gestational age 19 (D19). RNA was isolated from fetal whole brains and sequenced using Illumina HiSeq. HF+VS diet prevented maternal HF diet-induced decreases in placental weight at D19. Feeding of a maternal HF diet was associated with 79 differentially expressed genes (DEGs), while maternal vegetable substitution was associated with 131 DEGs. The vegetable substitution diet decreased Apold1 (P=.0319), Spata2l (P=.0404), and Celsr1 (P<.03) expression compared to HF diet. Enrichment analysis of HF vs. HF+VS DEGs identified that synapse organization and regulation of embryonic development were significantly represented. KEGG enrichment analysis identified a significant representation of DEGs in the ubiquitin mediated proteolysis pathway in HF vs. HF+VS, and chemokine signaling pathway in NF vs. HF. These findings suggest that at D19, in a rodent model, a maternal HF diet alters placental and fetal growth, and that vegetable supplementation renders a protective effect against these changes.

Timing of Diapause Initiation and Overwintering Conditions Alter Gene Expression Profiles in Megachile rotundata.

Within the United States and Canada, the primary pollinator of alfalfa is the alfalfa leafcutting bee (ALCB), Megachile rotundata. Our previous findings showed that overwintering conditions impacted gene expression profile in ALCB prepupae that entered diapause early in the season. However, ALCB are a bivoltine species, which begs the question of whether bees entering diapause later in the season also show this trend. To better understand the effects of the timing of diapause initiation, we analyzed mRNA copy number of genes known to be involved in diapause regulation in early and late season diapausing ALCB that were overwintered in field conditions or using current agricultural management conditions. We hypothesized that overwintering conditions for late diapausing bees also affects gene expression profiles. Our results showed that expression profiles were altered by both overwintering condition and timing of diapause initiation, with bees that entered diapause earlier in the season showing different expression patterns than those that entered diapause later in the season. This trend was seen in expression of members of the cyclin family and several targets of the insulin signaling pathway, including forkhead box protein O (FOXO), which is known to be important for diapause regulation and stress responses. But, of the genes screened, the proto-oncogene, Myc, was the most impacted by the timing of diapause initiation. Under field conditions, there were significant differences in Myc expression between the early and late season samples in all months except for November and February. This same general trend in Myc expression was also seen in the laboratory-maintained bees with significant difference in expression in all months except for November, February, and May. These results support previous conclusions from our research showing that the molecular regulation of diapause development in ALCB is not a simple singular cascade of gene expression but a highly plastic response that varies between bees depending upon their environmental history.

Performance of allergen testing in a survey of frozen meals and meals ready-to-eat (MREs).

A 7-plex immunoassay capable of detecting cashew, egg, hazelnut, milk, peanut, shrimp, and soy allergens was used to screen meals ready-to-eat (MREs) and frozen meals that contained meat or poultry. The same food matrices were also evaluated using single individual allergen immunoassays. Multiplex and single allergen test results were compared with the allergen declared on the food label, which was considered the standard. For both the frozen meals (n = 113) and MREs (n = 24) each analytical method failed to detect allergens that were declared on product labels, but only in frozen meals were allergens detected that were not declared on the label. Undeclared allergens were detected for egg in 1.8% (2/113) and for soy in 7.1% (8/113) of frozen meals. Labelled allergens were not detected in 0.9% (1/113) of milk, 4.4% (5/113) of egg, and 15% (17/113) of soy allergens in frozen meals. Assay performance for evaluating allergens in MREs was poor.

Overwintering conditions impact insulin pathway gene expression in diapausing Megachile rotundata.

Diapause is a non-feeding state that many insects undergo to survive the winter months. With fixed resources, overall metabolism and insulin signaling (IIS) are maintained at low levels, but whether those change in response to seasonal temperature fluctuations remains unknown. The focus of this study was to determine 1) how genes in the insulin signaling pathway vary throughout diapause and 2) if that variation changes in response to temperature. To test the hypothesis that expression of IIS pathway genes vary in response to temperature fluctuations during overwintering, alfalfa leafcutting bees, Megachile rotundata, were overwintered at either a constant 4 °C in the lab or in naturally fluctuating temperatures in the field. Expression levels of genes in the IIS pathway, cell cycle regulators, and transcription factors were measured. Overall our findings showed that a few key targets of the insulin signaling pathway, along with growth regulators, change during overwintering, suggesting that only cell cycle regulators, and not the IIS pathway as a whole, change across the phases of diapause. To answer our second question, we compared gene expression levels between temperature treatments at each month for a given gene. We observed significantly more differences in expression of IIS pathway targets, indicating that overwintering conditions impact insulin pathway gene expression and leads to altered expression profiles. With differences seen between temperature treatment groups, these findings indicate that constant temperatures like those used in agricultural storage protocols, lead to different expression profiles and possibly different diapause phenotypes for alfalfa leafcutting bees.

Thermoprofile Parameters Affect Survival of Megachile rotundata During Exposure to Low-Temperatures.

Insects exposed to low temperature stress can experience chill injury, but incorporating fluctuating thermoprofiles increases survival and blocks the development of sub-lethal effects. The specific parameters required for a protective thermoprofile are poorly understood, because most studies test a limited range of thermoprofiles. For example, thermoprofiles with a wave profile may perform better than a square profile, but these two profiles are rarely compared. In this study, two developmental stages of the alfalfa leafcutting bee, Megachile rotundata, eye-pigmented pupae, and emergence-ready adults, were exposed to one of eight thermoprofiles for up to 8 weeks. All the thermoprofiles had a base of 6°C and a peak temperature of either 12°C or 18°C. The duration at peak temperature varied depending on the shape of the thermoprofile, either square or wave form. Two other treatments acted as controls, a constant 6°C and a fluctuating thermal regime (FTR) with a base temperature of 6°C that was interrupted daily by a single, 1-h pulse at 20°C. Compared with constant 6°C, all the test thermoprofiles significantly improved survival. Compared with the FTR control, the thermoprofiles with a peak temperature of 18°C outperformed the 12°C profiles. Bees in the eye-pigmented stage exposed to the 18°C profiles separated into two groups based on the shape of the profile, with higher survival in the square profiles compared with the wave profiles. Bees in the emergence-ready stage exposed to 18°C profiles all had significantly higher survival than bees in the FTR controls. Counter to expectations, the least ecologically relevant thermoprofiles (square) had the highest survival rates and blocked the development of sub-lethal effects (delayed emergence).

Next-Generation Sequencing Identifies Polyunsaturated Fatty Acid Responsive Genes in the Juvenile Rat Cerebellum.

Dietary n-3 polyunsaturated fatty acids (PUFA) influence postnatal brain growth and development. However, little data exist regarding the impacts of dietary n-3 PUFA in juvenile animals post weaning, which is a time of rapid growth. We tested the hypothesis that depleting dietary n-3 PUFA would result in modifications to the cerebellar transcriptome of juvenile rats. To test this hypothesis, three week old male rats (an age that roughly corresponds to an 11 month old child in brain development) were fed diets containing either soybean oil (SO) providing 1.1% energy from α-linolenic acid (ALA; 18:3n-3; ALA-sufficient) or corn oil (CO) providing 0.13% energy from ALA (ALA-deficient) for four weeks. Fatty acids (FAs) in the cerebellum were analyzed and revealed a 4-fold increase in n-6 docosapentaenoic acid (DPA; 22:5n-6), increases in arachidonic acid (AA; 20:4n-6) and docosatetraenoic acid (DTA; 22:4n-6), but no decrease in docosahexaenoic acid (DHA; 22:6n-3), in animals fed CO versus SO. Transcript abundance was then characterized to identify differentially expressed genes (DEGs) between the two diets. Upper quartile (UQ) scaling and transcripts per million (TPM) data normalization identified 100 and 107 DEGs, respectively. Comparison of DEGs from the two normalization methods identified 70 genes that overlapped, with 90% having abundance differences less than 2-fold. Nr4a3, a transcriptional activator that plays roles in neuroprotection and learning, was elevated over 2-fold from the CO diet. These data indicate that expression of Nr4a3 in the juvenile rat cerebellum is responsive to dietary n-3 PUFA, but additional studies are needed clarify the neurodevelopmental relationships between n-3 PUFA and Nr4a3 and the resulting impacts.

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).

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.

Phytohormonal networks promote differentiation of fiber initials on pre-anthesis cotton ovules grown in vitro and in planta.

The number of cotton (Gossypium sp.) ovule epidermal cells differentiating into fiber initials is an important factor affecting cotton yield and fiber quality. Despite extensive efforts in determining the molecular mechanisms regulating fiber initial differentiation, only a few genes responsible for fiber initial differentiation have been discovered. To identify putative genes directly involved in the fiber initiation process, we used a cotton ovule culture technique that controls the timing of fiber initial differentiation by exogenous phytohormone application in combination with comparative expression analyses between wild type and three fiberless mutants. The addition of exogenous auxin and gibberellins to pre-anthesis wild type ovules that did not have visible fiber initials increased the expression of genes affecting auxin, ethylene, ABA and jasmonic acid signaling pathways within 1 h after treatment. Most transcripts expressed differentially by the phytohormone treatment in vitro were also differentially expressed in the ovules of wild type and fiberless mutants that were grown in planta. In addition to MYB25-like, a gene that was previously shown to be associated with the differentiation of fiber initials, several other differentially expressed genes, including auxin/indole-3-acetic acid (AUX/IAA) involved in auxin signaling, ACC oxidase involved in ethylene biosynthesis, and abscisic acid (ABA) 8'-hydroxylase an enzyme that controls the rate of ABA catabolism, were co-regulated in the pre-anthesis ovules of both wild type and fiberless mutants. These results support the hypothesis that phytohormonal signaling networks regulate the temporal expression of genes responsible for differentiation of cotton fiber initials in vitro and in planta.

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.

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.

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.).

BACKGROUND: Cotton 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. RESULTS: Two 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. CONCLUSIONS: In 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.

Influences from Long-Term Crop Rotation, Soil Tillage, and Fertility on the Severity of Rice Grain Smuts.

False smut (Ustilaginoidea virens) and kernel smut (Neovossia horrida) are diseases of rice (Oryza sativa) that reduce both grain yield and quality. Susceptible rice cultivars are in widespread use on production acreage in the United States, and the effects from crop management practices on smut control are poorly understood. We studied the long-term effects of crop rotation, soil tillage, and fertility level on rice smut severity. The highest levels of false smut observed in this study were on cultivars grown in rotation with soybean, on traditionally tilled soils, with high fertilizer treatments. The highest levels of kernel smut were observed in a rice-soybean rotation with winter wheat grown between summer crops. These rotations are commonly used in rice-growing regions of the southern United States. Using combinations of crop rotation, soil tillage, and fertility rate, several alternative crop-management practices were identified that provided effective control of smuts in susceptible rice cultivars. The most effective method for controlling both false smut and kernel smut was in 3-year rotations of rice, soybean, and corn. Regardless of rotation order or tillage and fertility treatments within the rotations, rotating out of rice for 2 years was the most effective approach for smut control.

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.

Effect of Furrow Irrigation on the Severity of False Smut in Susceptible Rice Varieties.

False smut (Ustilaginoidea virens) is an important emerging disease of rice (Oryza sativa) in the southern United States, where all major rice cultivars and hybrids are susceptible to the disease. False smut susceptibility was evaluated in traditional paddy-rice fields and under furrow-irrigated conditions to determine the effects of alternative agricultural practices on the severity of this disease. Highly effective false smut suppression was observed in furrow-irrigated rice, where the disease was nearly eliminated in susceptible rice entries. False smut suppression was observed for two hybrids and one conventional rice cultivar, demonstrating that suppression was not limited to specific germplasm sources. Kernel smut severity was also monitored, but no effect on this disease was observed from the irrigation treatments. Therefore, suppression of disease severity in nonflooded rice appears to be a phenomenon unique to the rice-false smut pathosystem, which can be exploited to achieve effective field resistance to this disease.

Heterogeneity of vaginal microbial communities within individuals.

Recent culture-independent studies have revealed that a healthy vaginal ecosystem harbors a surprisingly complex assemblage of microorganisms. However, the spatial distribution and composition of vaginal microbial populations have not been investigated using molecular methods. Here, we evaluated site-specific microbial composition within the vaginal ecosystem and examined the influence of sampling technique in detection of the vaginal microbiota. 16S rRNA gene clone libraries were prepared from samples obtained from different locations (cervix, fornix, outer vaginal canal) and by different methods (swabbing, scraping, lavaging) from the vaginal tracts of eight clinically healthy, asymptomatic women. The data reveal that the vaginal microbiota is not homogenous throughout the vaginal tract but differs significantly within an individual with regard to anatomical site and sampling method used. Thus, this study illuminates the complex structure of the vaginal ecosystem and calls for the consideration of microenvironments when sampling vaginal microbiota as a clinical predictor of vaginal health.

Effect of Cultural Management Practices on the Severity of False Smut and Kernel Smut of Rice.

False smut (Ustilaginoidea virens) and kernel smut (Neovossia horrida) are diseases of rice (Oryza sativa) that reduce both grain yield and quality. False smut is an emerging disease worldwide that is rapidly gaining in importance, whereas kernel smut has historically been a chronic minor disease with sporadic outbreaks that cause considerable losses. Highly effective disease control was obtained for susceptible cultivars by employing conservation tillage (69% reduction in false smut), continuous rice cropping (88% reduction in false smut), and moderate nitrogen fertility rates (34 and 60% reductions in false smut and kernel smut, respectively). Combining these treatments nearly eliminated smuts from cultivars that were fully susceptible under conventional cultivation practices. Furthermore, using a nursery designed to promote smut diseases, two rice hybrids were identified that possessed kernel smut resistance under the most favorable disease conditions. The genetic basis of the resistance is unknown. However, the utility for disease control is great because hybrids occupy significant portions of production rice acreage.

Temporal analysis of Candida albicans gene expression during biofilm development.

Microarrays were used to identify changes in gene expression associated with Candida albicans biofilm development. Two biofilm substrates (denture and catheter), and two C. albicans strains for each substrate, were tested to remove model- and strain-dependent variability from the overall dataset. Three biofilm developmental phases were examined: early (6 h), intermediate (12 h), and mature (48 h). Planktonic specimens were collected at the same time points. Data analysis focused primarily on gene expression changes over the time-course of biofilm development. Glycolytic and non-glycolytic carbohydrate assimilation, amino acid metabolism, and intracellular transport mechanisms were important during the early phase of biofilm formation. These early events increase intracellular pools of pyruvate, pentoses and amino acids, which prepare the biofilm for the large biomass increase that begins around 12 h of development. This developmental stage demands energy and utilizes specific transporters for amino acids, sugars, ions, oligopeptides and lactate/pyruvate. At mature phase (48 h), few genes were differentially expressed compared with the 12 h time point, suggesting a relative lack of initiation of new metabolic activity. Data analysis to assess biofilm model-specific gene expression showed more dynamic changes in the denture model than in the catheter model. Data analysis to identify gene expression changes that are associated with each strain/substrate combination identified the same types of genes that were identified in the analysis of the entire dataset. Collectively, these data suggest that genes belonging to different, but interconnected, functional categories regulate the morphology and phenotype of C. albicans biofilm.