Diploid males support a two-step mechanism of endosymbiont-induced thelytoky in a parasitoid wasp
Ma, W.J. ; Pannebakker, B.A. ; Zande, L. van de; Schwander, T. ; Wertheim, B. ; Beukeboom, L.W. - \ 2015
BMC Evolutionary Biology 15 (2015). - ISSN 1471-2148
leptopilina-clavipes hymenoptera - sex-determination - parthenogenetic populations - quantitative pcr - insect sex - wolbachia - host - reproduction - braconidae - determines
Background Haplodiploidy, where females develop from diploid, fertilized eggs and males from haploid, unfertilized eggs, is abundant in some insect lineages. Some species in these lineages reproduce by thelytoky that is caused by infection with endosymbionts: infected females lay haploid eggs that undergo diploidization and develop into females, while males are very rare or absent. It is generally assumed that in thelytokous wasps, endosymbionts merely diploidize the unfertilized eggs, which would then trigger female development. Results We found that females in the parasitoid wasp Asobara japonica infected with thelytoky-inducing Wolbachia produce 0.7–1.2 % male offspring. Seven to 39 % of these males are diploid, indicating that diploidization and female development can be uncoupled in A. japonica. Wolbachia titer in adults was correlated with their ploidy and sex: diploids carried much higher Wolbachia titers than haploids, and diploid females carried more Wolbachia than diploid males. Data from introgression lines indicated that the development of diploid individuals into males instead of females is not caused by malfunction-mutations in the host genome but that diploid males are most likely produced when the endosymbiont fails to activate the female sex determination pathway. Our data therefore support a two-step mechanism by which endosymbionts induce thelytoky in A. japonica: diploidization of the unfertilized egg is followed by feminization, whereby each step correlates with a threshold of endosymbiont titer during wasp development. Conclusions Our new model of endosymbiont-induced thelytoky overthrows the view that certain sex determination mechanisms constrain the evolution of endosymbiont-induced thelytoky in hymenopteran insects. Endosymbionts can cause parthenogenesis through feminization, even in groups in which endosymbiont-diploidized eggs would develop into males following the hosts’ sex determination mechanism. In addition, our model broadens our understanding of the mechanisms by which endosymbionts induce thelytoky to enhance their transmission to the next generation. Importantly, it also provides a novel window to study the yet-poorly known haplodiploid sex determination mechanisms in haplodiploid insects.
AIL and HDG proteins act antagonistically to control cell proliferation
Horstman, A. ; Fukuoka, H. ; Muino Acuna, J.M. ; Nitsch, L.M.C. ; Guo, Changhao ; Passarinho, P.A. ; Sanchez Perez, G.F. ; Immink, R.G.H. ; Angenent, G.C. ; Boutilier, K.A. - \ 2015
Development 142 (2015). - ISSN 0950-1991 - p. 454 - 464.
arabidopsis-thaliana - transcription factors - plant transformation - ectopic expression - quantitative pcr - chip-seq - differentiation - genes - plethora - growth
AINTEGUMENTA-LIKE (AIL) transcription factors are key regulators of cell proliferation and meristem identity. Although AIL functions have been well described, the direct signalling components of this pathway are largely unknown.We show that BABY BOOM(BBM) and other AIL proteins physically interact with multiple members of the L1-expressed HOMEODOMAIN GLABROUS (HDG) transcription factor family, including HDG1, HDG11 and HDG12. Overexpression of HDG1, HDG11 and HDG12 restricts growth due to root and shoot meristem arrest, which is associated with reduced expression of genes involved in meristem development and cell proliferation pathways, whereas downregulation of multiple HDG genes promotes cell overproliferation. These results suggest a role for HDG proteins in promoting cell differentiation. We also reveal a transcriptional network in which BBM andHDG1regulate several common target genes, and whereBBM/AIL and HDG regulate the expression of each other. Taken together, these results suggest opposite roles for AIL and HDG proteins, with AILs promoting cell proliferation and HDGs stimulating cell differentiation, and that these functions are mediated at both the protein-protein interaction and transcriptional level.
A high-throughput method for GMO multi-detection using a microfluidic dynamic array
Brod, F.C.A. ; Dijk, J.P. van; Voorhuijzen, M.M. ; Dinon, A.Z. ; Guimarães, L.H.S. ; Scholtens, I.M.J. ; Arisi, A.C.M. ; Kok, E.J. - \ 2014
Analytical and Bioanalytical Chemistry 406 (2014)5. - ISSN 1618-2642 - p. 1397 - 1410.
genetically-modified maize - real-time pcr - polymerase-chain-reaction - modified organisms - reference molecules - quantitative pcr - screening-assay - digital pcr - zea-mays - validation
The ever-increasing production of genetically modified crops generates a demand for high-throughput DNAbased methods for the enforcement of genetically modified organisms (GMO) labelling requirements. The application of standard real-time PCR will become increasingly costly with the growth of the number of GMOs that is potentially present in an individual sample. The present work presents the results of an innovative approach in genetically modified crops analysis byDNA basedmethods, which is the use of a microfluidic dynamic array as a high throughput multi-detection system. In order to evaluate the system, six test samples with an increasing degree of complexity were prepared, preamplified and subsequently analysed in the Fluidigm system. Twenty-eight assays targeting different DNA elements, GM events and species-specific reference genes were used in the experiment. The large majority of the assays tested presented expected results. The power of low level detection was assessed and elements present at concentrations as low as 0.06 % were successfully detected. The approach proposed in this work presents the Fluidigm system as a suitable and promising platform for GMO multi-detection.
Effect of galactooligosaccharides and Bifidobacterium animalis Bb-12 on growth of Lactobacillus amylovorus DSM 16698, microbial community structure, and metabolite production in an in vitro colonic model set up with human or pig microbiota
Martinez, R.C.R. ; Cardarelli, H. ; Borst, W. ; Albrecht, S.A. ; Schols, H.A. ; Gutierrez, O. ; Maathuis, A. ; Melo Franco, B.D. de; Martinis, E.C.P. de; Zoetendal, E.G. ; Venema, K. ; Saad, S.M.I. ; Smidt, H. - \ 2013
FEMS microbiology ecology 84 (2013)1. - ISSN 0168-6496 - p. 110 - 123.
gradient gel-electrophoresis - 16s ribosomal-rna - large-intestine - fecal samples - human feces - gastrointestinal-tract - quantitative pcr - dietary-fibers - gut bacteria - double-blind
A validated in vitro model of the large intestine (TIM-2), set up with human or pig faeces, was used to evaluate the impact of potentially probiotic Lactobacillus amylovorus DSM 16698, administered alone (i), in the presence of prebiotic galactooligosaccharides (GOS) (ii), and co-administered with probiotic Bifidobacterium animalis ssp. lactis Bb-12 (Bb-12) (iii) on GOS degradation, microbial growth (L. amylovorus, lactobacilli, bifidobacteria and total bacteria) and metabolite production. High performance anion exchange chromatography revealed that GOS degradation was more pronounced in TIM-2 inoculated with pig faeces than with human faeces. Denaturing gradient gel electrophoresis profiling of PCR-amplified 16S rRNA genes detected a more complex Lactobacillus spp. community in pig faecal material than in human faecal inoculum. According to 16S rRNA gene-targeted qPCR, GOS stimulated the growth of lactobacilli and bifidobacteria in faecal material from both materials. The cumulative production of short chain fatty acids and ammonia was higher (P <0.05) for pig than for human faeces. However, lactate accumulation was higher (P <0.05) in the human model and increased after co-administration with GOS and Bb-12. This study reinforced the notion that differences in microbiota composition between target host organisms need to be considered when animal data are extrapolated to human, as is often done with pre- and probiotic intervention studies
Role of "Dehalococcoides" spp. in the anaerobic transformation of Hexachlorobenzene in European rivers
Tas, N. ; Schraa, G. ; Vos, W.M. de; Smidt, H. - \ 2011
Applied and Environmental Microbiology 77 (2011)13. - ISSN 0099-2240 - p. 4437 - 4445.
sp strain cbdb1 - dehalogenase-homologous genes - reductive dechlorination - chlorinated benzenes - enrichment culture - transcriptional regulators - microbial-communities - quantitative pcr - genome sequence - ethenogenes
The diffuse pollution by chlorinated organic compounds in river basins is a concern, due to their potential adverse effects on human health and the environment. Organohalides, like hexachlorobenzene (HCB), are recalcitrant to aerobic microbial degradation, and "Dehalococcoides" spp. are the only known microorganisms capable of anaerobic transformation of these compounds coupled to their growth. In this study, sediments from four European rivers were studied in order to determine their HCB dechlorination capacities and the role of Dehalococcoides spp. in this process. Only a weak correlation was observed between Dehalococcoides species abundance and HCB transformation rates from different locations. In one of these locations, in the Ebro River sediment, HCB dechlorination could be linked to Dehalococcoides species growth and activity by 16S rRNA-based molecular methods. Furthermore, HCB dechlorination activity in this sediment was found over the full range of ambient temperatures that this sediment can be exposed to during different seasons throughout the year. The sediment contained several reductive dehalogenase (rdh) genes, and analysis of their transcription revealed the dominance of cbrA, previously shown to encode a trichlorobenzene reductive dehalogenase. This study investigated the role of Dehalococcoides spp. in HCB dechlorination in river sediments and evaluated if the current knowledge of rdh genes could be used to assess HCB bioremediation potential
Telomere Length and Mortality in Elderly Men: The Zutphen Elderly Study
Houben, J.M.J. ; Giltay, E.J. ; Rius-Ottenheim, N. ; Hageman, G. ; Kromhout, D. - \ 2011
Journals of Gerontology. Series A: Biological Sciences & Medical Sciences 66 (2011)1. - ISSN 1079-5006 - p. 38 - 44.
peripheral-blood cells - coronary-heart-disease - oxidative stress - oldest-old - cardiovascular-disease - physical-activity - quantitative pcr - risk - cancer - association
Telomere shortening is a marker of aging and therefore telomere length might be related to disease progression and survival. To address these questions, we measured leukocyte telomere length (LTL) in male participants from the Zutphen Elderly Study. LTL was measured by quantitative polymerase chain reaction in 203 men: mean aged 78 years in 1993 and 75 surviving participants mean aged 83 years in 2000. During 7 years of follow-up, 105 men died. Cox proportional hazards models were used to estimate hazard ratios for all-cause and cause-specific mortality. We found that LTL declined with a mean of 40.2 bp/year, and LTL values measured in 1993 and 2000 correlated significantly (r = .51, p <.001). Longer telomeres at baseline were not predictive for all-cause mortality, cardiovascular mortality, or cancer mortality. These results suggest that LTL decreases with increasing age and that LTL is not related to mortality in men aged more than 70 years.
Concurrent hexachlorobenzene and chloroethene transformation by endogenous dechlorinating microorganisms in the Ebro River sediment
Tas, N. ; Heilig, G.H.J. ; Eekert, M.H.A. van; Schraa, G. ; Vos, W.M. de; Smidt, H. - \ 2010
FEMS microbiology ecology 74 (2010)3. - ISSN 0168-6496 - p. 682 - 692.
dehalococcoides sp strain - reductive dehalogenase genes - vinyl-chloride reductase - ribosomal-rna - chlorinated benzenes - anaerobic bacterium - enrichment culture - electron-acceptors - quantitative pcr - genome sequence
The ability of Dehalococcoides spp. to reduce chlorinated compounds offers a great potential for bioremediation and/or bioaugmentation of contaminated environments. So far, however, our knowledge of the activity of Dehalococcoides spp. in situ is limited to only a few subsurface environments. The aim of this study was to broaden this knowledge to other environments, and we investigated the role of Dehalococcoides spp. in the transformation of chlorinated benzenes and chlorinated ethenes in the Ebro River (Spain) sediments. Lab-scale batch microcosms were used to follow the growth and abundance of Dehalococcoides spp. during the transformation of selected chlorinated compounds. We applied biomolecular tools targeting the 16S rRNA, the 16S rRNA gene and several functional genes involved in dechlorination in combination with chemical measurements. The growth of Dehalococcoides spp. and the differential expression of several reductive dehalogenase genes during the dechlorination process could be demonstrated. Furthermore, 16S rRNA gene-based clone libraries of dechlorinating river sediment showed a complex community structure and indicated the involvement of several additional bacterial genera in the transformation process, underlining the remarkable potential of this rivers' sediment to transform different halo-organic pollutants
Exploiting the ecogenomics toolbox for environmental diagnostics of organohalide-respiring bacteria
Maphosa, F. ; Vos, W.M. de; Smidt, H. - \ 2010
Trends in Biotechnology 28 (2010)6. - ISSN 0167-7799 - p. 308 - 316.
dehalococcoides sp strain - reductive dehalogenase genes - real-time pcr - vinyl-chloride reductase - quantitative pcr - desulfitobacterium-hafniense - genome sequence - transcriptional regulator - contaminated groundwater - microbial-communities
Various ‘omics’ methods have enabled environmental probing at the molecular level and have created an important new paradigm in bioremediation design and management. Ecogenomics – the application of genomics to ecological and environmental sciences – defines phylogenetic and functional biodiversity at the DNA, RNA and protein levels. It capitalizes on this knowledge to elucidate functions and interactions of organisms at the ecosystem level in relation to ecological and evolutionary processes. Effective bioremediation of widespread halo-organic pollutants in anaerobic environments requires knowledge of catabolic potential and in situ dynamics of organohalide-respiring and co-metabolizing microorganisms. Here, we discuss the potential of ecogenomics approaches in developing high-throughput methods for detecting and monitoring organohalide respirers, and for providing improvements to selection, specificity and sensitivity of target biomarkers and their application to evaluate bioremediation strategies
Accurate Quantification of Microorganisms in PCR-Inhibiting Environmental DNA Extracts by a Novel Internal Amplification Control Approach Using Biotrove OpenArrays
Doorn, R. van; Klerks, M.M. ; Gent-Pelzer, M.P.E. van; Speksnijder, A. ; Kowalchuk, G.A. ; Schoen, C.D. - \ 2009
Applied and Environmental Microbiology 75 (2009)22. - ISSN 0099-2240 - p. 7253 - 7260.
polymerase-chain-reaction - real-time pcr - diagnostic pcr - salmonella-enterica - humic substances - quantitative pcr - positive control - soil - assays - purification
PCR-based detection assays are prone to inhibition by substances present in environmental samples, thereby potentially leading to inaccurate target quantification or false-negative results. Internal amplification controls (IACs) have been developed to help alleviate this problem but are generally applied in a single concentration, thereby yielding less-than-optimal results across the wide range of microbial gene target concentrations possible in environmental samples (J. Hoorfar, B. Malorny, A. Abdulmawjood, N. Cook, M. Wagner, and P. Fach, J. Clin. Microbiol. 42: 1863-1868, 2004). Increasing the number of IACs for each quantitative PCR (qPCR) sample individually, however, typically reduces sensitivity and, more importantly, the reliability of quantification. Fortunately, current advances in high-throughput qPCR platforms offer the possibility of multiple reactions for a single sample simultaneously, thereby allowing the implementation of more than one IAC concentration per sample. Here, we describe the development of a novel IAC approach that is specifically designed for the state-of-the-art Biotrove OpenArray platform. Different IAC targets were applied at a range of concentrations, yielding a calibration IAC curve for each individual DNA sample. The developed IACs were optimized, tested, and validated by using more than 5,000 unique qPCR amplifications, allowing accurate quantification of microorganisms when applied to soil DNA extracts containing various levels of PCR-inhibiting compounds. To our knowledge, this is the first study using a suite of IACs at different target concentrations to monitor PCR inhibition across a wide target range, thereby allowing reliable and accurate quantification of microorganisms in PCR-inhibiting DNA extracts. The developed IAC is ideally suited for high-throughput screenings of, for example, ecological and agricultural samples on next-generation qPCR platforms.
A TaqMan PCR method for routine diagnosis of the quarantine fungus guignardia citricarpa on citrus fruit
Gent-Pelzer, M.P.E. van; Brouwershaven, I.R. van; Kox, L.F.F. ; Bonants, P.J.M. - \ 2007
Journal of Phytopathology 155 (2007)6. - ISSN 0931-1785 - p. 357 - 363.
real-time pcr - black spot fungus - quantitative pcr - dna extraction - mangiferae - endophyte - plants - detect
With respect to disease risk for the quarantine fungus Guignardia citricarpa on citrus fruit an accurate diagnosis for routine analysis is required. Also, when inspections have to be performed on imported citrus fruits, a fast detection method is urgently needed. A fast automated DNA extraction method based on magnetic beads combined with a real-time PCR assay was optimized to improve and advance the routine diagnosis of citrus black spot disease. Real-time PCR was used for detection of the pathogen G. citricarpa in planta. A specific primer/TaqMan probe combination that discriminates between G. citricarpa and the harmless citrus endophyte Guignardia mangiferae, was designed based on the internal transcribed spacer region of the multi-copy rDNA gene. Co-amplification of target DNA along with an internal competitor DNA fragment made the diagnostic assay more reliable to check for false negatives. The real-time PCR was specific, since no cross reaction was observed with a series of citrus pathogens and related species. The diagnostic assay was performed on lesions dissected from imported diseased oranges. Comparison between the conventional PCR and the real-time PCR methods showed that the TaqMan method was more sensitive.
Real-time PCR for detection and quantification of fungal and oomycete tomato pathogens in plant and soil samples
Lievens, B. ; Brouwer, M. ; Vanachter, A.C.R.C. ; Cammue, B.P.A. ; Thomma, B.P.H.J. - \ 2006
Plant Science 171 (2006)1. - ISSN 0168-9452 - p. 155 - 165.
polymerase-chain-reaction - verticillium-dahliae - quantitative pcr - pythium - identification - roots - dna - microsclerotia - phytophthora - diagnostics
Although new, rapid detection and identification technologies are becoming available more and more for various plant pathogens, pathogen quantification remains one of the main challenges in the disease management of many crops. Currently, real-time polymerase chain reaction (PCR) is the most straightforward technique to quantify pathogen presence. This manuscript describes the use of real-time PCR to quantitatively assess the presence of a number of economically important fungal and oomycete tomato pathogens in biological samples. We demonstrate that pathogen DNA can be accurately quantified over at least four orders of magnitude. Additionally, we demonstrate the feasibility of the technique to quantify pathogen biomass in complex biological samples.
Real-time PCR detection of lactic acid bacteria in cecal contents of Eimeria tenella-infected broilers fed soybean oligosaccharides and soluble soybean polysaccharides
Lan, Y. ; Xun, S. ; Tamminga, S. ; Williams, B.A. ; Verstegen, M.W.A. ; Erdi, G. - \ 2004
Poultry Science 83 (2004)10. - ISSN 0032-5791 - p. 1696 - 1702.
competitive-exclusion - quantitative pcr - inulin - lactobacillus - oligofructose - differentiation - colonization - fluorescence - performance - chickens
This experiment was conducted to test whether dietary soybean meal oligosaccharides (SMO) and water-soluble polysaccharides (SMP) can assist broiler chickens in resisting Eimeria tenella, and to determine the survival of lactic acid bacteria in cecal contents postinfection. All birds received a soybean meal-free diet. The 6 experimental treatments were as follows: positive (COR) and negative (COW) control groups, 2 groups fed diets containing either 1% SMO or 0.5% SMP from 1 to 11 d of age; a vaccinated group (VAC), and an anticoccidial medicated group (ANT). Chickens of all treatments except COW were orally infected with 1000 sporulated oocysts of E. tenella on d 15. Fecal oocyst shedding was monitored per treatment group between d 5 and 13 postinfection. Lactic acid bacteria (LAB) in cecal contents were evaluated by a real-time PCR technique on d 7 postinfection. The results showed that the SMO and SMP groups had a lower number of oocysts per gram of feces during the monitoring period than the COR group. Threshold cycles were 22.21, 27.68, 13.99, 14.92, 12.97, and 14.85, for COW, COR, SMO, SMP, VAC, and ANT groups, respectively; specific PCR products were confirmed by the results of melting curve analysis and agarose gel electrophoresis. The results suggest that these LAB communities were promoted by SMO and SMP and have a competitive exclusion function when broiler chickens are infected with E. tenella