Methods for Assessment of Viability and Germination of Plasmodiophora brassicae Resting Spores

Clubroot caused by the obligate biotrophic parasite Plasmodiophora brassicae is a destructive soil borne disease of cruciferous crops. Resting spores of P. brassicae can survive in the soil for a long period without hosts or external stimulants. The viability and germination rate of resting spores are crucial factors of the inoculum potential in the field. The accurate assessment of viability and germination rate is the foundation to evaluate the effect of control methods. In this study, we evaluated several methods for the assessment of viability and germination rate of P. brassicae resting spores. Dual staining with calcofluor white-propidium iodide (CFW-PI) or single stain with Evans blue showed reliable accuracy in estimating viability. CFW-PI was capable of reliably determining the viability within 10 min, while Evans blue required overnight incubation to obtain accurate results. Due to DNA degradation of heat treatments, acetone was selected to evaluate the efficiency of propidium monoazide (PMA)–quantitative PCR (qPCR) used for the quantification of DNA from viable cells. The staining with 4,6-Diamidine-2-phenylindole dihydrochloride (DAPI) and the use of differential interference contrast microscopy were suitable for the determination of resting spore germination rates. The latter method also allowed recording individual germination states of spores. Alternatively, dual staining with CFW-Nile red was successfully used to assess the germination rate of resting spores with a lethal pre-treatment. This study evaluates and confirms the suitability of various microscopic and molecular genetic methods for the determination of viability and germination of P. brassicae resting spores. Such methods are required to study factors in the soil regulating survival, dormancy and germination of P. brassicae resting spores causing clubroot disease in Brassicaceae hosts and therefore are fundamental to develop novel strategies of control.

Effects of α-crystallin gene knockout on zebrafish lens development

The α-crystallin small heat shock proteins contribute to the transparency and refractive properties of the vertebrate eye lens and prevent the protein aggregation that would otherwise produce lens cataract, the leading cause of human blindness. There are conflicting data in the literature as to what role the α-crystallins may play in early lens development. In this study we used CRISPR gene editing to produce zebrafish lines with null mutations for each of the three α-crystallin genes (cryaa, cryaba and cryabb). Absence of protein was confirmed by mass spectrometry and lens phenotypes were assessed with differential interference contrast microscopy and histology. Loss of αA-crystallin produced a variety of lens defects with varying severity in larval lenses at 3 and 4 dpf, but little significant change in normal fiber cell denucleation. Loss of either αBa- or αBb-crystallin produced no significant lens defects. Mutation of each α-crystallin gene did not alter the expression levels of the remaining two, suggesting a lack of genetic compensation. These data confirm a developmental role for αA-crystallin in lens development, but the range of phenotype severity suggests its loss simply increases the chance for defect, and that the protein is not essential. Our finding that cryaba and cryabb null mutants lack noticeable lens defects is congruent with insignificant transcript levels in lens epithelial and fiber cells. Future experiments can explore the molecular consequences of cryaa mutation and causes of lens defects in this null mutant, as well as the roles of other genes in lens development and function.

Oncholaimus tripapillatus sp. nov., a New Free-Living Marine Nematode of the Genus Oncholaimus Dujardin, 1845 (Nematoda: Enoplida: Oncholaimidae) from the Subtidal Sediment of Dokdo Island, East Sea, Korea, with a New Record of O. qingdaoensis Zhang & Platt, 1983

During a survey of the free-living marine nematodes of Korea, two species belonging to the genus Oncholaimus Dujardin, 1845 were discovered. A new species, Oncholaimus tripapillatus sp. nov. and a newly recorded species, Oncholaimus qingdaoensis Zhang & Platt, 1983, are reported. Oncholaimus tripapillatus sp. nov. was collected from the washing of subtidal coarse sediments around Dokdo Island of the East Sea, Korea. Oncholaimus tripapillatus sp. nov. is characterized by a relatively long (4071–4435 µm in males and 4514–4661 µm in females) and slender body, a slightly constricted head region, relatively long cephalic setae (10–12 µm), males having a precloacal sexual protuberance bearing two small cone-shaped supplementary spines, five pairs of long cloacal setae (three pairs of precloacal and two pairs of postcloacal setae in subventral position), and three remarkable papillae near the end of the tail, with two pairs of subventral setae. The Korean specimens of Oncholaimus qingdaoensis Zhang & Platt, 1983 are almost identical to the Chinese specimens of the original description from the intertidal sand of Qingdao, China. However, the Korean specimens differ from the Chinese specimens in the longer body length in males (3379–3715 µm vs. 2380–2640 µm), the larger spicule length (47–52 µm vs. 34–36 µm), and the presence of ventral tail papillae situated around 14–16 µm from the tail tip. Detailed morphological features and illustrations of two Oncholaimus species from Korea were obtained by differential interference contrast microscopy and scanning electron microscopy. A pictorial key to the species group with distinct tail papillae among the genus Oncholaimus is also provided.

Statistical image processing quantifies the changes in cytoplasmic texture associated with aging in Caenorhabditis elegans oocytes

BackgroundOocyte quality decreases with aging, thereby increasing errors in fertilization, chromosome segregation, and embryonic cleavage. Oocyte appearance also changes with aging, suggesting a functional relationship between oocyte quality and appearance. However, no methods are available to objectively quantify age-associated changes in oocyte appearance.ResultsWe show that statistical image processing of Nomarski differential interference contrast microscopy images can be used to quantify age-associated changes in oocyte appearance in the nematodeCaenorhabditis elegans. Max–min value (mean difference between the maximum and minimum intensities within each moving window) quantitatively characterized the difference in oocyte cytoplasmic texture between 1- and 3-day-old adults (Day 1 and Day 3 oocytes, respectively). With an appropriate parameter set, the gray level co-occurrence matrix (GLCM)-based texture featureCorrelation(COR) more sensitively characterized this difference than the Max–min Value. Manipulating the smoothness of and/or adding irregular structures to the cytoplasmic texture of Day 1 oocyte images reproduced the difference in Max–min Value but not in COR between Day 1 and Day 3 oocytes. Increasing the size of granules in synthetic images recapitulated the age-associated changes in COR. Manual measurements validated that the cytoplasmic granules in oocytes become larger with aging.ConclusionsThe Max–min value and COR objectively quantify age-related changes inC. elegansoocyte in Nomarski DIC microscopy images. Our methods provide new opportunities for understanding the mechanism underlying oocyte aging.

Autonomous apomixis in Praxelis clematidea (Asteraceae: Eupatorieae), an invasive alien plant

Background and Aims Invasion by alien species poses serious threats to ecosystem services, biodiversity, and economic development. Understanding the reproductive mechanisms of invasive alien species can lay the foundation for effective control measures. Praxelis clematidea R.M. King & H. Rob is a triploid neotropical Asteraceae species that is invasive in China and other countries. However, few studies have focused on its reproductive biology. Methods In this study, flow cytometric seed screening (FCSS) was used to identify and confirm the reproductive mode of the species. The development of ovules, anthers, and mega- and microgametophytes was observed using a clearing method and differential interference contrast microscopy. Pollen viability was measured using the Benzidine test and Alexander’s stain. Pollen morphology was observed via fluorescence microscopy after sectioning the disk florets and staining with water-soluble aniline blue or 4’6-diamidino-2-phenylindole nuclei dyes. Controlled pollination experiments were conducted on four populations in China to examine the breeding system and to confirm autonomous apomixis. Key Results The reproductive mode was found to be autonomous apomixis without pseudogamy, according to FCSS. Megaspore mother cells developed directly into eight-nucleate megagametophytes without meiosis, conforming to Antennaria-type diplospory. The unreduced egg cells developed into embryos through parthenogenesis, while the endosperm was formed by the fusion of two unreduced polar nuclei. Pollen viability was very low (0.82 ± 0.57% and 0.36 ± 0.44% as measured by the Benzidine test and Alexander’s stain, respectively). The majority of the pollen grains were empty and had neither cytoplasm nor nuclei. The seed set was >90% for all treatments of open-pollination, bagging, and emasculated capitula. Mature cypselae developed in capitula that were emasculated before flowering, which confirmed that the breeding system of P. clematidea was autonomous apomixis. Conclusions The present study is the first report of autonomous apomixis in P. clematidea in China. Antennaria-type autonomous apomixis in P. clematidea greatly increases the probability of successful colonisation and dispersal of P. clematidea into new areas, which likely contributes to its high invasion potential. Effective control measures should be implemented to prevent autonomous (pollen-independent) seed production.

Transcriptomic and Morphological Analysis of Cells Derived from Porcine Buccal Mucosa—Studies on an In Vitro Model

Transcriptional analysis and live-cell imaging are a powerful tool to investigate the dynamics of complex biological systems. In vitro expanded porcine oral mucosal cells, consisting of populations of epithelial and connective lineages, are interesting and complex systems for study via microarray transcriptomic assays to analyze gene expression profile. The transcriptomic analysis included 56 ontological groups with particular focus on 7 gene ontology groups that are related to the processes of differentiation and development. Most analyzed genes were upregulated after 7 days and downregulated after 15 and 30 days of in vitro culture. The performed transcriptomic analysis was then extended to include automated analysis of differential interference contrast microscopy (DIC) images obtained during in vitro culture. The analysis of DIC imaging allowed to identify the different populations of keratinocytes and fibroblasts during seven days of in vitro culture, and it was possible to evaluate the proportion of these two populations of cells. Porcine mucosa may be a suitable model for reference research on human tissues. In addition, it can provide a reference point for research on the use of cells, scaffolds, or tissues derived from transgenic animals for applications in human tissues reconstruction.