Continuous,high-resolution biospeckle imaging reveals a discrete zone of activity at the root apex that responds to contact with obstacles

Background and Aims

Shining a laser onto biological material produces light speckles termed biospeckles. Patterns of biospeckle activity reflect changes in cell biochemistry, developmental processes and responses to the environment. The aim of this work was to develop methods to investigate the biospeckle activity in roots and to characterize the distribution of its intensity and response to thigmostimuli.

Methods

Biospeckle activity in roots of Zea mays, and also Jatropha curcas and Citrus limonia, was imaged live and in situ using a portable laser and a digital microscope with a spatial resolution of 10 μm per pixel and the ability to capture images every 0·080 s. A procedure incorporating a Fujii algorithm, image restoration using median and Gaussian filters, image segmentation using maximum-entropy threshold methods and the extraction of features using a tracing algorithm followed by spline fitting were developed to obtain quantitative information from images of biospeckle activity. A wavelet transform algorithm was used for spectral decomposition of biospeckle activity and generalized additive models were used to attribute statistical significance to changes in patterns of biospeckle activity.

Key Results

The intensity of biospeckle activity was greatest close to the root apex. Higher frequencies (3–6 Hz) contributed most to the total intensity of biospeckle activity. When a root encountered an obstacle, the intensity of biospeckle activity decreased abruptly throughout the root system. The response became attenuated with repeated thigmostimuli.

Conclusions

The data suggest that at least one component of root biospeckle activity resulted from a biological process, which is located in the zone of cell division and responds to thigmostimuli. However, neither individual cell division events nor root elongation is likely to be responsible for the patterns of biospeckle activity.     

The biology and methodology of assisted reproduction in deer mice (Peromyscus maniculatus)

Although laboratory-reared species of the genus Peromyscus—including deer mice—are used as model animals in a wide range of research, routine manipulation of Peromyscus embryogenesis and reproduction has been lagging. The objective of the present study was to optimize conditions for oocyte and/or embryo retrieval and for in vitro culturing. On average, 6.4 oocytes per mouse were recovered when two doses of 15 IU of pregnant mare serum gonadotropin (PMSG) were given 24 h apart, followed by 15 IU of hCG 48 h later. Following this hormone priming, females mated overnight with a fertile male yielded an average of 9.1 two-cell stage embryos. Although two-cell stage embryos developed to 8-cell stage in Potassium Simplex Optimized Medium (KSOM; Millipore-Chemicon, Billerica, MA, USA) in vitro, but not further, embryos recovered at the 8- to 16-cell stages developed into fully expanded blastocysts when cultured in M16 media in vitro. These blastocysts had full potential to develop into late stage fetuses and possibly into live pups. As a result of the present work, all stages of Peromyscus preimplantation development are now obtainable in numbers sufficient for molecular or other analyses. These advances provide the opportunity for routine studies involving embryo transfer (e.g., chimeras, transgenics), and preservation of genetic lines by cryopreservation.     

Principles and levels of laterality in unimanual and bimanual stone handling patterns by Japanese macaques

The preferential use of one hand over the other is considered the primary behavioral expression of structural and functional asymmetry in cerebral structures, which is a decisive factor in human evolution. We present the first analysis of manual laterality in a form of object play—stone handling (SH) behavior—in a free-ranging group of Japanese macaques. Defined as a stone-directed manipulative activity, and comprised of multiple one-handed SH patterns (e.g., grabbing a stone in one hand and cradling it against its chest), as well as coordinated two-handed SH patterns with manual role differentiation (e.g., holding a stone with one hand and rubbing it with the other), SH behavior is a good candidate for the study of hand lateralization. We systematically followed the methodological framework developed by McGrew and Marchant (1997) to measure and analyze the presence, strength, and direction of manual preference in the performance of SH behavior and in various SH patterns, both at the individual and group level. Some individuals showed a significant manual lateral bias on a single SH pattern (hand preference), whereas others showed consistency in laterality across all or most of the SH patterns they performed (hand specialization). At the group level, we found that, although their collective distribution of left versus right remained random, most subjects were either significantly but incompletely lateralized, or completely lateralized within particular SH patterns (pattern specialization), but not across all SH patterns (no handedness for SH behavior as a whole). As predicted by the task-complexity model, hand specialization and handedness were stronger in the coordinated bimanual SH patterns than in the unimanual patterns. We discuss the implications of our findings for the evolution of manual preferences in noninstrumental object manipulation versus stone tool use in nonhuman primates and hominins.     

Asymmetrical coexistence of Nosema ceranae and Nosema apis in honey bees

Globalization has provided opportunities for parasites/pathogens to cross geographic boundaries and expand to new hosts. Recent studies showed that Nosema ceranae, originally considered a microsporidian parasite of Eastern honey bees, Apis cerana, is a disease agent of nosemosis in European honey bees, Apis mellifera, along with the resident species, Nosema apis. Further studies indicated that disease caused by N. ceranae in European honey bees is far more prevalent than that caused by N. apis. In order to gain more insight into the epidemiology of Nosema parasitism in honey bees, we conducted studies to investigate infection of Nosema in its original host, Eastern honey bees, using conventional PCR and duplex real time quantitative PCR methods. Our results showed that A. cerana was infected not only with N. ceranae as previously reported [Fries, I., Feng, F., Silva, A.D., Slemenda, S.B., Pieniazek, N.J., 1996. Nosema ceranae n. sp. (Microspora, Nosematidae), morphological and molecular characterization of a microsporidian parasite of the Asian honey bee Apis cerana (Hymenoptera, Apidae). Eur. J. Protistol. 32, 356-365], but also with N. apis. Both microsporidia produced single and mixed infections. Overall and at each location alone, the prevalence of N. ceranae was higher than that of N. apis. In all cases of mixed infections, the number of N. ceranae gene copies (corresponding to the parasite load) significantly out numbered those of N. apis. Phylogenetic analysis based on a variable region of small subunit ribosomal RNA (SSUrRNA) showed four distinct clades of N. apis and five clades of N. ceranae and that geographical distance does not appear to influence the genetic diversity of Nosema populations. The results from this study demonstrated that duplex real-time qPCR assay developed in this study is a valuable tool for quantitative measurement of Nosema and can be used to monitor the progression of microsprodian infections of honey bees in a timely and cost efficient manner.     

Delineating metamorphic pathways in the ascidian Ciona intestinalis

In most ascidians, metamorphosis of tadpole-like swimming larvae is accompanied by dynamic changes in their shape to form sessile adults. The mechanisms underlying ascidian metamorphosis have been debated for a long time. Although recent molecular studies have revealed the presence of various molecules involving in this process, the basic mechanism of the metamorphic events is still unclear. For example, it has not been solved whether all metamorphic events are organized by the same single pathway or by multiple, independent pathways. In the present study, we approached this question using the ascidian Ciona intestinalis. When the papillae and preoral lobes of the larvae were cut off, the papillae-cut larvae initiated certain trunk metamorphic events such as the formation of an ampulla, body axis rotation and adult organ growth without other metamorphic events. This observation indicates that metamorphic events can be divided into at least two groups, events initiated in the papillae-cut larva and events not initiated in this larva. In addition to this observation, we have isolated a novel mutant, tail regression failed (trf), which shows similar phenotypes to those of papillae-cut larvae. The phenotypes of trf mutants are basically different from those of swimming juvenile mutants (Sasakura, Y., Nakashima, K., Awazu, S., Matsuoka, T., Nakayama, A., Azuma, J., Satoh, N., 2005. Transposon-mediated insertional mutagenesis revealed the functions of animal cellulose synthase in the ascidian Ciona intestinalis. Proc. Natl. Acad. Sci. U. S. A. 102, 15134-15139.), which also show abnormal metamorphosis. These findings suggest a model by which ascidian metamorphic events can be classified into four groups initiated by different pathways.     

A Simulation Model of Heterodera schachtii Infecting Beta vulgaris

A simulation model of a single sugarbeet, Beta vulgaris L., plant infected by the sugarbeet cyst nematode, Heterodera schachtii Schmidt, was developed using published information. The model is an interactive computer simulation programmed in FORTRAN. Given initial population densities of the nematode at planting, the model simulates nematode population dynamics and the growth of plant tap and fibrous roots. The driving variable for nematode development and plant growth is temperature.     

Mixed infection, Trichinella spiralis and Trichinella britovi, in a wild boar hunted in the Province of Cáceres (Spain)

The etiological agents of human trichinellosis are distributed worldwide in domestic and wild animals. In Spain, two morphologically indistinguishable Trichinella species have been described—Trichinella spiralis and Trichinella britovi—that are perpetuated in both domestic and sylvatic cycles. The present work reports a double natural infection involving these species in a wild boar killed by hunters in the Province of Cáceres, Spain. After artificial digestion of the boar’s muscles, nine larvae/g were collected. These were characterized by multiplex-PCR and Western-blotting using the Trichinella-specific monoclonal antibodies US5 and US9, and both T. spiralis and T. britovi were detected. The mechanism by which this wild boar came to acquire a mixed infection remains unclear.     

Polymorphism of Paramecium pentaurelia (Ciliophora, Oligohymenophorea) strains revealed by rDNA and mtDNA sequences

Paramecium pentaurelia is one of 15 known sibling species of the Paramecium aurelia complex. It is recognized as a species showing no intra-specific differentiation on the basis of molecular fingerprint analyses, whereas the majority of other species are polymorphic. This study aimed at assessing genetic polymorphism within P. pentaurelia including new strains recently found in Poland (originating from two water bodies, different years, seasons, and clones of one strain) as well as strains collected from distant habitats (USA, Europe, Asia), and strains representing other species of the complex. We compared two DNA fragments: partial sequences (349 bp) of the LSU rDNA and partial sequences (618 bp) of cytochrome B gene. A correlation between the geographical origin of the strains and the genetic characteristics of their genotypes was not observed. Different genotypes were found in Kraków in two types of water bodies (Opatkowice—natural pond; Jordan's Park—artificial pond). Haplotype diversity within a single water body was not recorded. Likewise, seasonal haplotype differences between the strains within the artificial water body, as well as differences between clones originating from one strain, were not detected. The clustering of some strains belonging to different species was observed in the phylogenies.     

Traffic optimization in railroad networks using an algorithm mimicking an amoeba-like organism, Physarum plasmodium

Traffic optimization of railroad networks was considered using an algorithm that was biologically inspired by an amoeba-like organism, plasmodium of the true slime mold, Physarum polycephalum. The organism developed a transportation network consisting of a tubular structure to transport protoplasm. It was reported that plasmodium can find the shortest path interconnecting multiple food sites during an adaptation process (Nakagaki et al., 2001. Biophys. Chem. 92, 47-52). By mimicking the adaptation process a path finding algorithm was developed by Tero et al. (2007). In this paper, the algorithm is newly modified for applications of traffic distribution optimization in transportation networks of infrastructure such as railroads under the constraint that the network topology is given. Application of the algorithm to a railroad in metropolitan Tokyo, Japan is demonstrated. The results are evaluated using three performance functions related to cost, traveling efficiency, and network weakness. The traffic distribution suggests that the modified Physarum algorithm balances the performances under a certain parameter range, indicating a biological process.     

An Escherichia coli expression system for glutamyl endopeptidases optimized by complete suppression of autodegradation

V8 protease (GluV8), a member of the glutamyl endopeptidase I family isolated from the V8 strain of Staphylococcus aureus, is widely used for proteome analysis because of its unique substrate specificity and resistance to detergents. We recently developed an Escherichia coli expression system for the production of GluV8 based on a technique that suppresses the autoproteolysis—the use of the prosequence of its homologue (GluSE) from Staphylococcus epidermidis as a chimeric form or the introduction of four substitutions in the prosequence of GluV8. In the current study, we refined this technique through five amino acid substitutions within the prosequence of GluV8 for complete suppression of the autodegradation. As a result, the recovery of GluV8 proform was enhanced to 20 fg/cell, which was comparable to the level of a constitutive inactive form of GluV8, indicating complete suppression of the autoproteolysis. This mutated propeptide was also effective for the expression of the mature sequence of the glutamyl endopeptidase from Staphylococcus warneri. The recombinant proteins were successfully converted to their active forms through a common cleavage mechanism mediated by thermolysin in vitro. This strategy may shed light on the way for the expression of the proteases that have been scarcely produced in E. coli to date.     

Molecular phylogenetic evidence supports a new family of octocorals and a new genus of Alcyoniidae (Octocorallia,Alcyonacea)

Molecular phylogenetic evidence indicates that the octocoral family Alcyoniidae is highly polyphyletic, with genera distributed across Octocorallia in more than 10 separate clades. Most alcyoniid taxa belong to the large and poorly resolved Holaxonia–Alcyoniina clade of octocorals, but members of at least four genera of Alcyoniidae fall outside of that group. As a first step towards revision of the family, we describe a new genus, Parasphaerasclera gen. n., and family, Parasphaerascleridae fam. n., of Alcyonacea to accommodate species of Eleutherobia Pütter, 1900 and Alcyonium Linnaeus, 1758 that have digitiform to digitate or lobate growth forms, completely lack sclerites in the polyps, and have radiates or spheroidal sclerites in the colony surface and interior. Parasphaerascleridae fam. n. constitutes a well-supported clade that is phylogenetically distinct from all other octocoral taxa. We also describe a new genus of Alcyoniidae, Sphaerasclera gen. n., for a species of Eleutherobia with a unique capitate growth form. Sphaerasclera gen. n. is a member of the Anthomastus–Corallium clade of octocorals, but is morphologically and genetically distinct from Anthomastus Verrill, 1878 and Paraminabea Williams & Alderslade, 1999, two similar but dimorphic genera of Alcyoniidae that are its sister taxa. In addition, we have re-assigned two species of Eleutherobia that have clavate to capitate growth forms, polyp sclerites arranged to form a collaret and points, and spindles in the colony interior to Alcyonium, a move that is supported by both morphological and molecular phylogenetic evidence.     

Symbiodinium clades A and D differentially predispose Acropora cytherea to disease and Vibrio spp. colonization

Coral disease outbreaks have increased over the last three decades, but their causal agents remain mostly unclear (e.g., bacteria, viruses, fungi, protists). This study details a 14‐month‐long survey of coral colonies in which observations of the development of disease was observed in nearly half of the sampled colonies. A bimonthly qPCR method was used to quantitatively and qualitatively evaluate Symbiodinium assemblages of tagged colonies, and to detect the presence of Vibrio spp. Firstly, our data showed that predisposition to disease development in general, and, more specifically, infection by Vibrio spp. in Acropora cytherea depended on which clades of Symbiodinium were harbored. In both cases, harboring clade D rather than A was beneficial to the coral host. Secondly, the detection of Vibrio spp. in only colonies that developed disease strongly suggests opportunistic traits of the bacteria. Finally, even if sporadic cases of switching and probably shuffling were observed, this long‐term survey does not suggest specific‐clade recruitment in response to stressors. Altogether, our results demonstrate that the fitness of the coral holobiont depends on its initial consortium of Symbiodinium, which is distinct among colonies, rather than a temporary adaptation achieved through acquiring different Symbiodinium clades.     

Biofunctionalization of cellulosic fibres with l-cysteine: Assessment of antibacterial properties and mechanism of action against Staphylococcus aureus and Klebsiella pneumoniae

The main purpose of this work is to obtain a cotton-based textile material functionalized with l-cysteine (l-cys) to achieve an antimicrobial effect with potential application in biomedical, geriatric or pediatric textiles. The binding capacity of l-cys to cotton fibres was assessed through different functionalization strategies—surface activation and exhaustion processes. A subsequent analysis of the possible antibacterial action against Staphylococcus aureus and Klebsiella pneumoniae was performed according with the Japanese International standard ( JISL, 2008). To determine the mechanism of action of l-cys on the selected strains, flow cytometry was used.     

Genetic variation and relationships in Laetiporus sulphureus s. lat., as determined by ITS rDNA sequences and in vitro growth rate

The aim of this study was to characterise the genetic variation and molecular relationships of the brown rot polypore, Laetiporus sulphureus s. lat., from Europe, South America, Africa, and Asia, using ITS sequences of the nu-rDNA and by comparing the growth rate in vitro. In a NJ analysis of the sequences of 130 individuals of L. sulphureus s. lat., eight distinct clusters emerged, supported by BS values of 70-100 %. Within each cluster, the ITS rDNA sequence variation was below 3 %. The sequences were also analysed together with Laetiporus sequences available from GenBank. Results demonstrated the possible presence of L. huroniensis in Europe (invalidly named L. montanus) and L. gilbertsonii in South America, and provided more information on the Pan-American and European distribution of one of the clades, currently known in North America as L. sulphureus. L. conifericola formed a separate distinct clade. Moreover, the analysis revealed two unknown Laetiporus taxa in Korea, one in South Africa, and one in Europe. As L. sulphureus is described from Europe (France), and we show that more than one taxon exist here, it is presently not possible to define L. sulphureus s. str. Certain biological differences between some of the clades (in vitro growth rates, chemical composition, and pigmentation) were demonstrated and discussed.     

A novel selective growth medium-PCR assay to isolate and detect Sphingomonas in environmental samples

Sphingomonas species can be found ubiquitously in the environment and can be frequently found in surface biofilms. Some Sphingomonas strains are well known for metabolizing complex organic pollutants but some are opportunistic human pathogens. Despite the importance of the Sphingomonas species, a reliable system to isolate this group of bacteria from the environment has not been developed. In this study, a combined streptomycin-piperacillin selective growth medium/polymerase chain reaction (PCR) detection approach is developed to isolate and identify the Sphingomonas bacteria. A total of 72 known Sphingomonas strains (including 21 different Sphingomonas species type strains) and 14 non-Sphingomonas species were tested using a new Sphingomonas-specific growth medium containing 100 and 50 µg/ml streptomycin and piperacillin, respectively. All the Sphingomonas strains showed positive growth on the selective medium and no growth was shown by the non-Sphingomonas species. In addition, two sets of PCR primers targeting the serine palmitoyltransferase gene (spt), a crucial sphingolipid biosynthesis gene, were developed. With the exception of the Sphingomonas subarctica type strain, 71 of the 72 known Sphingomonas samples were amplified positively by either one or both of the spt-specific primers. None of the non-Sphingomonas bacteria were amplified by the spt primers. To verify the effectiveness of this novel approach for use in environmental screening applications the Sphingomonas selective medium was used to isolate 165 potential Sphingomonas isolates, including 101 yellow, 4 orange and 58 unpigmented isolates, from the influent water and biofilm samples of a pulp and paper mill in Northwestern Ontario. Screening of these isolates with the two Sphingomonas spt-PCR primer sets showed that 98% of the yellow isolates and 100% of the orange isolates were positive to the spt-PCR test. None of the unpigmented isolates was positive to the spt-PCR assay. The 16S rDNA of 17% of the spt + ve and − ve isolates were sequenced and analyzed. All of the yellow and orange pigmented isolates were Sphingomonas while none of the unpigmented isolates were Sphingomonas. REP-PCR was performed on 79 Sphingomonas samples randomly selected from the paper mill and hospital isolates and showed that a diverse group of Sphingomonas can be grown or isolated by our Sphingomonas selective growth medium. Therefore, by using the streptomycin-piperacillin selective growth medium in combination with the colour pigmentation and the positive spt-PCR reactions of the isolates, a diverse population of Sphingomonas strains can be isolated and identified from complex microbial communities with high accuracy.     

Evaluation of FT-IR spectroscopy as a tool to quantify bacteria in binary mixed cultures

Fourier-transform infrared (FT-IR) spectroscopy is known as a high-resolution method for the rapid identification of pure cultures of microorganisms. Here, we evaluated FT-IR as a method for the quantification of bacterial populations in binary mixed cultures consisting of Pseudomonas putida and Rhodococcus ruber. A calibration procedure based on Principal Component Regression was developed for estimating the ratio of the bacterial species. Data for method calibration were gained from pure cultures and artificially assembled communities of known ratios of the two member populations. Moreover, to account for physiological variability, FT-IR measurements were performed with organisms sampled at different growth phases. Measurements and data analyses were subsequently applied to growing mixed cultures revealing that growth of R. ruber was almost completely suppressed in co-culture with P. putida. Population ratios obtained by fatty acid analysis as an independent reference method were in high agreement with the FT-IR derived ratios.     

Termination Structures in the Escherichia coli Chromosome Replication Fork Trap

The Escherichia coli chromosome contains two opposed sets of unidirectional DNA replication pause (Ter) sites that, according to the replication fork trap theory, control the termination of chromosome replication by restricting replication fork fusion to the terminus region. In contrast, a recent hypothesis suggested that termination occurs at the dif locus instead. Using two-dimensional agarose gel electrophoresis, we examined DNA replication intermediates at the Ter sites and at dif in wild-type cells. Two definitive signatures of site-specific termination—specific replication fork arrest and converging replication forks—were clearly detected at Ter sites, but not at dif. We also detected a significant pause during the latter stages of replication fork convergence at Ter sites. Quantification of fork pausing at the Ter sites in both their native chromosomal context and the plasmid context further supported the fork trap model.