Glenn M. Harper

The effect of Pediococcus acidilactici on the gut microbiota and immune status of on-growing red tilapia (Oreochromis niloticus)

Aim:  To assess Pediococcus acidilactici as a dietary supplement for on‐growing red tilapia (Oreochromis niloticus).

Dynamics of formation and secretion of heterococcoliths by Coccolithus pelagicus ssp. braarudii

The formation and secretion of heterococcoliths by the non-motile life phase of the coccolithophore Coccolithus pelagicus was investigated using electron microscopy and time-lapse bright field imaging. Coccolithogenesis in C. pelagicus exhibited sequential mineralization of single coccoliths in Golgi-derived and nuclear-associated vesicles, a pattern similar to the formation of heterococcoliths in Emiliania huxleyi. Our TEM data show that only on maturation does the single coccolith vesicle migrate away from the nucleus before secretion. A reticular body, distinct from the Golgi body, was also clearly visible at the distal surface of the developing coccolith vesicle, suggesting this is a common structural feature in placolith cells that mineralize and secrete coccoliths one at a time. Time-lapse imaging revealed that the coccolith secretion process is rapid, taking 60–190 seconds, and involves considerable contractile activity to eject and position the coccolith on the surface of the cell. An intact flagellar root apparatus was discovered at the anterior pole of this non-motile cell from which polarized secretion of coccoliths occurs, which may indicate a novel role for such cytoskeletal structures. Freeze-fracture preparations revealed columnar deposits and adhesions linking the scales and coccolith baseplates to the cell, across the periplasmic space providing points of attachment for cellular movement. Rotatory movements of the cell relative to external coccoliths were exhibited by all actively calcifying cells. These movements enable the cell, while exhibiting morphologically polarized secretion, to locate and secrete a mature coccolith in a spatially well-defined manner. Finally, the time-lapse imaging approach described here provides an opportunity to quantify the regulation of coccolith production in single cells with high temporal resolution allowing responses of calcification to rapidly fluctuating environmental conditions such as light–dark transitions to be examined in detail, which has not been possible with bulk calcification studies.

Effects of a dietary β-(1,3)(1,6)-D-glucan supplementation on intestinal microbial communities and intestinal ultrastructure of mirror carp (Cyprinus carpio L.).

To assess the effects of dietary Saccharomyces cerevisiae β‐(1,3)(1,6)‐d‐glucan supplementation (MacroGard®) on mirror carp (Cyprinus carpio L.) intestinal microbiota and ultrastructure of the enterocyte apical brush border.

An ex vivo approach to studying the interactions of probiotic Pediococcus acidilactici and Vibrio (Listonella) anguillarum in the anterior intestine of rainbow trout Oncorhynchus mykiss

The aim of the present study was to observe the antagonistic relationship of the probiotic Pediococcus acidilactici and the pathogen Vibrio (Listonella) anguillarum in the intestine of rainbow trout (Oncorhynchus mykiss) by using an ex vivo intestinal sac method. Rainbow trout (240-250g) were fed either a control diet (commercial diet: 43% protein, 20% lipid) or a probiotic diet (control diet supplemented with P. acidilactici (Bactocell) at log 7 CFU g -1 ) for two weeks. The anterior intestine was then isolated for an ex vivo challenge study and intestinal sacs formed by shutting one end of the intestinal portion. The sacs were filled with PBS solutions containing either no bacteria (as a control), the probiotic (P. acidilactici), the pathogen (V. anguillarum), or probiotic+pathogen and incubated for one hour. At the end of the exposure bacterial levels in the lumen were determined by culture based approaches and colonisation of the mucosa assessed with PCR-DGGE and electron microscopy (EM). Intestinal morphology (observation of gross morphological damage, ultrstructural differences and the quantification of goblet cells and intra epithelial lecuocyte numbers) and bacterial infection/translocation was assessed using light microscopy (LM) and EM. Results revealed that V. anguillarum caused extensive histological damage to the gut but P. acidilactici did not. No signs of translocation of either the probiotic or pathogen were observed. Microbiological analyses indicated that P. acidilactici was able to outcompete V. anguillarum in the rainbow trout intestine and also to populate or colonise the mucosa. Additionally, elevated leucocyte levels and goblet cells in the epithelium of P. acidilactici fed fish, and intestines exposed to P. acidilactici, suggests that P. acidilactici might have potential use in controlling vibriosis. In vivo disease-challenge studies are warranted to ascertain if V. anguillarum infections can be controlled in rainbow trout using dietary applications of P. acidilactici.

Application of the rainbow trout derived intestinal cell line (RTgutGC) for ecotoxicological studies: molecular and cellular responses following exposure to copper

There is an acknowledged need for in vitro fish intestinal model to help understand dietary exposure to chemicals in the aquatic environment. The presence and use of such models is however largely restrictive due to technical difficulties in the culturing of enterocytes in general and the availability of appropriate established cell lines in particular. In this study, the rainbow trout (Oncorhynchus mykiss) intestinal derived cell line (RTgutGC) was used as a surrogate for the “gut sac” method. To facilitate comparison, RTgutGC cells were grown as monolayers (double-seeded) on permeable Transwell supports leading to a two-compartment intestinal model consisting of polarised epithelium. This two-compartment model divides the system into an upper apical (lumen) and a lower basolateral (portal blood) compartment. In our studies, these cells stained weakly for mucosubstances, expressed the tight junction protein ZO-1 in addition to E-cadherin and revealed the presence of polarised epithelium in addition to microvilli protrusions. The cells also revealed a comparable transepithelial electrical resistance (TEER) to the in vivo situation. Importantly, the cell line tolerated apical saline (1:1 ratio) thus mimicking the intact organ to allow assessment of uptake of compounds across the intestine. Following an exposure over 72 h, our study demonstrated that the RTgutGC cell line under sub-lethal concentrations of copper sulphate (Cu) and modified saline solutions demonstrated uptake of the metal with saturation levels comparable to short term ex situ gut sac preparations. Gene expression analysis revealed no significant influence of pH or time on mRNA expression levels of key stress related genes (i.e. CYP3A, GST, mtA, Pgp and SOD) in the Transwell model. However, significant positive correlations were found between all genes investigated suggesting a co-operative relationship amongst the genes studied. When the outlined characteristics of the cell line are combined with the division of compartments, the RTgutGC double seeded model represents a potential animal replacement model for ecotoxicological studies. Overall, this model could be used to study the effects and predict aquatic gastrointestinal permeability of metals and other environmentally relevant contaminants in a cost effective and high throughput manner.

Vesicular glutamate release from central axons contributes to myelin damage

The axon myelin sheath is prone to injury associated with N-methyl-d-aspartate (NMDA)-type glutamate receptor activation but the source of glutamate in this context is unknown. Myelin damage results in permanent action potential loss and severe functional deficit in the white matter of the CNS, for example in ischemic stroke. Here, we show that in rats and mice, ischemic conditions trigger activation of myelinic NMDA receptors incorporating GluN2C/D subunits following release of axonal vesicular glutamate into the peri-axonal space under the myelin sheath. Glial sources of glutamate such as reverse transport did not contribute significantly to this phenomenon. We demonstrate selective myelin uptake and retention of a GluN2C/D NMDA receptor negative allosteric modulator that shields myelin from ischemic injury. The findings potentially support a rational approach toward a low-impact prophylactic therapy to protect patients at risk of stroke and other forms of excitotoxic injury.Neuronal activity can lead to vesicular release of glutamate. Here the authors demonstrate that vesicular release of glutamate occurs in axons during ischemic conditions, and that an allosteric modulator of GluN2C/D is protective in models of ischemic injury.

Phaeoviruses discovered in kelp (Laminariales)

Phaeoviruses are latent double-stranded DNA viruses that insert their genomes into those of their brown algal (Phaeophyceae) hosts. So far these viruses are known only from members of the Ectocarpales, which are small and short-lived macroalgae. Here we report molecular and morphological evidence for a new Phaeovirus cluster, referred to as sub-group C, infecting kelps (Laminariales) of the genera Laminaria and Saccharina, which are ecologically and commercially important seaweeds. Epifluorescence and TEM observations indicate that the Laminaria digitata Virus (LdigV), the type species of sub-group C, targets the host nucleus for its genome replication, followed by gradual degradation of the chloroplast and assembly of virions in the cytoplasm of both vegetative and reproductive cells. This study is the first to describe phaeoviruses in kelp. In the field, these viruses infected two thirds of their host populations; however, their biological impact remains unknown.