Cell death induced by different virus isolates of aquabirnaviruses.

<p>Flow cytometric analysis of FITC-Annexin (a) and PI (b) staining of cells infected with different isolates. The charts show the percentage of FITC-annexin stained cells (apoptosis) and PI stained cells (cells with compromised membranes) after subtracting the percentage of the FITC-annexin and PI positive percentages obtained from uninfected cells, respectively. The data represent the means of three independent experiments ±SD.</p

Isolates used in the study.

<p>Isolates used in the study.</p

Type I IFN response induced by different isolates of aquabirnaviruses.

<p>Normalized gene expression of A) IFNα, B) PKR C) Mx1, and D) TNFα2 at different time points post infection ± S.E.M.. *P<0.05; **P<0.01; ***P<0.001. N = 3.</p

Replication curves for different aquabirnaviruses in CHSE-214 cells.

<p>Cell monolayers were infected with different IPNV isolates at 20pfu/cell. Amounts of virus in A) intracellular or B) supernatant at different time points post infections were determined by titration in CHSE-214 cells. The data are presented as mean ±S.E.M. log₁₀ TCID50/ml of minimum of 5 replicated taken from at least two independent experiments.</p

CPE development in CHSE-214 cells after infection with different aquabirnavirus isolates.

<p>Phase-contrast microscopical images at different times post infection.</p

Effect of boric acid on the integrity of <i>Saprolegnia</i> spore membranes.

<p>Fluorescence microscopy analysis of Propidium Iodide (PI) uptake by <i>Saprolegnia</i> spores. a) non-treated spores kept in water, were able to germinate and to form hyphae that only flourecent green with SYTO 9 (a1) without PI uptake (a2). b) Boric acid treated spores, neither germinate (b1) nor absorb the PI dye (b2). c) Non-viable, bronopol treated <i>Saprolegnia</i> spores showing uptake of SYTO 9 (c1) and PI dye (c2).</p

Effect of boric acid on the nuclear division and germination of <i>Saprolegnia</i> spores.

<p>Confocal laser scanning microscopy images of <i>Saprolegnia</i> spores stained with the nucleic acid dye DAPI. a1–a4) Spore germination in non-treated water control group. Note the movement of the nucleus towards the newly developing germ tube following 2 and 4 h incubation (a1 and a2). Development of multinuclear hyphae indicating growth and viability is shown in image a3 and a4. b1–b4) Gradual reduction of fluorescence intensity of <i>Saprolegnia</i> spores treated with boric acid following 2, 4, 6 and 8 h of incubation, b1, b2, b3 and b4 respectively. No nuclear division was observed in the treated group.</p

Effect of boric acid on <i>Saprolegnia</i> mitochondria using confocal laser scanning microscopy.

<p>Confocal laser scanning microscopy showing the effect of the boric acid on <i>Saprolegnia</i> spore (a) and hyphal (b) mitochondrial activity using MitoTracker Red. a1) Accumulation of the stain in the non-treated control. Gradual reduction in the number of mitochondria in treated spores 4 (a2), 12 (a3), and 24 (a4) hours after boric acid treatment. b1) <i>Saprolegnia</i> hyphae with densely distributed mitochondria indicating high activity in the non-treated control. Pronounced degradation of hyphal mitochondria 4 (b2), 12 (b3), and 24 (b4) hours post boric acid treatment. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110343#pone-0110343-g003" target="_blank">Figure 3 c and d</a> show the average fluorescence intensity of BA treated <i>Saprolegnia</i> spores (c) and hyphae (d) compared to the non-treated control following 24 h exposure.</p

Effect of boric acid on <i>Saprolegnia</i> mitochondria using fluorescence microscopy.

<p>Fluorescence microscope image showing the concentration of tetramethyl rhodamine (TMRE) staining in healthy non-treated <i>Saprolegnia</i> hyphae (a1) compared to boric acid treated hyphae (b1) where the depolarized mitochondria exhibit reduced red fluorescence. Figure (a2) is showing ROS level in the non-treated control compared to treated <i>Saprolegnia</i> hyphae (b2). TMRE and ROS staining are merged in a3 and b3.</p

Alterations in <i>Saprolegnia</i> spores following boric acid treatment.

<p>Transmission electron microscopy image of an untreated <i>Saprolegnia</i> spore (a) and a <i>Saprolegnia</i> spore treated with boric acid (1 g/L) for 4 h (b). Normal, well defined mitochondrial structure is seen in the non-treated spores (a1 and a2) compared to the spore that has been exposed to boric acid (b1 and b2). Different degrees of degenerative changes were observed in the mitochondria of the treated spore (circle). The condensed nucleus (N) with disintegrated nuclear membrane is seen in the treated spore (b1), but this was not a consistent finding and seen only in a few spores.</p