Three-dimensional reconstruction of black tiger prawn (Penaeus monodon) spermatozoa using serial block-face scanning electron microscopy

Serial Block-Face Scanning Electron Microscopy (SBF-SEM) was used in this study to examine the ultrastructural morphology of Penaeus monodon spermatozoa. SBF-SEM provided a large dataset of sequential electron-microscopic-level images that facilitated comprehensive ultrastructural observations and three-dimensional reconstructions of the sperm cell. Reconstruction divulged a nuclear region of the spermatophoral spermatozoon filled with decondensed chromatin but with two apparent levels of packaging density. In addition, the nuclear region contained, not only numerous filamentous chromatin elements with dense microregions, but also large centrally gathered granular masses. Analysis of the sperm cytoplasm revealed the presence of degenerated mitochondria and membrane-less dense granules. A large electron-lucent vesicle and "arch-like" structures were apparent in the subacrosomal area, and an acrosomal core was found in the acrosomal vesicle. The spermatozoal spike arose from the inner membrane of the acrosomal vesicle, which was slightly bulbous in the middle region of the acrosomal vesicle, but then extended distally into a broad dense plate and to a sharp point proximally. This study has demonstrated that SBF-SEM is a powerful technique for the 3D ultrastructural reconstruction of prawn spermatozoa, that will no doubt be informative for further studies of sperm assessment, reproductive pathology and the spermiocladistics of penaeid prawns, and other decapod crustaceans. J. Morphol., 2016. (c) 2016 Wiley Periodicals, Inc.

A convenient sample preparation protocol for scanning electron microscope examination of xylem-occluding bacterial biofilm on cut flowers and foliage

Microbes and their exopolysaccharides (EPS) can block xylem vessels, thereby increasing the hydraulic resistance and decreasing the vase life of cut flowers and foliage. Scanning electron microscopy (SEM) provides a powerful tool for investigation of bacteria-induced xylem occlusion. However, conventional preparation protocols for SEM involving chemicals can cause loss of hydrated EPS material, and thereby damage the bacterial biofilms during dehydration. A modified chemical fixation protocol involving pre-fixation with 75 mM lysine plus 2.5% glutaraldehyde followed by the normal fixation in 3% glutaraldehyde was, therefore, tested for improved preservation of bacterial biofilm at the stem-ends of cut Acacia holosericea foliage stems. Stem-end segments with different stages of bacterial growth were obtained from stems stood into water. The lysine-based protocol was compared with four other processing protocols of critical point drying (CPD) without fixation (control), freeze-drying (FD), conventional chemical fixation followed by drying with hexamethyldisilazane (HMDS), and conventional chemical fixation with CPD. The non-fixed control. FD and the glutaraldehyde fixation with HMDS drying gave poor preservation of hydrated material, including bacterial EPS. Conventional glutaraldehyde fixation followed by CPD was superior to these three methods in terms of better preserving the EPS. However, this fourth method gave condensation of biofilms during dehydration. In contrast, the modified lysine-based protocol resulted in superior preservation of EPS and biofilm structure. Thus, this fifth method was the most appropriate for examination of bacterial stem-end blockage in cut ornamentals. (C) 2012 Elsevier B.V. All rights reserved.

Banana peel: an effective biosorbent for aflatoxins

This work reports the application of banana peel as a novel bioadsorbent for in vitro removal of five mycotoxins (aflatoxins (AFB1, AFB2, AFG1, AFG2) and ochratoxin A). The effect of operational parameters including initial pH, adsorbent dose, contact time and temperature were studied in batch adsorption experiments. Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and point of zero charge (pHpzc) analysis were used to characterise the adsorbent material. Aflatoxins’ adsorption equilibrium was achieved in 15 min, with highest adsorption at alkaline pH (6–8), while ochratoxin has not shown any significant adsorption due to surface charge repulsion. The experimental equilibrium data were tested by Langmuir, Freundlich and Hill isotherms. The Langmuir isotherm was found to be the best fitted model for aflatoxins, and the maximum monolayer coverage (Q0) was determined to be 8.4, 9.5, 0.4 and 1.1 ng mg−1 for AFB1, AFB2, AFG1 and AFG2 respectively. Thermodynamic parameters including changes in free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) were determined for the four aflatoxins. Free energy change and enthalpy change demonstrated that the adsorption process was exothermic and spontaneous. Adsorption and desorption study at different pH further demonstrated that the sorption of toxins was strong enough to sustain pH changes that would be experienced in the gastrointestinal tract. This study suggests that biosorption of aflatoxins by dried banana peel may be an effective low-cost decontamination method for incorporation in animal feed diets. © 2016 Informa UK Limited, trading as Taylor & Francis Group.

Impact of low temperature storage on active and storage forms of folate in choy sum (Brassica rapa subsp parachinensis)

Choy sum (Brassica rapa subsp. parachinensis) is a dark green leafy vegetable that contains high folate (vitamin B9) levels comparable to spinach. Folate is essential for the maintenance of human health and is obtained solely through dietary means. Analysis of the edible portion of choy sum by both microbiological assay and LC-MS/MS indicated that total folate activity remained significantly unchanged over 3 weeks storage at 4 degrees C. Inedible fractions consisted primarily of outer leaves, which showed signs of rotting after 14d, and a combination of rotting and yellowing after 21 d, contributing to 20% and 40% of product removal, respectively. Following deconjugation of the folate present in choy sum to monoglutamate and diglutamate derivatives, the principal forms (vitamers) of folate detected in choy sum were 5-methyltetrahydrofolate and 5-formyl tetrahydrofolate, followed by tetrahydrofolate (THF), 5,10-methenyl-THF, and 10-formyl folic acid. During storage, a significant decline in 5-formyl-THF was observed, with a slight but not significant increase in the combined 5-methyl-THF derivatives. The decline in 5-formyl-THF in relation to the other folate vitamers present may indicate that 5-formyl-THF is being utilised as a folate storage reserve, being interconverted to more metabolically active forms of folate, such as 5-methyl-THF. Although folate vitamer profile changed over the storage period, total folate activity did not significantly change. From a human nutritional perspective this is important, as while particular folate vitamers (e.g. 5-methyl-THF) are necessary for maintaining vital aspects of plant metabolism, it is less important to the human diet, as humans can absorb and interconvert multiple forms of folate. The current trial indicates that it is possible to store choy sum for up to 3 weeks at 4 degrees C without significantly affecting total folate concentration of the edible portion. Crown Copyright (C) 2012 Published by Elsevier B.V. All rights reserved.

Aiming for the management of the small hive beetle, Aethina tumida, using relative humidity and diatomaceous earth

Small hive beetles (SHBs) are a global pest of European honeybee colonies. In the laboratory, the survival of adult SHBs was evaluated in relation to relative humidity (RH = 56, 64, 73, 82 and 96 %) and treatment with diatomaceous earth (DE) across 4 days. Low RH reduced survival. The application of DE reduced survival in addition to RH. Adults treated with corn flour (control) showed no difference in survival from untreated beetles. Scanning electron microscopy images showed no scarification of adult beetle cuticle after exposure to DE; therefore, water loss is likely facilitated through non-abrasive means such as the adsorption of cuticular lipids. The data agree with the hypothesis that DE causes mortality through water loss from treated insects. Egress, ingress, mortality and the egg-laying behaviours of beetles were observed in relation to a popular in-hive trench trap with and without the addition of DE. Traps filled with DE resulted in 100 % mortality of beetles compared with 8.6 % mortality when no DE was present. A simple method for visually determining beetle sex was used and documented.

Low temperature exposure of root system and inflorescence affected flowering and fruit set in 'Chardonnay' grapevines (Vitis vinifera)

The mechanisms by which low temperature affects flowering and fruit set of grapevines are poorly understood, as is the specific response of the grapevine root system and inflorescence to low temperature effects that reduce fruit set. This study aimed to determine the responses of the root system and inflorescence of the grapevine 'Chardonnay' to low temperature (10 degrees C) during flowering, and considered the possible mechanisms of low temperature effects on those parts. Temperature treatments of 10 degrees C or 20 degrees C were imposed to potted 'Chardonnay' grapevines in a glasshouse for up to two weeks during the early stages of flowering. When the root system alone was exposed to 10 degrees C (with the rest of the plant at 20 degrees C) during flowering, the number of attached berries and percentage fruit set were significantly reduced by 50 % than when the root system alone was exposed to 20 degrees C. Whereas, exposure of the inflorescence alone to 10 degrees C (with the rest of the plant at 20 degrees C) delayed flowering, allowed rachis to grow longer, and increased both the number of attached berries (from 22 to 62 per vine) and fruit set (from 8 % to, 20 %), than when the inflorescence alone was exposed to 20 degrees C. This study will enhance our understanding of the possible mechanisms of low temperature effects on grapevine fruit set and productivity.