'Candidatus Xiphinematincola pachtaicus' gen. nov., sp. nov., an endosymbiotic bacterium associated with nematode species of the genus Xiphinema (Nematoda, Longidoridae).

An intracellular bacterium, strain IAST, was observed to infect several species of the plant-parasitic nematode genus Xiphinema (Xiphinema astaregiense, Xiphinema incertum, Xiphinema madeirense, Xiphinema pachtaicum, Xiphinema parapachydermum and Xiphinema vallense). The bacterium could not be recovered on axenic medium. The 16S rRNA gene sequence of IAST was found to be new, being related to the family Burkholderiaceae, class Betaproteobacteria. Fungal endosymbionts Mycoavidus cysteinexigens B1-EBT (92.9 % sequence identity) and 'Candidatus Glomeribacter gigasporarum' BEG34 (89.8 % identity) are the closest taxa and form a separate phylogenetic clade inside Burkholderiaceae. Other genes (atpD, lepA and recA) also separated this species from its closest relatives using a multilocus sequence analysis approach. These genes were obtained using a partial genome of this bacterium. The localization of the bacterium (via light and fluorescence in situ hybridization microscopy) is in the X. pachtaicum females clustered around the developing oocytes, primarily found embedded inside the epithelial wall cells of the ovaries, from where they are dispersed in the intestine. Transmission electron microscopy (TEM) observations supported the presence of bacteria inside the nematode body, where they occupy ovaries and occur inside the intestinal epithelium. Ultrastructural analysis of the bacterium showed cells that appear as mostly irregular, slightly curved rods with rounded ends, 0.8-1.2 µm wide and 2.5-6.0 µm long, possessing a typical Gram-negative cell wall. The peptidoglycan layer is, however, evident only occasionally and not detectable by TEM in most cells. Another irregularly occurring shell surrounding the endosymbiont cells or the cell clusters was also revealed, probably originating from the host cell membrane. Flagella or spore-like cells do not occur and the nucleoid is diffusely distributed throughout the cell. This endosymbiont is transmitted vertically through nematode generations. These results support the proposal of IAST as a new species, although its obligate intracellular and obligate endosymbiont nature prevented isolation of a definitive type strain. Strain IAST is therefore proposed as representing 'Candidatus Xiphinematincola pachtaicus' gen. nov., sp. nov.

Structure, development, and evolutive patterns of spermatozoa in rhabditid nematodes (Nematoda: Rhabditida).

Spermatogenesis of five rhabditid nematodes was studied using transmission electron microscopy and is described herein. Structure and development of nematode sperm in all available representatives of the extensive order Rhabditida have been analysed and the main characteristics of each infraorder are discussed. The ancestral sperm of the order Rhabditida was reconstructed using maximum likelihood and Bayesian methods based on 44 ultrastructural sperm characters. The hypothetical ancestral spermatogenesis of the order Rhabditida agrees with the previously suggested "rhabditid" pattern and appears to be conserved throughout the order Rhabditida. Despite the enormous variation of rhabditid nematodes, few groups deviate from the ancestral pattern. This conserved pattern can be informative within the phylum Nematoda at order level, but poses limitations when used in taxonomic and phylogenetic analysis within Rhabditida.

Self-assembly of Tween 80 micelles as nanocargos for oregano and trans-cinnamaldehyde plant-derived compounds.

The self-assembly of Tween 80 (T80) micelles loaded with plant-based oregano essential oil (OR) and trans-cinnamaldehyde (TCA) was studied. The effect of different factors, including the surfactant to oil ratio, the presence of sodium chloride, thermal treatment, and dilution on their formation and physicochemical stability was evaluated. The creation of nano-cargos was confirmed by TEM. The self-associated structures had z-average droplet diameters of 92 to 337 nm without any energy input. Whereas addition of 10% (w/v) NaCl prevented the formation of oregano essential oil nano-assemblies of T80, swollen micelles containing TCA were successfully produced. Moreover, the OR or TCA loaded-micelles had only a slight droplet size variation upon thermal treatment. Ultimately, their antibacterial activity analysis against some food pathogens revealed that the encapsulation of OR and TCA within micelles crucially improved their antibacterial activity. These straightforward and cost-effective designed systems can be applicable in different products, including foods and agrochemicals.

Mechanisms for Color Convergence in a Mimetic Radiation of Poison Frogs.

In animals, bright colors often evolve to mimic other species when a resemblance is selectively favored. Understanding the proximate mechanisms underlying such color mimicry can give insights into how mimicry evolves-for example, whether color convergence evolves from a shared set of mechanisms or through the evolution of novel color production mechanisms. We studied color production mechanisms in poison frogs (Dendrobatidae), focusing on the mimicry complex of Ranitomeya imitator. Using reflectance spectrometry, skin pigment analysis, electron microscopy, and color modeling, we found that the bright colors of these frogs, both within and outside the mimicry complex, are largely structural and produced by iridophores but that color production depends crucially on interactions with pigments. Color variation and mimicry are regulated predominantly by iridophore platelet thickness and, to a lesser extent, concentration of the red pteridine pigment drosopterin. Compared with each of the four morphs of model species that it resembles, R. imitator displays greater variation in both structural and pigmentary mechanisms, which may have facilitated phenotypic divergence in this species. Analyses of nonmimetic dendrobatids in other genera demonstrate that these mechanisms are widespread within the family and that poison frogs share a complex physiological "color palette" that can produce diverse and highly reflective colors.

Fundamental Study on the Salt Tolerance of Oregano Essential Oil-in-Water Nanoemulsions Containing Tween 80.

This study provides fundamental information about the influence of salt on the physicochemical stability of oregano essential oil (EO) and its main components incorporated in a nanoemulsion delivery system containing Tween 80 (T80) emulsifier. The emulsion stability was found to be strongly correlated with the lipid phase composition and the type of salts. The oregano essential oil nanoemulsions remained stable for several weeks in the absence of salts. Moreover, they were insensitive to tetrabutylammonium bromide, whereas similar to carvacrol emulsions, they exhibited a rapid phase separation and oiling-off in the presence of sodium chloride. On the other hand, high oleic sunflower oil (HOSO) and p-cymene emulsions remained stable in the presence of NaCl. Addition of 70 and 80% HOSO to the lipid phase of oregano EO and carvacrol, respectively, was found to be sufficient for the formation of emulsions with a high stability to 1.7 M NaCl. Hereby, the morphology of the oregano EO emulsions after 30 days of storage in the presence of NaCl was visualized using a transmission electron microscope. The determination of the surface load and area per surfactant molecule by interfacial tension (IFT) measurements and quartz crystal microbalance with dissipation revealed the dehydration of the polyoxyethylene groups of T80 in the presence of salt. The thickness of the T80 adsorbed layer onto solid hydrophobic and hydrophilic surfaces was significantly lower (p < 0.05) in the presence of sodium chloride. It is hypothesized that a combination of Ostwald ripening and coalescence due to an IFT increase and dehydration was responsible for the instability of the emulsions containing the more polar oregano EO and carvacrol in the presence of salt. The results obtained in this study could be useful for the formulation of essential oil nanoemulsions in the presence of salts applicable in food, pharmaceutical, and personal care products.

Self-assembly, functionality, and in-vitro properties of quercetin loaded nanoparticles based on shellac-almond gum biological macromolecules.

Quercetin-fortified nanoparticles were prepared from almond gum (AG), a novel biological macromolecule, and Tween 80 (T80) as stabilizers and shellac (SH) as core material using an antisolvent precipitation method. The final nanoparticles were prepared by 0.67% SH, 0.02% Q, 0.5% AG and 0.1% w/v T80 using the stirring speed of 750 rpm at a dosing rate of 0.5 ml/min. The morphology of the particles was characterized using Cryo-SEM and TEM microscopy. The average particle size was 135 ±â€¯8 nm with a polydispersity index of 0.252 ±â€¯0.01 and an encapsulation efficiency of 97.7 ±â€¯1.2%. At pH 7.4 (intestinal pH), quercetin-loaded nanoparticles showed significantly (p < 0.05) higher antioxidant activity compared to free quercetin while the degradation of quercetin was lower in the nanoparticles compared to free quercetin at the similar pH. Quercetin loaded in nanoparticles was successfully found to be 2 times more available for uptake than free quercetin at pH 7.4. MTT and SRB assays revealed that no significant (p > 0.05) toxicity was observed for Caco-2 cells treated with quercetin-loaded nanoparticles with a dilution factor of 100. This study provides information about the formulation of promising nanocarriers using biological macromolecules for oral delivery of bioactive compounds.

Antimicrobial peptides in frog poisons constitute a molecular toxin delivery system against predators.

Animals using toxic peptides and proteins for predation or defense typically depend on specialized morphological structures, like fangs, spines, or a stinger, for effective intoxication. Here we show that amphibian poisons instead incorporate their own molecular system for toxin delivery to attacking predators. Skin-secreted peptides, generally considered part of the amphibian immune system, permeabilize oral epithelial tissue and enable fast access of cosecreted toxins to the predator's bloodstream and organs. This absorption-enhancing system exists in at least three distantly related frog lineages and is likely to be a widespread adaptation, determining the outcome of predator-prey encounters in hundreds of species.

Ultrastructural morphology of the envelope of Dover sole Solea solea eggs from fertilization until hatching with emphasis on sample preparation.

This study is the first to describe the ultrastructural morphology of the envelope of Solea solea eggs from fertilisation until hatching. Defining the ultrastructural morphology of fish eggs is important for species identification and may assist in predicting the effect of external influences on these early life stages. In first instance, various fixation and embedding protocols were assessed to explore the morphology of the egg envelope, whereby the encountered difficulties were highlighted. The successful protocol for SEM proved to be combined fixation with 4% glutaraldehyde in 0.1M cacodylate buffer for minimum 4h with post-fixation of 2h with 1% OsO4 in 0.1M cacodylate buffer. For TEM, puncturing the egg envelope during the first steps of the fixation protocol was necessary to allow the embedding medium to penetrate through the egg envelope. Based on both scanning and transmission electron microscopical examination, three distinct layers were discerned in the egg envelope. During the development of the fish embryo, a change in the outer structure of the egg was observed. Scanning electron microscopical examination of one day post-fertilisation eggs (DPF) revealed a homogeneous outer layer, displaying a large number of pores uniformly distributed on the surface of the egg envelope. Starting from 2 DPF parts of the outermost layer or two outer layers peeled off. The second deeper layer showed larger pores, with less defined edges. In the third innermost layer irregular indentations were noted. On transmission electron microscopy the first outermost layer of 1 DPF eggs clearly folded into the pores. The second layer was more electron dense, had a uniform appearance and did not cover the surface of the pores. The third innermost layer was much thicker and possessed indentations. A total number of 12 undulating zones were discriminated based on different degrees of electron density. Prior to hatching, the compact structure of the innermost layer was distorted by dispersed holes and tears.

Ultrastructural immunogold localization of major sperm protein (MSP) in spermatogenic cells of the nematode Acrobeles complexus (Nematoda, Rhabditida).

The nematode spermatozoa represent a highly modified (aberrant) type of male gametes that lack a flagellum but for which the process of spermatogenesis culminates in the production of a crawling spermatozoon on the basis of the cytoskeletal component known as "major sperm protein", or MSP. MSP is also known as an important hormone triggering oocyte maturation and ovulation in the model nematode Caenorhabditis elegans, where this protein was first identified. However, direct evidence of MSP localization and of its fate in nematode spermatogenic cells is rare. In this study, the spermatogenesis and sperm structure in the rhabditid nematode Acrobeles complexus (Rhabditida: Tylenchina: Cephalobomorpha: Cephaloboidea: Cephalobidae) has been examined with electron microscopy. Morphological observations were followed by high-pressure freezing and freeze-substitution fixation which allows post-embedding immunogold localization of MSP in all stages of sperm development using antibodies raised for MSP of C. elegans. In spermatocytes, synthetic activity results in the development of specific cellular components, fibrous bodies (FB) and membranous organelles (MO), which appear as FB-MO complexes where the filamentous matter of FB has been MSP-labeled. The spermatids subdivide into a residual body with superfluous cytoplasm, and a main cell body which contains nucleus, mitochondria and FB-MO complexes. These complexes dissociate into individual components, MO and FB, with the MSP being localized in FB. Immature spermatozoa from testes are opaque cells where a centrally located nucleus is surrounded by mitochondria, MO and FB clustered together, the MSP still being localized only in FB. Cytoplasm of mature spermatozoa from spermatheca is segregated into external pseudopods lacking organelles and a central cluster of mitochondria with intact MO surrounding the central nucleus. The FB ultimately disappear, and the MSP labeling becomes concentrated in the filamentous content of pseudopods and cytoplasm of the main cell body. Although the spermatogenesis and sperm structure of A. complexus is similar to that of many other rhabditid nematodes, their intact MO makes the morphology of the mature spermatozoa distinct from the "rhabditid pattern" and may be considered as a synapomorphy. The MSP localization in spermatogenic cells of A. complexus also follows the "rhabditid pattern" described in C. elegans and Ascaris spp. Our results and techniques of MSP labeling of A. complexus spermatogeneous cells reveal new possibilities to elucidate different research questions on MSP localization in nematodes related to C. elegans. Furthermore, the laboratory-cultured A. complexus strains can be used as a new and fascinating model to study MO and MSP functions in nematode reproduction.

High-pressure freezing and freeze-substitution fixation reveal the ultrastructure of immature and mature spermatozoa of the plant-parasitic nematode Trichodorus similis (Nematoda; Triplonchida; Trichodoridae).

The spermatozoa from testis and spermatheca of the plant-parasitic nematode Trichodorus similis Seinhorst, 1963 (Nematoda; Triplonchida; Trichodoridae) were studied with transmission electron microscopy (TEM), being the first study on spermatogenesis of a representative of the order Triplonchida and important to unravel nematode sperm evolution. Comprehensive results could only be obtained using high-pressure freezing (HPF) and freeze-substitution instead of chemical fixation, demonstrating the importance of cryo-fixation for nematode ultrastructural research. The spermatozoa from the testis (immature spermatozoa) are unpolarized cells covered by numerous filopodia. They contain a centrally-located nucleus without a nuclear envelope, surrounded by mitochondria. Specific fibrous bodies (FB) as long parallel bundles of filaments occupy the peripheral cytoplasm. No structures resembling membranous organelles (MO), as found in the sperm of many other nematodes, were observed in immature spermatozoa of T. similis. The spermatozoa from the uterus (mature or activated spermatozoa) are bipolar cells with an anterior pseudopod and posterior main cell body (MCB), which include a nucleus, mitochondria and MO appearing as large vesicles with finger-like invaginations of the outer cell membrane, or as large vesicles connected to the inner cell membrane. The peripheral MO open to the exterior via pores. In the mature sperm, neither FBs nor filopodia were observed. An important feature of T. similis spermatozoa is the late formation of MO; they first appear in mature spermatozoa. This pattern of MO formation is known for several other orders of the nematode class Enoplea: Enoplida, Mermithida, Dioctophymatida, Trichinellida but has never been observed in the class Chromadorea.

Transmission electron microscopy sample preparation protocols for the ultrastructural study of cysts of free-living protozoa.

Cysts of free-living protozoa have an impact on the ecology and epidemiology of bacteria because they may act as a transmission vector or shelter the bacteria against hash environmental conditions. Detection and localization of intracystic bacteria and examination of the en- and excystment dynamics is a major challenge because no detailed protocols for ultrastructural analysis of cysts are currently available. Transmission electron microscopy (TEM) is ideally suited for those analyses; however, conventional TEM protocols are not satisfactory for cysts of free-living protozoa. Here we report on the design and testing of four protocols for TEM sample preparation of cysts. Two protocols, one based on chemical fixation in coated well plates and one on high-pressure freezing, were selected as the most effective for TEM-based ultrastructural studies of cysts. Our protocols will enable improved analysis of cyst structure and a better understanding of bacterial survival mechanisms in cysts.

Interaction of Aspergillus fumigatus conidia with Acanthamoeba castellanii parallels macrophage-fungus interactions.

Aspergillus fumigatus and free-living amoebae are common inhabitants of soil. Mechanisms of A. fumigatus to circumvent the amoeba's digestion may facilitate overcoming the vertebrate macrophage defence mechanisms. We performed co-culture experiments using A. fumigatus conidia and the amoeba Acanthamoeba castellanii. Approximately 25% of the amoebae ingested A. fumigatus conidia after 1 h of contact. During intra-amoebal passage, part of the ingested conidia was able to escape the food vacuole and to germinate inside the cytoplasm of A. castellanii. Fungal release into the extra-protozoan environment by exocytosis of conidia or by germination was observed with light and transmission electron microscopy. These processes resulted in structural changes in A. castellanii, leading to amoebal permeabilization without cell lysis. In conclusion, A. castellanii internalizes A. fumigatus conidia, resulting in fungal intracellular germination and subsequent amoebal death. As such, this interaction highly resembles that of A. fumigatus with mammalian and avian macrophages. This suggests that A. fumigatus virulence mechanisms to evade macrophage killing may be acquired by co-evolutionary interactions among A. fumigatus and environmental amoebae.

Soft dentin results in unique flexible teeth in scraping catfishes.

Teeth are generally used for actions in which they experience mainly compressive forces acting toward the base. The ordered tooth enamel(oid) and dentin structures contribute to the high compressive strength but also to the minor shear and tensile strengths. Some vertebrates, however, use their teeth for scraping, with teeth experiencing forces directed mostly normal to their long axis. Some scraping suckermouth catfishes (Loricariidae) even appear to have flexible teeth, which have not been found in any other vertebrate taxon. Considering the mineralized nature of tooth tissues, the notion of flexible teeth seems paradoxical. We studied teeth of five species, testing and measuring tooth flexibility, and investigating tooth (micro)structure using transmission electron microscopy, staining, computed tomography scanning, and scanning electron microscopy-energy-dispersive spectrometry. We quantified the extreme bending capacity of single teeth (up to 180°) and show that reorganizations of the tooth (micro)structure and extreme hypomineralization of the dentin are adaptations preventing breaking by allowing flexibility. Tooth shape and internal structure appear to be optimized for bending in one direction, which is expected to occur frequently when feeding (scraping) under natural conditions. Not all loricariid catfishes possess flexible teeth, with the trait potentially having evolved more than once. Flexible teeth surely rank among the most extreme evolutionary novelties in known mineralized biological materials and might yield a better understanding of the processes of dentin formation and (hypo)mineralization in vertebrates, including humans.

An endosymbiotic bacterium in a plant-parasitic nematode: member of a new Wolbachia supergroup.

Wolbachia is an endosymbiotic bacterium widely present in arthropods and animal-parasitic nematodes. Despite previous efforts, it has never been identified in plant-parasitic nematodes. Random sequencing of genes expressed by the burrowing nematode Radopholus similis resulted in several sequences with similarity to Wolbachia genes. The presence of a Wolbachia-like endosymbiont in this plant-parasitic nematode was investigated using both morphological and molecular approaches. Transmission electronmicroscopy, fluorescent immunolocalisation and staining with DAPI confirmed the presence of the endosymbiont within the reproductive tract of female adults. 16S rDNA, ftsZ and groEL gene sequences showed that the endosymbiont of R. similis is distantly related to the known Wolbachia supergroups. Finally, based on our initial success in finding sequences of this endosymbiont by screening an expressed sequence tag (EST) dataset, all nematode ESTs were mined for Wolbachia-like sequences. Although the retained sequences belonged to six different nematode species, R. similis was the only plant-parasitic nematode with traces of Wolbachia. Based on our phylogenetic study and the current literature we designate the endosymbiont of R. similis to a new supergroup (supergroup I) rather than considering it as a new species. Although its role remains unknown, the endosymbiont was found in all individuals tested, pointing towards an essential function of the bacteria.

Ontogeny of the complex sperm in the macrostomid flatworm Macrostomum lignano (Macrostomorpha, Rhabditophora).

Spermiogenesis in Macrostomum lignano (Macrostomorpha, Rhabditophora) is described using light- and electron microscopy of the successive stages in sperm development. Ovoid spermatids develop to highly complex, elongated sperm possessing an undulating distal (anterior) process (or "feeler"), bristles, and a proximal (posterior) brush. In particular, we present a detailed account of the morphology and ontogeny of the bristles, describing for the first time the formation of a highly specialized bristle complex consisting of several parts. This complex is ultimately reduced when sperm are mature. The implications of the development of this bristle complex on both sperm maturation and the evolution and function of the bristles are discussed. The assumed homology between bristles and flagellae questioned.

Unusual intestinal lamellae in the nematode Rhabditophanes sp. KR3021 (Nematoda: Alloinematidae).

The free-living nematode Rhabditophanes sp. has recently been placed in a clade of animal parasites and may be a unique example of a reversal to a nonparasitic lifestyle. Detailed morphological analysis of the intestine reveals the unusual and unique structure of splitting microlamellae forming a meshwork with cavities along the entire intestinal tract. Secretion vesicles were observed along the whole tract and along the length of the lamellae. It is suggested that these lamellae are adaptations to a different digestive strategy where low food availability and a low absorption surface are compensated for by maximizing the nutrient uptake efficiency along the entire length of the intestine. The likely reversal to a free-living life cycle may have caused drastic changes in diet, providing the necessary driving forces to such morphological changes.

A new class of ubiquitin extension proteins secreted by the dorsal pharyngeal gland in plant parasitic cyst nematodes.

By performing cDNA AFLP on pre- and early parasitic juveniles, we identified genes encoding a novel type of ubiquitin extension proteins secreted by the dorsal pharyngeal gland in the cyst nematode Heterodera schachtii. The proteins consist of three domains, a signal peptide for secretion, a mono-ubiquitin domain, and a short C-terminal positively charged domain. A gfp-fusion of this protein is targeted to the nucleolus in tobacco BY-2 cells. We hypothesize that the C-terminal peptide might have a regulatory function during syncytium formation in plant roots.

The apical disposition of the Caenorhabditis elegans intestinal terminal web is maintained by LET-413.

We wish to understand how organ-specific structures assemble during embryonic development. In the present paper, we consider what determines the subapical position of the terminal web in the intestinal cells of the nematode Caenorhabditis elegans. The terminal web refers to the organelle-depleted, intermediate filament-rich layer of cytoplasm that underlies the apical microvilli of polarized epithelial cells. It is generally regarded as the anchor for actin rootlets protruding from the microvillar cores. We demonstrate that: (i) the widely used monoclonal antibody MH33 reacts (only) with the gut-specific intermediate filament protein encoded by the ifb-2 gene; (ii) IFB-2 protein accumulates near the gut lumen beginning at the lima bean stage of embryogenesis and remains associated with the gut lumen into adulthood; and (iii) as revealed by immunoelectron microscopy, IFB-2 protein is confined to a discrete circumferential subapical layer within the intestinal terminal web (known in nematodes as the "endotube"); this layer joins directly to the apical junction complexes that connect adjacent gut cells. To investigate what determines the disposition of the IFB-2-containing structure as the terminal web assembles during development, RNAi was used to remove the functions of gene products previously shown to be involved in the overall apicobasal polarity of the developing gut cell. Removal of dlg-1, ajm-1, or hmp-1 function has little effect on the overall position or continuity of the terminal web IFB-2-containing layer. In contrast, removal of the function of the let-413 gene leads to a basolateral expansion of the terminal web, to the point where it can now extend around the entire circumference of the gut cell. The same treatment also leads to concordant basolateral expansion of both gut cell cortical actin and the actin-associated protein ERM-1. LET-413 has previously been shown to be basolaterally located and to prevent the basolateral expansion of several individual apical proteins. In the present context, we conclude that LET-413 is also necessary to maintain the entire terminal web or brush border assembly at the apical surface of C. elegans gut cells, a dramatic example of the so-called "fence" function ascribed to epithelial cell junctions. On the other hand, LET-413 is not necessary to establish this apical location during early development. Finally, the distance at which the terminal web intermediate filament layer lies beneath the gut cell surface (both apical and basolateral) must be determined independently of apical junction position.