Thick Cuticle Layer Research Articles

Secretory Cells in Halla parthenopeia (Oenonidae): Potential Implications for the Feeding and Defence Strategies of a Carnivorous Burrowing Polychaete.

Carnivorous polychaetes are known to bear diversified and often unique anatomical and behavioural adaptations for predation and defence. Halla parthenopeia, a species known to be a specialized predator of clams, thrives in the soft bottoms of the Mediterranean Sea, holding potential for polyculture and biotechnology due to the secretion of bioactive compounds. Our objective was to provide a comprehensive description of H. parthenopeia's anatomy and microanatomy, shedding light on the relation between morphology and habitat, chemical defences, and feeding behaviour. The pharynx, housing maxillae and mandibles connected to an extensive mucus gland, occupies a considerable portion of the worm's length, reaching beyond the oesophagus. This unique gland is responsible for secreting the feeding mucus, which immobilizes and aids in the digestion of clams probably acting as a vehicle of bioactive compounds synthesized by specialized serous cells in the mouth. Moreover, H. parthenopeia combines behavioural tactics, such as burrowing, and anatomical defences to evade predators. Examination of its epidermis revealed a thick cuticle layer and abundant mucocytes secreting locomotion mucus, both of which save the worm from mechanical harm during movement. When it is preyed upon, the worm can release a substantial amount of Hallachrome, a toxic anthraquinone produced by specific cells in its distal region. This pigment, with its known antimicrobial properties, likely acts as a chemical shield in case of injury. The results suggest that the ability of H. parthenopeia to prey on bivalves and to provide mechanical protection plus defence against pathogens rely on its ability to secrete distinct types of mucus. The interplay between highly specialized microanatomical features and complex behaviours underscores its adaptation as a predator in marine benthic environments.

Rediscovery, identity, and conservation strategies of a critically endangered endemic plant, <em>Hedyotis quinquinervia</em> Thwaites (Rubiaceae) in Sri Lanka

Sri Lanka, a biodiversity hotspot in Asia, records 30 Hedyotis species of which 25 species and a variety are endemic. Among these 25 species, seven Hedyotis species were categorized as critically endangered (CR), and 13 as endangered (EN). During our field survey in 2014, an extremely attractive plant belonging to the genus Hedyotis was discovered from Mount Thotupola, Sri Lanka. The plant was tentatively identified as H. quinquinervia. For accurate species identification, morphological characters were compared with voucher specimens, and identification keys were also used. In addition, DNA barcoding using the sequence of the internal transcribed spacer region of the nuclear ribosomal DNA (rDNA-ITS) region was performed, followed by molecular phylogenetic analysis. A simple method to remove a thick cuticle layer on the leaves was employed to obtain a sufficient amount of DNA suitable for Polymerase Chain Reaction. A comparison with its protologue and type specimen along with molecular phylogenetic analysis confirmed that the unidentified plant was H. quinquinervia Thwaites. The National Red List of Sri Lanka (2020) had revised H. quinquinervia as a CR species upon the rediscovery confirmed after the lapse of a century. Habitat characteristics, ex situ and in situ conservation measures for H. quinquinervia, and general conservation strategies applicable to threatened heterostylous plants are also discussed.

Chromosome-level genome assembly of the diploid blueberry Vaccinium darrowii provides insights into its subtropical adaptation and cuticle synthesis

Vaccinium darrowii is a subtropical wild blueberry species that has been used to breed economically important southern highbush cultivars. The adaptive traits of V. darrowii to subtropical climates can provide valuable information for breeding blueberry and perhaps other plants, especially against the background of global warming. Here, we assembled the V. darrowii genome into 12 pseudochromosomes using Oxford Nanopore long reads complemented with Hi-C scaffolding technologies, and we predicted 41 815 genes using RNA-sequencing evidence. Syntenic analysis across three Vaccinium species revealed a highly conserved genome structure, with the highest collinearity between V. darrowii and Vaccinium corymbosum. This conserved genome structure may explain the high fertility observed during crossbreeding of V. darrowii with other blueberry cultivars. Analysis of gene expansion and tandem duplication indicated possible roles for defense- and flowering-associated genes in the adaptation of V. darrowii to the subtropics. Putative SOC1 genes in V. darrowii were identified based on phylogeny and expression analysis. Blueberries are covered in a thick cuticle layer and contain anthocyanins, which confer their powdery blue color. Using RNA sequencing, we delineated the cuticle biosynthesis pathways of Vaccinium species in V. darrowii. This result can serve as a reference for breeding berries whose colors are appealing to customers. The V. darrowii reference genome, together with the unique traits of this species, including its diploid genome, short vegetative phase, and high compatibility in hybridization with other blueberries, make V. darrowii a potential research model for blueberry species.

Morfoanatomi Hoya coronaria Berbunga Kuning pada Perlakuan Intensitas Cahaya Berbeda

Knowledge on the anatomical structure of Hoya coronaria Blume can help to understand the various function of the plant to support the success of Hoya cultivation outside its natural habitat. This study aims to examine the effect of differences in light intensity on the anatomical characteristics of yellow-flowered H. coronaria to evaluate the adaptation of H. coronaria outside its natural habitat. The experiment used a one-factor factorial completely randomized design with two light intensity treatments (highest daily light intensity 15 Klux) and no shade with the highest daily light intensity average 59 Klux). The preparation of the leaf paradermal incision was made using the whole preparation method while the transverse incision was made using the paraffin method. Based on the analysis of the anatomical structure of the leaves, H. coronaria is a plant with C3 photosynthesis type. Leaf anatomy is composed of a thick cuticle layer, a single layer of the epidermis, mesophyll tissue which is differentiated into palisade and sponges, has a wide intercellular air cavity, and there is a transport network with undeveloped vascular sheath cells. H. coronaria growing under higher light intensity had thicker upper and lower cuticle, lower epidermis, mesophyll layer, and spongy layer compared to lower light intensity. The chlorophyll content was not significantly different between those grown at low and high light intensities. Based on these results, it can be concluded that H. coronaria adapted to high light intensity (without shade) or locations exposed to direct sunlight.

Anatomy of Consolida orientalis (Gay) Schröd. (Ranunculaceae): Root, stem and leaf

Background and Aims: Consolida orientalis (J.Gay.) Schrödinger belonging to the Ranunculaceae family, which is called “morçiçek” in Turkey. It can be recognized by its laciniae linear leaves, intense violet flower, and follicle fruit. The aim of the present study is to elucidate the anatomical properties of C. orientalis, as one of the medically important plant species of Ranunculaceae family. Methods: Plant material was collected from Kahramankazan (Ankara-Turkey). The samples were protected in 70% alcohol. The transverse and surface sections were investigated by light microscope and photographed. Results: The root is characterized by a periderm, pericyclic sclerenchymatous ring, and vessels embedded in sclerenchymatous pith cells. It was observed that in the anatomical features of the stem, the adaxial walls of epidermis cells curved outward and were covered with a thick cuticle layer. Moreover, stomata were observed in the epidermis and the stem had hairs only at the above parts, while other parts were bare. These hairs consisted of both glandular (with a long stalk) and non-glandular (unicellular, silicified) types. The leaf is broadly v-shaped, monofacial and has a 1-layer epidermis. Ranunculaceous stomata and non-glandular (unicellular, silicified and slightly bowed) hairs were observed on both surfaces. Conclusion: The root, stem, and leaf anatomy of C. orientalis was studied for the first time. The anatomical features described in detail with reference reports could be helpful in future taxonomic studies.

Study of some properties and antibacterial activity of (Alhagi maurorum Medik) collected from the desert of Al – Najaf

In traditional medicine, Alhagi maurorum is one member of medicinal plants which is used for many diseases therapy, that it is used as diuretic and Kidney stones treatments. This study was founded to identify some morphological characters and antibacterial activity of methanolic extract of A. maurorum. The morphological properties studied with macro and microscopical examination while the antibacterial activity by screening with well diffusion method against Bacillus cereus and Proteus mirabilis. The results showed that the plant is shrubby herb with evergreen appearance and its leaves are small obovate shape and the polygonal epidermal cells covered with thick cuticle layer. Stomata are frequently Paracytic type in addition to a few anomocytic type with unicellular covering trichome and prismatic crystals. The plant did not showed antibacterial activity against studied bacteria ( Bacillus cereus and Proteus mirabilis ) with concentrations up to 200 mg / ml.

Morphological, anatomical and cytological investigations on three taxa of Centaurea L. (Asteraceae) from Turkey

Morphological, anatomical and cytological features of three Turkish taxa of Centaurea L., viz. C. polypodiifolia Boiss. var. polypodiifolia, C. urvillei DC. subsp. urvillei and C. urvillei subsp. armata Wagenitz were investigated. Stem anatomy revealed the presence of a thick cuticle layer outside the stem, and epidermis with dense hairs was observed in the lower part. Investigation on leaf anatomy showed that different types of hairs on the outside of the leaf were very intense. Palisade parenchyma was observed below the upper and lower epidermis. In Centaurea polypodiifolia var. polypodiifolia, chromosome number was found to be 2n=16, while in C. urvillei subsp. urvillei and C. urvillei subsp. armata 2n=20. Total karyotype length of C. polypodiifolia var. polypodiifolia, C. urvillei subsp. urvillei and C. urvillei subsp. armata was 22.9 µm, 37.84 µm and 40.01 µm, respectively. Among the investigated taxa the karyotype asymmetry index was found lowest in C. urvillei subsp. armata. Satellite was detected in C. urvillei subsp. urvillei and subsp. armata, while it was absent in C. polypodiifolia Boiss. var. polypodiifolia.

Studies on some anatomical features of selected plant species grown in sand dune areas of North Sinai, Egypt

The present investigation aims at studying some anatomical features of some selected native plant species in arid environments of sand dunes at North Sinai, Egypt. Plant samples were collected from three localities; Coastal sand dunes of Balouza (Zygophyllum album, Zygophyllaceae; root, stem and leaf); sand sheets of El-Arish city (stems of Anabasis articulata, Amaranthaceae and Salsola tetrandra, Chenopodiaceae) and El-Owga interdune areas (Fagonia indica, Zygophyllaceae; stem and root, Zygophyllum album, stem). Anatomical adaptations in leaves of Zygophyllum album include the presence of palisade layers on the adaxial and abaxial surfaces, thick cuticle layer cover the epidermis, spongy mesophyll and the presence of water storage cells. Stem anatomical adaptations in different plant species as general observations were the presence of thick cuticle, multiepidermal layers, palisade shape chlorenchyma, patches of sclerenchymatous fiber in the cortex and the precipitation of raphid and druse crystals of different sizes in plant tissues. Root adaptations in Zygophyllum album and Fagonia indica include the presence of periderm layers composed of compacted and elongated cells of different origins and increase the number of small xylem vessels which can guarantee the continuity of water uptake from soil in the case of xylem embolism.

A theoretical approach to the relationship between wettability and surface microstructures of epidermal cells and structured cuticles of flower petals.

The epidermal surface of a flower petal is composed of convex cells covered with a structured cuticle, and the roughness of the surface is related to the wettability of the petal. If the surface remains wet for an excessive amount of time the attractiveness of the petal to floral visitors may be impaired, and adhesion of pathogens may be promoted. However, it remains unclear how the epidermal cells and structured cuticle contribute to surface wettability of a petal. By considering the additive effects of the epidermal cells and structured cuticle on petal wettability, a thermodynamic model was developed to predict the wetting mode and contact angle of a water droplet at a minimum free energy. Quantitative relationships between petal wettability and the geometries of the epidermal cells and the structured cuticle were then estimated. Measurements of contact angles and anatomical traits of petals were made on seven herbaceous species commonly found in alpine habitats in eastern Nepal, and the measured wettability values were compared with those predicted by the model using the measured geometries of the epidermal cells and structured cuticles. The model indicated that surface wettability depends on the height and interval between cuticular steps, and on a height-to-width ratio for epidermal cells if a thick hydrophobic cuticle layer covers the surface. For a petal epidermis consisting of lenticular cells, a repellent surface results when the cuticular step height is greater than 0·85 µm and the height-to-width ratio of the epidermal cells is greater than 0·3. For an epidermis consisting of papillate cells, a height-to-width ratio of greater than 1·1 produces a repellent surface. In contrast, if the surface is covered with a thin cuticle layer, the petal is highly wettable (hydrophilic) irrespective of the roughness of the surface. These predictions were supported by the measurements of petal wettability made on flowers of alpine species. The results indicate that surface roughness caused by epidermal cells and a structured cuticle produces a wide range of petal wettability, and that this can be successfully modelled using a thermodynamic approach.

Observation and comparison of structure changes in wheat caryopsis maternal tissues and endosperm

During the caryopsis development, maternal tissues provide protection and nutrients for the endosperm. Wheat caryopses were observed using different microscopies to investigate structure characteristics of wheat caryopsis maternal tissues and endosperm. The results were as the following: (1) Maternal tissues, especially nucellar cells and pericarp parenchyma cells, degenerated to supply space to the endosperm growth. A thick cuticle layer adhered to the outer integument existed till the endosperm matured. The thick cuticle layer not only made sure that nutrients from the vascular system got into the endosperm through the chalaza but also was probably the key reason why integuments could affect the endosperm growth. (2) The green pericarp contained chloroplasts and perhaps played an important part in nursing other maternal tissues and limiting nutrients from phloem to be transported toward pericarp parenchyma cells. (3) Sieve tubes of the vascular system increased in response to the enhanced endosperm sink strength. Chalazal cell walls and inclusions probably changed to regulate the flux of nutrients transported into the endosperm. In one word, structure changes of maternal transport tissues had a close relationship with the endosperm development.

Biodiversity characteristics of Teucrium polium species in Saudi Arabia

Teucrium (Lamiaceae) is a large and polymorphic genus distributed mainly in Europe, North Africa and in the temperate parts of Asia. In this study, the anatomical features of the leaf and stem of Teucrium polium are investigated. Teucrium has 19 taxa in Iran, which mainly grow in the Irano-Turanian region between 700 and 2000m above sea level. T. polium belonging to sect. Polium, is a perennial herb growing on Lorestan province. The leaves clearly exhibit xeromorphy due to features such as thick cuticle layer, thick outer epidermal cell wall, high density of trichomes and thick palisade layer of the mesophyll. Anatomical studies on T. polium revealed that the stem shares the general characteristics of the Labiatae family. The aim of our approach was to study the morphological and taxonomical parameters for T. polium in Saudi Arabia. The results of this study showed that there was no influence of environment on the structure of stomata and trichomes as studying species with T.polium. In conclusion our study shows we have studied the geographical distribution of the species in Saudi Arabia and in the world.

The relationship between the anatomical structure of the tomato fruit pericarp (Lycopersicon esculentum Mill.) and its mechanical properties

The anatomical structure of the tomato (<i>Lycopersicon esculentum</i> Mill.) pericarp was studied in the fruits of five determinant field cultivars. The fruits of the examined cultivars differed in their mechanical properties. Fruits of cultivars which are firm and resistant to injury (Kecskemeti 886j-2, Campbell 28), moderately firm and resistant (Atma, Wenus) and soft, susceptible to injury (line No 155/84) were examined. The pericarp of the cultivars which have fruits resistant to injury had an approx. 200 µm thick covering layer (epidermis plus cuticle plus hypodermis) whereas that of fruits susceptible to injury was only 100 µm thick. In the fruits resistant to injury the cuticle penetrated deeply into the hypodermis, the epidermal cells were flattened and the hypodermis was composed of 4 - 5 rows of cells. The tomato fruits resistant to injury had a thick cuticle layer on their tangential walls - whereas those more susceptible had a thick cuticle layer on these walls.

Anatomy of Iranian species Teucrium polium (Lamiaceae)

Teucrium (Lamiaceae) is a large and polymorphic genus distributed mainly in the Europe, North Africa and in the temperate parts of Asia. In Iran, 12 species are native. In this study, the anatomical features of the leaf and stem Teucrium polium are investigated. T. polium belonging to sect. Polium is a perennial herb growing on Lorestan province. The anatomical studies on T. polium revealed that the stem shares the general characteristics of the Labiatae family. The leaves clearly exhibit xeromorphy due to features such as the thick cuticle layer, thick outer epidermal cell wall, a high density of trichomes and thick palisade layer of the mesophyll.

The structural and histochemical analyses and chemical characters of the cuticle and epidermal walls of cotyledon in ungerminated seeds of Zostera marina L.

How Zostera marina L. adapts to environmental stresses is of major interest to researchers wanting to obtain a better understanding of how to preserve this ecologically important seagrass. We have examined the structure and chemical properties of the cuticle and epidermal walls of Z. marina cotyledons at the pre-germination stage by microscopy, histochemistry, and chemical analyses. The epidermal cells, which have a smooth plasma membrane and slight wall-ingrowth, are surrounded by thickened outer tangential walls consisting of cellulose and pectic substances. The thick cuticle layer covers the outer surface of the outer tangential walls; the former were observed here to be much thicker than has been reported earlier for the leaves and sheaths of Z. marina. Chemical analysis of the isolated cuticle by attenuated total reflectance–infrared Fourier transform spectrometry and gas chromatography–mass spectrometry detected C16 and C18 fatty acids, ω-hydroxy fatty acids and β-sitosterol as components of the wax and cutin. The outermost cuticle layer was also observed to be covered with a slimy layer consisting of some polysaccharides. These results suggest that the extremely thick cuticle and slimy layer could play a significant role in protecting the cotyledons from environmental stresses at the pre-emergence stage and just after emergence.

Caracterização anatômica e química de folhas de Jacaranda puberula (Bignoniaceae) presente na Mata Atlântica

A espécie Jacaranda puberula (Bignoniaceae) é conhecida popularmente como "carobinha" sendo utilizada na medicina popular em comunidades tradicionais. O objetivo deste trabalho foi realizar a caracterização anatômica e química das folhas de J. puberula. Para a análise anatômica utilizou-se a microscopia de luz e microscopia eletrônica de varredura (MEV). Para a análise química utilizou-se a Cromatografia Líquida de Alta Eficiência (CLAE). O mesofilo é dorsiventral, a epiderme unisseriada com cutícula espessa, tricomas glandulares do tipo peltado e tricomas tectores em ambas as faces da epiderme, parênquima clorofiliano com duas a três camadas de células paliçádicas e quatro a cinco camadas de células formando o parênquima lacunoso, de tamanho pequeno, com muitos espaços intercelulares, hipoestomática com nervura pinada. Os estudos através de MEV evidenciaram a epiderme recoberta com cera epicuticular e glândulas peltadas com maior incidência na epiderme da face abaxial, constituídas por oitos células secretoras apicais. A análise cromatográfica do extrato etanólico evidenciou a presença de fitoquinóides e flavonóides.

Anatomy, palynology and nutlet micromorphology of Turkish endemic Teucrium sandrasicum (Lamiaceae)

In this study, the anatomical features of the leaf and stem, besides the pollen and nutlet characteristics of Teucrium sandrasicum are investigated. T. sandrasicum, belonging to sect. Teucrium, is an endemic perennial herb growing on serpentine around Mugla province. The anatomical studies on T. sandrasicum revealed that the stem shares the general characteristics of the Labiatae family. The leaves clearly exhibit xeromorphy due to features such as the distribution of stomata on the lower surface (hipostomatic), the occurrence of guard cells below the epidermis (xeromorphic type), inrolled margins, thick cuticle layer, thick outer epidermal cell wall, a high density of trichomes and thick palisade layer of the mesophyll. The anatomical studies showed that the upper epidermal cells of the leaf include many spherocrystals. The pollen grains are prolate, medium in size, 3-colpate with verrucate ornamentation. The nutlets are ellipsoid with a reticulate-verrucate surface. The results have proven that T. sandrasicum is different from the other species of the sect. Teucrium because of the branched trichomes on the stem and the lack of eglandular trichomes on the nutlets.

The female abdomen of the viviparous earwig Hemimerus vosseleri (Insecta: Dermaptera: Hemimeridae), with a discussion of the postgenital abdomen of Insecta

The viviparous, epizoic African earwigs of the genus Hemimerus are currently regarded as the sister taxon of the remaining Dermaptera (Forficulina). Exoskeleton, musculature, and part of the nervous system of the female abdomen, from segment IV on, are described. The morphological interpretation and homology relations of most components are discussed, using previous and original data on Forficulina, Zygentoma, Ephemeroptera, Orthoptera and Dictyoptera as a comparative framework. In the mid-abdominal segments some interesting similarities with Zygentoma are indicated. Focal issues in the postgenital abdomen are the terminal dorsal sclerites, the cercal muscles, and the paraprocts and associated muscles. Earlier hypotheses on the dermapteran postabdomen (opisthomere and pseudocercus hypotheses) and results from ontogenetic studies are scrutinized. Some interesting features detected in female Hemimerus are the immobilization of terga VIII–X by means of a thick internal cuticle layer, the lack of dorsal muscles on these terga, the shift of some insertions of cercal and rectal muscles from tergum X to tergum IX, and minute pits on the venters IX and X that could be spiracle vestiges. Some of these features occur also in other Dermaptera. Some abdominal characters suggest that Hemimerus is nested within the Forficulina. The lack of the clasper-shape in the cerci is not a strong argument against this.

The female abdomen of the viviparous earwig Hemimerus vosseleri (Insecta: Dermaptera: Hemimeridae), with a discussion of the postgenital abdomen of Insecta

The viviparous, epizoic African earwigs of the genus Hemimerus are currently regarded as the sister taxon of the remaining Dermaptera (Forficulina). Exoskeleton, musculature, and part of the nervous system of the female abdomen, from segment IV on, are described. The morphological interpretation and homology relations of most components are discussed, using previous and original data on Forficulina, Zygentoma, Ephemeroptera, Orthoptera and Dictyoptera as a comparative framework. In the mid-abdominal segments some interesting similarities with Zygentoma are indicated. Focal issues in the postgenital abdomen are the terminal dorsal sclerites, the cercal muscles, and the paraprocts and associated muscles. Earlier hypotheses on the dermapteran postabdomen (opisthomere and pseudocercus hypotheses) and results from ontogenetic studies are scrutinized. Some interesting features detected in female Hemimerus are the immobilization of terga VIII–X by means of a thick internal cuticle layer, the lack of dorsal muscles on these terga, the shift of some insertions of cercal and rectal muscles from tergum X to tergum IX, and minute pits on the venters IX and X that could be spiracle vestiges. Some of these features occur also in other Dermaptera. Some abdominal characters suggest that Hemimerus is nested within the Forficulina. The lack of the clasper-shape in the cerci is not a strong argument against this.

806 PB 118 CHARACTERIZATION OF BERRY CUTICLE AND EPICUTICULAR WAX OF GRAPE CULTIVARS

The cuticle and epicuticular wax layer of grape berries provides a first line of defense against fungal pathogens. A relatively thick cuticle and wax layer may result in enhanced resistance to Botrytis cinerea Pers. The structure of epicuticular wax also has been postulated to play a role in Botryris resistance. To examine the role of cuticle and wax in disease resistance, berries of diverse grape cultivars were sampled to quantify the cuticle and wax layers. Wax surface structure was examined by scanning electron microscopy. Significant differences among cultivars were found for both cuticle and wax measurements. Environmental conditions may influence development of these layers, some cultivars had significantly greater cuticle and wax layers in berries that developed in full sunlight. Visual differences in surface wax appearance were apparent among cultivars. Size, density and orientation of wax platelets varied among cultivars.

Two ultrastructurally distinct types of transporting tissues, the branchiostegal and the gill epithelia, in an estuarine tanaid, Sinelobus stanfordi (Crustacea, Peracarida)

On each lateral side of the cephalothorax segments, the adult Sinelobus stanfordi has a branchial chamber which contains an elongated bag-shaped gill and is covered by a thick branchiostegite. The ultrastructural study revealed that the inner surface of the branchiostegite is composed of a transporting-type epithelium which is morphologically distinct from the gill epithelium. Both epithelia are covered by extremely thin (about 80 nm) cuticle layers, suggesting high permeability to gases, ions, and water. In contrast, the outer surface of the branchiostegite consists of ordinary epithelium covered by a very thick cuticle layer in common with the body surface. The inner branchiostegal epithelium (4–10 μm thick) has a shallow (about 1 μm deep) apical infolding system of the cell membrane (AIS) and an extensive three-dimensional tubular network (about 120 nm in diameter) which is formed by the invagination of the basolateral cell membrane (TNB). The TNB is associated with slender mitochondria and occupies the majority of the cytoplasmic area of the epithelial cell. The gill epithelium, on the other hand, is about 10 μm thick and characterized by an abundance of oval mitochondria, well-developed (4–5 μm deep) AIS, and a smooth basal cell membrane lacking any infoldings. These morphological features indicate that not only the gill epithelia, but also the inner branchiostegal epithelia, are involved in the ion-transporting processes. The ultrastructural differences between these two kinds of epithelia also suggest their different roles in the osmoregulation of this animal, since it inhabits estuaries which are subject to extreme changes in salinity.