The surface morphology of locust hindgut cuticle*

The surface architecture of hindgut cuticle in Locusta migratoria is described. Infundibulate pores are present in the midrectal pads, the ileum and the posterior rectum. It is suggested that they lead to canals traversing the cuticle. Larger occluded crater-like pores are present on the posterior rectal pads and may be glandular. The cuticle of the ileum and colon is invested with numerous spikes pointing backwards, and the interpad region of the rectum with upright spikes pointing inwards. These spikes probably serve to direct the faeces backwards from the ileum and colon, and to impale the faecal pellet during dehydration in the rectum.     

Anatomy and fine structure of the alimentary canal of the spittlebug Lepyronia coleopterata (L.) (Hemiptera: Cercopoidea)

The alimentary canal of the spittlebug Lepyronia coleopterata (L.) differentiates into esophagus, filter chamber, midgut (conical segment, tubular midgut), and hindgut (ileum, rectum). The filter chamber is composed of the anterior extremity of the midgut, posterior extremity of the midgut, proximal Malpighian tubules, and proximal ileum; it is externally enveloped by a thin cellular sheath and thick muscle layers. The sac-like anterior extremity of the midgut is coiled around by the posterior extremity of the midgut and proximal Malpighian tubules. The tubular midgut is subdivided into an anterior tubular midgut, mid-midgut, posterior tubular midgut, and distal tubular midgut. Four Malpighian tubules run alongside the ileum, and each terminates in a rod closely attached to the rectum. Ultrastructurally, the esophagus is lined with a cuticle and enveloped by circular muscles; its cytoplasm contains virus-like fine granules of high electron-density. The anterior extremity of the midgut consists of two cellular types: (1) thin epithelia with well-developed and regularly arranged microvilli, and (2) large cuboidal cells with short and sparse microvilli. Cells of the posterior extremity of the midgut have regularly arranged microvilli and shallow basal infoldings devoid of mitochondria. Cells of the proximal Malpighian tubule possess concentric granules of different electron-density. The internal proximal ileum lined with a cuticle facing the lumen and contains secretory vesicles in its cytoplasm. Dense and long microvilli at the apical border of the conical segment cells are coated with abundant electron-dense fine granules. Cells of the anterior tubular midgut contain spherical secretory granules, oval secretory vesicles of different size, and autophagic vacuoles. Ferritin-like granules exist in the mid-midgut cells. The posterior tubular midgut consists of two cellular types: 1) cells with shallow and bulb-shaped basal infoldings containing numerous mitochondria, homocentric secretory granules, and fine electron-dense granules, and 2) cells with well-developed basal infoldings and regularly-arranged apical microvilli containing vesicles filled with fine granular materials. Cells of the distal tubular midgut are similar to those of the conical segment, but lack electron-dense fine granules coating the microvilli apex. Filamentous materials coat the microvilli of the conical segment, anterior and posterior extremities of the midgut, which are possibly the perimicrovillar membrane closely related to the nutrient absorption. The lumen of the hindgut is lined with a cuticle, beneath which are cells with poorly-developed infoldings possessing numerous mitochondria. Single-membraned or double-membraned microorganisms exist in the anterior and posterior extremities of the midgut, proximal Malpighian tubule and ileum; these are probably symbiotic.     

Morphology and ultrastructure of the alimentary canal of the cicada Platypleura kaempferi (Hemiptera: Cicadidae)

The alimentary canal of cicada Platypleura kaempferi is described. It comprises the oesophagus, filter chamber, external midgut section and hindgut. The elongate oesophagus expands posteriorly, with its posterior end constricting to become a bulb. The filter chamber consists of two parts: a very thin sheath and a filter organ. The filter organ is composed of the anterior and posterior ends of the midgut (internal midgut section), and the internal proximal ends of the Malpighian tubules. The external midgut section differentiates into a collapsed sac and a midgut loop. The latter is divided into three distinct segments. The hindgut contains a dilated rectum and a long narrow ileum. The distal portions of the four Malpighian tubules are enclosed in a peritoneal sheath together with the distal ileum before reaching to the rectum. Ultrastructurally, the oesophagus and the hindgut are lined with a cuticle. The filter chamber sheath consists of cells with large irregular nuclei. Filamentous substances coat the microvilli of the cells of the internal midgut section. The posterior end of the midgut comprises two types of cells, with the first type of cells containing many vesicles and scattered elements of rough endoplasmic reticulum. The anterior and posterior segments of the midgut loop cells have ferritin‐like granules. The ileum cells have well‐developed apical leaflets associated with mitochondria. Accumulations of virus‐like particles enclosed in the membrane are observed in the esophagus, conical segment, mid‐ and posterior segments of the midgut loop.     

Formation of the hindgut cuticular lining during embryonic development of Porcellio scaber (Crustacea,Isopoda)

The hindgut and foregut in terrestrial isopod crustaceans are ectodermal parts of the digestive system and are lined by cuticle, an apical extracellular matrix secreted by epithelial cells. Morphogenesis of the digestive system was reported in previous studies, but differentiation of the gut cuticle was not followed in detail. This study is focused on ultrastructural analyses of hindgut apical matrices and cuticle in selected intramarsupial developmental stages of the terrestrial isopod Porcellio scaber in comparison to adult animals to obtain data on the hindgut cuticular lining differentiation. Our results show that in late embryos of stages 16 and 18 the apical matrix in the hindgut consists of loose material overlaid by a thin intensely ruffled electron dense lamina facing the lumen. The ultrastructural resemblance to the embryonic epidermal matrices described in several arthropods suggests a common principle in chitinous matrix differentiation. The hindgut matrix in the prehatching embryo of stage 19 shows characteristics of the hindgut cuticle, specifically alignment to the apical epithelial surface and a prominent electron dense layer of epicuticle. In the preceding embryonic stage – stage 18 – an electron dense lamina, closely apposed to the apical cell membrane, is evident and is considered as the first epicuticle formation. In marsupial mancae the advanced features of the hindgut cuticle and epithelium are evident: a more prominent epicuticular layer, formation of cuticular spines and an extensive apical labyrinth. In comparison to the hindgut cuticle of adults, the hindgut cuticle of marsupial manca and in particular the electron dense epicuticular layer are much thinner and the difference between cuticle architecture in the anterior chamber and in the papillate region is not yet distinguishable. Differences from the hindgut cuticle in adults imply not fully developed structure and function of the hindgut cuticle in marsupial manca, possibly related also to different environments, as mancae develop in marsupial fluid. Bacteria, evenly distributed within the homogenous electron dense material in the hindgut lumen, were observed only in one specimen of early marsupial manca. The morphological features of gut cuticle renewal are evident in the late marsupial mancae, and are similar to those observed in the exoskeleton.     

Adaptation to hypoosmotic challenge in brachyuran crabs: a microanatomical and electrophysiological characterization of the intestinal epithelia

Besides its role in digestion and nutrient absorption, the crustacean gut participates in osmo/ionic regulation. We investigate microanatomy, ionic permeability and transepithelial electrophysiological parameters in the mid- and hindguts of three hyperosmoregulating crabs that inhabit estuarine waters (Chasmagnathus granulata), brackish mangrove swamp (Sesarma rectum) or freshwater (Dilocarcinus pagei). The abdominal hindguts are cuticle lined, the single-layered epithelia consisting of narrow, columnar cells exhibiting apically dense, unvesiculated cytoplasm. In the saltwater species, the thoracic midgut epithelium consists of tall, narrow, columnar cells displaying numerous, apical microvilli above dense apical cytoplasm. However, the corresponding gut segment in the hololimnetic species, D. pagei, consists of squat cells lacking apical microvilli, overlain by a heavy cuticle, constituting a thoracic or anterior hindgut. The midgut/thoracic hindgut epithelia in all three crabs, and abdominal (posterior) hindgut of D. pagei, exhibit similar, small, lumen-negative voltages when perfused symmetrically with hemolymph-like salines. The hindguts of the saltwater species show similar, small, lumen-positive voltages. Small short-circuit currents are detectable after voltage clamping. Washout and/or addition of luminal glucose or amino acids do not alter current or conductance, suggesting the absence of active, electrogenic nutrient absorption. Ion substitution did not disclose active, electrogenic absorption or secretion of Na+ and/or Cl-. The midguts of the saltwater species exhibit similar conductances, greater than in D. pagei, but no ion selectivity; hindgut conductance is low, the epithelia showing moderate anion selectivity. The thoracic (anterior) and abdominal (posterior) hindgut epithelia of D. pagei, the freshwater species, exhibit similar, low conductances, and are ion selective. These findings reveal that active, electrogenic, salt and nutrient transport is undetectably low or absent. The reduced transepithelial conductances and notable ion selectivities in the abdominal and thoracic hindguts of D. pagei may reduce passive salt losses in fresh water, contributing to osmotic and ionic regulation.     

Description of Dorylaimoides punctatus n. sp. and Paractinolaimus acutus n. sp. (Nematoda: Dorylaimida) from Korea

Soil samples collected from Taebak Changkunbong and Undal mountains of Kyungbuk Province, yielded two new species of the order Dorylaimida which are described and illustrated below. Dorylaimoides punctatus n. sp. (Nematoda: Dorylaimida) is 1.2–1.4 mm long; b = 5.9–6.9; c = 14.6–17.7; odontostyle 7.3–8.5 μm; spicules 33.5–36.5 μm; ventromedian supplements 5–6 and is characterized by having zig-zag lines on body cuticle, continuous lip region, elongate conoid tail and comes close to Dorylaimoides micoletzkyi (De Man, 1921) Thorne & Swanger, 1936 and Dorylaimoides leptus Husain & Khan, 1968. Paractinolaimus acutus n. sp. (Nematoda: Dorylaimida) is 2.1–2.4 mm long; b = 4.3–4.9; c = 10–12; odontostyle 25–27 μm and characterized by having continuous lip region, longitudinally oval vulva and long filiform tail with acute tip and comes close to Parctinolaimus aruprus Khan et al., 1994 and Parctinolaimus baldus Thorne, 1967.     

合哑蝉成虫消化道形态、组织结构及超微结构（英文）

【目的】本研究通过合哑蝉Karenia caelatata成虫消化道的形态学、组织学和超微结构研究,进一步了解蝉科(Cicadidae)代表种类的消化道形态和功能分化。【方法】利用光学显微镜和透射电子显微镜技术,对合哑蝉雄成虫消化道的整体形态以及食道、滤室(中肠前端及后端、马氏管基部、后肠基部)、滤室外中肠(锥形体、中肠环)、后肠(回肠、直肠)的一般形态和超微结构进行了详细观察,同时对滤室的组织结构进行了研究。【结果】结果表明,合哑蝉消化道由食道、滤室、滤室外中肠及后肠组成。食道狭长,被有上表皮和内表皮。中肠前端、中肠后端、马氏管基部以及后肠基部被一肌肉鞘包围形成滤室构造。组成中肠前端和后端的细胞基膜高度内褶,顶端的微绒毛发达。中肠后端分布许多线粒体和高电子密度的分泌颗粒。滤室外的中肠包括膨大的锥形体、中肠环。其中,锥形体由两种细胞组成;中肠环分为前、中、后3个不同的区段。前中肠细胞包含大量的分泌颗粒、线粒体、粗面内质网和溶酶体;中中肠细胞含有分泌颗粒;后中肠细胞包括许多低电子密度的分泌颗粒和滑面内质网。类铁蛋白颗粒零星分布于中肠环的前、中区段。组成锥形体和中肠环前端的细胞顶端微绒毛被丝状物质覆盖。后肠被有一层表皮。食道、中肠环中段、直肠细胞中含有微生物。【结论】本研究获得的合哑蝉消化道形态、组织结构和超微结构方面的信息为其功能分化研究提供了重要信息。同时,相关微生物的发现为进一步探讨共生菌与蝉总科昆虫的协同进化提供了信息。     

Ultrastructure and phylogeny of Glugea arabica n. sp. (Microsporidia), infecting the marine fish Epinephelus polyphekadion from the Red Sea

A new microsporidian species, Glugea arabica n. sp., is reported infecting the intestinal wall of the marine teleost Epinephelus polyphekadion (=microdon) collected from the Red Sea coast off Saudi Arabia, and described on the basis of microscopic and molecular procedures. Spherical blackish xenomas formed parasitophorous vacuoles completely packed with several parasitic developmental stages, including spores. The nuclei were monokaryotic in all developmental stages. Spores were ellipsoidal to pyriform and measured 6.3 ± 0.3 (5.9–6.6) μm in length and 3.3 ± 0.4 (2.9–3.7) μm in width. A lamellar polaroplast surrounded the uncoiled portion of the polar filament, which extended into the spore's posterior pole and formed 27–29 coils organized in three or four rows. The posterior vacuole, located at the spore's posterior pole, appeared surrounded by the polar filament coils and displayed an irregular matrix composed of light material, in which was located the posterosome. Molecular analysis of the rRNA genes, including the ITS region, was performed using maximum parsimony, neighbor-joining and maximum likelihood methodologies. The ultrastructural features observed, in combination with the molecular data analysed, suggests the parasite to be a new species of the genus Glugea.     

Ultrastructure of the water-movement-sensitive sensilla in the medicinal leech

Behavioral and physiological experiments have shown that medicinal leeches are able to detect low amplitude surface waves, and further, that the transduction of this stimulus modality occurs primarily, if not exclusively, at the annular sensilla (Young, Dedwylder, and Friesen, 1981; Friesen, 1981). Here we examine the morphology of these specialized sensory structures using light, scanning electron, and transmission electron microscopes. We found that three types of ciliated sensory cells occur at the sensilla: (1) a uniciliate cell, with an axial cilium that projects at least 12 μm beyond the cuticle; (2) a multiciliate cell with from two to four grouped cilia that extend 1–3 μm beyond the cuticle; and (3) a second multiciliate cell, whose cilia project parallel to the body surface but remain within the cuticle. The cilia of all three cell types arise from the cuplike depressions which form the apices of slender, elongated cells (approximately 2 μm diameter × 50 μm length). A complexly interconnected ring of microvilli surrounds the cilium of the uniciliate cells. The morphology of the uniciliate cells closely resembles the structure of vibration-sensitive sensory neurons found in other species. We propose, based on previous results and our new findings, that the uniciliate receptor cells are the sensillar movement receptors which mediate leech sensitivity to water movements.     

Pupal cuticle formation by Manduca sexta epidermis in vitro: Patterns of ecdysone sensitivity

During the larval-pupal transformation, various regions of the epidermis of Manduca sexta larvae have previously been found to require different lengths of exposure to the prothoracic glands in order to form pupal cuticle. To distinguish between requirements for differing threshold concentrations of ecdysone and those for differing durations of exposure to ecdysone, wandering stage larval epidermis was cultured in Grace's medium. When most of the thick larval cuticle was removed, the epidermis responded to concentrations of β-ecdysone of 1.0 μ/ml or greater for 4 days by forming cysts which later formed tanned pupal cuticle. No fat body or protein supplement was required. When the larval integument was explanted intact, similar requirements for cuticle formation and for tanning were found. All regions of the fifth abdominal segment required similar concentrations of β-ecdysone (0.4–0.6 μg/ml) for 4 days for 50% to form pupal cuticle, but gin trap epidermis required the least exposure to a threshold concentration of ecdysone (1.5 days in 0.9 μg/ml). The anterior dorsal intersegmental region required about 0.5 day longer, followed by the posterior intersegmental and the dorsal intrasegmental regions. Thus, the duration of exposure seemed more important. About 1 day longer of exposure to ecdysone was required for subsequent tanning of the new cuticle than for cuticle formation, yet tanning of the cuticle did not occur with prolonged exposure to ecdysone.     

Acanthodasys paurocactus sp. n., a new species of Thaumastodermatidae (Gastrotricha, Macrodasyida) with multiple scale types from Capron Shoal, Florida

A new species of Acanthodasys (Gastrotricha, Macrodasyida, Thaumastodermatidae) is described from sublittoral sediments off the Atlantic coast of Florida. Acanthodasys paurocactussp. n. is a relatively small species (to 450 μm long) with a strap-shaped outline, a series of anterior, lateral, and ventrolateral adhesive tubes, paired caudal pedicles with posterior adhesive tubes, and a morphologically diverse cuticle. The cuticle contains both spined and unspined scales. Unspined scales are present in two general shapes: lanceolate and eye shaped, with some transitional shapes. All scales have a thickened rim and depressed central region; some scales of both shapes bear either one or more central bumps, a parallel ridge, or a perpendicular ridge that gives the appearance of a cross-shaped pattern under transmitted light. Spined scales are somewhat quadrangular in shape and bear uniancres to 15 μm long with a cross-shaped sectional profile. The new species is now one of five described species to possess both spined and spineless scales, and only one of two species to possess two types of spineless scales (the second species is an incompletely described specimen from Norway).     

A new species of Uronychia (Spirotrichea: Euplotida) from Argentina

Uronychia clapsae sp. n. was discovered in an artificial channel that drains an endorheic area from the “sandy Pampa” into the upper basin of Salado River, Buenos Aires, Argentina. This euplotid measures 56–112 μm × 42–70 μm in vivo, is oval in shape and the buccal field is enormous, occupying ca. 80% of body length. It is characterized by having two macronuclear nodules and one micronucleus; usually 10 anterior and invariably three posterior membranelles; right end of paroral hook-like; buccal cirrus base about 3.5–6.0 μm long; invariably four frontal, two ventral, three left marginal, four transverse, and three caudal cirri; six dorsal kineties, kinety 1 with 15–22 dikinetids. Most Uronychia species were recorded in marine habitats, while this new isolate was found in a slightly saline, inland water body. Taxonomic and nomenclatorial concerns on some species assigned to Uronychia are also discussed.     

A new species of Pleistophora (Microsporida: Pleistophoridae) parasitic in the shrimp Palaemonetes pugio

Pleistophora sp. Sprague, 1970, in the muscle of Palaemonetes pugio was studied. Formalin fixed spores were ellipsoidal, 2.5–3.3 × 1.4–2.0 μm, and with large anterior and posterior clear areas representing the polaroplast and posterior vacuole. This species, after comparison with four other species of Pleistophora in decapod crustacea, was found to be new. It is named Pleistophora lintoni n. sp.     

Control of cuticle formation by wing imaginal discs in vitro.

Wing discs from late final-instar Ephestia larvae form only pupal cuticle when immediately implanted into pupae which subsequently undergo metamorphosis. However, either pupal or adult structures are made in vitro depending on (1) the ecdysterone dose and/or (2) disc cell proliferation. Continuous culture in ecdysterone (0.5–5.0 μg/ml) results in the appearance of transparent cuticle. On the basis of several criteria, this untanned cuticle is postulated to be scaleless adult cuticle. Discs pulsed with 0.5 μg/ml ecdysterone for 48–120 hr, or with 5.0 μg/ml for 24 hr, formed tanned cuticle. Lower doses of ecdysterone (i.e., 0.5 μg/ml for 24 hr or continuous exposure to 0.05 μg/ml) trigger adult scale formation. Enhancement of [³H]thymidine incorporation by these latter doses suggests the occurrence of disc cell divisions and polyploidization. The choice between pupal and adult pathways by wing discs of this age can be controlled exclusively by ecdysterone; juvenile hormone need not be involved in vitro.     

Taxonomy,phylogeny and host-parasite interaction of two novel Myxobolus species infecting Brycon orthotaenia from the São Francisco River,Brazil

Two new Myxobolus species were described infecting Brycon orthotaenia from the São Francisco River, in the state of Minas Gerais, Brazil. From a total of 39 B. orthotaenia collected, two specimens (5.1%) exhibited infection of the ovary and 12 specimens (30.8%) displayed infection of the liver. The plasmodia of both Myxobolus species were white and spherical measuring around 1 mm in length. The plasmodium found in the ovary showed mature myxospores, which were oval shaped from the frontal view and measured 9.2–11.0 (9.8 ± 0.4) μm in length, 5.9–6.9 (6.5 ± 0.3) μm in width and 4.6–5 (4.9 ± 0.1) μm in diameter. The two polar capsules were the same size and measured 3.9–6.2 (4.7 ± 0.5) μm in length and 1.8–2.4 (2.1 ± 0.2) μm in width. The polar tubules had 9 coils. The plasmodium found in the liver showed mature myxospores which were ellipsoidal in shape from the frontal view and measured 10.0–11.4 (10.7 ± 0.5) μm in length, 7.3–8.6 (8.1 ± 0.4) μm in width and 5.3–7.0 (6.8 ± 0.4) μm in diameter. The two polar capsules were the same size and measured 4.2–5.4 (4.9 ± 0.3) μm in length and 1.9–2.9 (2.7 ± 0.3) μm in width. The polar tubules had 8 coils. Ultrastructural analysis revealed an asynchronous sporogenesis process, with young developmental myxospore stages more often found in the periphery of the plasmodium and mature myxospores in the centre of the plasmodium. The plasmodial wall was formed by a single membrane which was not surrounded by a layer of host tissue. A thick layer of fibrous material was found in the peripheral ectoplasm close to the plasmodial wall of the plasmodium found in the ovary. Phylogenetic analysis based on the small-subunit ribosomal DNA – ssrDNA sequences and using the closest myxozoan sequences to each one of the species studied here based on previous GenBank data and Henneguya/Myxobolus/Thelohanellus species parasitizing fish from South American, revealed that the new species are grouped in a subclade together with other Myxobolus species parasitizing bryconid hosts.     

Cuticular control of diffusional water permeability

The diffusional water permeability (P_d) in various gut structures of the fleshfly, Sarcophaga bullata, was measured using tritiated water. Water P_d in the larval hindgut was 3.91 × 10^?6 cm/sec, whereas in the adult hindgut it was 4.4 × 10^?4 cm/sec. The presence of cuticle in various parts of the gut apparently controls the water permeabilities of these structures. Furthermore, the water permeability of the cuticle may be correlated with the mechanism for the production of a hyperosmotic excretion in the hindgut.     

Foregut microspines in four families of cockroaches (Blattaria)

The microspines of the cockroach foregut were investigated in order to determine their fine structure, variation, patterns of distribution, and possible functions. The following were studied: Blaberidae (4 species), Blatellidae (3 species), Blattidae (2 species), and Cryptocercidae (one species). Elongate microspines (over 30 μm), usually several attached to a basal plate, were found in the buccal cavity and anterior and posterior pharynx of blaberids and blattids, whereas moderate (15–20 μm) to short (1–5 μm) microospines were found in 2 regions in the blattellid and cryptocercid cockroaches. Short microspines (1–5 gmm) individually attached to a basal plate occurred in the esophagus, crop, proventriculus and stomodeal valve regions in all families studied. Microspines appear to be useful in understanding systematics and evolution of cockroaches. The possible role of elongate microspines in retaining food particles during regurgitation behavior is postulated.     

Host dietary changes and the hindgut fauna of cockroaches

Peregrine Pauline C. 1974. Host dietary changes and the hindgut fauna of cockroaches. International Journal for Parasitology4: 645–656. The effects of changes in the diet of Periplaneta americana on Hammerschmidtiella diesingi (Nematoda), nematode larvae, Nyctotherus ovalis (Ciliata), Endamoeba blattae (Lobosa), Lophomonas striata (Zooflagellata) and Monocercomonoides orthopterorum (Zooflagellata) have been studied. All species are found in the hindgut of the host and appear to be supported to advantage by slightly different dietary fractions thus enhancing coexistence. Interspecific competition seems to be reduced by both spatial and dietary factors although most species tend to aggregate in a ‘preferred zone’ 30–45 per cent along the length of the hindgut. Under conditions of stress, distribution often expands. Intraspecific competition also appears to be partially controlled by dietary preferences, male, female and larval nematodes responding to differing dietary fractions. Effects on H. diesingi are closely paralleled by those on Thelastoma attenuatum (Nematoda), also parasitic in the hindgut, indicating a very finely balanced system of coexistence. Some of the parameters which may be involved are discussed. Almost all parasites were adversely affected by synthetic diets.     

Expression patterns of genes encoding proteins with peritrophin A domains and protein localization in Mamestra configurata

Genes encoding three proteins (McPPAD1-3) with peritrophin A chitin-binding domains (PADs) were identified from a Mamestra configurata larval midgut cDNA library. In addition to midgut, McPPAD1-3 and a previously identified gene encoding the peritrophin, McPM1, were expressed in foregut, hindgut, Malpighian tubules, tracheae, fat body and cuticle; however, the corresponding McPPAD proteins exhibited different localization patterns. McPPAD1 was restricted to the digestive tract and Malpighian tubules, McPPAD2 to Malpighian tubules, and McPPAD3 to the foregut, midgut, hindgut, tracheae and cuticle. Protein fold recognition analysis using tachycitin as a guide structure modelled the McPPAD1 PADs, but not McPPAD2 or McPPAD3 PADs. The McPPAD1 PADs were predicted to contain three anti-parallel β-sheets and a hevein-like fold that form a chitin-binding pocket containing two hydrophobic R-groups in a sandwich-like orientation.