The musculature of Squatinella rostrum (Milne, 1886) (Rotifera: Lepadellidae) as revealed by confocal laser scanning microscopy with additional new data on its trophi and overall morphology

Wilts, E.F., Wulfken, D., Ahlrichs, W.H. and Martínez Arbizu, P. 2012. The musculature of Squatinella rostrum (Milne, 1886) (Rotifera: Lepadellidae) as revealed by confocal laser scanning microscopy with additional new data on its trophi and overall morphology.—Acta Zoologica (Stockholm) 93 : 14–27. The monogonont rotifer Squatinella rostrum was investigated with light, scanning electron and confocal laser scanning microscopy to reveal new morphological data on its inner and outer anatomy. In total, the visualized somatic musculature displays five paired longitudinal muscles (musculi longitudinales I–V) and nine circular muscles (musculi circulares I–IX). Compared to other species, S. rostrum is characterized by the absence of several longitudinal and circular muscles (e.g. musculus longitudinalis capitis, corona sphincter and pars coronalis). A reconstruction of the mastax musculature revealed a total number of seven paired and two unpaired mastax muscles. Possibly homologous somatic and mastax muscles in other, thus far investigated rotifers are discussed. Moreover, we provide a phylogenetic evaluation of the revealed morphological characters and suggest possible autapomorphic characters supporting Squatinella and Lepadellidae. Finally, we refer to some striking similarities in the morphology, ecology and way of movement of Squatinella and Bryceella that may indicate a closer relationship of both taxa.     

Study of architectonics of rotifer musculature by the method of fluorescence with use of confocal microscopy

Musculature of two species of rotifers Testudinella patina (Testudinellidae) and Platyias patulus (Brachiomidae) was studied in confocal laser scanning microscope (CLSM) using fluorescent-labeled phalloidin. It includes cutaneous, visceral, and cutaneus-visceral musculature. The common pattern of structure of the cutaneous musculature is represented by postcoronal circular or transverse muscles and connected with them 2–3 pairs of retractors of the trunk, dorsolateral muscles (17-4), two pairs or bundles of lateral retractors of the corona, circular muscles of the foot, and 10-2 retractors of the foot. Visceral musculature includes muscles of the mastax of both kinds. Spiral-like muscle of cloaca of the T. patina and associated with it V-shaped one as well as strong dorsolateral retractors consisting of 6 longitudinal muscle bundles are typical of Testudinellidae only. Three pairs of cutaneus-visceral muscles bind the musculature of mastax with the body surface in T. patina. Differences in localization and thickness of some elements of musculature of these species are determined by morphological peculiarities of structure of the corona, mastax, and foot, as well as by the rotifer body shape.     

The somatic musculature of Bryceella stylata (Milne, 1886) (Rotifera: Proalidae) as revealed by confocal laser scanning microscopy with additional new data on its trophi and overall morphology

The monogonont rotifer Bryceella stylata was investigated with light, electron and confocal laser scanning (CLSM) microscopy to provide detailed insights into its anatomy and new information for future phylogenetic analyses of the group. Results from CLSM and phalloidin staining revealed a total of six paired longitudinal muscles (musculi longitudinales I-VI) and eight circular muscles (musculi circulares I-VIII) as well a complex network of mostly fine visceral muscles. In comparison with other rotifer species that have been investigated so far, B. stylata shares the presence of the circular and longitudinal muscles: musculus longitudinalis ventralis, musculus longitudinalis lateralis inferior, musculus longitudinalis dorsalis, musculus longitudinalis capitis and musculus circumpedalis. However, the species lacks lateral and dorsolateral longitudinal muscles and some circular muscles (e.g., corona sphincter, musculus pars coronalis). With light and electron microscopy, we were able to document the precise number of pseudosegments and the arrangement of the chambers comprising the trophi elements. Furthermore, our observations revealed several new morphological characteristics, including a shield-like epidermal projection covering the dorsal antenna, an epidermal projection restricting the corona caudally and an unpaired hypopharynx with distinct shovel-like structures.     

Neuromuscular organization of the freshwater colonial rotifer, Sinantherina socialis, and its implications for understanding the evolution of coloniality in Rotifera

Coloniality among rotifers is rare, and while the adaptive significance of the lifestyle has been explored previously, there are few details about how it may have influenced the morphology of colony members. In this study, we use confocal laser scanning microscopy combined with cyto- and immunohistochemistry to determine if the colonial rotifer, Sinantherina socialis, differs in neuromuscular organization relative to other colonial and solitary forms. Our observations indicate that the patterns of serotonergic neurons and somatic muscles in S. socialis are broadly similar to other rotifers. At the neuronal level, the distribution of serotonergic (5HT-IR) neurons in S. socialis is most similar to colony-forming species of Conochilus: (1) paired nerve cords extend the length of the body; (2) the cerebral ganglion innervates the corona with paired neurites that form a neuronal ring; and (3) a single pair of neurites innervates the ventral sensory antennae. Unique to S. socialis is the presence of two additional neuronal rings that supply the corona and may function to modulate ciliary beat. At the muscular level, S. socialis displays a typical pattern of somatic muscle organization similar to other rotifers, but diverges from the pattern in three significant ways: (1) somatic circular muscles form complete rings; (2) circular muscles are distinguishable based on their size and position in the body; and (3) transverse muscles are present within the corona and presumably function to modify its shape for feeding and defensive purposes. These differences in neuromuscular organization may be adaptations to the colonial and/or sessile lifestyle, both of which are characteristic of S. socialis.     

Musculature in three species of <Emphasis Type="Italic">Proales</Emphasis> (Monogononta,Rotifera) stained with phalloidin-labeled fluorescent dye

The musculature in the rotifer species Proales daphnicola, P. reinhardti and P. fallaciosa was stained with phalloidin-labeled fluorescent dye and compared using confocal laser scanning microscopy. All three species share several homologous muscle systems, but each systems detailed morphology varies among the species both concerning appearance, number and location. The obtained results were compared with data from other rotifers and it was concluded that the muscles pars coronalis and the corona sphincter probably represent conditions in Ploima or Monogononta, while incomplete circular muscles and dorsal and ventral trunk retractors might be part of the eurotatorian ground pattern.     

绣球属(Hydrangea L.)及近缘属花粉形态的研究

为深入研究绣球属植物花粉形态的分类学价值和系统学意义,厘清绣球属与近缘属之间的系统发育关系,该文利用扫描电子显微镜(SEM,scanning electron microscope)对国产绣球属及其近缘属41种绣球花科(Hydrangeaceae)植物的花粉形态以及表面纹饰进行了观察。结果表明:绣球属及其近缘属的花粉为三孔沟;形状多数为长圆体形或近球体形;赤道面观为椭圆形或圆形;极面观多为圆形,少数为三角形或圆三角形。花粉外壁纹饰可分为网状和孔穴状。网眼内的三级纹饰可分为光滑和具颗粒状突起。根据花粉形状和外壁纹饰类型将上述物种划分为4个组,即花粉的形状为长圆体形,表面纹饰为孔穴纹饰;花粉的形状为长圆体形,表面纹饰为网状纹饰;花粉的形状为近球体形,表面纹饰为孔穴纹饰;花粉的形状为近球体形,表面纹饰为网状纹饰。以上可进一步细分为8个类型。上述表明花粉形态证据可为绣球属及其近缘属的属下分类和种间界定提供重要佐证;但结合前人的系统发育重建分析该属植物花粉形态的系统学意义相对有限,如花粉形态证据对于该属及其近缘种属系统发育树上大支的界定难以提供有力的证据。     

富宁附地菜果实形态特征及其分类学意义

紫草科(Boraginaceae)附地菜属(Trigonotis)的果实形态具有重要的分类学意义,是该属组级水平划分的重要依据.而该属之中的中国特有种富宁附地菜(T.funingensis)果实形态特征尚不清楚,其组级水平分类学地位的归属尚存疑问.野外考察在广西那坡采集到该种成熟果实,在解剖镜和扫描电镜下观察形态特征并...     

In Vitro Model of Bacterial Biofilm Formation on Polyvinyl Chloride Biomaterial

The aim of the study was to establish an in vitro model of Staphylococcus epidermidis biofilms on polyvinyl chloride (PVC) material, and to investigate bacterial biofilm formation and its structure using the combined approach of confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM). Staphylococcus epidermidis bacteria (stain RP62A) were incubated with PVC pieces in Tris buffered saline to form biofilms. Biofilm formation was examined at 6, 12, 18, 24, 30, and 48 h. Thicknesses of these biofilms and the number, and percentage of viable cells in biofilms were measured. CT scan images of biofilms were obtained using CLSM and environmental SEM. The results of this study showed that Staphylococcus epidermidis biofilm is a highly organized multi-cellular structure. The biofilm is constituted of large number of viable and dead bacterial cells. Bacterial biofilm formation on the surface of PVC material was found to be a dynamic process with maximal thickness being attained at 12–18 h. These biofilms became mature by 24 h. There was significant difference in the percentage of viable cells along with interior, middle, and outer layers of biofilms (P < 0.05). Staphylococcus epidermidis biofilm is sophisticated in structure and the combination method involving CLSM and SEM was ideal for investigation of biofilms on PVC material.     

The organization of the muscle system of the causative agent of dicrocoeliosis,Dicrocoelium dendriticum

The musculature of parasitic flatworms plays a central role in locomotory movement, attachment to the host, and in the function of the digestive, reproductive, and excretory systems. We examine for the first time the muscle system of the flatworm Dicrocoelium dendriticum, a causative agent of the parasitic disease dicrocoeliosis, by use of fluorescently labeled phalloidin and confocal laser scanning microscopy. Somatic musculature of D. dendriticum consists of the circular, longitudinal, and diagonal muscles. The distribution of the muscle fibers in the body wall differed among the anterior, middle, and posterior body regions of the worm. The musculature of the attachment organs, the oral and ventral suckers, includes several types of muscles: the external equatorial and meridional muscles, internal circular and semicircular muscles, and radial muscles. Inside of the ventral sucker the diagonally located muscles were revealed and the supplementary u-shaped muscles were found adjoined to the base of the sucker from outside. The musculature of the internal organs composed of the excretory, reproductive, and digestive systems were characterized. Our results increase our knowledge of the morphology of trematodes and the arrangement of their muscle system.     

中华白花丹参与其近缘种的形态分类鉴定

该研究采用光学显微镜和扫描电镜相结合的方法,对中华白花丹参(Salvia sinealba)与其近缘种丹参(S.miltiorrhiza)及山东丹参(S.shandongensis)根的解剖学、叶表皮、花及花粉粒进行了系统地比较研究。结果表明:中华白花丹参(S.sinealba)根的中央部位有多数薄壁细胞,在根横切面射线薄壁细胞间及韧皮部外侧与次生皮层部位有多数裂隙;叶片上表皮细胞外平周壁具有细密平行丝状褶皱(SEM);花冠白色;花粉粒椭圆形,外壁具网状雕纹,网眼较大,多角形,少为单穿孔。上述这些显著的特征,不仅为中华白花丹参在植物分类学上的地位提供了形态解剖学依据,而且为近缘种丹参及山东丹参的分类鉴定提供了佐证,同时也为中华白花丹参这一特有新资源的保护和开发利用提供了资料。     

Preparation of chromosome spreads for electron (TEM,SEM, STEM), light and confocal microscopy

In the past, ultrastructural studies on chromosome morphology have been carried out using light microscopy, scanning electron microscopy and transmission electron microscopy of whole mounted or sectioned samples. Until now, however, it has not been possible to use all of these techniques on the same specimen. In this paper we describe a specimen preparation method that allows one to study the same chromosomes by transmission, scanning-transmission and scanning electron microscopy, as well as by standard light microscopy and confocal microscopy. Chromosome plates are obtained on a carbon coated glass slide. The carbon film carrying the chromosomes is then transferred to electron microscopy grids, subjected to various treatments and observed. The results show a consistent morphological correspondence between the different methods. This method could be very useful and important because it makes possible a direct comparison between the various techniques used in chromosome studies such as banding, in situ hybridization, fluorescent probe localization, ultrastructural analysis, and colloidal gold cytochemical reactionsAbbreviations CLSM confocal laser scanning microscope - EM electron microscopy - kV kilovolt(s) - LM light microscope - SEM scanning electron microscope - STEM scanning-transmission electron microscope - TEM transmission electron microscope     

Structure and occurrence of cyphonautes larvae (bryozoa,ectoprocta)

We have studied larvae of the freshwater ctenostome Hislopia malayensis with scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and LM of serial sections. Some additional observations on larvae of M. membranacea using SEM and CLSM are also reported. The overall configuration of muscles, nerves, and cilia of the two larvae are identical. However, the larva of H. malayensis is much smaller than that of M. membranacea, which may explain most of the differences observed. Although all major nerves and muscle strands are present in H. malayensis, they are generally composed of fewer fibers. The H. malayensis larva lacks the anterior and posterior intervalve cilia. Its pyriform organ is unciliated with only a small central depression. The adhesive epithelium is not invaginated as an adhesive sac and lacks the large muscles interpreted as adhesive sac muscles in the M. membranacea larva. The velum carries two rows of ciliated cells, though the lower “row” consists of only one or two cells. Both rows of ciliated cells are innervated by nerves, which have not been detected in the M. membranacea larva. The ciliated ridge of H. malayensis lacks the frontal cilia. The planktotrophic cyphonautes larvae in a number of ctenostome clades and in the “basal” cheilostome clade Malacostega (and probably in the earliest cheilostomes) support the idea that the cyphonautes larva is the ancestral larval type of the Eurystomata. It may even represent the ancestral larval type of the bryozoans (= ectoprocts). J. Morphol. 271:1094‐1109, 2010. © 2010 Wiley‐Liss, Inc.     

Do Rotifer Jaws Grow After Hatching?

The hard articulated jaws of some pseudocoelomate metazoans were recently used in reconstructing their phylogenetic relationships, but we still do not know if these structures could change in size and shape during the life of individuals, and experimental data are lacking on their post-embryonic development. Rotifers are one of the groups in which hard articulated jaws, called trophi, are well known, and are widely used taxonomically. Here we report on SEM study of trophi of rotifers of different ages, to determine if the trophi structures change in shape and/or in size during post-embryonic development. We used linear measurements and geometric morphometrics analyses from scanning electron microscopic pictures of trophi of Cupelopagis vorax, Dicranophorus forcipatus, Macrotrachela quadricornifera, Notommata glyphura, Rotaria macrura, R. neptunoida, and R. tardigrada. Results for these species show that trophi do not change after hatching, either in size or in shape. In contrast, data on Asplanchna priodonta reveal trophi growth after hatching.     

Cadophora finlandia and Phialocephala fortinii: Agrobacterium-mediated transformation and functional GFP expression

Hygromycin B resistance was transferred to the sterile mycelia of Cadophora finlandia and Phialocephala fortinii by co-cultivation with Agrobacterium tumefaciens. Constitutively expressed green fluorescent protein (GFP) was also introduced using the same vector. Confocal laser scanning microscopy (CLSM) revealed strong fluorescence of transformants. Both traits were mitotically stable during one year of subculturing on non-selective growth medium. Southern blot analysis showed that the majority of the transformants contained single-copy integrations at random sites in the genome.     

Structure of first- and second-stage mineralized elements in teeth of the sea urchin Lytechinus variegatus

Microstructure of the teeth of the sea urchin Lytechinus variegatus was investigated using optical microscopy, SEM (scanning electron microscopy) and SIMS (secondary ion mass spectroscopy). The study focused on the internal structure of the first-stage mineral structures of high Mg calcite (primary, secondary and carinar process plates, prisms) and on morphology of the columns of second-stage mineral (very high Mg calcite) that cement the first-stage material together. Optical micrographs under polarized light revealed contrast in the centers (midlines) of carinar process plates and in prisms in polished sections; staining of primary and carinar process plates revealed significant dye uptake at the plate centers. Demineralization with and without fixation revealed that the midlines of primary and carinar process plates (but not secondary plates) and the centers of prisms differed from the rest of the plate or prism, and SIMS showed proteins concentrated in these plate centers. SEM was used to study the morphology of columns, the fracture surfaces of mature teeth and the 3D morphology of prisms. These observations of internal structures in plates and prisms offer new insight into the mineralization process and suggest an important role for protein inclusions within the first-stage mineral. Some of the 3D structures not reported previously, such as twisted prisms and stacks of carinar process plates with nested wrinkles, may represent structural strengthening strategies.     

Organisation of body musculature in Encentrum mucronatum Wulfert, 1936, Dicranophorus forcipatus (O. F. Müller, 1786) and in the ground pattern of Ploima (Rotifera: Monogononta)

Fluorescence-labelled phalloidin in combination with confocal laser scanning microscopy (cLSM) has been used to reconstruct the body musculature in Encentrum mucronatum and Dicranophorus forcipatus in order to gain insight into the architecture of body musculature in representatives of the hitherto uninvestigated Dicranophoridae.

In both species, a system of outer circular and inner longitudinal muscles has been found. In E. mucronatum, seven circular muscles (musculi circulares I–VII) and six paired longitudinal muscles (musculi longitudinales I–VI) have been identified. In D. forcipatus, eight circular muscles (musculi circulares I–VIII) and nine paired longitudinal muscles (musculi longitudinales I–IX) are present. In both species, some of the longitudinal muscles span the whole specimen, while others are shorter and connect head and trunk or foot and trunk. Differences in shape and extension of the circular muscles in both species are related to differences in structure of the trunk integument.

Surveying the literature on rotifer musculature, muscles identified in this study are homologised across Rotifera and given individual names. Based on the study of E. mucronatum and D. forcipatus and previous studies on other rotifers, a system of musculature in the ground pattern of Ploima comprising at least three circular muscles (pars coronalis, corona sphincter, musculus circumpedalis) and three pairs of longitudinal muscles (musculi longitudinales ventrales, musculi longitudinales dorsales and musculi longitudinales capitum) is suggested.     

中国五种仙茅科植物叶形态及其分类学意义

为澄清仙茅科属间界限不清的分类学问题,该文以中国仙茅科3属5种植物为研究对象,利用显微镜、扫描电镜和石蜡切片技术,观察了其叶形态、叶表皮显微特征和叶解剖特征。结果表明:(1)叶形态有小型平整叶、中型波状叶、大型折扇状叶三种。(2)叶表皮毛状体结构为单细胞单列,可分为长柔毛、糙伏毛和星状柔毛三类。(3)叶表皮细胞有六边形和五边形,气孔为平列型和椭圆形,气孔大小和气扎密度呈反比。(4)叶表皮蜡质纹饰有光滑、颗粒、屑状和壳状四类。(5)叶中脉横切面分为平整型和龙骨型,维管束有圆形和椭圆形,叶表皮厚度与表皮细胞具有正相关性。对5种植物的叶形态和叶解剖特征比较分析认为,一些特征组合有助于理解仙茅科属间的亲缘关系和物种鉴定,支持大叶仙茅属独立于仙茅属。     

Comparative morphology of the somatic musculature in species of Hexarthra and Polyarthra (Rotifera, Monogononta): Its function in appendage movement and escape behavior

Species of Hexarthra and Polyarthra are freshwater rotifers with well-known escape behaviors that result from interactions with planktonic predators. Both rotifers bear a suite of mobile appendages that function in evasive maneuvers and saltatory jumps through the water column, but the anatomical and functional bases of these actions are poorly understood. Here, we use a combination of phalloidin staining, confocal laser scanning microscopy, and video analysis to describe the morphology of the somatic muscles that supply the mobile appendages in order to understand how they function in escape behavior. Results show that species of Hexarthra, which bear six radially distributed limbs, possess a highly complex trunk musculature that supplies the inside of each limb with its own abductor and adductor muscles, i.e., a direct muscle supply. The singular dorsal and ventral limbs each receive a pair of large abductor and adductor muscles (four muscles total per limb), while the paired dorsolateral and ventrolateral limbs each receives three muscles (two abductors, one adductor per limb). Contraction of the abductor muscles creates a power stroke in the form of an anterior sweep of the limbs, which leads to a three-dimensional tumbling of the rotifer through the water column. Alternatively, species of Polyarthra possess 12 blade-like appendages that are arranged into four equal bundles; each bundle receives an indirect muscle supply that attaches to the shoulder of the paddles. A single longitudinal paddle muscle supplies each dorsolateral bundle, while a pair of longitudinal paddle muscles supplies each ventrolateral bundle. Contraction of these muscles, whether singly or in concert, functions to abduct the paddles in a power stroke, leading to rotation of the body and movement of the rotifer. The recovery stroke is hypothesized to be a multi-step process that begins with reorientation of the appendages prior to adduction, followed by contraction of various muscles to antagonize the paddle muscles. In total, these observations reveal novel complexities in the rotifer muscular system that aids our understanding of the biophysics of predator avoidance in appendage-bearing rotifers.     

黑龙江悬钩子属植物叶表皮形态结构的研究

利用扫描电子显微镜对黑龙江悬钩子属植物的叶表皮形态结构进行比较研究。结果显示:(1)悬钩子属植物叶的上表皮细胞呈多边形,垂周壁平直,或无规则形,垂周壁浅波纹;下表皮细胞无规则形,垂周壁浅波纹或深波纹。(2)表皮毛类型有单细胞直立不分支、卷曲不分支,头状腺毛和盾状腺毛四种类型。(3)气孔器均分布于下表皮,且气孔器类型为无规则形;气孔外拱盖单层、内缘平滑或不规则波状。研究表明,黑龙江悬钩子属植物的叶表皮微形态学特征表现出一定差异性,对种间的划分和鉴定具有一定的分类学意义。     

Three-dimensional reconstruction of the naupliar musculature and a scanning electron microscopy atlas of nauplius development of Balanus improvisus (Crustacea: Cirripedia: Thoracica)

An atlas of the naupliar development of the cirripede Balanus improvisus Darwin, 1854 using scanning electron microscopy (SEM) is provided. Existing spikes on the hindbody increase in number with each moult and are an applicable character for identification of the different nauplius stages, as is the setation pattern of the first antennae. The naupliar musculature of B. improvisus was stained with phalloidin to visualise F-actin, followed by analysis using confocal laser scanning microscopy (CLSM) with subsequent application of 3D imaging software. The larval musculature is already fully established in the first nauplius stage and remains largely unchanged during all the six nauplius stages. The musculature associated with the feeding apparatus is highly elaborated and the labrum possesses lateral muscles and distal F-actin-positive structures. The alimentary tract is entirely surrounded by circular muscles. The extrinsic limb musculature comprises muscles originating from the dorsal and the ventral sides of the head shield, respectively. The hindbody shows very prominent postero-lateral muscles that insert on the dorso-lateral side of the head shield and bend towards ventro-posterior. We conclude that the key features of the naupliar gross anatomy and muscular architecture of B. improvisus are important characters for phylogenetic inferences if analysed in a comparative evolutionary framework.