Ultrastructure of the extraordinary pedal gland in Asplanchna aff. herricki (Rotifera: Monogononta).

Rotifers possess complex morphologies despite their microscopic size and simple appearance. Part of this complexity is hidden in the structure of their organs, which may be cellular or syncytial. Surprisingly, organs that are cellular in one taxon can be syncytial in another. Pedal glands are widespread across Rotifera and function in substrate attachment and/or egg brooding. These glands are normally absent in Asplanchna, which lack feet and toes that function as outlets for pedal glandular secretions in other rotifers. Here, we describe the ultrastructure of a pedal gland that is singular and syncytial in Asplanchna aff. herricki, but is normally paired and cellular in all other rotifers. Asplanchna aff. herricki has a single large pedal gland that is active and secretory; it has a bipartite, binucleate, syncytial body and a cytosol filled with rough endoplasmic reticulum, Golgi, and several types of secretory vesicles. The most abundant vesicle type is large and contains a spherical electron-dense secretion that appears to be produced through homotypic fusion of condensing vesicles produced by the Golgi. The vesicles appear to undergo a phase transition from condensed to decondensed along their pathway toward the gland lumen. Decondensation changes the contents to a mucin-like matrix that is eventually exocytosed in a "kiss-and-run" fashion with the plasma membrane of the gland lumen. Exocytosed mucus enters the gland lumen and exits through an epithelial duct that is an extension of the syncytial integument. This results in mucus that extends from the rotifer as a long string as the animal swims through the water. The function of this mucus is unknown, but we speculate it may function in temporary attachment, prey capture, or floatation.

Comparison of the life-history parameters and competition outcome with Moina macrocopa between two morphs of Brachionus forficula.

In rotifers, the costs of morphological defenses, especially the development of long spines, have been investigated for several decades. However, the obtained results were inconsistent and the underlying reasons were complicated. Investigations on more species might be helpful to find out the reasons. In the present study, Brachionus forficula was selected as the model organism. The differences in developmental durations, life-table demography, starvation resistant time and the competitive ability with Moina macrocopa were compared between B. forficula with long (LPS) and short (SPS) posterior spines. The results showed that LPS showed relatively longer durations of juvenile stage at 1.0 × 106, 2.0 × 106 and 4.0 × 106 cells/ml Scenedesmus obliquus, and longer embryo stage at 2.0 × 106 cells/ml S. obliquus than SPS. The intrinsic rate of population increase and net reproduction rate were lower in LPS than SPS, suggesting the energy input to reproduction decreased. The starvation resistant time was also reduced in LPS, in comparison to SPS, further supporting that LPS consumed more energy, which might be directed to the development of long spines. All these results revealed that LPS spent more energy for individual growth than SPS, which might be used to develop long spines. Moreover, the maximum population density and population growth rate of LPS were always lower than those of SPS, suggesting that LPS might have a weaker competition ability with M. macrocope than SPS.

Epibiotic rotifers of Gammarus pulex (L.) (Crustacea, Amphipoda), with descriptions of two new species and notes on the terminology of the trophi.

The epibiotic rotifers of a Gammarus pulex (L.) collection from the Ardèche, France are reported. Four species were found, of which Cephalodella jersabeki n. sp. and Proales gammaricola n. sp. are new to science and described. The other species, Dicranophorus cambari Wulfert and Embata laticeps (Murray), are commented upon. SEM micrographs of the trophi are presented, and a new or amended terminology is proposed for some diagnostic trophi parts.

Polychromatic polarization microscope: bringing colors to a colorless world.

Interference of two combined white light beams produces Newton colors if one of the beams is retarded relative to the other by from 400 nm to 2000 nm. In this case the corresponding interfering spectral components are added as two scalars at the beam combination. If the retardance is below 400 nm the two-beam interference produces grey shades only. The interference colors are widely used for analyzing birefringent samples in mineralogy. However, many of biological structures have retardance <100 nm. Therefore, cells and tissues under a regular polarization microscope are seen as grey image, which contrast disappears at certain orientations. Here we are proposing for the first time using vector interference of polarized light in which the full spectrum colors are created at retardance of several nanometers, with the hue determined by orientation of the birefringent structure. The previously colorless birefringent images of organelles, cells, and tissues become vividly colored. This approach can open up new possibilities for the study of biological specimens with weak birefringent structures, diagnosing various diseases, imaging low birefringent crystals, and creating new methods for controlling colors of the light beam.

Spermatozoon of the freshwater rotifer Brachionus calyciflorus (Rotifera, Monogononta): Advances in morphological and ultrastructural studies.

The morphological and ultrastructural features of the spermatozoon in Brachionus calyciflorus are described using light, fluorescence and transmission electron microscopy (TEM). The mature spermatozoon, which appears to be thread-like, is composed of a slightly expanded anterior of cell body region and a flagellum region without acrosome. The cell body region and flagellum region are respectively 16-27µm and 20-33µm in length (n=60). The spermatozoon is characterized by a mass of dense tubular materials, which occupy most of the cell. Some mitochondria are distributed around the nuclear region in the anterior of the cell body region, while in the posterior portion of cell body, the chromatin often contains a single lobated nucleus arranged at the center of cell. The flagellum contains the classic axoneme (9×2+2) and possesses lateral undulating membrane. Mature B. calyciflorus males have no germ cell stages earlier than the spermatids in the testis. TEM examination reveals rigid rods as well as predominant typical spermatozoon in the testis. Observations, based on successive photographs and videos, enabled a first-time recording of the unique inverted movement of the spermatozoon, which indicated that the movement of the spermatozoon is driven by the flagellum. Our study also provides further supplementary insights into the phylogenetic systematics of the Rotifera.

Morphological alterations in the freshwater rotifer Brachionus calyciflorus Pallas 1766 (Rotifera: Monogononta) caused by vinclozolin chronic exposure.

Vinclozolin (VZ) is a dicarboximide fungicide widely used on fruits, vegetables and wines, effective against fungi plagues. In this study we characterized the effects of VZ using a 4-day reproductive chronic assay with the freshwater rotifer Brachionus calyciflorus. The assay included observations of several features of asexual and sexual reproduction. Our results indicate that VZ: (a) increased asexual and sexual reproduction, (b) caused severe abnormality in females and (c) these abnormalities were inherited by sexual and asexual reproduction. At 1.2 mg/L three abnormal females were found out of 457 total females (0.66 %). This low percentage is consistent and reproducible according to further analysis, where we increased the number of replicates and total females exposed to 1.2 mg/L of VZ, and found 18 abnormal females out of 2868 total females (0.63 % abnormality). Interestingly, abnormal females found at 5.6 mg/L VZ exposure, were able to show mating behavior. Our results suggest that VZ behaves as a strong endocrine disruptor whose effects show the characteristic inverted-U-shape exposure concentration response curve regarding the intrinsic population increase and the percentage of abnormalities as endpoints.

Morphology and ultrastructure of the freshwater rotifer Brachionus bidentatus (Monogononta: Brachionidae) using scanning and transmission electron microscopy / Morfología y ultraestructura del rotífero de agua dulce Brachionus bidentatus (Monogononta: Brachionidae), utilizando microscopía electrónica de barrido y transmisión

The study of sexual reproductive behavior supported by ultrastructural evidence is important in rotifers to describe differences among potential cryptic species. In this research, the morphology of the rotifer Brachionus bidentatus is described at the ultrastructural level, using electronic microscopy, together with a brief description and discussion of its sexual reproductive behavior. The characteristics of the (a) male,(b) the female, (c) the sexual egg or cyst, (d) the partenogenic egg, (e) the no-fecundated sexual egg (male egg), and (f) the trophi, were described. Another part of this research is dedicated to the ultrastructure of the sex cells of the male rotifer B. bidentatus. Samples were obtained from La Punta pond in Cosio, Aguascalientes, Mexico (22°08’ N - 102°24’ W), and a culture was maintained in the laboratory. Fifty organisms, from different stages of the rotifer Brachionus bidentatus, were fixed in Formol at 4% and then prepared; besides, for the trophi, 25 female rotifer Brachionus bidentatus were prepared for observation in a JEOL 5900 LV scanning electronic microscope. In addition, for the observation of male sex cells, 500 males of Brachionus bidentatus were isolated, fixed and observed in a JEOL 1010 transmission microscope. Females of B. bidentatus in laboratory cultures had a lifespan of five days (mean±one SD=4.69±0.48; N=13), and produced 4.5+3.67 (N=6) parthenogenetic eggs during such lifespan. In the case of non-fertilized sexual eggs, they produced up to 18 eggs (mean±one SD=13±4.93; N=7). Sexual females produced a single cyst on average (mean±one SD=1±0; N=20). For the sexual cycle, the time of copulation between male and female ranged from 10 to 40 seconds (mean±one SD=17.33±10.55, N=7). The spermatozoa are composed of a celular body and a flagellum, the size of the body is of 300nm while the flagellum measures 1 700nm. The rods have a double membrane. Their mean length is almost 2.45µm±0.74, N=6; and their mean wide is 0.773µm±0.241, N=11. The evidence on the specific ultrastructural characteristics of the rotifer B. bidentatus is notorious, even more in the male and in the cyst cell. Regarding the ultrastructure of the spermatozoa and the rods, compared to other species they only differ in size, despite their structural resemblance. Our study of the ultraestructure of this species adds useful information that along with molecular data will help clarify the taxonomy of brachionid rotifers. Rev. Biol. Trop. 61 (4): 1737-1745. Epub 2013 December 01.

El estudio del comportamiento reproductivo sexual apoyado en evidencias ultraestructurales en rotíferos, es importante para describir diferencias entre especies potencialmente crípticas. En este trabajo se describe a nivel ultraestructural la morfología del rotífero Brachionus bidentatus, usando microscopía electrónica, junto con una breve descripción y discusión de su comportamiento sexual reproductivo. Se presentan las características del: (a) macho, (b) hembra, (c) huevo partenogenético, (d) huevo sexual no fecundado, (e) trofos. También se muestra un apartado sobre la ultra estructura de las células sexuales de rotíferos macho B. bidentatus. Para el estudio se utilizó una cepa proveniente del bordo La Punta (22°08’ N - 102°24’ W), ubicado en Cosió, estado de Aguascalientes, México y se cultivaron en el laboratorio. Para el procesamiento de las muestras se tomaron 50 organismos fijados en Formol al 4%, de los diferentes estadios del rotífero Brachionus bidentatus, mientras que para el trofos se tomaron 25 organismos hembra del rotífero Brachionis bidentatus y se prepararon para observarse en un microscopio electrónico de barrido JEOL 5900 LV, mientras que para las observaciones de las células sexuales del macho se aislaron 500 organismos machos del rotífero Brachionus bidentatus, se fijaron e incluyeron en resina epóxica (EPON) para su observación en un microscopio electrónico de transmisión JEOL 1010 operado a 80kv. Los análisis obtenidos de hembras de B. bidentatus en cultivos de laboratorio demuestran un ciclo de vida de cinco días (4.69±0.48; N=13), y una producción de 4.5±3.67 (N=6) huevos partenogenéticos. En el caso de los huevos no fertilizados, la hembra produce más de 18 huevos (13±4.93; N=7). Para los huevos sexuales solo se produce uno solo por hembra (1±0; N=20). En el ciclo sexual, los tiempos de copula entre el macho y la hembra están en el rango de 10 a 40 segundos (17.33±10.55, N=7). Los espermatozoides se componen de un cuerpo celular y un flagelo, el tamaño del cuerpo celular es de 300nm mientras que el flagelo mide 1 700nm. Los bastones presentan una doble membrana y su tamaño a lo largo va de 2.45µm±0.74; N=6 mientras que el ancho es de 0.773µm±0.241; N=11. La evidencia sobre las características específicas ultraestructurales del rotífero B. bidentatus son notorias, más aun en el macho y en el quiste. En cuanto a la ultraestructura de los espermatozoides y los bastones respecto a otras especies sólo difieren en su tamaño, a pesar de las similitudes estructurales las estructuras de los bastones se evidencian con mayor claridad y dan más evidencias sobre su funcionalidad. Nuestro estudio de la ultraesturctura de esta especie añade información útil que junto con un análisis molecular ayudarán a clarificar la taxonomía de rotíferos brachionidos.

Morphology and ultrastructure of the freshwater rotifer Brachionus bidentatus (Monogononta: Brachionidae) using scanning and transmission electron microscopy.

The study of sexual reproductive behavior supported by ultrastructural evidence is important in rotifers to describe differences among potential cryptic species. In this research, the morphology of the rotifer Brachionus bidentatus is described at the ultrastructural level, using electronic microscopy, together with a brief description and discussion of its sexual reproductive behavior. The characteristics of the (a) male, (b) the female, (c) the sexual egg or cyst, (d) the partenogenic egg, (e) the no-fecundated sexual egg (male egg), and (f) the trophi, were described. Another part of this research is dedicated to the ultrastructure of the sex cells of the male rotifer B. bidentatus. Samples were obtained from La Punta pond in Cosio, Aguascalientes, Mexico (22 degrees 08' N - 102 degrees 24' W), and a culture was maintained in the laboratory. Fifty organisms, from different stages of the rotifer Brachionus bidentatus, were fixed in Formol at 4% and then prepared; besides, for the trophi, 25 female rotifer Brachionus bidentatus were prepared for observation in a JEOL 5900 LV scanning electronic microscope. In addition, for the observation of male sex cells, 500 males of Brachionus bidentatus were isolated, fixed and observed in a JEOL 1010 transmission microscope. Females of B. bidentatus in laboratory cultures had a lifespan of five days (mean+one SD = 4.69 +/- 0.48; N=13), and produced 4.5 +/- 3.67 (N=6) parthenogenetic eggs during such lifespan. In the case of non-fertilized sexual eggs, they produced up to 18 eggs (mean+one SD = 13 +/- 4.93; N=7). Sexual females produced a single cyst on average (mean +/- one SD = I +/- 0; N=20). For the sexual cycle, the time of copulation between male and female ranged from 10 to 40 seconds (mean +/- one SD = 17.33 +/- 10.55, N=7). The spermatozoa are composed of a celular body and a flagellum, the size of the body is of 300 nm while the flagellum measures 1 700nm. The rods have a double membrane. Their mean length is almost 2.45 microm +/- 0.74, N=6; and their mean wide is 0.773 microm +/- 0.241, N=11. The evidence on the specific ultrastructural characteristics of the rotifer B. bidentatus is notorious, even more in the male and in the cyst cell. Regarding the ultrastructure of the spermatozoa and the rods, compared to other species they only differ in size, despite their structural resemblance. Our study of the ultraestructure of this species adds useful information that along with molecular data will help clarify the taxonomy of brachionid rotifers.

Surviving starvation: changes accompanying starvation tolerance in a bdelloid rotifer.

Bdelloid rotifers survive desiccation and starvation by halting activity and entering a kind of dormancy. To understand the mechanisms of survival in the absence of food source, we studied the anatomical and ultrastructural changes occurring in a bdelloid species, Macrotrachela quadricornifera Milne 1886, after starvation for different periods. The starved rotifers present a progressive reduction of body size accompanied with a consistent reduction of the volume of the stomach syncytium, where lipid inclusions and digestive vacuoles tend to fade with prolonged starvation. Similar reduction occurs in the vitellarium gland, in which yolk granules progressively decrease in number and size. The changes observed in the syncytia of the stomach and the vitellarium suggest that during starvation M. quadricornifera uses resources diverted from the stomach syncytium first and from the vitellarium syncytium later, resources that are normally allocated to reproduction. The fine structure of starved bdelloids is compared with that of anhydrobiotic bdelloids, revealing that survival during either forms of dormancy is sustained by different physiological mechanisms.

Dry and survive: morphological changes during anhydrobiosis in a bdelloid rotifer.

Bdelloid rotifers are aquatic microinvertebrates able to cope with the loss of environmental water by entering dormancy, and are thus capable of living in temporary habitats. When water is evaporating, bdelloids contract into "tuns", silence metabolism and lose water from the body, a condition known as anhydrobiosis. Under controlled conditions, a bdelloid species (Macrotrachela quadricornifera) was made anhydrobiotic, and its morphology was studied by light, confocal and electron microscopy. A compact anatomy characterizes the anhydrobiotic rotifer, resulting in a considerable reduction of its body volume: the internal organs, precisely packed together, occupy the body cavity almost completely and the lumen of hollow organs disappears. Remarkable ultrastructural changes characterize the anhydrobiotic condition. The mitochondria are wholly surrounded by a ring of electron-dense particles, and the epidermal pores, open in the hydrated specimens, become gradually closed by structures similar to epithelial junctions. The cilia are densely packed: microtubules are still identifiable, but the axonemal organization appears disrupted. This is the first extensive comparative study on the morphological changes associated with the anhydrobiosis process in a rotifer, providing the basis for an improved understanding of the processes involved in this extreme adaptation.

Ultrastructure and function of the mastax in Dicranophorus forcipatus (Rotifera: Monogononta).

Rotifers are characterized by a complex set of cuticularized jaw elements in the pharynx. The fine structure of the jaw elements has been the subject of SEM studies for some time, but only very limited information exists on the ultrastructure of the jaw elements and their function beyond taxonomic considerations. Drawing on SEM and TEM techniques, the present study presents a detailed analysis of the mastax in Dicranophorus forcipatus, a carnivorous monogonont rotifer species from freshwater habitats characterized by an extrusible, grasping jaw apparatus. Based on ultrathin serial sections, the jaw elements are reconstructed and, in total, nine paired and two unpaired muscles identified. Possibly homologous muscles in other rotifer species are discussed and functional considerations of the forcipate mastax are suggested.

Volume and morphology changes of a bdelloid rotifer species (Macrotrachela quadricornifera) during anhydrobiosis.

Following a study on the changes occurring in a bdelloid species (Macrotrachela quadricornifera, Rotifera, Bdelloidea) when entering anhydrobiosis, we investigated the changes in morphology, including weight and volume during the transition from the active hydrated to the dormant anhydrobiotic state by scanning electron microscopy, confocal microscopy and light microscopy. We compared sizes and morphologies of hydrated extended, hydrated contracted and anhydrobiotic specimens. Bdelloid musculature is defined: longitudinal muscles are contracted in the hydrated contracted animal (head and foot are retracted inside the trunk), but appear loose in the anhydrobiotic animal. When anhydrobiotic, M. quadricornifera appears much smaller in size, with a volume reduction of about 60% of the hydrated volume, and its internal organization undergoes remarkable modifications. Internal body cavities, clearly distinguishable in the hydrated extended and contracted specimens, are no longer visible in the anhydrobiotic specimen. Concomitantly, M. quadricornifera loses more than 95% of its weight when anhydrobiotic; this is more than expected from the volume reduction data and could indicate the presence of space-filling molecular species in the dehydrated animal. We estimate that the majority of body mass loss and volume reduction can be ascribed to the water loss from the body cavity during desiccation.

Independently evolving species in asexual bdelloid rotifers.

Asexuals are an important test case for theories of why species exist. If asexual clades displayed the same pattern of discrete variation as sexual clades, this would challenge the traditional view that sex is necessary for diversification into species. However, critical evidence has been lacking: all putative examples have involved organisms with recent or ongoing histories of recombination and have relied on visual interpretation of patterns of genetic and phenotypic variation rather than on formal tests of alternative evolutionary scenarios. Here we show that a classic asexual clade, the bdelloid rotifers, has diversified into distinct evolutionary species. Intensive sampling of the genus Rotaria reveals the presence of well-separated genetic clusters indicative of independent evolution. Moreover, combined genetic and morphological analyses reveal divergent selection in feeding morphology, indicative of niche divergence. Some of the morphologically coherent groups experiencing divergent selection contain several genetic clusters, in common with findings of cryptic species in sexual organisms. Our results show that the main causes of speciation in sexual organisms, population isolation and divergent selection, have the same qualitative effects in an asexual clade. The study also demonstrates how combined molecular and morphological analyses can shed new light on the evolutionary nature of species.

A modern approach to rotiferan phylogeny: combining morphological and molecular data.

The phylogeny of selected members of the phylum Rotifera is examined based on analyses under parsimony direct optimization and Bayesian inference of phylogeny. Species of the higher metazoan lineages Acanthocephala, Micrognathozoa, Cycliophora, and potential outgroups are included to test rotiferan monophyly. The data include 74 morphological characters combined with DNA sequence data from four molecular loci, including the nuclear 18S rRNA, 28S rRNA, histone H3, and the mitochondrial cytochrome c oxidase subunit I. The combined molecular and total evidence analyses support the inclusion of Acanthocephala as a rotiferan ingroup, but do not support the inclusion of Micrognathozoa and Cycliophora. Within Rotifera, the monophyletic Monogononta is sister group to a clade consisting of Acanthocephala, Seisonidea, and Bdelloidea-for which we propose the name Hemirotifera. We also formally propose the inclusion of Acanthocephala within Rotifera, but maintaining the name Rotifera for the new expanded phylum. Within Monogononta, Gnesiotrocha and Ploima are also supported by the data. The relationships within Ploima remain unstable to parameter variation or to the method of phylogeny reconstruction and poorly supported, and the analyses showed that monophyly was questionable for the families Dicranophoridae, Notommatidae, and Brachionidae, and for the genus Proales. Otherwise, monophyly was generally supported for the represented ploimid families and genera.

Morphological response of a bdelloid rotifer to desiccation.

We desiccated bdelloid rotifers (Macrotrachela quadricornifera), submitting the animals to four desiccation procedures (protocols A, B, C, D) that differed in the rate of water evaporation, in the time of desiccation, and in the substrates provided. We observed external morphological changes of the rotifer bodies during drying with scanning electron microscopy and, in parallel, assessed rates of recovery after a 7-day period of dormancy. Two protocols produced disorganized morphologies of the anhydrobiotic animals, with no (A) or very poor (B) recovery. Protocols C and D gave rather high rates of recovery and dry rotifers appeared unaltered and well organized. The different protocols affected rotifer morphology during the 7-day anhydrobiosis and rates of recovery after the 7-day anhydrobiosis; high recovery rates corresponded to well-organized morphologies of anhydrobiotic bdelloids, suggesting that a proper contraction of the body into a tun shape and probably a rigorous packing of internal structures are necessary for survival after anhydrobiosis. These features are affected by the time between water shortage and full desiccation, but also by the surrounding relative humidity and by the nature of the substrate. Possible adaptations of anhydrobiotic rotifers are discussed.

Ectosymbiotic bacteria on ciliated cells of a rotifer.

During the examination of Brachionus plicatilis (Rotifera, Monogononta), ectosymbiotic bacteria were found in the ciliated buccal region. These have about the same dimensions as the cilia and possess a specific apical region that apparently serves to attach the bacterium to its host cell. This apical region resembles a vesicle but is interpreted as a specialized region of the bacterial nucleocytoplasm devoid of ribosomes and chromatin strands but containing a crystalline rod. The bacterium has two membranes (as have other Gram-negative bacteria) also over the apical region. Thin strands join the two membranes and similar strands extend from the bacterium to the cell membrane of the host cell. No intracellular bacteria were found in this study.

Specific behavioural responses triggered by identified mechanosensory receptor cells in the apical field of the giant rotifer Asplanchna sieboldi.

The giant rotifer Asplanchna sieboldi swims by the propulsive effect of thousands of cilia arrayed in clusters around the apical field, which has several mechanosensory structures (sensilla) located at defined positions. Males and females differ in both their patterns of behaviour and their sensory receptor equipment. Unstimulated males swim straight with occasional spontaneous changes in direction until they hit an obstacle with their apical field. Depending on the direction and the strength of the mechanical interference, the animals show different behavioural responses. To analyse the effect of excitation of the apical mechanosensitive sensilla on these responses, males were held on microcapillaries, and the sensitivity of individual sensilla was assayed using micromanipulator-mediated mechanical stimulation. Stimulation of each of the four different types of sensillum triggered a specific and well-defined initial behavioural response. Individual animals behaved identically with respect to the receptor specificity of the responses. The behaviour of free-swimming males upon contact with obstacles or females is discussed on the basis of these results.

The hard parts (trophi) of the rotifer mastax do contain chitin: evidence from studies on Brachionus plicatilis.

The jaws (trophi) of the rotifer Brachionus plicatilis are soluble in strong acids but are resistant to long treatments by strong alkali. They show the same buoyant density as chitin and also as the chitin-containing layers of rotifer egg-shells. The presence of chitin in these structures was confirmed using the following techniques: chitosan-tests, thin-layer chromatography of trophi-hydrolysates which revealed glucosamine, by dissolving trophi with chitinase and electron microscopic WGA/gold-labelling. The content of chitin in the trophi was estimated by two different methods to be approx. 64% (50-75%).