On the serotonergic nervous system of two planktonic rotifers, Conochilus coenobasis and C. dossuarius (Monogononta, Flosculariacea, Conochilidae)

The serotonergic nervous systems of two non-colonial species of Conochilus were examined to obtain the first immunohistochemical insights into the neuroanatomy of species of Flosculariacea (Rotifera, Monogononta). Species of Conochilus, subgenus Conochiloides, were examined using serotonin (5-HT) immunohistochemistry, epifluorescence and confocal laser scanning microscopy, and 3D computer imaging software. In specimens of C. coenobasis and C. dossuarius, the serotonergic nervous system is defined by a dorsal cerebral ganglion, apically directed cerebral neurites, and paired nerve cords. The cerebral ganglion contains approximately four pairs of small 5-HT-immunoreactive perikarya; one pair innervates the posterior nerve cords and three pairs innervate the apical field. The most dorsal pair innervates a coronal nerve ring that encircles the apical field. Within the apical field is a second nerve ring that outlines the inner border of the coronal cilia. Together, both the inner and outer nerve rings may function to modulate ciliary activity of the corona. The other two pairs of perikarya innervate a region around the mouth. Specific differences in the distribution of serotonergic neurons between species of Conochilus and previously examined ploimate rotifers include the following: (a) a lack of immunoreactivity in the mastax; (b) a greater number of apically directed serotonergic neurites; and (c) a complete innervation of the corona in both species of Conochilus. These differences in nervous system immunohistochemistry are discussed in reference to the phylogeny of the Monogononta.     

Three‐dimensional reconstruction and neural map of the serotonergic brain of Asplanchna brightwellii (Rotifera,Monogononta)

The basic organization of the rotifer brain has been known for nearly a century; yet, fine details on its structure and organization remain limited despite the importance of rotifers in studies of evolution and population biology. To gain insight into the structure of the rotifer brain, and provide a foundation for future neurophysiologic and neurophylogenetic research, the brain of Asplanchna brightwellii was studied with immunohistochemistry, confocal laser scanning microscopy, and computer modeling. A three‐dimensional map of serotonergic connections reveals a complex network of approximately 28 mostly unipolar, cerebral perikarya and associated neurites. Cells and their projections display symmetry in quantity, size, connections, and pathways between cerebral hemispheres within and among individuals. Most immunopositive cells are distributed close to the brain midline. Three pairs of neurites form decussations at the brain midline and may innervate sensory receptors in the corona. A single neuronal pathway appears to connect both the lateral horns and dorsolateral apical receptors, suggesting that convergence of synaptic connections may be common in the afferent sensory systems of rotifers. Results show that the neural map of A. brightwellii is much more intricate than that of other monogonont rotifers; nevertheless, the consistency in neural circuits provides opportunities to identify homologous neurons, distinguish functional regions based on neurotransmitter phenotype, and explore new avenues of neurophylogeny in Rotifera. J. Morphol. 2009. © 2008 Wiley‐Liss, Inc.     

Behavior,metamorphosis, and muscular organization of the predatory rotifer Acyclus inquietus (Rotifera,Monogononta)

Abstract. The atrochid rotifer, Acyclus inquietus, is a sedentary predator that lives within the colonies of its prey, the rotifer Sinantherina socialis. After larvae infiltrate and become associated with the colony, they secrete a permanent gelatinous tube and undergo metamorphosis to the adult stage. We followed settlement and metamorphosis using bright-field microscopy to document specific larval behaviors after eclosion, and used epifluorescence and confocal microscopy of phalloidin-labeled specimens to visualize some of the morphological changes that occur during metamorphosis. Upon eclosion, larvae possess paired eyespots and a ciliated corona that functions strictly in locomotion. After leaving the parent's gelatinous tube, larvae eventually settle on unoccupied colonies of S. socialis or on other substrates if colonies are unavailable. Settlement involves a period of gliding among colony members before attachment with the foot and the secretion of a gelatinous tube. After settlement, there is a drastic reconfiguration of the corona that involves loss of the eyespots, loss of the coronal cilia, and the formation of the cup-shaped infundibulum, a deep depression in the anterior of the head that leads to the mouth. The development of the infundibulum involves the expansion of tissues around the mouth and is accompanied by a reorientation of the underlying musculature that supplies the infundibulum and allows its use in prey capture. The arrangement of the muscles in the trunk and foot regions, which contain outer circular (complete and incomplete) and inner longitudinal bands, remains unchanged between ontogenetic stages, and reflects the condition characteristic of other rotifers.     

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.     

Presence of anisotropic (birefringent) crystalline structures in embryonic and juvenile monogonont rotifers

A systematic survey for the presence of birefringent (anisotropic) structures in rotifers was undertaken. Several common features of rotifers exhibit anisotropism (e.g. trophi & muscles). However, unusual anisotropic crystalline structures (ACS) were found in late stage embryos (i.e. possessing eyespots and trophi, and showing movement). ACS were found in 18 of 26 species of monogonont rotifers (comprising 11 genera of 5 families). In Sinantherina socialis, ACS were present in the lower gut as compact, spherical masses of minute crystals that slowly broke apart and disappeared within 20 hours of hatching. Although several authors have described the existence of refractive bodies in rotifers, to my knowledge this is the first report of their birefringent properties.     

Ultrastructure of the rotifer integument: peculiarities of Sinantherina socialis (Monogononta: Gnesiotrocha)

The rotifer integument is a well‐described syncytium that contains an apical intracytoplasmic lamina (ICL) that functions for both skeletal support and muscle insertion. To date, there is limited information on the structure of the integument in species of Gnesiotrocha, a diverse subclade of Monogononta that consists of solitary, colonial, sessile, and planktonic species. In this study, we examined the ultrastructure of the integument in the colonial rotifer Sinantherina socialis to determine how it corresponds to that of other monogononts. The integument of S. socialis was broadly similar to that of other rotifers, consisting of a thickened glycocalyx, multilaminate ICL, and syncytial epidermis. However, it was different in several regards. The ICL consisted of three distinct layers from apical to basal: layer 1 consisted of at least two electron‐dense laminae; layer 2 was a thickened matrix of amorphous, electron‐dense material or was fibrous; and layer 3 was an electron‐dense lamina of varying thickness that covered the underlying syncytium. Significantly, layers 1 and 2 formed a ridge‐and‐groove like system of finger‐like projections across the trunk surface that has not been observed in other rotifers. A voluminous syncytial cytoplasm (up to 3 μm thick) was present beneath the ICL and was mostly electron lucent and with few organelles. Bundles of potential microtubules were scattered throughout the syncytium. We hypothesize that the voluminous cytoplasm with microtubules serves as skeletal support for the rotifer's sessile lifestyle, while the external ridges may function as a texture‐based deterrent to predators, or serves to trap secretions from the species' defensive glands. Basally, the epidermis was highly folded and bordered by a thin basal lamina that separated the plasmalemma from the blastocoel. Membrane‐bound vesicles were present throughout the integument's cytoplasm and are hypothesized to function in the secretion of extracellular matrix and in the maintenance of the ICL.     

Inflated organelle genomes and a circular-mapping mtDNA probably existed at the origin of coloniality in volvocine green algae

The volvocine lineage is a monophyletic grouping of unicellular, colonial and multicellular algae, and a model for studying the evolution of multicellularity. In addition to being morphologically diverse, volvocine algae boast a surprising amount of organelle genomic variation. Moreover, volvocine organelle genome complexity appears to scale positively with organismal complexity. However, the organelle DNA architecture at the origin of colonial living is not known. To examine this issue, we sequenced the plastid and mitochondrial DNAs (ptDNA and mtDNA) of the 4-celled alga Tetrabaena socialis, which is basal to the colonial and multicellular volvocines.

Tetrabaena socialis has a circular-mapping mitochondrial genome, contrasting with the linear mtDNA architecture of its relative Chlamydomonas reinhardtii. This suggests that a circular-mapping mtDNA conformation emerged at or near the transition to group living in the volvocines, or represents the ancestral state of the lineage as a whole. The T. socialis ptDNA is very large (>405 kb) and dense with repeats, supporting the idea that a shift from a unicellular to a colonial existence coincided with organelle genomic expansion, potentially as a result of increased random genetic drift. These data reinforce the idea that volvocine algae harbour some of the most expanded plastid chromosomes from the eukaryotic tree of life. Circular-mapping mtDNAs are turning out to be more common within volvocines than originally thought, particularly for colonial and multicellular species. Altogether, volvocine organelle genomes became markedly more inflated during the evolution of multicellularity, but complex organelle genomes appear to have existed at the very beginning of colonial living.     

Topology of the nervous system of Notommata copeus (Rotifera: Monogononta) revealed with anti-FMRFamide, -SCPb, and -serotonin (5-HT) immunohistochemistry

Abstract. The nervous system of the benthic freshwater rotifer, Notommata copeus , was examined using antibody probes, epifluorescence and confocal laser scanning microscopy, and digital imaging to highlight similarities with other monogonont rotifers. Immunoreactivity to anti-FMRFamide (Phe–Met–Arg–Phe–NH₂), -SCPb (small cardioactive peptide b), and -serotonin (5-HT, 5-hydroxytryptamine) was present in the central, peripheral, and stomatogastric nervous system. Specifically, anti-FMRFamide and -SCPb staining was abundant in perikarya and neurites of the cerebral ganglion, ventrolateral nerve cords, and mastax. In addition, a single loop-like neurite was present in between the nerve cords at the posterior end of the body. Serotonergic neurites were also abundant, and highlighted several cerebral pathways that included connections to the nerve cords and possibly the mastax. Novel neural pathways were also present in the posterior trunk region, where serotonergic neurites innervated the foot and lateral body wall. The results presented herein also highlight the utility of 3D visualization software to gain further insights into the organization and architecture of the rotifer cerebral ganglion.     

First description of the serotonergic nervous system in a bdelloid rotifer: Macrotrachela quadricornifera Milne 1886 (Philodinidae)

Class Bdelloidea of phylum Rotifera comprises aquatic microinvertebrates that are known for both obligate parthenogenesis and for resisting desiccation through a dormant reversible state. In the frame of an investigation about the role of the nervous system in controlling life cycle, reproduction and dormancy, we describe the serotonergic system of a bdelloid, Macrotrachela quadricornifera, using serotonin immunohistochemistry and confocal laser scanning microscopy. Serotonin immunoreactivity is present in the cerebral ganglion, lateral nerve cords and peripheral neurites. The cerebral ganglion consists of perikarya that send neurites cephalically to the rostrum and corona. A pair of neurites exits the cerebral ganglion as lateral nerve cords, and proceeds caudally to the pedal ganglion where additional neurites enter the foot. Based on the location of serotonergic immunoreactivity, we hypothesize that the neurotransmitter is involved in both motor activity (e.g., ciliary beating, inchworm-like locomotion) and sensory activity. A comparison between the serotonergic nervous systems of M. quadricornifera and species of Monogononta reveals differences in the numbers and patterns of cerebral perikarya, peripheral perikarya, and periperhal neurites. These differences may have functional significance for understanding adaptations to specific environments and/or systematic significance for reconstructing the rotiferan ground pattern.     

Peptidergic motoneurons in the buccal ganglia of Aplysia californica Immunocytochemical,morphological, and physiological characterizations

Summary We used physiological recordings, intracellular dye injections and immunocytochemistry to further identify and characterize neurons in the buccal ganglia of Aplysia calif ornica expressing Small Cardioactive Peptide-like immunoreactivity (SCP-LI). Neurons were identified based upon soma size and position, input from premotor cells B4 and B5, axonal projections, muscle innervation patterns, and neuromuscular synaptic properties. SCP-LI was observed in several large ventral neurons including B6, B7, B9, B10, and B11, groups of s1 and s2 cluster cells, at least one cell located at a branch point of buccal nerve n2, and the previously characterized neurons B1, B2 and B15.B6, B7, B9, B10 and B11 are motoneurons to intrinsic muscles of the buccal mass, each displaying a unique innervation pattern and neuromuscular plasticity. Combined, these motoneurons innervate all major intrinsic buccal muscles (I1/I3, I2, I4, I5, I6). Correspondingly, SCP-LI processes were observed on all of these muscles. Innervation of multiple nonhomologous buccal muscles by individual motoneurons having extremely plastic neuromuscular synapses, represents a unique form of neuromuscular organization which is prevalent in this system. Our results show numerous SCPergic buccal motoneurons with widespread ganglionic processes and buccal muscle innervation, and support extensive use of SCPs in the control of feeding musculature.Abbreviations SCP-LI small cardioactive peptide-like immunoreactivity - PSC postsynaptic current - EPSP excitatory postsynaptic potential - IPSP inhibitory postsynaptic potential - FI facilitation index - TMR time to maximal response     

Cytochrome oxidase activity as a measure of synaptogenesis by mutifunctional neuron L7 of Aplysia

Neuron L7 of the marine mollusc, Aplysia californica, is unique in that it innervates five different target tissues in the animal. We show that when L7 is grown in vitro with two of these targets, that is, muscle cells isolated from the auricle or the gill vein, newly formed L7 neurites contact the muscle cells. Chemical synapses are formed since intracellular stimulation of L7 elicits contraction of individual muscle cells. Interestingly, auricle muscles are also innervated by neuron RBhe and co-cultures of RBhe and auricle muscle cells also exhibit synapse formation. To explore the molecular basis for synaptogenesis between L7 and its targets, it would be useful to quantify the extent of synapse formation in vitro, that is, to determine how many muscle cells can be innervated by a single L7. We show that this can be attained by staining for cytochrome oxidase activity. Cultures of auricle and gill vein muscles were exposed to the appropriate neurotransmitter in order to elicit contraction. The cells were then fixed and stained. In both cases, only cells that contracted were stained and electron microscopy showed reaction product associated with the cristae of mitochondria. When this procedure was applied to cultures of L7 and muscle cells, 38 ± 2.8% (S.E.M.;n=7) of the cells on the neurites were stained and therefore responded to L7 stimulation. Thus, part of the L7-RBhe circuit can be assembled in vitro and the extent of synaptogenesis can be accurately quantitated.     

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.     

External Morphology and Muscle Arrangement of Brachionus urceolaris, Floscularia ringens, Hexarthra mira and Notommata glyphura (Rotifera, Monogononta)

We studied four monogonont rotifers (Brachionus urceolaris, Floscularia ringens, Hexarthra mira, Notommata glyphura) using two different techniques of microscopy: (1) the presence of filamentous actin was examined using phalloidin-fluorescent labelled specimens and a confocal laser scanning microscope (CLSM); (2) external morphology was investigated using a scanning electron microscope (SEM). B. urceolaris, F. ringens, and N. glyphura showed similar patterns of muscle distribution: a set of longitudinal muscles acting as head and foot retractors, and a set of circular muscles. However, the size and distribution of circular muscles differed among these species. H. mira differed from the other species in that it lacked circular muscles but possessed strong muscles that extended into each arm. The study showed that using both CLSM and SEM provides better resolution of the anatomy and external morphology of rotifers than using one of these techniques alone. This can facilitate better understanding of the complicated anatomy of these animals.     

Serial homologies of the motor neurons of the dorsal intersegmental muscles of the cockroach,Periplaneta americana (L.)

The types and locations of serially homologous motor neurons of the dorsal muscles in the cockroach Periplaneta americana remain rather constant regardless of the various adaptations of their muscles or the fusion of ganglia. However, the size and number of neurons do vary according to the development of the muscles they innervate. Neurons in four distinctive locations, two ipsisegmental and two antesegmental, innervate the dorsal longitudinal (DL) muscles in most segments. One of the ipsisegmental neurons (DLC) is common to all of the DL muscles of a segment and probably has a modulatory function. The dorsal oblique (DO) muscles of most segments have neurons in two antesegmental positions. One of these, an antesegmental, contralateral neuron, innervates both DO and DL muscles in each segment and is also probably modulatory. One neuron (DOC) of the prothoracic ganglion is the principal exception to the constancy of these serially homologous neurons. This neuron appears to be homologous to the DLC neurons of other segments but innervates the DO rather than the DL muscles.     

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.     

Serotonin immunoreactivity in the nervous system of the free‐swimming larvae and sessile adult females of Stephanoceros fimbriatus (Rotifera: Gnesiotrocha)

Unlike most rotifers (Rotifera), which are planktonic and direct developers, many gnesiotrochan rotifers (Monogononta: Gnesiotrocha) are sessile and have indirect development. Few details exist on larval metamorphosis in most gnesiotrochans, and considering the drastic transformation that takes place at metamorphosis—the replacement of the ciliated corona with a new head that bears ciliated tentacles (the infundibulum)—it is perhaps surprising that there are limited data on the process. Here, we document part of this metamorphosis by examining the presence and distribution of neurons with serotonin immunoreactivity in the nervous system of both planktonic larvae and sessile adult females. Using antibodies against serotonin combined with confocal laser‐scanning microscopy (CLSM) and 3D reconstruction software, we mapped the immunoreactive cell bodies and neurites in both life stages and found that relatively few changes occurred during metamorphosis. The larvae possessed a total of eight perikarya with serotonergic immunoreactivity (5HT‐IR) in the brain, with at least two pairs of perikarya outside the brain in the region of the corona. Cells with 5HT‐IR in the brain innervated the larval corona and also sent neurites to the trunk via the nerve cords. During metamorphosis, the corona was replaced by the infundibulum, which emerged from the larval mouth to become the new functional head. This change led to a posterior displacement of the brain and also involved the loss of 5HT‐IR in the lateral brain perikarya and the gain of two perikarya with 5HT‐IR in the anterior brain region. The innervation of the anterior end was retained in the adult; neurites that extended anteriorly to the mouth of the larva formed a distinct neural ring that encircled the infundibulum after metamorphosis. Significantly, there was no innervation of the infundibular tentacles by neurites with 5HT‐IR, which suggests that ciliary control is unlikely to be modulated by serotonin within the tentacles themselves.     

Rotifer digestive enzymes: direct detection using the ELF technique

Hydrolytic enzymes involved in rotifer digestive processes were investigated directly at the sites of enzyme action using the ELF (Enzyme Labelled Fluorescence) technique. After enzymatic hydrolysis of an artificial ELF substrate, the fluorescent product ELF alcohol (ELFA) marked the sites of enzyme action. The time development of ELFA labelling was studied at different incubation times. Phosphatases, β-N-acetylhexosaminidases and lipases were examined in Brachionus angularis, B. calyciflorus, Keratella cochlearis and Lecane closterocerca from fed-batch cultures. We detected activities of all studied enzymes mostly in the stomach and intestine of rotifers. L. closterocerca was the only species showing enzyme activity at the mastax. Lipase activity was observed in the stomach and intestine of all species and in the mastax of L. closterocerca. Phosphatases were frequently located at the corona of B. calyciflorus. In other cases, both phosphatases and β-N-acetylhexosaminidases were rarely detected at the corona, and on the lorica and epidermis of some species. Guest editors: S. S. S. Sarma, R. D. Gulati, R. L. Wallace, S. Nandini, H. J. Dumont and R. Rico-Martínez Advances in Rotifer Research     

Epizoic and parasitic rotifers

Many rotifer species live in close association with plants or other animals. Most of these associations are of a commensal or synoecious nature, some rotifer species having lost the ability to live independently. Few rotifers are true parasites, actually harming their hosts.The Seisonidae, Monogononta and Bdelloidea include epizoic and parasitic species. The most widely known are probably the parasites of colonial and filamentous algae (e.g. Volvox, Vaucheria). However, rotifers are also found on a wide range of invertebrates: colonial, sessile Protozoa; Porifera; Rotifera; Annelida; Bryozoa; Echinodermata; Mollusca, especially on the shells and egg masses of aquatic gastropods; Crustacea, including the lower forms (e.g. Daphnia, Asellus, Gammarus) and in the gill chambers of Astacus and Chasmagnathus; the aquatic larvae of insects. There appear to be few records of epizoic or parasitic rotifers among vertebrates, apart from Encentrum kozminskii on carp, Limnias ceratophylli on the Amazonian crocodile, Melanosuchus niger, and an unidentified Bdelloid apparently living as a pathogenic rotifer in Man.