Ultrastructural and cytochemical features of the supramedullary neurons of the pufferfish Diodon holacanthus (L.) (Osteichthyes)

Exceptionally high DNA contents were found in supramedullary neuron (SN) nuclei of the pufferfish Diodon holacanthus by quantitative microfluorimetric assay. This phenomenon has been explained by endoreplication, the functional significance of which is still unclear. In this view, the peptidergic nature and large dimensions make the teleostean clustered SN an interesting model for investigating the relationships between endoreplication, nuclear morphology and biosynthetic cellular activity. In this paper, we present a cytochemical and ultrastructural study on the SN of D. holacanthus (Tetraodontiformes). The nucleolar and nucleus structures suggest an intense production of ribosomal components in order to satisfy high cellular demands for protein synthesis. Accordingly, the cytoplasmic compartment presents an extensive rough endoplasmic reticulum, well-developed Golgi apparatus and a remarkable vesicular traffic. These features suggest that SN are engaged in an intense process of protein biosynthesis. The SN are completely surrounded by processes of different types of glial cells. The glial cells may be considered part of the SN cluster.     

Glial cells: basic components of clusters of supramedullary neurons in pufferfish

In this paper a cytochemical and ultrastructural study of clustered supramedullary neurons (SN) of Tetraodon fluviatilis (Tetraodontiformes) is presented. SN are large-sized nerve cells that have a high metabolic rate and are intensely engaged in protein biosynthesis. The SN are completely surrounded by two types of glial cell, which have been ultrastructurally, histochemically and immunohistochemically identified as astrocyte-like cells and microglial cells. The glial cells are located very close to the SN and sometimes contact them, which suggests that they do not only provide mechanical support but are also trophic for the SN and serve their functioning. We consider these glial cells as a constitutive and functional part of the cluster of SN, and therefore propose that the SN cluster constitute a suitable model to study in detail the morphological and functional relationships between neurons and glial cells in Anamnia.     

Acid phosphatase activity in the supramedullary neurons of Coris julis (L.).

This work describes the acid phosphatase activity in supramedullary neurons of Coris julis, analyzed by a cytochemical method. The presence of both acid phosphatase-positive and -negative membrane bound granules indicates that only a part of the numerous electrodense granules in the supramedullary neurons can be interpreted as lysosomes. The great number of lysosomes in these large neurons of young animals is indicative of the rapid turnover of cell structures, which may be correlated with the high rate of synthesis. The electrodense granules showing no acid phosphatase activity are postulated to be vesicles containing gastrin/CCK-like peptide or precursors of this neuromediator.     

Occurrence of Kudoa sp. (Myxozoa) in Trachurus trachurus L. (osteichthyes) in Portugal

Specimens of Trachurus trachurus L., obtained monthly in a fish market of Oporto from October 1998 to August 1999, were examined for the presence of Kudoa in the muscle. Kudoa sp. spores were found in 84.7% of the specimens (n = 209). The prevalence was not significantly different between seasons and was not related to the host's length. The infection does not seem to have negative effects on the fish quality once no macroscopic pseudocysts or myoliquefaction were detected.     

Analysis of DNA endoreplication in the brain neurons in the terrestrial slug, Limax valentianus

DNA endoreplication is the DNA synthesis without cell division, resulting in the generation of a nucleus containing a larger amount of genomic DNA compared to a normal diploid genome. There are many such giant neurons in the molluscan brain that are generated as a result of repeated endoreplication. However, it has been controversial whether the endoreplication is the whole genome replication (polyploidy) or the local amplification of the genes that are necessary for the neuron's function (polyteny/polysomy). Here in this study, we investigated these two possibilities by (1) immunohistochemical analysis of the distribution of 5'-bromodeoxyuridine incorporated into the nuclei of the brain neurons, and by (2) quantitative genomic PCR directed to two different genes expressed in specific brain regions. Our data supported the view that the DNA endoreplication is the whole genome replication rather than the local amplification of a specific genomic region.     

Ultrastructural features of supramedullary neurons in Crenilabrus quinquemaculatus

The ultrastructural study of the supramedullary neurons in Crenilabrus quinquemaculatus shows that these cells are engaged in intense synthetic activity as is testified by the nuclear morphology, the plentiful rough endoplasmic reticulum and polysomes, and finally the remarkably developed Golgi complexes, many of them active. Moreover the cytoplasm of the supramedullary neurons shows numerous membrane-bounded bodies (1,800-4,000 A) containing electron dense material, more or less finely granular. Research is presently being carried out to establish the meaning of these electron dense bodies.     

Biological role of endoreplication in the process of spermatogenesis inChara vulgaris L.

Summary During cell division in antheridial filaments ofChara vulgaris an increase in DNA content occurs in both shield cells and manubria within an antheridium, reaching 16C–64C and 8C–32C levels, respectively. Endoreplication ceases prior to the formation of spermatids and initiation of spermiogenesis, probably as a result of symplasmic isolation of the antheridium from the thallus. As the DNA content of the nuclei increases, the shield cells³H-leucine incorporation increases, and they grow intensively in the tangential plane. Translation decreases considerably after termination of shield cell growth. DNA content of mature manubria is half of that in shield cells, although their size is 10 times that of manubria. Translational activity of manubria also increases as DNA content rises and cells grow. However, during spermiogenesis, this activity remains at its maximum, which is associated with the secretory function of the manubria. Spermiogenesis is also accompanied by far-reaching ultrastructural changes within the manubrial cytoplasm.The level of endopolyploidy in both shield cells and manubria of antheridia formed in the spring is higher by one replication cycle, than in autumnal antheridia. AMO-1618, at a concentration of 10^–5M reduces the DNA content in the autumnal manubria. The higher the manubrial level of endopolyploidy in spermiogenesis, the greater their size, and the higher the translational activity and number of joined spermatids. The number of spermatozoids in the antheridium is also positively correlated with the internal volume of an antheridium, which is itself dependent on the endopolyploidy level of shield cells.The results obtained confirm the assumption that endoreplication favours the higher growth dynamics and potential translational activity, which occurs in the dynamic growth phase only in shield cells, while in manubria, i.e. cells producing substances necessary to spermatozoids development, it remains high until the end of spermiogenesis.     

Behavior of delayed current under voltage clamp in the supramedullary neurons of puffer

Depolarizations applied to voltage-clamped cells bathed in the normal solution disclose an initial inward current followed by a delayed outward current. The maximum slope conductance for the peak initial current is about 30 times the leak conductance, but the maximum slope conductance for the delayed current is only about 10 times the leak conductance. During depolarizations for as long as 30 sec, the outward current does not maintain a steady level, but declines first exponentially with a time constant of about 6 msec; it then tends to increase for the next few seconds; finally, it declines slowly with a half-time of about 5 sec. Concomitant with the changes of the outward current, the membrane conductance changes, although virtually no change in electromotive force occurs. Thus, the changes in the membrane conductance represent two phases of K inactivation, one rapidly developing, the other slowly occurring, and a phase of K reactivation, which is interposed between the two inactivations. In isosmotic KCl solution after a conditioning hyperpolarization there occurs an increase in K permeability upon depolarization. When the depolarizations are maintained, the increase of K permeability undergoes changes similar to those observed in the normal medium. The significance of the K inactivation is discussed in relation to the after-potential of the nerve cells.     

The supramedullary cells of the teleost Coris julis (L.): a noradrenergic neuronal system

This study, carried out on Coris julis (Labridae), is a contribution to the immunocytochemical characterization of fish supramedullary neurons. The significance of these giant cells has been debated since the beginning of the twentieth century. Our research provides the first evidence for a noradrenergic feature of this neuronal system. The possible role of supramedullary neurons as components of the autonomic nervous system is discussed. Moreover, the present results, taken together with our previous studies, surmise that this the first known case of colocalization of a neuropeptide (gastrin/CCK-like) and noradrenaline in the nervous system of teleosts.     

Isolation and identification of novel protein kinase genes from the round-spotted pufferfish(Tetraodon fluviatilis) genomic DNA

The round-spotted pufferfishTetraodon fluviatilis has a genome size of 380 Mb which is slightly smaller than that of another pufferfish,Fugu rubripes rubripes (Fugu). Due to their compact genome and small introns, both pufferfishes have been proposed as model organisms for genome studies. In this study, we have used genomic DNA as template to perform PCR to screen for protein kinase (pk) genes. Forty-oneT. fluviatilis pk genes encoding 7 receptor tyrosine kinases, 14 nonreceptor tyrosine kinases, 16 serine/threonine kinases, 1 dual kinase and 3 novel kinases have been identified. The success of this approach depends on the size and location of the introns. Most of the identifiedpk gene fragments contain introns, ranging from 71 to 300 bp, with an average of 120 bp. It is noteworthy that the intron/exon boundaries of certain genes which belong to the same family are identical. We also analyzed by specific RT-PCR primers the expression profile of those 3 novel genes as well as some selectedpk genes in a variety of tissues. We found thaterbB3,pku , mrk, CaMK I,CaMKII, and two novel kinase genes (133 and 3–26) are expressed in all tissues examined. However, the novel clone 146 is strongly expressed in the brain and weakly in the intestine, kidney and heart.     

Morphological, cytochemical, and cytofluorimetric features of supramedullary neurons of the fish Solea ocellata

Various teleost species belonging to different orders possess a particular neuronal system formed by giant supramedullary neurons (SNs). In some species, SNs are scattered along the spinal cord; in others they are organized in a compacted and well-defined cluster located at the boundary between the medulla oblongata and spinal cord. In addition to the many morphological, physiological, and histochemical studies performed both in vivo and in vitro by several authors since the end of the 19th century, quantitative microfluorometric evaluation of the DNA content of SNs has showed that clustered SNs but not aligned SNs have a DNA content much greater than the normal value of 2C. Such a high DNA content is exceptional for vertebrate neurons. In the present study, we extend this analysis of SNs to the fish Solea ocellata. Our results show that the organization of the SNs of S. ocellata is neither strictly aligned nor clustered, but somewhere in between, and that this is also true of both their morphological characteristics and DNA content values. Interspecific differences in the distribution and morphology of SNs may reflect functional differences, possibly related to environmental or behavioral differences among species. In addition, the possible functional significance of endoreplication in SNs is discussed.     

Biomechanics of swimming in the pufferfish Diodon holocanthus: propulsive momentum enhancement is an adaptation for thrust production in an undulatory median and paired-fin swimmer

A form of large-amplitude elongated-body theory appropriate for the analysis of undulatory fins attached to a rigid body of elliptical section suggests a benefit due to momentum enhancement relative to the fins on their own. This theoretical prediction is experimentally confirmed for the first time. Theoretical momentum enhancement factors for Diodon holocanthus (2.2 and 2.7 for the median and pectoral fins, respectively) compared well to inferred thrust values determined from particle-image velocimetry (PIV) wake measurements (2.2-2.4 and 2.7-2.9). Caudal fin mean theoretical thrust was not significantly different from measured (PIV) values (n = 24, P > 0.05), implying no momentum enhancement. Pectoral-fin thrust was half that of the median and caudal fins due to high fin-jet angles, low circulation and momentum. Average total fin thrust and fish drag were not significantly different (n = 24, P > 0.05). Vortex rings generated by the fins were elliptical, with size dependent on fin chord and stroke amplitude. Hydrodynamic advantages (thrust enhancement at no cost to hydrodynamic efficiency, reduction of side forces minimizing energy wasting yawing motions and body drag) are probably common among rigid-bodied organisms propelled by undulatory fins. A trade-off between momentum enhancement and the rate of momentum generation (thrust force) sets a practical limit to the former. For small fins whilst momentum enhancement is high, absolute thrust is low. In addition, previously suggested limitations on thrust enhancement set by reductions in propulsive force associated with progressive reductions in fin wavelength are found to be biologically unrealistic.     

Karyotypes and nuclear DNA amounts in Polypodium L. (Polypodiaceae)

Karyotype studies in several species of Polypodium show that telocentric chromosomes are the most common with acrocentrics forming the remainder of the complement. The relative numbers of these chromosome types can be used as indicators of species relationships although direct comparisons are difficult to make due to the large number of similar-sized chromosomes. The karyotype data support the theory that P. interjectum is a polyploid derived from the hybridization of, P. australe and P. vulgare. Measurements of nuclear DNA content show that the four diploid species P. australe, P. scouleri, P. virginianum and P. glycyrrhiza all have very similar amounts of DNA. The tetraploid P. vulgare has one-and-a-half times the DNA content of the diploids and the hexaploid P. interjectum has two times the DNA content of the diploids. The chromosomes of the tetraploid and hexaploid are smaller than those of the diploids and evolution in Polypodium appears to have been accompanied by either a loss or gain of nuclear DNA; the direction of the change cannot be ascertained by the present study.     

Evidence for differential DNA endoreplication during the development of a molluscan brain

The DNA content of neurons in the cerebral ganglion of Achatina fulica was determined by the two-wavelength method of microspectrophotometry using Feulgen-stained sections. DNA measurements of mouse hepatocytes were used as a control of the methods. All sampled neurons with a nuclear diameter greater than 7 microns were polyploid. The shape of the frequency histogram of DNA contents is not compatible with an interpretation that assumes one or more simple duplications of the genome. It is suggested, instead, that the results are due to either the underreplication of some DNA sequences or the selective amplification of genes. Additional experiments using [3H]-thymidine autoradiography showed that the incidence of neuronal DNA synthesis is highest during the period of the animal's greatest growth and then declines rapidly near the onset of sexual maturity. The mesocerebrum incorporated greater amounts of [3H]-thymidine than the rest of the brain, whereas the procerebrum remained diploid throughout the course of the study.