Meiotic behavior and karyotypic variation in Acledra (Pentatomidae, Heteroptera).

Acledra comprises 15 taxonomically identified species, most of which are crop pests. This is the first cytogenetic study of species of this genus. Acledra kinbergii and A. modesta showed the modal number of the Pentatomidae (2n = 14 = 12 + XY), while A. bonariensis had a reduced complement (2n = 12 = 10 + XY), with a markedly larger autosomal pair. Meiotic behavior follows the general pattern of the family; the autosomes divide pre-reductionally, the sex chromosomes are achiasmatic and divide post-reductionally, and at metaphase II the autosomes show a ring-shaped configuration with the pseudobivalent at the center. However, the configuration at metaphase I varies; A. modesta shows the typical arrangement (ring of bivalents with the sex chromosomes lying at its center). In A. kinbergii, the sex chromosomes are part of the ring or only the Y chromosome is at the center. In A. bonariensis, the ring arrangement is not well defined. There are also differences at the diffuse stage; chromatin strands of different width are observed in A. bonariensis and A. modesta, whereas bivalents do not entirely lose their identity in A. kinbergii. In A. bonariensis, the reduced complement may have originated from the fusion of the two larger non-homologous autosomes, which could characterize the ancestral karyotype of this genus. The presence of secondary constrictions in the larger pair of A. modesta and A. bonariensis may support this hypothesis. Since secondary constrictions are uncommon in the holokinetic chromosomes of heteropterans, their presence in these species may indicate that it is a plesiomorphic character of the genus.

Occurrence of two NOS isoforms in the developing gut of sea bass Dicentrarchus labrax (L.).

In this work we have examined the appearance and distribution of nitric oxide synthase (NOS), with histochemical, immunohistochemical and biochemical methods, during development of the sea bass (Dicentrarchus labrax) gut. The data showed that both the calcium-calmodulin dependent neuronal isoform (nNOS) and calcium-independent inducible isoform (iNOS) are present in the larval gut of sea bass. The nNOS-immunoreactivity was present in the epithelial cells and enteric nerve cells of gut both in the 8-day-old specimens and in the 24-day-old-larvae. In the adult nNOS-immunoreactivity disappeared from epithelial cells, remaining in the wall intramural neurons and fibers. The iNOS-immunoreactivity was present in the epithelial cells of 24-day-old-larvae and was not detectable in the adult gut. Western blot analysis and determination of NOS activity also demonstrated the presence of the two NOS isoforms, nNOS and iNOS, in the gut of 24-day-old specimens. The presumably different roles played by the two isoforms of enzyme are discussed. The presence of nNOS isoform in the gut enteric neurons of the same larval stages of D. labrax in which we previously demonstrated the presence of substance P and Vasoactive Intestinal Polypeptide (VIP), may suggest that all these three components of the motility control system are already present in the larval phase. Nitric oxide (NO) may be also involved in the early immune response. The present results on the occurrence of iNOS isoform in epithelial gut cells of the same regions in which the gut-associated lymphoid tissue (GALT) will differentiate, may suggest for NO a role in early defence mechanisms, before the establishment of immune responses in GALT. Finally, the developmental and regional differences in nNOS and iNOS expression also suggest a regulatory role in development and differentiation of the sea bass gut.

Occurrence of nitric oxide synthase in Megoura viciae Buckton (Homoptera, Aphididae): an histochemical and immunohistochemical localisation.

Nitric oxide (NO) is known to be involved in many physiological reactions of insects. We analysed NOS localisation in aphids of the species Megoura viciae by means of histochemical reaction for the NADPH-diaphorase activity and immunohistochemical methods for uNOS, nNOS and iNOS. The obtained data provided a complex and peculiar pattern of NOS distribution in cells and tissue of M. viciae. The histochemical reaction for NADPH-diaphorase was an indicative, but not exact marker of NOS localisation in aphids. The use of anti uNOS antiserum (frequently applied in insects) was of limited value in our specimens, whereas more satisfactory results were obtained with anti nNOS and iNOS antisera of human origin. The results of Western blot analysis confirmed the immunohistochemical ones, showing an aphid protein that reacted strongly with the polyclonal antibody anti-iNOS and anti-nNOS while a similar protein band was weakly immunoreactive with the polyclonal antibody anti-uNOS. Our results suggest that NO, prevalently synthesised by calcium/calmodulin-dependent isoform, plays important physiological roles both in adult and embryological stages of aphids. The data of principal interest was NOS presence in bacteriocytes, cells that host symbiotic prokaryotes belonging to the species Buchnera aphidicola, and in nuclei of adipocytes and gut cells.

Immunolocalisation of vasoactive intestinal peptide and substance P in the developing gut of Dicentrarchus labrax (L.).

This study was carried out on the sea bass (Dicentrarchus labrax) to follow, during development, the appearance and distribution of substance P (SP) and vasoactive intestinal peptide (VIP), which act on gut motility. The results suggest that SP and VIP play an important role as neuromodulators, influencing the motility of the digestive tract starting from the early stages of gut development, even prior to exotrophic feeding. In the peptidergic nervous system, the appearance of immunoreactivity to SP began at the rectum and followed a distal to proximal gradient, whereas for VIP, it began proximally and progressed along a proximal to distal gradient. The two peptides also appeared in gut epithelial cells. In some regions, all the cells were positive. From this distribution of positive cells, we conclude that these peptides may also have other roles, besides being neurotransmitters in the enteric nervous system and hormones of the gastro-entero-pancreatic system. VIP and SP might have paracrine and/or autocrine activity in the physiological maturation of the gut epithelium, as it has already been hypothesised for other peptides.

Behaviour of ring bivalents in holokinetic systems: alternative sites of spindle attachment in Pachylis argentinus and Nezara viridula (Heteroptera).

Heteropteran chromosomes are holokinetic; during mitosis, sister chromatids segregate parallel to each other but, during meiosis, kinetic activity is restricted to one pair of telomeric regions. This meiotic behaviour has been corroborated for all rod bivalents. For ring bivalents, we have previously proposed that one of the two chiasmata releases first, and a telokinetic activity is also achieved. In the present work we analyse the meiotic behaviour of ring bivalents in Pachylis argentinus (Coreidae) and Nezara viridula (Pentatomidae) and we describe for the first time the chromosome complement and male meiosis of the former (2n = 12 + 2m + X0, pre-reduction of the X). Both species possess a large chromosome pair with a secondary constriction which is a nucleolus organizer region as revealed by in-situ hybridization. Here we propose a new mode of segregation for ring bivalents: when the chromosome pair bears a secondary constriction, it is not essential that one of the chiasmata releases first since these regions or repetitive DNA sequences adjacent to them become functional as alternative sites for microtubule attachment and they undertake chromosome segregation to the poles during anaphase I.

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.

DNA endoreplication in the clustered supramedullary neurons of the pufferfish Diodon holacanthus L. (osteichthyes).

DNA contents, ranging from 4C to more than 500C, were determined by a quantitative microfluorimetric assay in supramedullary neuron nuclei of the pufferfish Diodon holacanthus. The distribution of C values after ethidium bromide staining indicates an inter- and intra-individual variation in DNA contents which do not correspond to duplications of the total genome, suggesting that DNA replication is specific for particular genes (endoreplication). Moreover, the DNA content appears to be correlated with nuclear size. A relationship between the DNA amounts and the presence of AT- and GC-rich sequences has been shown. This work demonstrates, for the first time, DNA endoreplication in a specific neuronal type in vertebrates.

Meiotic studies in Dysdercus Guérin Meneville 1831 (Heteroptera: Pyrrhocoridae). I. Neo-XY in Dysdercus albofasciatus Berg 1878, a new sex chromosome determining system in Heteroptera.

The genus Dysdercus Guérin Méneville 1831 represents the only taxon within the family Pyrrhocoridae in the New World. Based on morphological features, it has been suggested that American species derived from immigrants from the Old World, most probably from the Ethiopian Region. So far, 10 species from Dysdercus, including six species from the Old World and four species from the Neotropical Region have been cytogenetically analyzed. As is characteristic of Heteroptera, they possess holokinetic chromosomes and a prereductional type of meiosis. While the X1X20 sex chromosome system has been reported in all cytologically analyzed species of Dysdercus from the Old World, the system X0 has been found in all but one species from the New World, regardless of the number of autosomes in the complement. In the present study the male meiosis of D. albofasciatus Berg 1878 was studied in specimens from four different populations from Argentina. The diploid chromosome number was found to be 2n = 10 + neo-XY. The neo-X shows at each subterminal region a positively heteropycnotic and DAPI-bright segment which corresponds to the ancestral X-chromosome. The origin of this neo-XY system involved, most probably, a subterminal insertion of the ancestral X chromosome in an autosome, followed by a large inversion, which included part of the original X chromosome.

Endogenous morphine levels increase in molluscan neural and immune tissues after physical trauma.

The aim of this study was to demonstrate by biochemical and immunocytochemical methods the presence of endogenous morphine in nervous and immune tissues of the freshwater snail, Planorbarius corneus. High performance liquid chromatography (HPLC) coupled to electrochemical detection performed on tissues from control snails, revealed that the CNS contains 6.20+/-2.0 pmol/g of the alkaloid, the foot tissue contains a much lower level, 0.30+/-0.03 pmol/g, whilst morphine is not detected in the hemolymph and hepatopancreas. In specimens that were traumatized, we detected a significant rise of the CNS morphine level 24 h later (43.7+/-5.2 pmol/g) and an initial decrease after 48 h (19.3+/-4.6 pmol/g). At the same times, we found the appearance of the opiate in the hemolymph (0.38+/-0.04 pmol/ml and 0.12+/-0.03 pmol/ml) but not in the hepatopancreas. Using indirect immunocytochemistry, a morphine-like molecule was localized to a number of neurons and a type of glial cell in the CNS, to some immunocytes in the hemolymph and to amoebocytes in the foot, as well as to fibers in the aorta wall. Simultaneously to the rise of morphine biochemical level following trauma, morphine-like immunoreactivity (MIR) increased in both intensity and the number of structures responding positively, i.e., neurons and fiber terminals. In another mollusc, the mussel Mytilus galloprovincialis, the same pattern of enhanced MIR was found after trauma. Taken together, the data suggest the presence of a morphinergic signaling in invertebrate neural and immune processes resembling those of classical messenger systems and an involvement in trauma response.

Comparative meiotic studies in Triatoma sordida (Stål) and T. guasayana Wygodzinsky & Abalos (Reduviidae, Heteroptera).

Triatoma sordida and T. guasayana are competent Trypanosoma cruzi vectors, with overlapping distribution areas in Argentina. Both species are morphologically similar, and their immature stages are hard to discriminate. Cytogenetic studies in the genus Triatoma reveal scarce karyotypic variations, being 2n = 20 + XY the most frequent diploid number in males. In the present work the meiotic behaviour of different Argentinian populations of T. sordida and T. guasayana has been analyzed; the meiotic karyotype of both species has also been compared. The species differ in total chromosome area and in the relative area of the sex chromosomes. These meiotic karyotypic differences constitute an additional tool for the taxonomic characterization of T. sordida and T. guasayana. The analysis of an interpopulation hybrid of T. sordida (Brazil x Argentina) reveals a regular meiotic behaviour; despite the presence of heteromorphic bivalents. Our observations support the hypothesis that karyotype variations through the gain or loss of heterochromatin can not be considered as a primary mechanism of reproductive isolation in Triatoma.

Comparative meiotic studies in Triatoma sordida (Stal) and T. guasayana Wygodzinsky & Abalos (Reduviidae, Heteroptera)

Triatoma sordida and T. guasayana are competent Trypanosoma cruzi vectors, with overlapping distribution areas in Argentina. Both species are morphologically similar, and their immature stages are hard to discriminate. Cytogenetic studies in the genus Triatoma reveal scarce karyotypic variations, being 2n=20 + XY the most frequent diploid number in males. In the present work the meiotic behaviour of different Argentinian populations of T. sordida and T. guasayana has been analyzed; the meiotic karyotype of both species has also been compared. The species differ in total chromosome area and the relative area of the sex chromosomes. These meiotic karyotypic differences constitute an additional tool for the taxonomic characterization of T. sordida and T. guasayana. The analysis of an interpopulation hybrid of T. sordida (Brazil x Argentina) reveals a regular meiotic behaviour, despite the presence of heteromorphic bivalents. Our observations support the hypothesis that karyotype variatons through the gain or loss of heterochromatin can not be considered as a primary mechanism of reproductive isolation in Triatoma.

Cytofluorometric evidence for differential genome endoreplication in the cluster neurons of Lophius piscatorius L. (Osteichthyes, Lophiiformes).

Quantitative microfluorometric evaluation indicated that, in Lophius piscatorius, the DNA content of large neurons of a cluster located at the boundary between the medulla oblongata and the spinal cord varied from 4C to more than 5000C. This finding must be considered exceptional for the nervous system of Vertebrates. DNA contents were correlated to both nuclear and animal size. Utilization of AT and GC specific fluorochromes showed that the increase in DNA content is due to differential genome amplification involving GC-rich DNA sequences.

Neuroanatomical and immunohistochemical studies on the dorsal neurons in the spinal cord of Trigla lucerna L. and Scorpaena porcus L. (Scorpaeniformes).

Peculiar dorsal neurons present in the dorsal spinal cord of Trigla lucerna L. and Scorpaena porcus L. were investigated by neuroanatomical and immunohistochemical methods. These neurons were previously defined as commissural cells, but have now been identified as supramedullary neurons. The following fundamental criteria for identifying the supramedullary neurons of Teleosts are proposed: a) dorsomedial location in the spinal cord; b) large size of the soma and axon; c) immunoreactivity to gastrin/CCK-like peptides.

Cytofluorimetric determination of DNA content in large neurons of Lophius piscatorius L. (Osteichthyes, Lophiiformes).

The central nervous system of Lophius piscatorius has a cluster of large neurons located at the boundary between the medulla oblongata and spinal cord. The DNA content of these neurons was evaluated by microfluorimetric methods. Results demonstrated a DNA content ranging from a minimum of 8C in the smaller to over 1000C in the larger neurons of L. piscatorius cluster. These data are exceptional for the nervous system of Vertebrates, and known only for the giant neurons of Mollusca.

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.

Preliminary findings on the nucleus of large neurons in Lophius piscatorius L. (Osteichthyes, Lophiiformes).

Some Teleosts belonging to the orders Batrachoidiformes, Lophiiformes, Perciformes and Tetraodontiformes show a cluster of large neurons dorsally located at the boundary between the spinal cord and medulla oblongata. These elements have traditionally been grouped with the supramedullary neurons aligned in the dorso-medial region of the spinal cord of other teleosts (see Marini and Benedetti, in press). However, recent morphological and immunohistochemical studies have suggested that the neurons grouped in a cluster on the spinal cord of Lophius piscatorius may not be of the same nature as the supramedullary neurons aligned within the dorsal gray matter of the spinal cord (Benedetti and Mola, 1988; Mola, 1990). Over the course of these previous investigations, it seemed that the nucleus of the neurons in the cluster showed more abundant chromatin than other neurons in the dorsal spinal cord. This prompted a series of investigations on the nucleus of these neurons.

Survey of neuropeptide-like immunoreactivity in supramedullary neurons of Coris julis (L.).

The supramedullary neurons of the marine teleost Coris julis (L.) were surveyed for neuropeptide-like immunoreactivity using antisera against 12 peptides. These neurons exhibit positive immunoreactivity to CCK-8, CCK-39 and gastrin(18-34). The presence of gastrin/CCK-like peptides in the supramedullary neurons is discussed.