A study of the spirocytes from the Ceriantharia and Actiniaria (Cnidaria: Anthozoa)

Summary The structure and ultrastructure of ceriantharian and actiniarian spirocysts were compared. In the ceriantharian spirocyst, the apical cap consisted of two layers and the wall consisted of three, whereas in the actiniarian spirocyst, the number of layers was reversed. The inner wall of the ceriantharian spirocyst was smooth in longitudinal section whilst that of the actiniarian was serrated. The apical ends of ceriantharian spirocytes posessed either a set of microvilli or an arrangement that included both microvilli and a flagellum. The actiniarian spirocyte possessed only microvilli. Fine tubules occupying the undischarged tube of the ceriantharian spirocysts were of both moderate and strong electron densities, whilst those of the actiniarian were consistently electrondense. Undischarged ceriantharian spirocytes possessed tubules situated in the pleats and in the centre of the tube, whilst the tubes of undischarged actiniarian spirocytes possessed only centrally placed tubules. Fine electron-dense filaments linking the cell membrane to the spirocyst were present at the apical ends of actiniarian spirocytes. A different electron-dense network of filaments was present in the apical end of ceriantharian spirocytes. All cnidae of ceriantharians lacked tripartite opercula whilst the nematocysts of actiniarians possessed them. Primitive features of ceriantharian cnidae are discussed.     

Effects of controlled treatment with trypsin on the functional characteristics of isolated nematocysts of Calliactis parasitica and Aiptasia mutabilis (Cnidaria,Actiniaria)

Glycerol-isolated basitrichs of Calliactis parasitica responsive to thioglycolate had open apical flaps, while unresponsive capsules isolated with Triton X100 or by freezing had closed apical flaps. Limited treatment with trypsin induced the apical flaps to open without causing discharge, suggesting that nematocysts can maintain the resting condition even with open flaps. Trypsin-treated basitrichs acquire a high responsiveness to thioglycolate. Microbasic mastigophores of Aiptasia mutabilis are more responsive to distilled water after controlled trypsin treatment but the apical flaps are unchanged. Ca²⁺ is inhibitory regardless of trypsin treatment. It is proposed that the capsule tip may control the penetration of the discharging agents rather than providing mechanical resistance to inner pressure.     

Geographic variations,cloning, and functional analyses of the venom acidic phospholipases A2 of Crotalus viridis viridis

Geographic venom samples of Crotalus viridis viridis were obtained from South Dakota, Wyoming, Colorado, Oklahoma, Texas, New Mexico, and Arizona. From these samples, the phospholipases A(2) (PLA(2)s) were purified and their N-terminal sequences, precise masses, and in vitro enzymatic activities were determined. We purified two to four distinct acidic PLA(2)s from each sample; some of them displayed different inhibition specificities toward mammalian platelets. One of the acidic PLA(2)s induced edema, but had no anti-platelet activity. There was also a common basic PLA(2) myotoxin in all the samples. We have cloned five acidic PLA(2)s and several hybrid-like nonexpressing PLA(2)s. Molecular masses and N-terminal sequences of the purified PLA(2)s were matched with those deduced from the cDNA sequences, and the complete amino acid sequences of five novel acidic PLA(2)s were thus solved. They share 78% or greater sequence identity, and a cladogram based on the sequences of many venom acidic PLA(2)s of New World pit vipers revealed at least two subtypes. The results contribute to a better understanding of the ecogenetic adaptation of rattlesnakes and the structure-activity relationships and evolution of the acidic PLA(2)s in pit viper venom.     

Immunolocalization of venom metalloproteases in venom glands of adult and of newborn snakes of Bothrops jararaca

Using immunoelectronmicroscopy we analyzed qualitative and quantitatively the intracellular distribution of bothropasin, hemorrhagic factor 2 (HF2) and hemorrhagic factor 3 (HF3) in the venom secretory cells from adult snakes in the active (7 days after venom extraction) and in the resting (without venom extraction for 40 days) stages of protein synthesis. Glands from the newborn Bothrops jararaca were also studied. The results lead to the conclusion that all the secretory cells and the secretory pathway in the cells are qualitatively alike in regard to their content of the three metalloproteases. Secretory cells from the resting glands, unlike the active ones and the newborn glands, did not present immunolabeling in the narrow intracisternal spaces of the rough endoplasmic reticulum (RER). The label intensity for bothropasin was greater than that for the other proteins in the adults. HF3 and HF2 labeling densities in the newborn were higher than in the adults and HF3 labeling was not different from that of bothropasin. Co-localization of the three metalloproteases was detected in the RER cisternae of the active gland secretory cells, implying that mixing of the proteases before co-packaging into secretory vesicles occurs at the beginning of protein synthesis in the RER cisternae.     

Actiniaria and Ceriantharia of the Azores (Cnidaria Anthozoa)

The common shallow water species of sea anemones (Actiniaria) and tube anemones (Ceriantharia) of the Azores are listed. Eight species of sea anemones are mentioned, the species Cereus pedunculatus and Sagartia affinis being new records for the archipelago. Both species of Ceriantharia, namely Arachnanthus nocturnus and Pachycerianthus solitarius, are recorded from the Azores for the first time. Arachnanthus nocturnus is also recorded from the Cape Verde Islands and from Madeira for the first time. Communicated by H.-D. Franke     

Embryogenesis and plantlet development in the bamboo Phyllostachys viridis (Young) McClure

Leaf explants of Phyllostachys viridis (Young) McClure were cultured on Murashige and Skoog medium supplemented with 9×10^-6 M 2,4-dichlorophenoxyacetic acid. Numerous embryoids were observed. On transfer to Murashige and Skoog medium lacking hormones, plantlets developed within two weeks and were later successfully transferred to the field.     

Variation in Coding (NADH Dehydrogenase Subunits 2, 3, and 6) and Noncoding Intergenic Spacer Regions of the Mitochondrial Genome in Octocorallia (Cnidaria: Anthozoa)

Low rates of evolution in cnidarian mitochondrial genes such as COI and 16S rDNA have hindered molecular systematic studies in this important invertebrate group. We sequenced fragments of 3 mitochondrial protein-coding genes (NADH dehydrogenase subunits ND2, ND3 and ND6) as well as the COI-COII intergenic spacer, the longest noncoding region found in the octocoral mitochondrial genome, to determine if any of these regions contain levels of variation sufficient for reconstruction of phylogenetic relationships among genera of the anthozoan subclass Octocorallia. Within and between the soft coral families Alcyoniidae and Xeniidae, sequence divergence in the genes ND2 (539 bp), ND3 (102 bp), and ND6 (444 bp) ranged from 0.5% to 12%, with the greatest pairwise distances between the 2 families. The COI-COII intergenic spacer varied in length from 106 to 122 bp, and pairwise sequence divergence values ranged from 0% to 20.4%. Phylogenetic trees constructed using each region separately were poorly resolved. Better phylogenetic resolution was obtained in a combined analysis using all 3 protein-coding regions (1085 bp total). Although relationships among some pairs of species and genera were well supported in the combined analysis, the base of the alcyoniid family tree remained an unresolved polytomy. We conclude that variation in the NADH subunit coding regions is adequate to resolve phylogenetic relationships among families and some genera of Octocorallia, but insufficient for most species - or population-level studies. Although the COI-COII intergenic spacer exhibits greater variability than the protein-coding regions and may contain useful species-specific markers, its short length limits its phylogenetic utility.     

Production of antibacterials from the freshwater alga Euglena viridis (Ehren)

The antibacterial properties of Euglena viridis, collected from a freshwater pond at the Central Institute of Freshwater Aquaculture (CIFA), Bhubaneshwar, India, were tested against various strains of virulent pathogens viz. Pseudomonas putida(PP1, PP2),P. aeruginosa (PA1, PA2, PA3, PA4), P. fluorescens (PF1, PF2, PF3, PF4), Aeromonas hydrophila (AH30, AH31, AH32, AH34), Edwardsiella tarda, Vibrio alginolyticus (VA1),V. anguillarum(VN1, VN2 & VN3), V. fluvialis (VF1), V. parahemolyticus (VP1) and V. harveyi (VH1) andEscherichia coli(O115, O1, O156, O164, O111 & O109). Four organic extracts viz. methanolic, ethanolic, acetone and acetone/ethanol of theE. viridis showed moderate to high antibacterial activity to all the bacterial pathogens. Rotavapor extraction products showed higher sensitivity in comparison to cold and hot extractions.     

Viper and cobra venom neutralization by β-sitosterol and stigmasterol isolated from the root extract of Pluchea indica Less. (Asteraceae)

We reported previously that the methanolic root extract of the Indian medicinal plant Pluchea indica Less. (Asteraceae) could neutralize viper venom-induced action [Alam, M.I., Auddy, B., Gomes, A., 1996. Viper venom neutralization by Indian medicinal plant (Hemidesmus indicus and P. indica) root extracts. Phytother. Res. 10, 58-61]. The present study reports the neutralization of viper and cobra venom by beta-sitosterol and stigmasterol isolated from the root extract of P. indica Less. (Asteraceae). The active fraction (containing the major compound beta-sitosterol and the minor compound stigmasterol) was isolated and purified by silica gel column chromatography and the structure was determined using spectroscopic analysis (EIMS, (1)H NMR, (13)C NMR). Anti-snake venom activity was studied in experimental animals. The active fraction was found to significantly neutralize viper venom-induced lethal, hemorrhagic, defibrinogenation, edema and PLA(2) activity. Cobra venom-induced lethality, cardiotoxicity, neurotoxicity, respiratory changes and PLA(2) activity were also antagonized by the active component. It potentiated commercial snake venom antiserum action against venom-induced lethality in male albino mice. The active fraction could antagonize venom-induced changes in lipid peroxidation and superoxide dismutase activity. This study suggests that beta-sitosterol and stigmasterol may play an important role, along with antiserum, in neutralizing snake venom-induced actions.     

Growth, reproduction and survival of a tropical sea anemone (Actiniaria): benefits of hosting anemonefish

The ecological performance of the sea anemone Heteractis magnifica was examined during a 36-month experiment with respect to season and the presence and numbers of a mutualist (orange-fin anemonefish Amphiprion chrysopterus). Anemones primarily grew during the autumn, with most asexual reproduction occurring in winter; mortality was not strongly seasonal. Individual growth rates did not differ between anemones harboring one or two anemonefish, but these rates were three times faster than for anemones lacking Amphiprion. Anemones with two anemonefish had the highest fission rate, whereas those without anemonefish had the lowest. By contrast, anemones that were not defended by anemonefish suffered higher-than-expected mortality. As a consequence, anemones with two Amphiprion had the greatest net increase in surface area, and those lacking anemonefish had a negligible gain that was statistically indistinguishable from zero after three years. Anemonefish not only enhanced anemone survivorship as previously believed, they also fostered faster growth and more frequent asexual reproduction.     

The life cycle of Laodicea indica (Laodiceidae,Leptomedusae, Cnidaria)

The life cycle of Laodicea indica is described. In the Bismarck Sea this species shows a normal alternation of generations in the wet season; but in the dry season the cycle is contracted, and planulae give rise to gonothecae without formation of a hydroid colony. Medusae are liberated about 3 d after planula settlement. This life cycle pattern is previously unreported in hydromedusae. The possible adaptive value of such a life cycle and the evolution of polyp reduction in hydromedusan life cycles are discussed.     

<Emphasis Type="Italic">Bathyphellia margaritacea</Emphasis> (Cnidaria: Actiniaria): the most northern species of the world

The sea anemone Bathyphellia margaritacea (Danielssen in Actinida. The Norwegian North-Atlantic expedition 1876–1878, Groendahl, Oslo, 1890) was collected by the research submersible MIR at the North Pole at a depth of 4,262 m and by the North Pole Drifting Station NP-22 in the American sector of Arctic Ocean covered by permanent ice. These widely separated records significantly increase the known geographic range of the species. B. margaritacea is highly plastic and has an ability to occupy different types of substrates. It appears to be the only species of sea anemone that is able to range in the high Arctic up to the North Pole and the only reliably identified species known from this part of the world.     

Inhibition of the myotoxic activity of Bothrops jararacussu venom and its two major myotoxins, BthTX-I and BthTX-II, by the aqueous extract of Tabernaemontana catharinensis A. DC. (Apocynaceae)

Partial neutralization of the myotoxic effect of Bothrops jararacussu venom (BV) and two of its myotoxins [bothropstoxin-I (BthTX-I), catalytically inactive, and II (BthTX-II), showing low PLA2 activity], by the lyophilized aqueous extract of Tabernaemontana catharinensis (AE), was studied in rat isolated soleus muscle preparations (in vitro) and through i.m. injection in the gastrocnemius muscle (in vivo) by determination of creatine kinase (CK) activity and histopathological analysis. Incubation of soleus muscle for 1 h with BV or toxins (20 microg/ml) plus AE (400 microg/ml) added immediately after BV, BthTX-I or BthTX-II reduced CK levels by 53%, 37% and 56%, respectively. The myonecrotic effects of BV (20 microg/ml) upon soleus muscle was reduced 24%, 35% and 36% when AE (400 microg/ml) was added 1 h after BV and CK was evaluated 30 min, 1 and 2 h later, respectively. For BthTX-I these values were 46%, 48% and 47%, while for BthTX-II no inhibitory effect was detected. Histological analysis of soleus muscle after incubation with AE (400 microg/ml, 1 h) did not reveal any change in muscle fibers, but severe necrosis induced by BV or toxins (20 microg/ml) was clearly in evidence, and decreased significantly when soleus muscle was protected by AE. This protection was also observed when AE was administered 1 h after BV or BthTX-I, but not after BthTX-II. AE did not inhibit the catalytic PLA2 activity of BthTX-II or BV and did not change the PAGE pattern of BV, BthTX-I or BthTX-II. In vivo assays were performed in 100-g rats and maximal CK release was attained at a dose of 100 microg of BV, 3 h after injection. AE was not effective when injected 20 s after BV or toxins. However, injecting BV or toxins (100 microg), which were pre-incubated with AE (2 mg) caused an inhibition of 57%, 59% and 51%, respectively, with zero time pre-incubation, but was less effective with 1 h pre-incubation. This plant represents a potential source of promising myotoxin inhibitors.     

The role of Pimpla hypochondriaca venom in the suppression of pupal Noctuid host immunity

When pupae of the Tomato moth (Lacanobia oleracea L.) (Lepidoptera: Noctuidae) are injected with venom from the endoparasitoid wasp Pimpla hypochondriaca Retzius (Hymenoptera: Ichneumonidae), they show an increased susceptibility to the fungal entomopathogen Metarhizium anisopliae Sorokin (Fungi Imperfecti: Deuteromycotina). This observation, coupled with the fact that wasp eggs are comparatively unlikely to be encapsulated when implanted into envenomated pupae, suggests that venom of P. hypochondriaca suppresses the cellular immune defense mechanisms of L. oleracea. Injection of host pupae with the venom of P. hypochondriaca has a rapid, adverse effect on the normal respiration pattern of L. oleracea. It is possible that severe disruption to host-metabolism may contribute to a general failure in the hosts' normal immune response repertoire.     

Toxoplasma gondii: effects of neuwiedase, a metalloproteinase from Bothrops neuwiedi snake venom, on the invasion and replication of human fibroblasts in vitro

The major aim to the present study was to determine the effects of neuwiedase, a metalloproteinase isolated from Bothrops neuwiedi snake venom, on invasion and replication of Toxoplasma gondii in human fibroblasts in vitro. Neuwiedase treatment was done on host cells previously infected with T. gondii or on parasite before fibroblast infection. When treatments were done after or before infection, infection rates were inhibited in 71% and 61%, respectively. Considering that therapy protocols currently used in T. gondii infection cause considerable side effects, particularly in immunocompromised individuals and pregnant women, the results of neuwiedase treatment described herein could be taken into account for the development of new synthetic therapeutic agents, mainly due to the capacity of this enzyme to degrade extracellular matrix components, such as laminin, fibronectin and type I collagen, which is important to interfere in T. gondii host cell invasion.     

Taxonomical investigations onHelleborus viridis s. l. (Ranunculaceae) in Northern Italy

The taxon commonly namedHelleborus viridis in Lombardy (NW. Italy) differs from both, subsp.viridis and subsp.occidentalis, and is shown to fall within the variation range ofH. odorus subsp.laxus which has been reported so far for NE. Italy only. TrueH. viridis within Italy grows only in the Maritime Alps.     

Acetylcholinesterase activity in Clytia hemisphaerica (Cnidaria)

Cholinesterase activity is known in representatives of all living organisms phyla but the origin of the cholinergic system as known in bilaterian animals is still undeciphered. In particular the implication of cholinesterases in the nervous system of non-bilaterian Metazoa is not well known. We thus chose to investigate this activity in the Clytia hemisphaerica (Cnidaria) medusa. In toto histochemical staining revealed an acetylcholinesterase activity in the tentacle bulbs but not in the nervous system. Sequences homologous to acetylcholinesterase were searched within Clytia ESTs and compared to other sequences found in public databases.