Developmental changes in the embryo, pronymph, and first molt of the scorpion Centruroides vittatus (scorpiones: buthidae)

For the first time the scanning electron microscope was used to compare developmental changes in scorpion embryos and the first and second stadia. In the buthid species of this study, Centruroides vittatus, and all other scorpions, the newborn climb up on their mother's back and remain there without feeding for several days. At this location, they undergo their first molt and in a few days they disperse, fully capable of foraging in the terrestrial environment. The results here support earlier suggestions that the first stadium (pronymph) is a continuation and extension of embryological development. The first molt results in a nymph with exoskeletal features much like those in the adult. In the first molt the metasoma becomes relatively longer, and the sting (aculeus) becomes sharp and functional. The metasomal segments are modified for dorsal flexion and sting use. The embryos and the pronymphs have spiracles that open into an invagination near the posterior margin of flap-like abdominal plates in segments 4-7 of the ventral mesosoma. The second instars have spiracles that lead to book lungs farther anterior in sternites. Tubular legs with cylindrical segments in embryos and pronymphs become more sculptured and oval in the transverse plane. Each leg in the pronymph has a blunt, cup-shaped tip while distal claws (ungues, dactyl) are present in the second instar and subsequent stages. There are some sharp bristles and primordial sensilla in the pronymphs, but the second stadium has adult-like surface features: rows of knobs or granulations (carinae), serrations on the inner surfaces of cheliceral and pedipalpal claws, filtering hairs at the mouthparts, peg sensilla on the pectines, and mechano- and chemoreceptor sensilla on the body and appendages. Scorpion embryos and pronymphs have some structures like fossil scorpions thought to have been aquatic. There is a gradual development of features that appear to be terrestrial adaptations. Evidence is provided for the formation of the sternum from third and fourth leg coxal primordia and possibly from the first abdominal segment. This study is the first to provide evidence for a forward shift of the gonopore along with other structures in the anterior abdomen.     

Identification of second lipolysis activating protein from scorpion Buthus occitanus tunetanus

Besides the previously described LVP1, a second protein, LVP2, inducing a lipolytic response in adipose cells, was purified from scorpion Buthus occitanus tunetanus venom. It represented 2% of crude venom proteins, with pHi = 6 and molecular mass of 16889 Da. The reduction and the alkylation of LVP2 revealed an heterodimeric structure. Isolated alpha and beta chains of LVP2 have a molecular weight (MW) of 8822 Da and 8902, respectively. This protein was not toxic to mice and stimulated lipolysis on freshly dissociated rat adipocytes in a dose-dependent manner with EC50 = 2 +/- 0.75 microg/ml. LVP2 subunits did not display any lipolytic activity. As previously described for venom and LVP1, beta adrenergic receptor (beta AR) antagonists interfere with LVP2 activity. Furthermore, it is shown that LVP2 competes with [3H] CGP 12177 (beta1/beta2 AR antagonist) for binding to adipocyte plasma membrane with an IC50 of about 10(-7)M. Thus, these results bring original information on the existence of proteins that are present in scorpion venoms and can exert a distinct biological activity on adipocyte lipolysis through a beta-type adreno-receptor pathway.     

Fine structure of scorpion trichobothria (Arachnida,Scorpiones)

Summary The fine structure of trichobothria in the scorpions Buthus occitanus (Amoureux, 1789) and Euscorpius carpathicus (Linné, 1767) was investigated by electron microscopy. In both species, cuticular and cellular characteristics are very similar. The articulation of the hair corresponds to that of other arachnid hair sensilla. The receptor endings are excentrically attached to the hair base. They consist of an enveloped S-shaped bundle of seven dendrites in B. occitanus and four in E. carpathicus. Neighbouring outer dendritic segments differ a great deal in diameter and ciliary modification. In B. occitanus, three enveloping cells and several additional secretory cells surround the inner dendritic segments. Structural characteristics are compared to those of other arachnid sensilla and their possible functional significance is discussed.     

The effects of temperature and moisture on survival capacity,cuticular permeability,hemolymph osmoregulation and metabolism in the scorpion,Centruroides hentzi (banks) (scorpiones,buthidae)

• 1.1. The temperature and water relations of Centruroides hentzi females were investigated. At 12 and 72% relative humidity (RH), the lower and upper Lt₅₀ were -4.5 and 43.7°C, and -4.7 and 45.1°C, respectively. When exposed to high temperature stress, survivorship was significantly greater under mesic conditions.
• 2.2. Cuticular water loss was higher under xeric conditions (12% RH), ranging from 0.061 mg/cm²/hr at 30°C to 0.211 at 41°C.
• 3.3. Exposure to dry air (0–5% RH) resulted in a significant increase in hemolymph osmolality: from 441 to 688 mOsm over a 5 day period.
• 4.4. Mean oxygen consumption rates increased from 161.7 mm³/g/hr at 34°C to 541.6 at 44°C. ATPase activity was significantly higher in animals acclimated and tested at 35°C.

     

Isolation and molecular characterization of LVP1 lipolysis activating peptide from scorpion Buthus occitanus tunetanus

LVP1, a novel protein inducing lipolytic response in adipose cells, was purified from scorpion Buthus occitanus tunetanus venom. It represented 1% of crude venom proteins, with pHi approximately 6 and molecular mass of 16170 Da. In contrast to well-characterized scorpion toxins, reduction and alkylation of LVP1 revealed an heterodimeric structure. Isolated alpha and beta chains of LVP1 have a respective molecular mass of 8877 and 8807 Da as determined by mass spectrometry. The N-terminal and some internal peptide sequences of LVP1alpha and beta were determined by Edman degradation. The full amino acid sequences of both chains were deduced from nucleotide sequences of the corresponding cDNAs prepared based on peptide sequences and the 3' and 5' RACE methodologies. LVP1alpha and beta cDNAs encode a signal peptide of 22 residues and a mature peptide of 69 and 73 residues, respectively. Each mature peptide contains seven cysteines, which are compatible with an interchain disulfide bridge. The cDNA deduced protein structures share a high similarity with those of some Na+ channel scorpion toxins. LVP1 was not toxic to mice after intracerebro-ventricular injection. LVP1 stimulated lipolysis on freshly dissociated rat adipocytes in a dose-dependent manner with EC50 of approximately 1+0.5 microg/ml. LVP1 subunits did not display any lipolytic activity. As previously described for venom, beta adrenergic receptor (beta AR) antagonists interfere with LVP1 activity. Furthermore, it is shown that LVP1 competes with [3H]-CGP 12177 (beta1/beta2 antagonist) for binding to adipocyte plasma membrane with an IC50 of about 10(-7) M. These results demonstrate the existence of a new type of scorpion venom nontoxic peptides that are structurally related to Na+ channel toxins but can exert a distinct biological activity on adipocyte lipolysis through a beta-type adrenoreceptor pathway.     

Characterisation of a novel toxin active on potassium apanaine channels, purified from Buthus occitanus tunetanus scorpion venom

A new peptidyl inhibitor of the small-conductance Ca(2+)-activated K+ channels (SKca) was purified to homogeneity from the venom of the Tunisian scorpion Buthus occitanus tunetanus. The molecular mass determined by SDS-PAGE, shows that it's a short peptide (3300 Da). The primary sequence of this toxin shows that it is a 31-residue polypeptide cross-linked by three disulfide bridges and structurally related to subfamily 5 of short scorpion toxins. This molecule shows similar pharmacological properties with this group of peptides inducing high toxicity in mice after intracerebro-ventricular injection, and competing with iodinated apamin for binding to its receptor site from rat brain synaptosomes (K0.5 = 4 nM).     

Fine structure of spermatozoa inAnthopharynx sacculipenis (Plathelminthes,Solenopharyngidae)

Summary The organisation of the spermatozoa ofAnthopharynx sacculipenis is described, based on electron-microscopical observations. The male gametes are fili-form in shape. They are totally enclosed by cortical microtubules and possess two free cilia. Special features are dot-like dense granules arranged in regular rows and terraced elaborations of the nuclear membrane. Such terraced elaborations are not known in any other species of flatworms whereas dot-like dense granules are described for some other taxa of the Rhabdocoela. Male gametes do not show synapomorphic correspondences between the Solenopharyngidae and the Prolecithiphora.Abbreviations ci cilia - ct cortical microtubules - db dense bodies - gl glycogen - mi mitochondrion - n nucleus - nt nuclear terraces     

Isolation of the first toxin from the scorpion Buthus occitanus israelis showing preference for Shaker potassium channels

We have purified BoiTx1, the first toxin from the venom of the Israeli scorpion, Buthus occitanus israelis, and studied its activity and genomic organization. BoiTx1 is a 37 amino acid-long peptide contained six conserved cysteines, and is classified as an alpha-KTx3.10 toxin. The pharmacological effects of BoiTx1 were studied on various cloned K(+) channels expressed in Xenopus laevis oocytes. BoiTx1 inhibited currents through Drosophila Shaker channels with an IC(50) value of 3.5+/-0.5nM, yet had much lesser effect on its mammalian orthologs. Thus, BoiTx1 is the first member of the alpha-KTx3 family that preferentially affects insect potassium channels.     

Isolation and some properties of a lectin from the venom of the Vietnamese scorpion Buthus occitanus sp

A lectin was isolated from the venom of scorpion Buthus occitanus sp. by means of Sephadex G-50 gel filtration and CM-cellulose ion exchange chromatography. The homogeneous lectin preparation consisted of homodimeric molecules with a subunit Mr of 9.3 kDa. Glycine, alanine, and serine dominated in the lectin amino acid composition. The lectin was a glycoprotein containing 20% carbohydrates (predominantly mannose and glucose). Trypsin-treated murine erythrocytes agglutinated at the lectin concentration of 32 micrograms/ml. Hemagglutination was inhibited by carbohydrates (L-fucose > D-glucose > L-rhamnose > D-xylose). The lectin revealed no phospholipase or hyaluronidase, nor toxic activity.     

Fine structure of the ladybird spermatozoa (Insecta,Coleoptera, Coccinellidae)

The sperm structure of several ladybird species belonging to different subfamilies of Coccinellidae was studied. Three main sperm types were clearly recognized, and were characterized by differences in acrosomal length, the presence of a dense coat around the acrosome, the length of the basal body, the amount of the centriole adjunct material, and the diameter of the mitochondrial derivatives. However, the whole group shares a pattern of the posterior sperm region uncommon for insects, in which the axoneme and other flagellar components are running parallel with the nucleus. As a general conclusion, this study has revealed an inconsistency between the sperm structure and the systematics of the group, indicating that the generic concepts within the group do not reflect a natural classification, a statement also shared by molecular studies.     

An equilibrium ELISA for the dosage of Androctonus australis garzonii (Aag) and Buthus occitanus tunetanus (Bot) scorpion venoms: set up and calibration

Scorpion stings are very frequent in Tunisia; yet a method for evaluating envenoming severity and consequently victim treatment, has never been adequately established nor has its efficiency been properly evaluated. Indeed, a management of envenomed patients requires the optimization of envenoming antivenom immunotherapy. This task requires either, an accurate evaluation of toxicokinetic parameters of scorpion venoms in absence and in presence of antivenom, using animals as models, and the establishment of a quantitative relationship between human blood scorption venom levels, envenoming severities and clinical symptoms. A performant sandwich ELISA was set up and calibrated for measuring scorpion venom levels in human and rabbit sera. This assays was performed with polyclonal F(ab')2 specific to the two North African scorpion (Androctonus australis garzonii; Aag and Buthus occitanus tunetanus: Bot) venoms. It is simple, rapid, very sensitive (detection limit = 0.9 ng/ml) and shows a good linearity for venom concentrations in human sera comprised between 0.5 and 15 ng/ml. The ELISA is also reproducible: the coefficient of variation, determined at different venom concentrations (low: 4 ng/ml; medium: 8 ng/ml and high: 12 ng/ml) prepared in a pool of sera collected from several healthy donors, were lower than 10%. Such an ELISA has been successfully used, either in experimental toxinokinetic and immunotherapeutic studies carried out in rabbits or for the quantification of Aag and Bot venom levels in the serum of human victims stung by these scorpion.     

Molecular cloning and nucleotide sequence analysis of encoded anti-insect toxin BotIT2 from the scorpion Buthus occitanus tunetanus venom

Numerous toxins from scorpion venoms are much more toxic to insects than to other animal classes, and possess high affinity to Na+ channels. Many of them active on insects were purified from the venom of Buthus occitanus tunetanus. Using amino acid sequences of BotIT2 and RACE-PCR amplification (Rapid amplification of cDNA ends) technique, we isolated, identified and sequenced the nucleotide sequence from the venom glands of the scorpion Buthus occitanus tunetanus. The cDNA encodes a precursor of an insect toxin of 60 amino acid residues. The deduced nucleotide sequence toxin was identical to the determined amino acid sequence of BotIT2. BotIT2 is more similar to the excitatory toxins in its mode of action and to the depressant toxins in its primary structure.     

Fine structure of the spermatozoa of Crassostrea gigas (Mollusca,Bivalvia)

We describe sperm ultrastructure and acrosome differentiation during spermiogenesis in Crassostrea gigas (Mollusca Bivalvia). The sperm cell is a uniflagellated cell of the primitive type. The head region contains a rounded or conical nucleus surmounted by small acrosome. This organelle consists of a membrane-bound acrosomal granule, the contents of which have a homogeneous density, except in the anterior region, which is positive for PTA. The acrosome also surrounds the perforatorium, which includes oriented fibrillar elements: this is the axial body. The middle piece contains four mitochondria encircling two perpendicular centrioles. The distal centriole is provided with a system of mechanical fixation to the plasma membrane, consisting of nine fibers in radial arrangement. The tail flagellum, about 50 m?m long, contains the usual microtubular axoneme. © 1993 Wiley-Liss, Inc.     

Purification and primary structure of low molecular mass peptides from scorpion (Buthus sindicus) venom

The primary structures of four low molecular mass peptides (Bs 6, 8, 10 and 14) from scorpion Buthus sindicus were elucidated via combination of Edman degradation and matrix-assisted laser desorption ionization mass spectrometry. Bs 8 and 14 are cysteine-rich, thermostable peptides composed of 35-36 residues with molecular weights of 3.7 and 3.4 kDa, respectively. These peptides show close sequence homologies (55-78%) with other scorpion chlorotoxin-like short-chain neurotoxins (SCNs) containing four intramolecular disulfide bridges. Despite the sequence variation between these two peptides (37% heterogeneity) their general structural organization is very similar as shown by their clearly related circular dichroism spectra. Furthermore, Bs6 is a minor component, composed of 38 residues (4.1 kDa) containing six half-cystine residues and having close sequence identities (40-80%) with charybdotoxin-like SCNs containing three disulfide bridges. The non-cysteinic, bacic and thermolabile Bs10 is composed of 34 amino acid residues (3.7 kDa), and belongs to a new class of peptides, with no sequence resemblance to any other so far reported sequence isolated from scorpions. Surprisingly, Bs10 shows some limited sequence analogy with oocyte zinc finger proteins. Results of these studies are discussed with respect to their structural similarities within the scorpion LCNs, SCNs and other biologically active peptides.     

Study of the protective capacity of scorpion venom Buthus occitanus tunetanus polymerised to glutaraldehyde in mice strains with different haplotypes

The immune response obtained against the toxic fraction of the scorpion venom Buthus occitanus tunetanus detoxified by polymerisation with glutaraldehyde, was analysed for low inbred mice having different haplotypes: C57BL/6 (H-2b) et BALB/c (H-2d) and the SWISS outbred mouse. This three strains of mice, immunized with the polymeric form of Bot-G50 are able to induce an immune response with bumoral mediation. The anti-polymers antibodies obtained from immunized mice, cross-react with the native Bot-G50 fraction. Indeed, in vitro protection experiments demonstrated that immune sera were neutralizing (between 150 and 235 micrograms of Bot-G50 ml). The in vivo protection assays showed that immunized mice could resist the challenge by high amount of toxic fraction (between 70 and 80 micrograms of Bot-G50). This protection was found to be long-lived, since immunized SWISS mice could resist the challenge by 4 DL50 of the toxic fraction (80 micrograms) six month after the start of the immunized program.     

Purification, characterization, and primary structure of four depressant insect-selective neurotoxin analogs from scorpion (Buthus sindicus) venom.

Four depressant insect-selective neurotoxin analogs (termed Bs-dprIT1 to 4) from the venom of the scorpion Buthus sindicus were purified to homogeneity in a single step using reverse-phase HPLC. The molecular masses of the purified toxins were 6820.9, 6892.4, 6714.7, and 6657.1 Da, respectively, as determined by mass spectrometry. These long-chain neurotoxins were potent against insects with half lethal dose values of 67, 81, 103, and 78 ng/100 mg larva and 138, 160, 163, and 142 ng/100 mg cockroach, respectively, but were not lethal to mice even at the highest applied dose of 10 microg/20 g mouse. When injected into blowfly larvae (Sarcophaga falculata), Bs-dprIT1 to 4 induced classical manifestations of depressant toxins, i.e., a slow depressant flaccid paralysis. The primary structures of Bs-dprIT 1 to 4 revealed high sequence homology (60-75%) with other depressant insect toxins isolated from scorpion venoms. Despite the high sequence conservation, Bs-dprIT1 to 4 showed some remarkable features such as (i) the presence of methionine (Met(6) in Bs-dprIT1 and Met(24) in Bs-dprIT2 to 4) and histidine (His(53) and His(57) in Bs-dprIT1) residues, i.e., amino acid residues that are uncommon to this type of toxin; (ii) the substitution of two highly conserved tryptophan residues (Trp43 --> Ala and Trp53 --> His) in the sequence of Bs-dprIT1; and (iii) the occurrence of more positively charged amino acid residues at the C-terminal end than in other depressant insect toxins. Multiple sequence alignment, sequence analysis, sequence-based structure prediction, and 3D homology modeling studies revealed a protein fold and secondary structural elements similar to those of other scorpion toxins affecting sodium channel activation. The electrostatic potential calculated on the surface of the predicted 3D model of Bs-dprIT1 revealed a significant positive patch in the region of the toxin that is supposed to bind to the sodium channel.     

Fine structure of boar spermatozoa

Summary The fine structure of epididymal spermatozoa of boars was studied, with special regard to the head cytoplasm, the neck, and the axial filament. Epon embedding and staining with heavy metals was used.The acrosome consists of a moderately opaque, homogeneous substance bounded by a single membrane. Within the distinct equatorial segment, the acrosome is very thin and separated from the nuclear membrane by a narrow rim of moderately dense material, which may be related to the perforatorium of rat spermatozoa.The postnuclear cap consists of a dense, homogenous substance inside the cell membrane and is stainable with phosphotungstic acid.The fibre structures of the neck are surrounded by folded extensions of the nuclear membrane. Two short, dark rods appear in the centre of the neck. The light segments of the coarse, peripheral fibres are merely deep notches in the fibre substance. The coarse peripheral fibres reach their maximal thickness at the anterior end of the middle piece. They taper rapidly anteriorly from this point and more gradually posteriorly. Irregular bridges connect them with each other in the anterior middle piece.The central 9+2 fibrils of the axial filament have distinct arms and spokes in the middle piece and main piece. The subfibrils connected with the arms and spokes appear to be solid, except in the neck and end pieces. The two central fibrils run through the neck to the wall of the proximal centriole. Acknowledgements. We wish to express our sincere gratitude to Dr. B. Afzelius, the Wenner-Gren Institute, Stockholm, for helpful discussions regarding tail fine structure, and to Dr. J. Luft, Department of Anatomy, University of Washington, Seattle, Wash., U.S.A., for the generous supply of ingredients for the Epon embedding procedure.     

Allozymic differentiation of Tunisian populations of Androctonus species and Buthus occitanus (Scorpiones: Buthidae)

We examined the population structure and phylogeny of Buthus occitanus (Amoreux, 1789), Androctonus amoreuxi (Audouin & Savigny 1812) and Androctonus australis (Linnaeus, 1758) in Tunisia, using horizontal starch gel and polyacrylamide gel electrophoresis to resolve allozymes at 18 loci. Populations of B. occitanus and A. amoreuxi showed little genetic variation, whereas A. australis populations showed moderate genetic variation. Moreover, despite the restricted dispersal abilities of these species, the allozyme analysis indicated very low levels of intraspecific population differentiation. The Island model of population differentiation best fitted the intraspecific variation, both for mainland populations and for the slightly more differentiated populations from two Mediterranean islands. No diagnostic, fixed alleles were observed between the species of Androctonus , which concurs with the results of nuclear ribosomal DNA analysis. Two subspecies of A. australis , A. a. garzonii Goyffon & Lamy, 1973 and A. a. hector Vachon, 1948, were not resolved by a phylogenetic analysis of the allozyme data. The most likely reasons for the slight population differentiation are either low mutation rates or recent range extensions promoted by human activities. The possibility of indirect gene flow between Androctonus species should also be explored.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 81 , 255–265.