Joachim Haupt

Distinct primary structures of the major peptide toxins from the venom of the spider Macrothele gigas that bind to sites 3 and 4 in the sodium channel1

Six peptide toxins (Magi 1–6) were isolated from the Hexathelidae spider Macrothele gigas. The amino acid sequences of Magi 1, 2, 5 and 6 have low similarities to the amino acid sequences of known spider toxins. The primary structure of Magi 3 is similar to the structure of the palmitoylated peptide named PlTx‐II from the North American spider Plectreurys tristis (Plectreuridae). Moreover, the amino acid sequence of Magi 4, which was revealed by cloning of its cDNA, displays similarities to the Na+ channel modifier δ‐atracotoxin from the Australian spider Atrax robustus (Hexathelidae). Competitive binding assays using several 125I‐labelled peptide toxins clearly demonstrated the specific binding affinity of Magi 1–5 to site 3 of the insect sodium channel and also that of Magi 5 to site 4 of the rat sodium channel. Only Magi 6 did not compete with the scorpion toxin LqhαIT in binding to site 3 despite high toxicity on lepidoptera larvae of 3.1 nmol/g. The K is of other toxins were between 50 pM for Magi 4 and 1747 nM for Magi 1. In addition, only Magi 5 binds to both site 3 in insects (K i=267 nM) and site 4 in rat brain synaptosomes (K i=1.2 nM), whereas it showed no affinities for either mammal binding site 3 or insect binding site 4. Magi 5 is the first spider toxin with binding affinity to site 4 of a mammalian sodium channel.

Trichobothrien und tastborsten der milbe Microcaeculus (Acari, Prostigmata, Caeculidae)

SummaryThe ultrastructure of trichobothria and normal mechanoreceptive bristles has been studied in Microcaeculus steineri delamarei. In spite of great differences in the outer appearance the inner structure of both kinds of sensilla is quite similar: In each case two dendrites are linked to the hair base by a system of fibrils. Therefore it may be suggested that two different directions may be distinguished by each sensillum. Apparently, the bristles may be bent in every direction, but at least the trichobothria show two favorite directions of bending. Certainly the oval shape of the hair base is the morphological reason for this pecularity. The fibrillar arrangement attaching the tubular bodies to the hair base deserves special interest concerning possible movements of the tubular bodies during the bending process of the sensory bristle. Some cytochemical studies are under investigation.ZusammenfassungAn Microcaeculus steineri delamarei wurde die Feinstruktur der Trichobothrien und der normalen mechanorezeptiven Borsten untersucht. Trotz der großen Unterschiede in der äußeren Erscheinung ist der innere Bau beider Sensillentypen recht ähnlich: In jedem Falle stehen zwei Dendriten über ein fibrilläres System mit der Haarbasis in Verbindung. Es ist daher anzunehmen, daß these Sensillen jeweils zwei verschiedene Richtungen unterscheiden können. Obwohl die Sinneshaare aufgrund ihrer Aufhängung in alle Richtungen ausgelenkt werden konnen, sind wenigatens bei den Trichobothrien zwei bevorzugte Auslenkrichtungen zu beobachten. Wahrscheinlich ist die leicht ovale Form der Sinneshaarbasis für these Besonderheit ausschlaggebend. Das fibrilläre System, das die Tubularkörper mit der Haarbasis verbindet, erscheint von besonderem Interesse im Hinblick auf mögliche Bewegungen der Tubularkörper bei Haarauslenkung. Weitere cytochemische Untersuchungen sind im Gange.

Moulting and morphogenesis of sensilla in a prostigmate mite (Acari, Actinotrichida, Actinedida: Caeculidae)

SummaryIn the prostigmate mite Microcaeculus steineri delamarei moulting and morphogenesis of mechanoreceptive sensilla were studied by electron microscopy and compared to corresponding sensilla of other arthropods. Dendritic contact with the cuticular parts of old sensilla breaks down during apolysis. Two groups of cells are engaged in the formation of new sensilla: 1) several trichogen and two tormogen cells in a semicircular arrangement, and 2) two sheath cells surrounding the mechanoreceptive dendrites. Cells ensheathing the dendrites do not play any part in the formation of bristles. These observations differ from those on insect sensilla during moulting.

A Trial of Mass Spectrometric Characterization of Femto‐Molar Amount from Subtropical Islands

Many kinds of venomous principles modulate physiological responses of mammalian signal transduction systems, on which they act selectively as enhancers, inhibitors or some other kind of effectors. These toxins have become useful tools for physiological research. We have characterized paralyzing toxins from the venom of spiders, scorpions, insects, jellyfishes and sea anemones in the subtropical region including the Ryukyu Islands. Venom profiles are screened by MALDI‐TOF fingerprinting analysis prior to purification of the venomous components, then marked target toxins of small molecular mass (1000–5000) are characterized directly by means of mass spectrometric techniques such as Frit‐FAB MS/MS, PSD/CID‐TOF MS, Capil. ‐HPLC/Q‐TOF MS/MS etc. The proteinous toxins of jellyfish or sea anemone are characterized by RT‐PCR technique by the information of the cleaved peptides after the protein was hydrolyzed by appropriate peptidase and the sequence of the cleaved peptide was determined by conventional methods. The venom of Araneid spider is mainly composed of a mixture of closely related acylpolyamines. More than 90 polyamine toxins were identified from one venom sac of the Madagascan spider, Nephilengys borbonica, by Frit‐Fab MS/MS employing charge remote fragmentation technique. A novel polyamine toxin was also found from the rare wondering spider, Macrothele gigas from Iriomote Island. The structure of the toxin is an analog of polyamine toxin found in trapdoor spiders. Many kinds of cystine‐rich peptides showing various types of ion channel antagonism have been isolated from spiders. A series of toxins possessing the same mode of cystine knots was recently isolated from the saliva of assassin bugs, Peirates turpis, Isyndus obscurus, Agriophodrus dohrni. These toxins act as calcium channel blocker. Most of the scorpion toxins reported are from scorpions hazardous to humans, and they belong to the major super family Buthoidea. Among them are the well‐known genera, such as Buthus, Androctonus, Centruroides, Leiurus, or Tytius. We have investigated the minor group of scorpions from the super family Chactoidea (Scorpionidae, Ishnuridae). The venoms of these scorpions, involving the genera Heterometrus, Pandinus, Opisthacanthus, and Isometrus, contain different kinds of peptide toxins. Fingerprinting peptide analysis of the toxin profiles for these scorpions showed some difference from the profiles of Buthoidea scorpions. These venoms contain linear pore‐forming peptides and 2‐cystine‐bridged toxins in addition to 4‐cystine‐bridged toxins. The most hazardous jellyfish in Okinawa, Chiropsalmus quadrigatus, and the related box jellyfishes, Carybdea rastoni, C. alata, contain quite labile proteinaceous toxins, CqTX, CrTX and CaTX, respectively. The toxins were inactivated by adding an organic solvent such as methanol or acetonitrile, by changing the pH of the toxin solution, dialyzing the toxin solution, storing the toxin in a refrigerator, or by lyophilizing the toxin solution. However, the toxic activity was retained in the presence of sodium chloride. We purified the jellyfish toxins by adding sodium chloride through all steps of the purification procedure and finally obtained the whole primary amino acid sequence of the toxin by RT‐PCR method. The toxic protein CqTX is homologous to the other box jelly fish toxin, CrTX and CaTX. These toxins belong to a new class of proteins since they show no homology to known proteins. Another notorious and dangerous specimen in the Ryukyu Islands is Phyllodiscus semori. The venom is composed of three kinds of proteins (PsTX‐20A, PsTX‐60A, PsTX‐60B). PsTX‐20A shows homology to the proteinaceous toxin actinoporin, a cytolytic protein isolated from the genus Actinia, but PsTX‐60s has no homology to any ever cloned proteins. Further elucidation of the mechanism of toxic action of these Coelenterates is in progress.

New products of defense secretion in south east Asian whip scorpions (Arachnida: Uropygi: Thelyphonida).

Abstract Secretion products from the opisthosomal defense gland of south east Asian whip scorpions were identified for the first time by gas-chromatography and mass-spectrometry. Specimens of the genera Hypoctonus, Typopeltis and Ginosigma were tested. While some ingredients are present in large concentrations, others are possibly only side products and may be synthesized more incidentally. For this reason no important functional role is attributed to them. There are considerable individual differences concerning the concentrations of various ingredients. While the secretion products of most species of the genus Typopeltis - similar to Mastigoproctus - are characterized by acetic and octanoic acid in large concentrations, the secretion product of Hypoctonus siamensis provides octanoic acid only in a very low concentration but it is characterized by hexyl acetate.

Chinese whip scorpion using 2-ketones in defense secretion (Arachnida: Uropygi)

SummaryNorth American as well as East Asian whip scorpions of the generaMastigoproctus andTypopeltis are known to use acetic acid and caprylic acid as a defense mechanism. In the secretion from defense glands of a ChineseTypopeltis species, 2-ketones (2-heptanone, 2-octanone, and 2-nonanone) were identified as constituents for the first time. While acetic acid is also present as a secretory product, no caprylic acid has been found.

The median eyes of Microcaeculus (Atari, Prostigmata, Caeculidae)

SummaryA large single “cornea” is situated on the frontal side of the naso of Microcaeculus. Caudally and nearly coaxial to the cornea separated by a hemolymphatic cavity, are the two median eyes on the other side of the naso. They face the bases of the cheliceres, directed caudally. The cuticle of the eye attains a thickness of up to only 0.24 μm. Sensory cells protrude from the eye cup in a dorsal direction delivering axons of different diameters bent caudally toward as yet undetermined centers.Each eye consists of 6 retinula cells, covered by a thin epithelial layer, and of an undetermined number of sheath cells. Different zones can be distinguished schematically in the sensory cells: 1. Rhabdomere Zone. Directly beneath the cuticle and the cuticle-forming cells, rhabdomeres are spread superficially; their corresponding plasmatic regions, also located here, contain much smooth- and rough-surfaced endoplasmic reticulum (ER), some multivesicular bodies, mitochondria, centrioles, and α-glycogen. 2. Microtubuli Zone. This zone is characterized by many microtubules. Few rough-surfaced ER, but many mitochondria, are present in this zone; voluminous ER cisterns are to be found placed in the cell area facing the hemolymphatic cavity. 3. Nucleus Zone. Due to an increased cell volume in this zone, the eyes overlap over a large area. In the sensory cells a conspicuous massing of glycogen rosettes, as well as several organelles, is to be found. 4. Axon Zone. The axons are of varied diameter (0.7–1.8 μm). Large amounts of glycogen are found only in thicker axons, cross-structured by single smooth ER-cisterns.Electron-microscopic investigations confirmed the findings of Coineau (1970). Additional probabilities are presented and discussed in regard to other functions of these eyes. As to the structure of the eyes, a perception of differences in light intensity is probable. Image vision seems an impossibility, as only 6 retinula cells exist in each eye. However, a certain “perception of light direction” may be possible since the field of view is limited by the body and its dorsal ledge.RésuméSur la face ventrale du naso, Microcaeculus possède une grande cornée impaire. A larrière, et dans laxe de Cette cornée, se trouvent, dans un pli profond, une paire dyeux médians. Its sent orientés vers larrière et séparés de la cornée dorsale par un espace interne rempli dhémolymphe. Lépaisseur de la cuticule de la cornée primaire est seulement de 0,24 μm.Les cellules sensorielles émergent des calottes oculaires, sélévent dans le naso puis se dirigent vers larrière. Elles constituent alors des axons dune épaisseur différente et se dirigent vers des centres optiques encore inconnus. Chaque œil est constitué de 6 cellules rétiniennes qui sont situées au-dessus dune mince couche épithéliale et dun nombre inconnu de cellules denveloppement. On pout distinguer, schématiquement, plusieurs régions dans ces cellules sensorielles: 1. La zone des rhabdomères. On trouve juste au-dessous de la cuticule, et des cellules qui la sécrétent, les rhabdomères qui sétalent superficiellement et également leurs régions plasmatiques correspondantes, qui contiennent beaucoup de RE à surface lisse on rugueuse, quelques corps multivésiculaires, des mitochondries, des centrioles et de lα-glycogène. 2. La zone des microtubules. Cette zone est caractérisée par la présence de nombreux microtubules. A côté dun pen de RE à surface rugueuse, on trouve dans cette zone un plus grand nombre de mitochondries et également de volumineuses citernes du RE, qui sent situées plus spécialement dans cette zone du côté de la cavité de lhémolymphe. 3. La zone nucléaire. Du fait du plus grand volume des cellules les yeux se trouvent en contact sur une grande surface dans cette zone. Dans les cellules visuelles il y a beaucoup dorganites cellulaires et une suprenante accumulation de rosettes de glycogène. 4. La zone des axones. Le diamètre des axones est diffèrent (0,7–1,8 μm). On ne trouve plus de glycogene, en grande quantité, quo dans les plus grands axones, qui sent caractérisés par la présence des citernes transversales de RE à surface lisse.Cette étude en microscopie électronique confirme les découvertes de Coineau (1970). A côté des cola, des fonctions hypothétiques de cet œil sont discutées. En raison de la structure des yeux, onpeut présumer que la perception des différences dintensité lumineuse est possible. La vision dune image semble impossible étant donné quo chaque œil ne possède quo 6 céllules rétiniennes. Toutefois, une certaine perception directionelle de la lumière est pout-être possible en raison de la limitation du champ par le corps.ZusammenfassungMicrocaeculus besitzt auf der frontal gelegenen Seite des Naso eine große, unpaare „Cornea”. Caudal und nahezu coaxial von ihr, durch einen Hämolymphraum getrennt, liegen auf der anderen Seite des Naso in einer tiefen Einfaltung 2 nach hinten gerichtete, sehr schwach rot pigmentierte Medianaugen. Die Dicke ihrer Cuticula beträgt nur 0,24 μm. Die Augenzellen ziehen zunächst noch innerhalb des Naso in dorsale Richtung und biegen dann unter Bildung unterschiedlich dicker Axone nach caudal zu den noch unbekannten optischen Zentren um.Jedes Auge besitzt 6 Retinulazellen, die unter einer diinnen Epidermisschicht liegen, sowie eine unbestimmte Auzahl von Hüllzellen. Die Sinneszellen lassen sich schematisch in mehrere Zonen untergliedern: 1. Die Rhabdomeren-Zone. Dicht unter der Cuticula und ihren Bildungszellen liegen die flach ausgebreiteten Rhabdomeren und deren angrenzende Plasmabereiche mit viol glattem und rauhem ER, einzelnen multivesikulären Körpern, Mitochondrion, Centriolen und α-Glykogen. 2. Die Mikrotubuli-Zone. Charakteristisch sind die zahlreichen Mikrotubuli. Es ist nur wenig rauhes ER vorhanden, dafür treten um so mehr Mitochondrien auf und, wie auch in anderen Zonen, voluminöse ER-Zisternen (Artefakte?). Diese liegen besonders auf der dem Hämolymphraum zugewandten Seite der entsprechenden Zellen. 3. Die Kernzone. Die im übrigen getrennten Augen stoßen in dieser Zone wegen ihres gröBeren Zellvolumens auf breiter Fläche aneinander. In den Sehzellen treten zahlreiche Organellen und auffällige Anhäufungen von Glykogen-Rosetten auf. 4. Die Axon-Zone. Die Axone haben einen unterschiedlichen Durchmesser (0,7–1,8 μm). Nur in den dickeren, durch einzelne, glatte ER-Zisternen querstrukturierten Axonen wurde bislang viel Glykogen gefunden. Befunde von Coineau (1970). Mit den vorhandenen Strukturen können sehr wahrscheinlich Lichtintensitätsuntersehiede wahrgenommen werden. Zwar machen die 6 Retinulazellen eines jeden Auges ein Bildsehen unmöglich, doch dürfte wegen der Einschränkung des Gesichtsfeldes durch den Körper und seine dorsale, dachartige Vorwölbung ein gewisses „Richtungssehen” gegeben sein. Darüber hinaus erscheinen weitere Funktionen möglich.

Taxonomy of Spiders

Among many arachnid orders like scorpions, sun spiders, harvestmen, pseudoscorpions, or mites, the araneae or web spiders include some of the most venomous species. For this reason taxonomy and phylogeny of this group are surveyed, at least in order to provide more attention to spider families and species whose venomous capabilities are unknown.