The morphology of mating plugs and its formation in scorpions: Implications for intersexual participation.

Mating plugs have been proposed as a mechanism that has evolved to avoid sperm competition. Their structure and composition vary across taxa and are related to the effectiveness of its function. This effectiveness could be related to different evolutionary interests of the sexes. Urophonius brachycentrus and Urophonius achalensis (Scorpiones, Bothriuridae) are highly suitable species to study mating plugs because both are monandrous species with specific morphological and physiological responses in the female's genitalia. Here, we analyze (a) the morphology and fine structure of the mating plugs of both species, (b) the site of production in males and the formation process of the mating plug, and (c) the changes that it undergoes over time in the female's reproductive tract. In both species, a complex mating plug obliterates the female's genital aperture and fills the genital atrium. We observed considerable interspecific variation in the mating plug morphology. A mating hemi-plug was found surrounding the capsular lobes of the hemispermatophore, which could have a mixed composition (involving portions of the hemispermatophore and glandular products). The glandular portion was transferred in a semi-solid state filling the female's genital atrium and then hardening. Changes that the plug undergoes in the female's genitalia (darkening and increase of the "distal" area of the plug) indicate a participation of the female to the formation of this type of plug. Our study provides new insights into the plugging phenomenon in scorpions, and we discussed the adaptive significance as a post-copulatory mechanism to avoid sperm competition.

Nested helicoids in biological microstructures.

Helicoidal formations often appear in natural microstructures such as bones and arthropods exoskeletons. Named Bouligands after their discoverer, these structures are angle-ply laminates that assemble from laminae of chitin or collagen fibers embedded in a proteinaceous matrix. High resolution electron microscope images of cross-sections through scorpion claws are presented here, uncovering structural features that are different than so-far assumed. These include in-plane twisting of laminae around their corners rather than through their centers, and a second orthogonal rotation angle which gradually tilts the laminae out-of-plane. The resulting Bouligand laminate unit (BLU) is highly warped, such that neighboring BLUs are intricately intertwined, tightly nested and mechanically interlocked. Using classical laminate analysis extended to laminae tilting, it is shown that tilting significantly enhances the laminate flexural stiffness and strength, and may improve toughness by diverting crack propagation. These observations may be extended to diverse biological species and potentially applied to synthetic structures.

Formation of the diverticular lumen that enables oocyte fertilization in katoikogenic scorpions, Heterometrus spinifer and Opistophthalmus boehmi (Scorpionidae).

The structure of diverticula of the female gonads was analyzed in two scorpions from the family Scorpionidae by means of standard microscopic techniques (light microscopy, histochemistry, transmission electron microscopy). In scorpions, the female gonad, termed the ovariuterus, participates in two consecutive processes: oogenesis and embryogenesis. In sexually reproducing scorpions, the ovariuterus is also involved in fertilization. Both scorpions under study reproduce sexually. They also represent the katoikogenic type of development, which means that oogenesis, fertilization, and embryogenesis take place in the diverticula, which are sac-like outpocketings of the ovariuterine tubules. Formation of a lumen in the diverticulum is indispensable for sperm entry to enable fertilization and subsequent embryogenesis. The aim of the study was to test our hypothesis that the diverticulum lumen forms due to the engagement of the centrally located stalk cells. In this report, we show that in two species of katoikogenic scorpions, at the final stages of oogenesis, the cytoplasm of the stalk cells contains secretory organelles. In the stages preceding fertilization, secretory activity of the stalk cells and fragmentation of their apical parts lead to formation of the diverticulum lumen by a process similar to "cord hollowing" that commonly occurs for lumen formation in other morphogenesis model systems.

Scorpion katoikogenic ovariuterus - Much more alike to apoikogenic type than it seemed to be.

Scorpions are viviparous matrotrophic arthropods. Both, fertilization and embryonic development occur in the female gonad called ovariuterus. Two distinct reproductive patterns are recognized among scorpions: apoikogenic and katoikogenic. In the ovariuterus of apoikogenic scorpions growing oocytes protrude from the ovarian wall and continue previtellogenic and vitellogenic growth on the gonad surface being accompanied by the follicular cells that cover the oocyte surface, and, in most families, the stalk cells that join the oocyte with the ovariuterus wall. In the katoikogenic ovariuterus the oocytes grow in outpocketings of the ovarian wall called diverticula. The aim of our study was to show the development and structure of the diverticula in two katoikogenic scorpions from the family Scorpionidae: Ophistothalmus boehmei and Heterometrus spinifer. We show that the somatic components of each diverticulum develop from the two epithelial layers of the ovariuterine wall. Before fertilization, the wall of the mature diverticula consists of two distinctive epithelial layers: an internal and an external one. Our observations reveal that the epithelial cells of the internal layer of the diverticulum show striking morphological resemblance to the follicular and stalk cells that accompany the growing oocytes in some apoikogenic scorpions. The external epithelial layer of the katoikogenic diverticulum seems to have no equivalents in the apoikogenic type. Functions of the somatic cells of the diverticulum are discussed.

Differentiation of somatic cells in the ovariuteri of the apoikogenic scorpion Euscorpius italicus (Chelicerata, Scorpiones, Euscorpiidae).

In apoikogenic scorpions, growing oocytes protrude from the gonad (ovariuterus) and develop in follicles exposed to the mesosomal (i.e. hemocoelic) cavity. During subsequent stages of oogenesis (previtellogenesis and vitellogenesis), the follicles are connected to the gonad surface by prominent somatic stalks. The aim of our study was to analyze the origin, structure and functioning of somatic cells accompanying protruding oocytes. We show that these cells differentiate into two morphologically distinct subpopulations: the follicular cells and stalk cells. The follicular cells gather on the hemocoelic (i.e. facing the hemocoel) surface of the oocyte, where they constitute a cuboidal epithelium. The arrangement of the follicular cells on the oocyte surface is not uniform; moreover, the actin cytoskeleton of these cells undergoes significant modifications during oocyte growth. During initial stages of the stalk formation the stalk cells elongate and form F-actin rich cytoplasmic processes by which the stalk cells are tightly connected to each other. Additionally, the stalk cells develop microvilli directed towards the growing oocyte. Our findings indicate that the follicular cells covering hemocoelic surfaces of the oocyte and the stalk cells represent two distinct subpopulations of epithelial cells, which differ in morphology, behavior and function.

Microwhip scorpions (Palpigradi) feed on heterotrophic cyanobacteria in Slovak caves--a curiosity among Arachnida.

To date, only morphological and anatomical descriptions of microwhip scorpions (Arachnida: Palpigradi) have been published. This very rare group is enigmatic not only in its relationships to other arachnids, but especially due to the fact that these animals dwell only underground (in caves, soil, and interstitial spaces). We observed the curious feeding habit of the microwhip scorpion Eukoenenia spelaea over the course of one year in Ardovská Cave, located in Slovakia's Karst region. We chose histology as our methodology in studying 17 specimens and based it upon Masson's triple staining, fluorescent light and confocal microscopy. Single-celled cyanobacteria (blue-green algae) were conspicuously predominant in the gut of all studied palpigrades. Digestibility of the consumed cyanobacteria was supported by the presence of guanine crystals, glycogen deposits and haemocytes inside the palpigrade body. Cyanobacteria, the oldest cellular organisms on Earth, are very resistant to severe conditions in caves, including even darkness. Therefore, the cyanobacteria are able to survive in dark caves as nearly heterotrophic organisms and are consumed by cave palpigrades. Such feeding habit is extraordinary within the almost wholly predacious orders of the class Arachnida, and particularly so due to the type of food observed.

A double role of sperm in scorpions: the mating plug of Euscorpius italicus (Scorpiones: Euscorpiidae) consists of sperm.

Mating plugs occluding the female gonopore after mating are a widespread phenomenon. In scorpions, two main types of mating plugs are found: sclerotized mating plugs being parts of the spermatophore that break off during mating, and gel-like mating plugs being gelatinous fluids that harden in the female genital tract. In this study, the gel-like mating plug of Euscorpius italicus was investigated with respect to its composition, fine structure, and changes over time. Sperm forms the major component of the mating plug, a phenomenon previously unknown in arachnids. Three parts of the mating plug can be distinguished. The part facing the outside of the female (outer part) contains sperm packages containing inactive spermatozoa. In this state, sperm is transferred. In the median part, the sperm packages get uncoiled to single spermatozoa. In the inner part, free sperm is embedded in a large amount of secretions. Fresh mating plugs are soft gelatinous, later they harden from outside toward inside. This process is completed after 3-5 days. Sperm from artificially triggered spermatophores could be activated by immersion in insect Ringer's solution indicating that the fluid condition in the females' genital tract or females' secretions causes sperm activation. Because of the male origin of the mating plug, it has likely evolved under sperm competition or sexual conflict. As females refused to remate irrespective of the presence or absence of a mating plug, females may have changed their mating behavior in the course of evolution from polyandry to monandry.

Complex meiotic configuration of the holocentric chromosomes: the intriguing case of the scorpion Tityus bahiensis.

Mitotic and meiotic chromosomes of Tityus bahiensis were investigated using light (LM) and transmission electron microscopy (TEM) to determine the chromosomal characteristics and disclose the mechanisms responsible for intraspecific variability in chromosome number and for the presence of complex chromosome association during meiosis. This species is endemic to Brazilian fauna and belongs to the family Buthidae, which is considered phylogenetically basal within the order Scorpiones. In the sample examined, four sympatric and distinct diploid numbers were observed: 2n = 5, 2n = 6, 2n = 9, and 2 = 10. The origin of this remarkable chromosome variability was attributed to chromosome fissions and/or fusions, considering that the decrease in chromosome number was concomitant with the increase in chromosome size and vice versa. The LM and TEM analyses showed the presence of chromosomes without localised centromere, the lack of chiasmata and recombination nodules in male meiosis, and two nucleolar organiser regions carrier chromosomes. Furthermore, male prophase I cells revealed multivalent chromosome associations and/or unsynapsed or distinctly associated chromosome regions (gaps, less-condensed chromatin, or loop-like structure) that were continuous with synapsed chromosome segments. All these data permitted us to suggest that the chromosomal rearrangements of T. bahiensis occurred in a heterozygous state. A combination of various factors, such as correct disjunction and balanced segregation of the chromosomes involved in complex meiotic pairing, system of achiasmate meiosis, holocentric nature of the chromosomes, population structure, and species dispersion patterns, could have contributed to the high level of chromosome rearrangements present in T. bahiensis.

Development of respiratory structures in embryos and first and second instars of the bark scorpion, Centruroides gracilis (Scorpiones: Buthidae).

The SEM was used to study the development of respiratory structures in successive stages in relation to the overall changes occurring in the scorpions. Book lung development is a slow process, starting with spiracles and a sac-like atrium in the early embryo and continuing lamellar formation to 150 or more in the adult. In the embryo, the primordial epithelial cells become aligned in a planar pattern as they secrete granules of material that aggregate spontaneously to form the cuticular walls of the lamellae. A blade-like structure is formed consisting of cells sandwiched within the two cuticle walls they secreted. These cells are in the primordial air channel. The adjacent hemolymph channel is nearly devoid of cells, but cross-bridges develop and help stabilize the cuticle walls and maintain the width of the channel. The cells in the primordial air channel undergo cytolysis, leaving it open for air except for cuticular cross-bridges. Development continues in the newborn (first instars); the air channels of some lamellae still contain cells and are not yet functional for gas exchange. The first instars are weak and relatively inactive. They climb up on the mother's dorsum until the first molt (about 8 days). With the cuticular walls of the lamellae in place, cells adhering to the wall in the hemolymph channel produce a thin, new tissue layer (epithelium) on the lamellar wall facing the hemolymph channel. This layer has many discontinuities as though it is slowly developing. Formation of the tissue layer and cytolysis of the cells in the air channels continue through the first molt in which little book lung cuticle is shed as exuvium. The air channels of the second instars (foraging nymphs) are now cell free and open for air passage except for the cross-bridges. The tissue layer is still incomplete and continues to be formed. It may provide the hypodermal primordium for cuticle replacement in later molts, but development was not studied beyond the second instar except for comparison with book lungs in the adult. The blade-like lamellae in the adult are larger and more numerous than in the second instar, but in the anterior book lung the shape of the cuticle wall and cross-bridges and the widths of the air and hemolymph channels are about the same as in the second instar. The air channels in the posterior part of the lamellae have distinctive, vein-like space-holders. The similarity of the adult anterior lamellae with those in the second instar suggests retention of this part through the 4-5 molts to maturation, and/or cell processes like those in the embryo are repeated, but this needs to be examined in further studies of cell and cuticle changes before and during the molts.

The pectine organs of the scorpion, Vaejovis spinigerus: structure and (glomerular) central projections.

The pectines of a new-world scorpion were studied as to their sensilla, nerve supply, and central nervous projections. (i) Pectines and sensilla in Vaejovis are similar to those examined in old-world species previously, although Vaejovis' pectines are larger and equipped with more receptors. The specialized peg sensilla show ultrastructural features characteristic of arthropod chemo- and mechanoreceptors, with the chemosensory exceeding the mechanosensory neuron population about 11-fold in number. (ii) The motoneuron supply of the pectines resembles that of other limbs and apparently conforms to a general arthropod plan. Motoneuron somata occur in three ventral groups, the anterior and posterior ipsilateral, and the contralateral groups. (iii) Pectine afferents terminate mainly in two ventromedial neuropil areas of the fused subesophageal ganglion mass. The larger posterior pectine neuropil shows a distinct glomerular and layered ("lobular") organization, reminiscent of insect antennal lobes and malacostracan olfactory lobes. Afferents enter the neuropil from its periphery, and output neurons leave through a central tract. Most projections show somatotopic organization, and several glomeruli exhibit GABA-like immunoreactivity, indicative of inhibitory synaptic interactions. The glomerular structure of the main pectine neuropil may indicate that such compartmentalisation is advantageous for the initial processing of chemosensory signals. The somatotopic projection of pectin receptors may be related to the use of the pectines in chemosensory orientation to substrate-bound chemicals, and in active sensing.

Immunocytochemical localization of scorpion digestive lipase.

The scorpion hepatopancreas consists of digestive diverticula and interstitial tissue. A digestive diverticulum is composed of two differentiated cell types: the secretory zymogene-like cells and the digestive cells which are the most abundant. The scorpion digestive lipase (SDL) has been previously purified from scorpion hepatopancreas, but its cellular localization has not yet been established. Polyclonal antibodies specific to SDL were prepared and used in immunofluorescence and immunogold techniques to determine the cellular location of SDL. Our results clearly established that SDL was detected intracellularly in specific vesicles tentatively named (SDL+) granules of the digestive cells. No immunolabelling was observed in secretory zymogene-like cells. This immunocytolocalization indicates that lipid digestion might occur in specific granules inside the digestive cells, as suggested by previous studies on the scorpion digestive process.

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.

Humicolous microcharmid scorpions: a new genus and species from Madagascar.

A new genus and species of humicolous microcharmid scorpion are described on the basis of a single specimen collected in the Ankarana Reserve, Madagascar. New considerations regarding the taxonomy and morphology of micro-buthoid Malagasy scorpions are proposed, based mainly on the study of the peg-shaped sensillae of the pectines by scanning electron microscopy.

Zinc is incorporated into cuticular "tools" after ecdysis: the time course of the zinc distribution in "tools" and whole bodies of an ant and a scorpion.

An understanding of the developmental course of specialized accumulations in the cuticular "tools" of arthropods will give clues to the chemical form, function and biology of these accumulations as well as to their evolutionary history. Specimens from individuals representing a range of developmental stages were examined using MeV - Ion microscopy. We found that zinc, manganese, calcium and chlorine began to accumulate in the mandibular teeth of the ant Tapinoma sessile after pre-ecdysial tanning, and the zinc mostly after eclosion; peak measured zinc concentrations reached 16% of dry mass. Accumulations in the pedipalp teeth, tarsal claws, cheliceral teeth and sting (aculeus) of the scorpion Vaejovis spinigeris also began after pre-ecdysial tanning and more than 48 h after ecdysis of the second instars. Zinc may be deposited in the fully formed cuticle through a network of nanometer scale canals that we observed only in the metal bearing cuticle of both the ants and scorpions. In addition to the elemental analyses of cuticular "tools", quantitative distribution maps for whole ants were obtained. The zinc content of the mandibular teeth was a small fraction of, and independent of, the total body content of zinc. We did not find specialized storage sites that were depleted when zinc was incorporated into the mandibular teeth. The similarities in the time course of zinc, manganese and calcium deposition in the cuticular "tools" of the ant (a hexapod arthropod) and those of the scorpion (a chelicerate arthropod) contribute to the evidence suggesting that heavy metal-halogen fortification evolved before these groups diverged.

[Study on the micro-identification of insect material medicine].

OBJECTIVE: To seek microstructure characters and method of insect material medicines. METHOD: Insect material medicines and Chinese patent medicines containing these medicines were observed and compared with OLYMPUS. RESULT: The texture of integumentary cells, the shape and size of hair socket and bristle were important characters for species identification. CONCLUSION: This method can effectively identify 8 insect medicines, which provides some data and foundation for Chinese patent medicines' identification containing insects medicines and making quality standard for new drugs.

Dispositions of junctional feet in muscles of invertebrates.

The structure and disposition of the feet occupying the junctions between sarcoplasmic reticulum (SR) and surface membrane/transverse tubules were studied in muscles from a variety of invertebrates. Feet were imaged by rotary shadowing of isolated junctional SR vesicles and by filtering of micrographs from grazing views of the junction in thin sections. The overall size and shape of invertebrate feet is the same as that of feet in skeletal and cardiac muscle of vertebrates. However, the arrangement of feet in invertebrate muscles differs from that in vertebrates. These findings are discussed in terms of known variations in properties of excitation-contraction coupling of the two phyla.

The ultrastructure of hemocytopoietic organs in the desert scorpion, Paruroctonus.

The light and electron microscopes were used to examine possible hemocytopoietic tissue in the desert scorpion, Paruroctonus mesaensis. Results agree with earlier light microscopic studies that cells are released into the blood from the two lateral lymphoid organs and the supraneural gland. The former are sacciform structures attached by their anterior ends to the diaphragm. The supraneural gland forms the thickened wall of the supraneural artery in the mesosoma from the first to the third abdominal ganglia. The lateral lymphoid glands have an acellular stroma in which are embedded granular and agranular cells. The stroma is apparently formed by specialized cells which release membranous cell fragments that become the matrix of the gland. Cells are released into the body cavity from the periphery of the two organs. The supraneural gland has a fibrous stroma in which are embedded a variety of cell types. The cells appear to be released in greatest abundance into the blood in the lumen of the gland. The gland has cells with opaque granules (0.9-1.4 micron diameter) and agranular cells of variable shape. The most abundant cell, possibly the stem-cell for the others, is about 10 micron diameter and often has processes of variable length. In addition, muscle cells at various stages of differentiation are found at the inner margin of the gland. These cells have thick and thin myofilaments (24-32 and 5-8 nm diameter) and dense bodies which sometimes become organized into sarcomeres with Z-bands before the cells are released into the gland lumen. The function of these muscle cells is unknown, but possibly they contribute to the maintenance of blood pressure and the release of cells into the blood from the inner margin of the gland.

Cytophysiological aspects of digestion and storage in the liver of a scorpion, Androctonus australis (Arachnida).

The liver of a scorpion, Androctonus australis (Arachnida), was examined electron-microscopically and cytochemically, emphasizing correlations between structure, cytochemistry and physiology. The liver consists of digestive diverticula and interstitial tissue. Digestive diverticula are composed of basophilic cells and digestive cells. Basophilic cells produce exoenzymes. Digestive cells ensure intracellular digestion of nutrients absorbed by pinocytosis and store glycogen, lipids and mineral salts; the wastes of the digestive process (guanine, uric acid, mineral elements, pigments) are concentrated in "brown body vacuoles" which are ejected into the lumen of the diverticula. The interstitial tissue stores glycogen and lipids; it contains many lysosome-like organelles rich in iron. Fasting induces a decrease of the ratio of the volume of the diverticula to that of the interstitial tissue, a slow disappearance of the reserves in both diverticula and interstitial tissue, an increase of synthesis in the basophilic cells, and a decrease of the number of vacuoles in the digestive cells. The digestive mode of the scorpions associates a primitive intracellular process with an advanced extracellular process. The interstitial tissue can be considered as homologous to the adipose tissue of insects and myriapods, although it is devoid of urate cells. The excretion of guanine and uric acid has a peculiar meaning, because these purine wastes do not come from endogenous catabolism.

Giant neurons and associated synapses in the peripheral nervous system of whip spiders.

Whip spiders (Amblypygi) are arachnids with a specialized first pair of legs. These legs are unusually long (20-25 cm) and are not used for walking. Instead their lengthy tarsi (7-8 cm) are covered with thousands of sensory hairs (mechano- and chemoreceptors). The legs thus resemble antennae of insects. Each sensory hair is associated with 4-40 neurons whose axons are grouped together to form two large tarsal nerves. The nerves contain about 23 000 sensory axons. Whereas most of the axons measure only 0.1-0.2 microns in diameter, a few are exceptionally large (3-20 microns). These are giant fibres. Their large somata are located in specific segments of the tarsi. The branched dendrites of the giant neurons receive hundreds of chemical synapses, presumably from the sensory axons of the hair sensilla. Since stimulation of the tarsal tip elicits fast withdrawal reaction (greater than or equal to 80 ms), it is likely that the giant fibres provide the pathway for the rapid conduction of nerve impulses to the motor centres of the C.N.S. The system is comparable to the giant fibre system of certain insects. In contrast, however, the giant interneurons and associated synapses of whip spiders are not located in the C.N.S., but lie some 20 cm removed in the periphery. Thus, some primary sensory information already becomes processed in the peripheral nervous system, before it reaches the C.N.S.

Ultrastructure of the flagellum in sperms of the scorpion Tityus bahiensis.

In spermatids of the scorpion Tityus bahiensis two centrioles appear coaxially aligned. The flagellum shows 9 peripheral doublets and a central rod (9 + 1 pattern). This central rod, of about 15 nm in diameter, is positively stained when a cytochemical method for histone staining is used. Longitudinal sections show that radial spokes are apparently in groups of three and showing a basic repeat of about 94 nm.