Diverticula in Male Lycorea halia Butterflies (Lepidoptera: Nymphalidae: Danaini: Itunina)-Support Organs for Everted Hairpencils with Unique Ultrastructure.

The involvement of the diverticula, a synapomorphy for Itunina, in protrusion and expansion of hairpencils by male Lycorea halia (Hübner, 1816) is demonstrated for the first time. They facilitate maintaining the haemolymph pressure necessary to keep the hairpencils everted. The diverticula are curved hook-like lobes, open to the body cavity and densely filled with tracheae and threads made by units of two staggered cells surrounding a central extracellular fibril bundle. Such complex structures, apparently metabolically active, have not been reported for insects previously and might indicate additional functions, but their functional role(s) remains a puzzle. When a male emerges from pupa, the diverticula are not yet formed; this happens only during the first protrusion of the hairpencils.

The eversible tentacle organs of Polyommatus caterpillars (Lepidoptera, Lycaenidae): Morphology, fine structure, sensory supply and functional aspects.

In their late (3rd and 4th) larval stages, caterpillars of the myrmecophilous lycaenid (Lepidoptera) species Polyommatus coridon and Polyommatus icarus, possess on their 8th abdominal segment two eversible so called tentacle organs (TOs). Previous histological and behavioural results have proposed that the TOs may release a volatile substance that elicits "excited runs" in attendant ants. In our study we investigated for the first time the temporal in- and eversion pattern of TOs. Using nerve tracing, Micro-CT, light- and electron microscopy techniques we studied (i) the histology of the 8th abdominal segment, (ii) the fine structure of the cuticular and cellular apparatus of the TOs, (iii) the attachment sites of the retractor muscle of each TO and (iv) the fine structure of the long slender tentacle hairs which are exposed to the outside, when the TOs are everted and fold back into the TO-sac during inversion. Our data show that the tentacle hairs are typical insect mechanoreceptors, each innervated by a small bipolar sensory cell with a tubular body in the tip of the outer dendritic segment. The latter is enclosed by a cuticular sheath previously called the "internal cuticular duct" and misinterpreted in earlier studies as the space, where the tentacle hairs actively secrete fluids. However, we found no glandular structures nearby or in the wall of the TO-sac. Also we did not reveal any conspicuous signs of secretory activity in one of the enveloping cells belonging to a tentacle hair. Although highly unusual features for an insect mechanoreceptor are: (a) the hair-shaft lumen of tentacle hairs contains flocculent material as well small vesicles and (b) the thin cuticular wall of the hair-shaft and its spines possess few tiny pores. Our data do not support the assumption of previous studies that volatile substances are released via the tentacle organs during their interactions with ants which in turn are supposed to cause excited runs in ants.

Mechanosensory pegs constitute stridulatory files in grasshoppers.

Stridulatory files on the inner face of hindleg femora were shown to consist of mechanosensory pegs in males and females of Syrbula montezuma (Saussure) and in males of Chorthippus biguttulus (L.). Females of Chorthippus had stiff protuberances on their stridulatory files, with an innervated tubercle instead of pegs. Pegs and tubercles of adult grasshoppers were shown to develop from innervated tubercular hairs present from the first instar onward in Chorthippus. In adults of Chorthippus, two sensory cells innervated each peg of males and each tubercle of females. Central projections of these afferents from the stridulatory files were very similar to those of the neighboring tactile hairs on the femur. The afferents from pegs in Syrbula responded to deflection and pressure introduced via the widened cuticular cap. In both species, selective stimulation of femoral cuticular receptors elicited antagonistic reflex responses in a coxal retractor muscle: pegs inhibited and neighboring hairs raised the efferent tonic discharges. Apparently, in these two distantly related grasshopper species, stridulatory files function as both sound-producing and proprioceptive organs.

The motor program for defensive kicking in crickets: performance and neural control

Crickets can repulse sources of mechanical touch to their wings, legs or to the posterior body by kicking backwards ipsilaterally with one hindleg. The main component of a kick is the rapid extension of the femoro-tibial (knee) joint. A kick as a defence against predators must occur instantly after the moment of touch. The cricket kick is completed within 60­100 ms, whereas in locusts 500­2000 ms elapses between the stimulus and the end of the kick. The rapid movement of the cricket hindleg was recorded with a high-resolution video technique. Cricket kicking is based on a dynamic co-contraction of the extensor and flexor tibiae muscles during the pre-kick knee flexion period, thus differing from the static co-contraction period seen in locusts. Biomechanically, the knee joint is specialized for kicking and jumping by the specific leverage of tendons inserting at the knee, by a femoral ridge that modifies the angle of attack for flexor muscular forces and by a cushion-like swelling on the flexor tibiae tendon. Because of these structural specialisations for rapid kicking, the neural control of the motor pattern of the muscles participating in the tibial movement can vary considerably, but still produce efficient kicks. Kicking is also an element of other complex behaviours.

'Campaniform' structures on lobster antennae are dermal glands.

The dome-shaped ('campaniform') cuticular structures found in small depressions on the lateral antennular flagella of the lobster (Homarus gammarus) were investigated by means of electron-microscopic methods. Each dome (diameter: 4-5 micron), which is surrounded by a 3-5 micron wide cuticular collar, is associated with a cell (soma size: long axis 90 micron, short axis 24 micron) showing fine-structural details characteristic of a dermal gland, e.g., a ductule lined by thin (cuticulin-like) cuticle and a well-developed granular endoplasmic reticulum. This ductule ends within the dome, which consists of spongious cuticle and, when seen from outside, resembles the cuticular cap of a typical campaniform sensillum of insects (Fig. 1 b). The fine-structural findings, especially the lack of any sensory element, are clear evidence against previous descriptions of these structures as representing 'campaniform sensilla of crustaceans'.

Development of the filiform hairs on the cerci of Gryllus bimaculatus deg. (saltatoria, gryllidae).

The filiform hairs, mechanoreceptors of Gryllus, pass through six developmental stages during the last larval stage. The cytoplasm of their sense cells suggests intensive synthesis of protein for cellular metabolism and intercytoplasmic exchange of material via glial evaginations. Ultrahistochemical tests demonstrated acid phosphatase in the lysosomes as well as in components of the Golgi apparatus. There was no significant change in the appearance of the sense cell cytoplasm, indicating a maintained functional state also during molting. The new cuticular apparatus is formed after apolysis by the three enveloping cells. Formation of the replacement hairs is initiated by a cytoplasmic outgrowth of the trichogen cell. During morphogenesis of the new hair, the microtubules serve as a cytoskeleton and probably control the flow of vesicles, which contain phenol oxidase, also demonstrated in the Golgi apparatus, and are incorporated into the new cuticle. Bundles of microfibrils are involved in the surface sculpturing of the replacement hair. The trichogen cell also forms a number of structural elements, e.g. the "cup" and "strut" marked geometric peculiarities of which indicate that they are important in the spatial orientation of the dendrite and thus also in transduction. Reduction of the apical cell membrane of the tormogen cell after apolysis permits unrestricted growth of the new hair into the exuvial space. The tormogen cell participates in the formation of the joint membrane, parts of the socket and the articulation of the hair.

Are the funnel-canal organs the 'campaniform sensilla' of the shore crab, Carcinus maenas (Decapoda, Crustacea)? II. Ultrastructure.

The funnel-canal organs on the dactyls of the shore crab, Carcinus maenas, are innervated by 3-24 sensory cells with unbranched dendrites, which attain a length of 500-1400 micron. The outer dendritic segments are enclosed in a dendritic sheath and pass through the cuticle within a canal. Two dendrite types can be distinguished according to ultrastructural criteria: Type I has a long ciliary segment, A-tubules with an osmiophilic core and arms, and a thick ciliary rootlet. Type II possesses only a short ciliary segment and a thin ciliary rootlet. Each funnel-canal organ contains two type-I dendrites. Their ciliary bases appear a few micron distal to those of the type-II dendrites (1 to 22 in number). Two inner and two to eight outer enveloping cells belong to a sensillum. The innermost enveloping cell contains a large scolopale. In the second enveloping cell single scolopale rods are present. Thus, the funnel-canal organs are characterized by structural features typical for mechanosensitive scolopidia, on the one hand, and for chemoreceptors, on the other. Therefore, the funnel-canal organs are very likely bimodal sensilla (contact chemoreceptors). A comparison with other arthropod sensilla shows that cuticular mechanoreceptors of aquatic crustaceans generally exhibit a 'scolopidial' organization.

Venom gland of the digger wasp Liris niger: morphology, ultrastructure, age-related changes and biochemical aspects.

Females of the parasitoid digger wasp species Liris niger hunt crickets as food for their future brood. The wasps paralyse the prey by injecting their venom directly into the CNS. The venom is produced in a gland consisting of two ramified glandular tubules terminating in a common reservoir. The reservoir contents enter the sting bulb via a ductus venatus. Secretory units of dermal gland type III line the two free gland tubules, the afferent ducts to the reservoir and the cap region within the reservoir. Secretion products of tubules reach the reservoir through the cuticle-lined central funnel. Secretory cells in the distal and middle parts of the tubules contain extensive rough endoplasmic reticulum and numerous electron-dense vesicles, whereas secretory cells of the afferent ducts and the cap region of the reservoir lack electron-dense vesicles and the endoplasmic reticulum is poorly developed. The secretory apparatus undergoes age-related changes. The secretory units in the venom gland tubules and inside the reservoir complete differentiation 1 day after imaginal ecdysis. After 30 days, massive autolytic processes occur in the secretory cells and in the epithelial cells of the reservoir. Analysis of the polypeptide composition demonstrates that the venom reservoir contains numerous proteins ranging from 3.4 to 200 kDa. A dominant component is a glycoprotein of about 90 kDa. In contrast the polypeptide composition of Dufour's gland is completely different and contains no glycoproteins. Comparison of the venom reservoir contents with the polypeptide pattern of venom droplets reveals that all of the major proteinaceous constituents become secreted. Thus the secreted venom contains exclusively proteins present in the soluble contents of the venom gland.

Arachnid oenocytes: ecdysone synthesis in the legs of harvestmen (Opilionidae).

Cells measuring up to 130 microns have been found in the proximal segments of the femora of all four pairs of walking legs in various species of harvestmen (Phalangium opilio, Leiobonum limbatum, Opilio parietinus, and Opilio ravennae). These cells exhibit all the fine-structural characteristics of insect oenocytes, in particular the conspicuous agranular endoplasmic reticulum. Radioimmunoassay after in vitro incubation of these cells has demonstrated the synthesis of alpha- and beta-ecdysone. These ecdysteroids have been found in the ovaries and tergites of the opisthosoma as well as in the oenocytes.

Morphogenesis of mechanoreceptor and epidermal cells of crickets during the last instar, and its relation to molting-hormone level.

(1) The fine structure of the cercal campaniform sensilla and epidermal cells of Gryllus bimaculatus Deg. (Saltatoria, Gryllidae) was examined, and the ecdysteroid level was monitored throughout the last larval instar. (2) The epidermal cells show changes in shape, cytoplasmic inclusions and differentiation of the apical cell membrane, coupled to the phases of buildup and breakdown of the (cercus) cuticle. (3) The imaginal epicuticle of the epidermal cells begins to form later (by about approximately 6h) than that of the campaniform sensilla. (4) The campaniform sensilla were studied with respect to (a) the morphogenesis of the cuticular apparatus, (b) the inclusion of phenol oxidases in the cuticular apparatus, and (c) changes in the sensory apparatus preparatory to molting. (5) After apolysis the folding of the tormogen-cell wall into microvilli transiently disappears. Microvilli re-form shortly before imaginal ecdysis, and at the same time an outer receptor-lymph space develops. The role of the tormogen-cell "plaques" is discussed. (6) The levels of alpha- and beta-ecdysone were determined separately by radioimmunoassay. (7) At the beginning of the instar the hormone level, especially that of beta-ecdysone, falls. Prior to apolysis, the concentration of alpha-ecdysone rises, reaching an intermediate peak after apolysis is complete. The maximum hormone concentration (approximately 2,000 ng/g) is reached after the cuticulin layer is deposited, primarily due to the increase in beta-ecdysone. While the proecdysial cuticle is forming, the hormone titer is reduced; at this time beta-ecdysone is its chief component. (8) The identification of the ecdysteroids monitored by radioimmunoassay was confirmed by gas chromatography.

Ultrastructure and mechanical properties of an insect mechanoreceptor: stimulus-transmitting structures and sensory apparatus of the cercal filiform hairs of Gryllus.

1. The following features of the cercal filiform hairs of the cricket Gryllus were investigated: (a) the ultrastructure and geometrical peculiarities of the various auxiliary structures in the region of the hair base, as well as those of (b) the stimulus-receiving outer segment of the dendrite (including the tubular body), and (c) the mechanical properties (directionality and linearity and frequency dependence of mobility) of the hair. 2. When stimulated by vibrations of the medium, the filiform hairs show regular or irregular oscillations depending on stimulus intensity. At higher stimulus intensities (xi > congruent to 100 microns at 100 Hz) the hairs flutter irregularly in various directions, at somewhat lower intensities preferentially in the plane of best mobility in even lesser intensities in the plane of stimulus vector. In the plane ob best mobility the maximal angle of deflection from the resting position is 5.3 +/- 1.4 degrees. 3. The dependence of hair mobility on stimulus frequency was tested in the range 20-1000 Hz. Best mobility was found in the range 100-200 Hz. 4. The directional characteristic of hair mobility has the form of a figure eight. Hairs can be grouped into three classes on the basis of direction (with respect to the long axis of the cercus) of best mobility: parallel (L-hairs), transverse (T-hairs), and diagonal (D-hairs). 5. The plane of best mobility corresponds with the plane symmetry of the hair base. The hair can be deflected furthest from the resting position in the direction of a cuticular peg at the hair base, which projects toward the lumen of the hair and marks the flat side of the tubular body within the terminal dendrite segment. Deflection of the hair shaft in the opposite direction is limited by a fibrous cushion, which exerts a counter-pressure. When the hair is deflected, the cuticular peg causes deformation of the tubular body. 6. The direction of best mobility of the hair is the direction in which the sensory cell is depolarized; the direction of depolarization can thus be determined entirely by morphological criteria.

K+ and Ca++ in the receptor lymph of arthropod cuticular mechanoreceptors.

Two types of cuticular strain detectors, the campaniform sensilla on the haltere of the blowfly, Calliphora vicina, and the slit sensilla on the tibia of the spider, Cupiennius salei, were investigated. In campaniform sensilla a transepithelial voltage (43.6 +/- 10.7 mV), which depends on an intact metabolism, occurs. In spider slit sensilla no transepithelial voltage exists. The occurrence and the lack of a transepithelial voltage is paralleled with differences in the ionic composition of the receptor lymph in the two arthropod sensilla. We used double-barrelled ion-selective microelectrodes to measure potassium and calcium content in the receptor lymph with respect to the hemolymph. The potassium concentration in campaniform sensilla (121 +/- 15 mM) is five times larger than that of the wing hemolymph (25 +/- 7 mM) and nine times larger than that of the haltere hemolymph (13 +/- 3 mM). These differences are statistically significant. The calcium concentration in campaniform sensilla (0.8 +/- 0.5 mM) does not differ significantly from that of the hemolymph (1.2 +/- 0.7 mM). In spider slit sensilla no significant difference occurs between the potassium concentration of the receptor lymph (9.5 mM +/- 5.5 mM) and that of the hemolymph (8 +/- 3 mM). The calcium concentration of the hemolymph (1.6 +/- 0.9 mM) is 3 times higher than that of the receptor lymph (0.6 +/- 0.3 mM). This difference is significant.

Macromolecules in the receptor lymph of campaniform sensilla.

The receptor lymph of campaniform sensilla on the halteres of the blowfly, Calliphora vicina, was analyzed histochemically. Acid mucopolysaccharides were demonstrated by a test for iron-binding capacity (Hale-reaction). Further characterization by enzyme treatment showed that the receptor lymph contains hyaluronic acid and/or chondroitin sulfate. Ultrahistochemical studies gave evidence for glycoproteins in the inner and outer receptor lymph space. The significance of acid mucopolysaccharides for arthropod sensilla is discussed.

Pheromone receptors in Bombyx mori and Antheraea pernyi. I. Reconstruction of the cellular organization of the sensilla trichodea.

The cellular organization of freeze-substituted antennal sensilla trichodea, which contain the sex pheromone receptors, was studied in male silkmoths of two species (Bombyx mori, Bombycidae; Antheraea pernyi, Saturniidae). The cellular architecture of these sensilla is complex, but very similar in both species. A three-dimensional reconstruction of a sensillum trichodeum of B. mori is presented. Two receptor cells (in A. pernyi 1-3) and three auxiliary cells are present. Of the latter, only the thecogen cell forms a true sheath around the receptor cells. A unique thecogen-receptor cell junction extends over the entire area of contact. Septate junctions occur between all sensillar cells apically, and in the region of the axonal origin basally. Gap junctions are also found between all cells except the receptor cells. The trichogen and tormogen cells show many structural indications of secretory activity and are thought to secrete the receptor lymph. Their apical membrane bordering the receptor-lymph space is enlarged by microvilli and microlamellae, but only those of the trichogen cell show regularly arranged membrane particles (portasomes), indicating secretory specialization among the auxiliary cells. Epidermal cells are found as slender pillars between sensilla, but extend apically along the non-sensillar cuticle and basally along the basal lamina.

Tormogen cell and receptor-lymph space in insect olfactory sensilla. Fine structure and histochemical properties in Calliphora.

(1) The basiconic sensilla on the antennae of Calliphora resemble other insect epidermal sensilla; one or several bipolar sense cells are surrounded by three non-neural cells. (2) The apical cell membrane of the tormogen cell (one of the three accessory cells) forms microvilli coated internally with particles. (3) In the (extracellular) outer receptor-lymph space hyaluronic acid can be demonstrated histochemically. (4) Demonstration of non-specific alkaline phosphatase, Mg2+-activated ATPase, and the presence of mitochondria in the apical part of the tormogen cell suggest active transport processes through these cells into the outer receptor-lymph space.

Digger wasp versus cricket: immediate actions of the predator's paralytic venom on the CNS of the prey.

The females of the palaearctic digger wasp species Liris niger hunt crickets (e.g., Acheta domesticus) as food for their future brood. The wasps paralyze the prey by injecting their venom directly into each of the three thoracic ganglia and the suboesophageal ganglion. This study describes the effects produced by the Liris venom at the level of the intact prey animal (by chronic electromyogram) and at the level of a dissected preparation (by extra- and intracellular records) during the immediate action. Natural or artificial injections of the Liris venom into various ganglia revealed that: (a) The venom injection induced an about 15- to 35-s long tonical discharge of the neurons located in the stung ganglion. This discharge is usually accompanied by convulsions of the prey's limbs. (b) Subsequently, the generation and propagation of action potentials are blocked for up to 30 min (total paralysis). (c) During total paralysis, the venom blocks synaptic transmission. (d) The effects of the venom are restricted to the stung ganglion. Responses of mechanoreceptors in the legs can be recorded from the peripheral nerves of the stung ganglion during the whole period of total paralysis. (e) The neurons almost completely recover after this period. The venom does not selectively affect leg motoneurons, but affects any neuron (e.g., internerneurons or neurosecretory neurons) in any part of the central nervous system of the prey where it was released.

Pheromone receptors in Bombyx mori and Antheraea pernyi. II. Morphometric analysis.

Sensilla trichodea of the silk moths, Antheraea pernyi and Bombyx mori, were reconstructed from serial sections after freeze substitution. The volume and surface area of the different sensillar cells were calculated from the area and circumference of consecutive section profiles. A. pernyi and B. mori differ largely in the size of the sensory hair and the larger outer dendritic segments as well as in the volume of the receptor lymph within the hair, while there are only small differences regarding inner dendritic segments, receptor-cell somata, trichogen and tormogen cells and the volume of the receptor lymph below the hair base. In each sensillum the two (or three) receptor-cell somata, dendrites, and initial axonal segments differ significantly in volume and surface. The apical cell membranes of the trichogen and tormogen cells, which border the receptor-lymph cavity and which are the presumed site of electrogenic cation pumps, are deeply invaginated and enlarged by microlamellae and microvilli, so that their area is twice that of the remaining basolateral cell membrane. In contrast to mechanoreceptors, the trichogen cell is the largest auxiliary cell and has the largest apical membrane surface. The morphometric data are discussed with regard to recent electrophysiological observations.

Digger wasp versus cricket: mechanisms underlying the total paralysis caused by the predator's venom.

The data presented here describe neurophysiological experiments addressing the question of cellular mechanisms underlying the total paralysis of locomotor behavior in crickets occurring after being stung by females of the digger wasp species Liris niger. The Liris venom effects have been studied by both in vivo recordings from identified neurons of the well-described giant fiber pathway and in vitro recordings from cultured neurons isolated from the terminal ganglion of crickets. The total paralysis of the prey is characterized by a general block of action potential generation as well as by a block of synaptic transmission. Intracellular recordings from neurons in intact ganglia under single electrode voltage-clamp conditions, as well as whole-cell patch-clamp recordings from cultured cricket neurons consistently show that the block of action potential generation by the Liris venom is due to a block of voltage-gated sodium inward currents in neurons of the stung ganglia. Furthermore, our data provide evidence that the Liris venom also blocks calcium currents in identified neurosecretory neurons. On the other hand, outward currents are not affected by the Liris venom. The in vitro recordings suggest that the Liris venom contains active venom components, which, at least for the observed block of inward currents, do not require a metabolic modification. Because venom application does not affect the ACh-induced EPSPs in giant interneurons, the Liris venom does not seem to influence the postsynaptic ACh receptors. The possible pre- and postsynaptic sites of venom action and the functional consequences on synaptic transmission within the giant fiber system are discussed.