Morphology and ultrastructure of the tarsal adhesive organs of the Madagascar hissing cockroach Gromphadorhina portentosa.

The present transmission and scanning electron microscopic study of the ultramorphology of the pliable attachment pads (arolium, euplantulae) of the Madagascar hissing cockroach Gromphadorhina portentosa reveals structural evidence for their function in producing, storing, and secreting an adhesion-mediating secretion and releasing it to the exterior. The exocrine epidermal tissue of both the arolium and the euplantula is significantly enlarged by numerous invaginations stretching into the hemolymph cavity. Its cells show large nuclei, numerous mitochondria, Golgi complexes, and a prominent rough-surfaced endoplasmic reticulum integrated within an electron-dense cytoplasm that contains numerous vesicles of diverse electron density and size. Invaginations of the cell membrane provide evidence for strong membrane turnover. The glandular epithelium of both the arolium and the euplantula releases the adhesion-mediating secretion into a subcuticular void from which it has to permeate the thick cuticle of the adhesive pads. The subcuticular void is compartmentalized by cuticle bands through which the adhesion-mediating secretion permeates via small canals. The secretion subsequently enters a larger storage reservoir before being received by a prominent sponge-like cuticle. The structural differences between the arolium and the euplantula consist of the number and length of the interdigitations spanning the hemolymph cavity, of the subdivision of the subcuticular reservoir by cuticle bands, and of the thickness of the sponge-like cuticle. The structural results are discussed with respect to the production of a chemically complex (emulsion-like) adhesive, its controlled release to the exterior, and the micromechanical properties of the cuticle of the pliable pad.

Not flying blind: a comparative study of photoreceptor function in flying and non-flying cockroaches.

Flying is often associated with superior visual performance, as good vision is crucial for detection and implementation of rapid visually guided aerial movements. To understand the evolution of insect visual systems it is therefore important to compare phylogenetically related species with different investments in flight capability. Here, we describe and compare morphological and electrophysiological properties of photoreceptors from the habitually flying green cockroach Panchlora nivea and the American cockroach Periplaneta americana, which flies only at high ambient temperatures. In contrast to Periplaneta, ommatidia in Panchlora were characterized by two-tiered rhabdom, which might facilitate detection of polarized light while flying in the dark. In patch-clamp experiments, we assessed the absolute sensitivity to light, elementary and macroscopic light-activated current and voltage responses, voltage-activated potassium (Kv) conductances, and information transfer. Both species are nocturnal, and their photoreceptors were similarly sensitive to light. However, a number of important differences were found, including the presence in Panchlora of a prominent transient Kv current and a generally low variability in photoreceptor properties. The maximal information rate in Panchlora was one-third higher than in Periplaneta, owing to a substantially higher gain and membrane corner frequency. The differences in performance could not be completely explained by dissimilarities in the light-activated or Kv conductances; instead, we suggest that the superior performance of Panchlora photoreceptors mainly originates from better synchronization of elementary responses. These findings raise the issue of whether the evolutionary tuning of photoreceptor properties to visual demands proceeded differently in Blattodea than in Diptera.

Effect of shear forces and ageing on the compliance of adhesive pads in adult cockroaches.

The flexibility of insect adhesive pads is crucial for their ability to attach on rough surfaces. Here, we used transparent substrates with micropillars to test in adult cockroaches (Nauphoeta cinerea) whether and how the stiffness of smooth adhesive pads changes when shear forces are applied, and whether the insect's age has any influence. We found that during pulls towards the body, the pad's ability to conform to the surface microstructures was improved in comparison to a contact without shear, suggesting that shear forces make the pad more compliant. The mechanism underlying this shear-dependent increase in compliance is still unclear. The effect was not explained by viscoelastic creep, changes in normal pressure, or shear-induced pad rolling, which brings new areas of cuticle into surface contact. Adhesive pads were significantly stiffer in older cockroaches. Stiffness increased most rapidly in cockroaches aged between 2.5 and 4 months. This increase is probably based on wear and repair of the delicate adhesive cuticle. Recent wear (visualised by Methylene Blue staining) was not age dependent, whereas permanent damage (visible as brown scars) accumulated with age, reducing the pads' flexibility.

Foot morphology and substrate adhesion in the Madagascan hissing cockroach, Gromphadorhina portentosa.

Insects are successful terrestrial organisms able to locomote over a wide range of obstacles and substrates. This study investigated how foot morphology (tarsal structure) correlates with substrate adhesion and ecological niche in the Madagascan hissing cockroach, Gromphadorhina portentosa Schaum (Blattaria: Blaberidae). Using light and scanning electron microscopy, the morphology of the different structures of the tarsus of G. portentosa was analysed. Using an Instron universal testing machine, a series of peak force experiments were then conducted to record the force required to lift the cockroaches off different substrates. G. portentosa was pulled off 10 different substrates, which consisted of smooth Perspex; Perspex scored at 1cm intervals; Perspex hatched at 1 cm, 0.5 cm, and 1 mm intervals; Perspex abraded with fine grade sandpaper; Perspex abraded with coarse grade sandpaper; wood; glass; and Teflon. A clear relationship was seen where an increase in scoring on the Perspex caused a decrease in adhesive ability of G. portentosa. This may be due to there being adequate contact area for the attachment of the pads and to allow the claws to engage. The results obtained suggest that to achieve the greatest adhesion to substrates, G. portentosa uses a combined effect of both adhesive pads and pretarsal claws. Adhesion to a wide range of substrates appears to be an adaptation to life as a wingless forest floor dweller.

Microtubules with 15 subunits in cockroach epidermal cells.

Since Ledbetter and Porter (1964) described the 13 subunits which are visible in cross sections of negatively stained plant microtubules, subsequent observations have generally confirmed this number. By using Mizuhira's fixative composed of tannic acid and glutaraldehyde, it is easyto demonstrate the subunits of microtublules without optical reinforcement Cytoplasmic microtubules and sperm axonemes, fixed with Mizuhira's fixtive, similarly show 13 subunits (Mizuhira's and Futaesaku, 1971, 1972; Futaesaky et al., 1972; Tilney et al., 1973). This paper will describe a particular type of microtubule in insect epidermal cells fixed with the above fixative. The number of the subunits is found to be 15 in tranverse sections.

The mechanism of sensory transduction in a mechanoreceptor. Functional stages in campaniform sensilla during the molting cycle.

This paper describes the ultrastructural modifications that cockroach campaniform sensilla undergo at three major stages in the molting cycle and finds that the sensilla are physiological functional at all developmental stages leading to ecdysis. Late stage animals on the verge of ecdysis have two completely separate cuticles. The campaniform sensillum sends a 220-mum extension of the sensory process through a hole in its cap in the new (inner) cuticle across a fluid-filled molting space to its functional insertion in the cap in the old (outer) cuticle. Mechanical stimulation of the old cap excites the sensillum. The ultrastructural geometry of late stage sensilla, coupled with the observation they are physiolgically functional, supports the hypotheses (a) that sensory transduction occurs at the tip of the sensory process, and (b) that cap identation causes the cap cuticle to pinch the tip of the sensory process, thereby stimulating the sensillum.

Friction ridges in cockroach climbing pads: anisotropy of shear stress measured on transparent, microstructured substrates.

The contact of adhesive structures to rough surfaces has been difficult to investigate as rough surfaces are usually irregular and opaque. Here we use transparent, microstructured surfaces to investigate the performance of tarsal euplantulae in cockroaches (Nauphoeta cinerea). These pads are mainly used for generating pushing forces away from the body. Despite this biological function, shear stress (force per unit area) measurements in immobilized pads showed no significant difference between pushing and pulling on smooth surfaces and on 1-microm high microstructured substrates, where pads made full contact. In contrast, on 4-mum high microstructured substrates, where pads made contact only to the top of the microstructures, shear stress was maximal during a push. This specific direction dependence is explained by the interlocking of the microstructures with nanometre-sized "friction ridges" on the euplantulae. Scanning electron microscopy and atomic force microscopy revealed that these ridges are anisotropic, with steep slopes facing distally and shallow slopes proximally. The absence of a significant direction dependence on smooth and 1-microm high microstructured surfaces suggests the effect of interlocking is masked by the stronger influence of adhesion on friction, which acts equally in both directions. Our findings show that cockroach euplantulae generate friction using both interlocking and adhesion.

Tight junctions in a fluid-transporting epithelium of an insect.

Occluding junctions have been found between the lateral cell borders at the base of the rectum of Periplaneta americana. They appear as punctate membrane appositions in thin sections, and after incubation in physiological solutions containing lanthanum before fixation the inward penetration of tracer is impeded in this same basal area. Moreover, freeze-fracture studies of this region reveal simple linear ridges on fracture face P and grooves on fracture face E, which are similar to the less complex vertebrate tight junctions. The luminal clefts, which permit free inward diffusion of tracers, present no tight junctions, but do have septate junctions. These results support the contention that, contrary to earlier speculation, arthropods do possess tight junctions; these, rather than septate junctions, appear to form the morphological basis of at least some of the permeability barriers observed in invertebrates.

Embryonic development of Eucorydia yasumatsui Asahina, with special reference to external morphology (Insecta: Blattodea, Corydiidae).

As the first step in the comparative embryological study of Blattodea, with the aim of reconstructing the groundplan and phylogeny of Dictyoptera and Polyneoptera, the embryonic development of a corydiid was examined and described in detail using Eucorydia yasumatsui. Ten to fifteen micropyles are localized on the ventral side of the egg, and aggregated symbiont bacterial "mycetomes" are found in the egg. The embryo is formed by the fusion of paired blastodermal regions, with higher cellular density on the ventral side of the egg. This type of embryo formation, regarded as one of the embryological autapomorphies of Polyneoptera, was first demonstrated for "Blattaria" in the present study. The embryo undergoes embryogenesis of the short germ band type, and elongates to its full length on the ventral side of the egg. The embryo undergoes katatrepsis and dorsal closure, and then finally, it acquires its definitive form, keeping its original position on the ventral side of the egg, with its anteroposterior axis never reversed throughout development. The information obtained was compared with that of previous studies on other insects. "Micropyles grouped on the ventral side of the egg" is thought to be a part of the groundplan of Dictyoptera, and "possession of bacteria in the form of mycetomes" to be an apomorphic groundplan of Blattodea. Corydiid embryos were revealed to perform blastokinesis of the "non-reversion type (N)", as reported in blaberoid cockroaches other than Corydiidae ("Ectobiidae," Blaberidae, etc.) and in Mantodea; the embryos of blattoid cockroaches (Blattidae and Cryptocercidae) and Isoptera undergo blastokinesis of the "reversion type (R)," in which the anteroposterior axis of the embryo is reversed during blastokinesis. Dictyopteran blastokinesis types can be summarized as "Mantodea (N) + Blattodea [= Blaberoidea (N) + Blattoidea (R) + Isoptera (R)]".

Development-dependent and -independent ubiquitin expression in divisions of the cockroach mushroom body.

It has been proposed that the alpha and beta divisions of the mushroom bodies support intermediate and long-term memory whereas the gamma lobes support short-term memory. Here we investigate developmentally dependent versus developmentally independent alterations of mushroom body structure with special emphasis on its lobes. We show that in the cockroach mushroom bodies there are two types of plastic remodeling. One is developmental, in which episodic addition of new circuitry to the alpha and beta lobes is accomplished by newly born Kenyon cells. The second is revealed as a persistent alteration of structure within the gamma lobe. In the alpha/beta lobes, newly generated Kenyon cell axons extend glutamate-immunoreactive collaterals across layers of the axons of mature Kenyon cells. At specific times in each developmental episode (instar) these collaterals express ubiquitin, undergo localized degeneration, and are scavenged by glial cells. In contrast, the mature Kenyon cells that comprise the gamma lobe express detectable ubiquitin throughout each developmental episode. This pattern of ubiquitin expression suggests that the gamma lobe circuitry undergoes continuous modification independent of development.

Cockroach homologs of praying mantis peripheral auditory system components.

This study identifies the cuticular metathoracic structures in earless cockroaches that are the homologs to the peripheral auditory components in their sister taxon, praying mantids, and defines the nature of the cuticular transition from earless to eared in the Dictyoptera. The single, midline ear of mantids comprises an auditory chamber with complex walls that contain the tympana and chordotonal transduction elements. The corresponding area in cockroaches, between the furcasternum and coxae, has many socketed hairs arranged in discrete fields and the Nerve 7 chordotonal organ, the homolog of the mantis tympanal organ. The Nerve 7 chordotonal organ attaches at the apex of the lateral ventropleurite (LVp), which has the same shape and general structure as an auditory chamber wall. High-speed video shows that when the coxa moves toward the midline, the LVp rotates medially to stimulate socketed hairs, and also moves like a triangular hinge giving the chordotonal organ maximal in-out stimulation. Formation of the mantis auditory chamber from the LVp and adjacent structures would involve only enlargement, a shift toward the midline, and a mild rotation. Almost all proprioceptive function would be lost, which may constitute the major cost of building and maintaining the mantis ear. Isolation from leg movement dictates the position of the mantis ear in the midline and the rigid frame, formed by the cuticular knobs, which protects the chordotonal organs.

The sperm structure of Cryptocercus punctulatus Scudder (Blattodea) and sperm evolution in Dictyoptera.

Sperm of the dictyopteran key taxon Cryptocercus punctulatus was examined. It has largely maintained a blattodean groundplan condition, with a three-layered acrosome, an elongate nucleus, a single centriole, a conspicuous centriole adjunct material, two connecting bands (=accessory bodies), and a long functional flagellum with a 9+9+2 axoneme provided with accessory tubules with 16 protofilaments and intertubular material. These sperm characters are shared with several other polyneopterans. The sperm of C. punctulatus is very similar to what is found in Periplaneta americana and species of other groups of roaches, including the sperm of Loboptera decipiens described here for the first time. The general sperm organization here described can be assumed for the groundplan of Insecta and Pterygota. The following evolutionary path can be suggested: after the split between Cryptocercidae and the common ancestor of Isoptera, the typical pattern of sperm formation was altered very distinctly, resulting in a duplication or multiplication (Mastotermitidae) of the centrioles. Mastotermes has maintained a certain sperm motility, but with a very unusual apparatus of multiple flagella with a 9+0 axoneme pattern. After the split into Mastotermitidae and the remaining Isoptera, sperm motility was completely abandoned, and different modifications of sperm components occurred, and even the loss of the sperm flagellum.

A morphological correlate of target recognition by regenerating motor axons in the cockroach.

A specific cell recognition process during regeneration of severed axons of identified cockroach motor neurons eventually leads to the reformation of the original innervation pattern of target muscles in the leg. This occurs even though, at early times after nerve crush, the multiple branches of each regenerating axon grow into both appropriate and inappropriate muscles. In this study, we sought to examine whether there are any structural differences between regenerating axon branches in appropriate and inappropriate muscles that could lead to an understanding of why only those in inappropriate muscles are eliminated. A neuron subset-specific monoclonal antibody, NSS-2A, which labels the inhibitory motor neurons, was used to make their axon branches visible at various times after nerve crush. In inappropriate muscles, these axons grow primarily parallel to the muscle fibers and are later eliminated. In the appropriate muscles, these axon branches initially also grow parallel to the muscle fibers, but subsequently grow many interstitial collaterals. The formation of the collateral branches is a morphological correlate of the specific interaction of a neuron with its appropriate muscle. The simultaneous occurrence of axonal elimination and collateral sprouting supports the idea that the two processes are causally related, as suggested by the sibling neurite bias hypothesis.

Structure, development, and evolution of insect auditory systems.

This paper provides an overview of insect peripheral auditory systems focusing on tympanate ears (pressure detectors) and emphasizing research during the last 15 years. The theme throughout is the evolution of hearing in insects. Ears have appeared independently no fewer than 19 times in the class Insecta and are located on various thoracic and abdominal body segments, on legs, on wings, and on mouth parts. All have fundamentally similar structures-a tympanum backed by a tracheal sac and a tympanal chordotonal organ-though they vary widely in size, ancillary structures, and number of chordotonal sensilla. Novel ears have recently been discovered in praying mantids, two families of beetles, and two families of flies. The tachinid flies are especially notable because they use a previously unknown mechanism for sound localization. Developmental and comparative studies have identified the evolutionary precursors of the tympanal chordotonal organs in several insects; they are uniformly chordotonal proprioceptors. Tympanate species fall into clusters determined by which of the embryologically defined chordotonal organ groups in each body segment served as precursor for the tympanal organ. This suggests that the many appearances of hearing could arise from changes in a small number of developmental modules. The nature of those developmental changes that lead to a functional insect ear is not yet known.

A simple procedure for mounting wrinkle-free sections on formvar-coated slot grids.

This paper describes a simple technique for consistent production of clean, unwrinkled, flat thin (500-1000 A) sections for TEM and thick (1/2-1 micron) sections for HVEM mounted on Formvar-covered slot grids for use in conventional and high voltage electron microscopy. The technique centers around use of a Formvar-covered aluminum supporting rack onto which slot grids that contain sections suspended in water are placed.

Inverted gap and other cell junctions in cockroach hemocyte capsules: a thin section and freeze-fracture study.

Freeze-fracture and thin-section studies were done on cockroach hemocytes that had encapsulated implanted pieces of Araldite. Desmosome-like junctions and 'B' type gap junction were described. Freeze-fractured gap junctions displayed fused and clustered, but not hexagonally arrayed intramembranous practicles (approximately 130 A) on the B face and pitted areas on the A face of the plasmalemma. Gap junctions were quite numerous and counts of gap and non-gap particles indicated at least a five-fold particle density increase (4000/mu2) compared with B face particle densities (approximately 800/mu2) from free circulating blood cells where gap junctions had not been formed.

Oenocyte differentiation correlated with the formation of ectodermal coating in the embryo of a cockroach.

The first signs of 'embryonic membrane' deposition could be observed at the 11th/12th stage of the embryonic development, while serosal apolysis occurs, and the first signs of oenocyte differentiation could be detected at the 15th stage. When pleuropodial cuticle deposition occurs, at the 16th stage, there is a rapid increase in the number of differentiating oenocytes. At the 19th stage there are some fully differentiated oenocytics, whereas, just before the cuticulin layer of the embryonic cuticle is laid down, another wave of oenocyte differentiation could be observed. The differentiation process of oenocytes and of vertebrate cells with a rapid cell membrane biogenesis (steroid secreting cells and hepatocytes) are compared. The correlation of oenocyte differentiation with ectodermal coating deposition, with molting hormone titer and with prothoracic gland differentiation is discussed.

Pre- and post-synaptic structures in insect CNS: intramembranous features and sites of alpha-bungarotoxin binding.

The central neuropile of thoracic ganglia in the central nervous system (CNS) of the cockroach Periplaneta americana contains synapses with characteristic pre- and post-synaptic membrane specializations and associated structures. These include dense pre-synaptic T-bars surrounded by synaptic vesicles, together with post-synaptic densities of varying electron opacity. Exocytotic release of synaptic vesicles is observed only rarely near presynaptic densities, but coated pits are seen at variable distances from them, and may be involved in membrane retrieval. After freeze-fracture, paralinear arrays of intramembranous articles (IMPs) are detected on the P face of many presynaptic terminals, with associated dimples indicative of vesicular release. The E face of these membranes exhibits protuberances complementary to the P face dimples, as well as scattered larger IMPs. Post-synaptic membranes possess dense IMP aggregates on the P face, some of which may represent receptor molecules. Electrophysiological studies with biotinylated alpha-bungarotoxin reveal that biotinylation does not inhibit the pharmacological effectiveness of the toxin in blocking acetylcholine receptors on an identified motoneurone in the metathoracic ganglion. Preliminary thin section ultrastructural analysis of this tissue post-treated with avidin-HRP or avidin-ferritin indicates that alpha-bungarotoxin-binding sites are localized at certain synapses in these insect thoracic ganglia.

The organization and isolating function of insect rectal sheath cells: a freeze-fracture study.

In termites and roaches the well-defined rectal papillae each comprise a layer of columnar principal cells specialized for active transport and a layer of basal cells. The whole cell group is entirely surrounded by several series of flattened 'sheath cells' (formerly termed 'junctional cells') which abut onto the basal components of the papilla. The sheath cells secrete a specialized sclerified cuticle which forms the framework of the papilla. Their regularly pleated apical membrane is closely apposed to the cuticle and contains parallel and closely spaced rows of intramembranous particles. at this level, no subcuticular space is present and hence the space associated with the apical surface of the principal cells is defined as an isolated compartment. Typical septate junctions are present between the sheath and basal cells; however those linking adjacent sheath cells are structurally unusual: they extend to the basal surface rather than being restricted to the apical zone, are frequently interrupted and in replicas are represented by relatively short and irregularly oriented particle rows. Moreover, lateral sheath cell contacts display two further peculiarities: absence of an apical desmosomal ring and paucity of gap junctions. Structural observations suggest that the sheath cells isolate the principal cells from communication with the hemolymph, consequently enhancing their efficiency in water and ionic regulation. Comparable cells have been described in a number of insects, but the 'isolation' system presents varying degrees of complexity, for which an evolutionary scheme is proposed.

Septate junctions in insects: comparison between intercellular and intramembranous structures.

Septate junctions have been studied in the hind-gut of Periplaneta americana, Incisitermes schwartzi and Thermobia domestica. The topographical disposition of intercellular septa revealed by lanthanum impregnation corresponds well with that of particle rows seen in freeze fracture preparations. However, there is no precise correspondence between the undulations of septa and the disposition of particles within a single row. The spacing of particles is variable and generally less than that of the undulation periodicity of septa. On the other hand, the disposition of septa, and of the rows of particles that correspond to them, appears to be variable: sometimes rectilinear and in close parallel, these may curve or form series of 'finger-print' loops. Moreover, the septa are evidently not deployed as continuous ribbons around the cell since intrruptions are freuqently observed. In addition to their mechanical role in intercellular cohesion, septate junctions apparently ensure a more or less complete closure of the intercellular space (i.e. provide a permeability barrier) and thus play a role comparable with that of tight junctions in epithelia of vertebrates.