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.

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)]".

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.

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.

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.

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.

The fine structure of colleterial glands in two cockroaches and three termites, including a detailed study of Cryptocercus punctulatus (Blattaria, Cryptocercidae) and Mastotermes darwiniensis (Isoptera, Mastotermitidae).

The colleterial glands of insects are organs associated with the female genital apparatus. In cockroaches, these glands produce secretions that cover two parallel rows of eggs during oviposition, and in oviparous species, these secretions become the tanned, sculpted, rigid outer casing of the ootheca. The goal of this study was to compare the gross anatomy of the colleterial glands and the ultrastructure of their component tubules in the phylogenetically significant genera Cryptocercus (Blattaria) and Mastotermes (Isoptera). Recent studies indicate that cockroaches in the genus Cryptocercus are the sister group of termites, and Mastotermes is the only termite known to produce a cockroach-like ootheca. One additional oviparous cockroach, Therea, and two additional termites, Zootermopsis and Pseudacanthotermes, were also examined. As in other cockroaches, the colleterial glands of Cryptocercus and Therea are asymmetrical, with a well developed bipartite left gland and a smaller right gland. In the termites Mastotermes, Zootermopsis, and Pseudacanthotermes, the colleterial glands are composed of a well-developed, paired, anterior gland and a small posterior gland; histological staining and cytological evidence suggest that these are homologues of the left and the right colleterial glands of cockroaches, respectively. At the ultrastructural level, colleterial gland tubules are made of cells belonging to a modified class 1 type cell in the cockroaches, in Mastotermes, and in Zootermopsis; the latter lays its eggs singly, without a surrounding ootheca-like structure. In the advanced termite Pseudacanthotermes, the tubules are made of secretory units belonging to the class 3 cell type. This study demonstrates that the cytological characteristics of colleterial glands in basal termites are similar to those of cockroaches, whether the termite secretes an oothecal casing that covers two parallel rows of eggs, as in Mastotermes, or lays its eggs singly, as in Zootermopsis. The function of colleterial glands in non-mastotermitid termites is unknown.

Effects of the juvenile hormone analog pyriproxyfen on German cockroach, Blattella germanica (L.), tergal gland development and production of tergal gland secretion proteins.

Male German cockroaches possess secretory glands that secrete fluid into a pair of transverse depressions on the seventh and eighth abdominal tergites. We investigated the effects of altered juvenoid titer during the first part of the last instar on tergal gland secretory tissue development and the production of tergal gland secretion proteins. Male fifth (last) instar nymphs (1-3 days post-emergent) were topically treated with the JH analog pyriproxyfen. Light and transmission electron microscopy demonstrated that treatment with pyriproxyfen produced a visible decrease in the amount of tergal gland tissue present, a deformation of the overall shape of the gland located on tergite seven, and a less orderly arrangement of the secretory cells in the tissue. The protein fraction of tergal gland secretion was examined in pyriproxyfen-treated and control insects 1, 5, and 15 days after the insects molted to the adult stage. Amounts of all tergal secretion proteins were reduced in treated insects.

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.

Ultrastructure of pigment-dispersing hormone-immunoreactive neurons in a three-dimensional model of the accessory medulla of the cockroach Leucophaea maderae.

Locomotor activity rhythms of the cockroach Leucophaea maderae are orchestrated by two bilaterally symmetric, mutually coupled, circadian pacemakers. They lie in the optic lobes of the brain and are confined to the accessory medulla (AMe), ventro-medially to the medulla. The AMe is innervated by approximately 12 pigment-dispersing hormone (PDH)-immunoreactive anterior medulla neurons (PDHMe), which are circadian pacemaker candidates in the fruitfly and the cockroach. We have developed a three-dimensional computer model of the AMe and associated structures as a framework for neuroanatomical studies. Our greatly improved understanding of this structure in space has allowed us further to subdivide the anterior PDHMe into three subgroups, i.e., large, medium-sized, and small anterior PDHMe. The synaptic connections of two of these subgroups have been examined within subcompartments of the AMe by light and electron microscopy. The large, intensely staining, anterior PDHMe contain medium-sized dense-core vesicles and form input and output synapses with profiles densely filled with clear vesicles primarily in the anterior and shell neuropil of the AMe. The medium-sized anterior PDHMe contain large dense-core vesicles and constitute input and output synapses either with profiles being densely filled with clear vesicles, or with profiles containing granular dense-core vesicles. The small, weakly staining anterior PDHMe belong to a morphological group different from the large and medium-sized PDHMe and cannot be further identified at the electron-microscopic level because of their weak PDH immunoreactivity.

Light and electron microscopic study of a Rickettsiella species from the cockroach Blatta orientalis.

An infection with Rickettsiella sp. was responsible for an illness causing heavy body swelling in the Oriental cockroach Blatta orientalis. Reproduction of the colony stagnated. Vacuoles with parasitic bacteria occurred mainly in the fat body, but also in nearly all other organs, such as gut epithelium, Malpighian tubules, blood cells, and ovarioles. The parasites clearly differed from the symbiotic bacteria of the genus Blattabacterium, which regularly occur in the mycetocytes of B. orientalis. The vacuoles contained four stages of Rickettsiella: (1) infectious, electron-dense, rod-like elementary bodies (mean size 300 x 145 nm); (2) an electron-dense, flat intermedium stage, called flat body (mean size 515 x 255 x 125 nm); (3) an electron-light, spherical intermedium stage, called condensing sphere (mean size 340 nm); portions of cytoplasm condensed crescent-like at the border or in the center of the cell; and (4) large, spherical, electron-light initial bodies that multiplied by binary fission (mean size 600 nm). The initial bodies had a three-layered cell boundary, but all other stages had a five-layered cell boundary. Elementary and flat bodies contained an electron-light, oblique lamella and an oval structure with an array of ribosome-like granules, respectively. In contrast to other species of Rickettsiella, crystal formation or multiple division did not occur. The described species of Rickettsiella is different from "R. blattae," which belongs to the R. popilliae group. Instead, it shares more similarities with the R. chironomi group. To avoid confusion, it was provisionally named "R. crassificans."

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.

Some aspects of intracellular symbiosis during embryo development of Mastotermes darwiniensis (Isoptera: Mastotermitidae).

All examined species of cockroaches have been shown to harbour intracellular bacteria in specialized cells (bacteriocytes) of the fat body. In termites, bacteria in specialized cells have been observed only in Mastotermes darwiniensis (Isoptera: Mastotermitidae). All of these bacteria have been assigned to the same eubacterial lineage, with the bacteria of M. darwiniensis as the sister group to the cockroach bacteria. While the main steps of the life cycle of cockroach bacteria have been described, little is known about the bacteria of M. darwiniensis. More specifically, no data are available on their behaviour during the development of this termite. Using both optical and electron microscopy methods, we examined embryos of M. darwiniensis at different developmental stages. Our results show that the integration of bacteria during the development of M. darwiniensis is implemented in the same way as in cockroaches. In particular, we observed the aggregation of a large amount of bacteria in a single mass in the yolk sac, with vitellophage-associated bacterial lysis. In cockroaches, a similar process has been described in detail for Periplaneta americana (Blattaria: Blattidae), where the bacterial mass is referred to as the transitory mycetome. The formation of a transitory mycetome could thus be regarded as an ancestral condition for cockroaches and termites.

Immunocytochemical localization of Na+/K(+)-ATPase and V-H(+)-ATPase in the salivary glands of the cockroach, Periplaneta americana.

The acinous salivary glands of the cockroach (Periplaneta americana) consist of four morphologically different cell types with different functions: the peripheral cells are thought to produce the fluid component of the primary saliva, the central cells secrete the proteinaceous components, the inner acinar duct cells stabilize the acini and secrete a cuticular intima, whereas the distal duct cells modify the primary saliva via the transport of water and electrolytes. Because there is no direct information available on the distribution of ion transporting enzymes in the salivary glands, we have mapped the distribution of two key transport enzymes, the Na+/K(+)-ATPase (sodium pump) and a vacuolar-type H(+)-ATPase, by immunocytochemical techniques. In the peripheral cells, the Na+/K(+)-ATPase is localized to the highly infolded apical membrane surface. The distal duct cells show large numbers of sodium pumps localized to the basolateral part of their plasma membrane, whereas their highly folded apical membranes have a vacuolar-type H(+)-ATPase. Our immunocytochemical data are supported by conventional electron microscopy, which shows electron-dense 10-nm particles (portasomes) on the cytoplasmic surface of the infoldings of the apical membranes of the distal duct cells. The apically localized Na+/K(+)-ATPase in the peripheral cells is probably directly involved in the formation of the Na(+)-rich primary saliva. The latter is modified by the distal duct cells by transport mechanisms energized by the proton motive force of the apically localized V-H(+)-ATPase.

Subcellular distribution and characterization of esterase isozymes from insecticide-resistant and -susceptible strains of German cockroach (Dictyoptera: Blattellidae).

Esterases from insecticide-resistant and -susceptible strains of German cockroach, Blattella germanica (L.), were isolated and analyzed for differences in activity. The isozymes E5, E6, E7, and E8 of resistant strains indicated about two- to fivefold increases in activity compared with those of the susceptible strain. These isozymes constituted approximately 60% of the total activity in the resistant strains, whereas they contributed only about 40% activity in the susceptible strain. Subcellular distribution studies showed that most esterase activity is present in the 100,000 x g fraction of the homogenate (cytosolic). Only a small portion of activity was membrane bound. Molecular weights of the esterase isozymes calculated based on their retardation coefficients ranged between 48 and 81 kd.

Structure-activity relationships in corpora allata of the cockroach Diploptera punctata: roles of mating and the ovary.

Morphometric studies were made on corpora allata of the cockroach Diploptera punctata from animals in which increasing gland size is not coupled to hormone synthesis (ovariectomized mated females; last-instar larvae) and in which gland size is coupled to hormone synthesis (normal mated and virgin females; penultimate-instar larvae). Cell number, gland volume, and juvenile hormone synthesis were measured. From electron micrographs, nuclear, cytoplasmic, and extracellular volumes; and cell membrane area were calculated; and fine structure described. Low-activity glands of ovariectomized mated females resembled high-activity glands from mated females in high cell number, large overall and cytoplasmic volume, and low nuclear-cytoplasmic ratio; they differed in having organelles typical of low-activity glands, mitochondria with dense matrices and large whorls of smooth endoplasmic reticulum. Inactive last-instar larval glands resembled mated ovariectomized female glands in increased cell number and organelles characteristic of inactive glands; however, their nuclear-cytoplasmic volume ratio was much higher. Penultimate larval glands with high activity per cell resembled active glands of normal mated females. Ovariectomy did not change morphometric parameters of virgin female glands; thus mating results in increase in size of adult female glands whereas the growing ovary is needed for changes in mitochondria and endoplasmic reticulum associated with high juvenile hormone synthesis.

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.