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Chronobiological analysis and mass spectrometric characterization of pigment-dispersing factor in the cockroach Leucophaea maderae.

Pigment-dispersing factor (PDF) is a neuropeptide that plays a prominent role in the circadian clock of several insects. The cockroach Leucophaea maderae was the first animal where the site of a biological clock could be located, and still is a focal point of circadian research. Although detailed studies on the action of pigment-dispersing factor and the distribution of PDF-like immunoreactivity in the L. maderae brain exist, a native pigment-dispersing factor of this species has not been characterized so far. The authentic Lem-PDF was isolated from L. maderae by a combination of high performance liquid chromatography, crab pigment-dispersion bioassay and an immunosorbent assay. Mass spectrometric characterization and the conserved sequence of pigment-dispersing factor in orthopteromorphan insects suggest that Lem-PDF has the sequence NSEXINSLLGLPKVLNDAa (where X= I or L). Lem-PDF is thus identical to either Periplaneta americana PDF or Acheta domesticus PDF. Detailed analysis of PDF-like immunofluorescence in different regions of the brain suggests that there are no drastic daily changes in the amount of pigment-dispersing factor as occur in Drosophila melanogaster, which might be explained by a lack of circadian pigment-dispersing factor release and production, or by phase differences between the pigment-dispersing factor neurons.

Amino Acid Sequence↗

Comparative sterilizing and ovicidal activity of fenoxycarb and hydroprene in adults and oothecae of the German cockroach (Dictyoptera: Blattellidae).

Adults and oothecae of the German cockroach, Blattella germanica (L.), were treated topically with the juvenoids fenoxycarb and hydroprene. Fenoxycarb suppressed reproduction when applied at 10 micrograms/microliters to virgin and fertilized females. Production of offspring also was reduced when males treated with 10 micrograms/microliters of fenoxycarb were mated with untreated females. Fenoxycarb (10 micrograms/microliters) also exhibited ovicidal activity, particularly when applied to 4-d-old oothecae. Ovicidal activity by fenoxycarb diminished when applied to oothecae older than 8 d. No reproductive or ovicidal effects were caused by 1 or 10 micrograms/microliters of hydroprene.

Animals↗

Volatile effects of insect growth regulators against the German cockroach (Dictyoptera: Blattellidae).

Third-instar German cockroach nymphs were held to maturity in continuous proximity to test surfaces treated with hydroprene and fenoxycarb, which they were unable to contact directly. Surfaces tested were unfinished plywood, fiberboard, vinyl tile (absorbent surfaces), glass, stainless steel, ceramic tile, and formica (nonabsorbent surfaces). Number of oothecae, percent egg hatch, and survival did not differ between any fenoxycarb treatment and the untreated controls, but there was significant wing twisting (76-94%) for all nonabsorbent surfaces. Number of oothecae was significantly lower and percent wing twisting was significantly higher for all hydroprene-treated surfaces with respect to the untreated controls, although survival was not affected. These effects were less pronounced for the nonabsorbent surfaces. These results indicate that hydroprene, but not fenoxycarb, has significant biological activity through volatile action when applied at current label rates. This activity is likely to have important consequences for control, especially for "crack and crevice" applications in confined spaces.

Animals↗

Pathogenicity and limited transoothecal transmission of Steinernema carpocapsae (Nematoda: Steinernematidae) in adult female German cockroaches (Dictyoptera: Blattellidae).

Nongravid female German cockroaches, Blattella germanica (L.), and females with young or old oothecae were exposed to infective stage juvenile (L3) Steinernema carpocapsae (Weiser) All strain by topical application and continuous exposure. Topically applied nematodes resulted in < or = 40% mortality 7 d after inoculation, whereas 100% of females continuously exposed to nematodes were dead within 7 d. Females carrying old oothecae had significantly lower LT50s (0.07 h) than females with young oothecae (42.71 h) and females without oothecae (38.49 h). Fully formed oothecae were not penetrated by the nematodes, even though infective juveniles were found in the vestibulum between the female genitalia and on the proximal end of the ootheca. Partially formed oothecae were infested (100%) by nematodes derived from the female. Topical application of nematodes to the proximal end, side, or keel of mature oothecae that were detached from females did not result in nematode infestation, and the nymphs emerged normally. Oothecae continuously exposed to nematodes were similarly not infested but exhibited significant mortality, probably caused by bacterial infection.

Animals↗

Adult German cockroach (Dictyoptera: Blattellidae) movement patterns and resource consumption in a laboratory arena.

Computerized moving-image analysis was used to determine movement behavior of adult German cockroaches among food, water, and harborage resource sites. Adults at different stages in the female reproductive cycle (nonmated females, mated nongravid females, and gravid females) and males were examined continually for 5 d under a photoperiod of 12:12 (L:D) h. Corresponding food and water consumption rates for these adult classes were also determined. Differences were evident among the female reproductive classes and males. Mated nongravid females were the most active overall, and consumed the most resources. Nonmated females were second to the mated nongravid females in consumption and were less active. Gravid females were the most inactive, spending the greatest time in the harborage and consuming the fewest resources. Males were second to females in the mated nongravid class regarding activity, but consumed significantly fewer resources than females in both the mated nongravid and nongravid females. Implications of these findings relating to the biology of these adult classes are discussed.

Animals↗

Two new reciprocal translocations in the German cockroach. Cytology and genetics of T(3;12) and T(7;12).

Two new reciprocal translocations in the German cockroach have been analyzed. They were identified cytologically to be T(3;12) and T(7;12). Linkage studies showed that groups XI, IX, and IV are on chromosomes 12, 3, and 7, respectively, and clearly demonstrated sex differences in recombination. Each of these chromosomes have distinctive morphological features that facilitate their identification, and permit breakpoint and centromere localization. A sex difference in fecundity is associated with T(7;12), but not T(3;12). About 40 percent mortality occurred when T(3;12) males or females and T(7;12) females were outcrossed. Outcrossing T(7;12) males produced the expected 50 percent mortality. Cell counts at metaphase I revealed that T(3;12) males exhibit directed segregation, while T(7;12) males do not. Tests for homozygosity indicated that the T(7;12) homozygote is viable. A map of chromosome 12 is presented showing the tentative placement of linkage group XI with respect to interchange breakpoints and chromosome morphology. The results are discussed in relation to possible sex differences in chiasma localization.

Animals↗

Synthesis and properties of a double translocation heterozygote involving a stable ring-of-six interchange in the German cockroach.

The synthesis and properties of a new double translocation heterozygote in B germanica, combining T (8;9) with the progressive interchange T (4;5;10), are described. Competitiveness and sterility data indicate that males are promising candidates for a "field" experiment in genetic control of a cockroach. Sterility from genetic load and embryonic trapping is complete. Disjunction properties of the parental interchanges are similar to those found in cells of the double males, but chiasma frequency of the interchanged chromosomes is increased. Evidence of a possible relationship between chiasma formation and disjunction type is discussed.

Animals↗

Differential effects of duplication-deficiency gametes from T (3;12) on embryonic development in the German cockroach.

Differential embryonic development, as seen within egg cases of the German cockroach, serves to distinguish matings of interchange heterozygotes from those of wild type. In T (3;12), one group of fertilized eggs ceases development during stage I of embryonic development; a second group, during stage VII. The frequency of the two groups correlates closely with that of adjacent-1 vs adjacent-2 disjunction. It also does not differ significantly from the expected frequency if zygotes fertilized by one of the two types of adjacent-1 gametes reach a more advanced stage of development than those of the other three types of duplication-deficiency gametes. The absence of a sex difference in the stages of embryonic death indicates that it makes little difference whether aneuploid gametes are of maternal or paternal origin.

Animals↗

Primary integumentary xenograft reactivity in the American cockroach, Periplaneta americana.

The nature of cell-mediated immunity in the American cockroach (Periplaneta americana) was studied by observing the events involved in graft reactivity. Adult roaches received integumentary xenografts from Blaberus giganteus, as well as control autografts. Grafts were scored histologically by counting the number of nuclei in a 100-mu-wide area under the center of the graft. The data showed that the reaction to the xenografts was characterized by quick recognition of foreignness by day 3 posttransplantation, followed by a slow decline in the cellular response, which lasted for more than 70 days. Control autografts did not appear to stimulate a reaction. Histological examination of xenografts revealed a disruption of the normal cytoarchitecture by a heavy cellular infiltrate. Our findings indicate that the outcome of the response to foreign integument in the roach is more characteristic of an encapsulation reaction than a classic rejection reaction.

Cockroaches↗

Primary integumentary allograft reactivity in the American cockroach, Periplaneta americana.

Previous reports have failed to demonstrate integumentary allograft rejection in insects. We realized however, that these studies may not have fully appreciated the structure of the insect exoskeleton. Since the subcuticlar epidermal layer constitutes the only living tissue associated with insect integument, its destruction would indicate that the animal recognized and responded to the foreign tissue. Thus, we investigated allograft reactivity in the American cockroach, Periplaneta americana, by observing the fate of the epidermal portion of the integument. Each animal in a pair received a 3 X 4-mm integumentary allograft from its partner, as well as a 3 X 4-mm control autograft. The transplants were then examined histologically for signs of epidermal destruction at 0, 1, 3, 5, 7, and 10-70 days (in 10-day increments) posttransplantation. The results indicated that significant rejection of the allografts began by day 3, with peak reactivity occurring by day 7 when 92% of the grafts were scored as rejected. At later periods (greater than 20 days), the graft sites showed signs of repopulation by host epidermal cells. The allograft reaction was found to lag behind the xenograft reaction, which showed peak activity after only 1 day posttransplantation. Even so, allograft rejection in this insect occurred quite rapidly (as compared with some other invertebrates), and would appear to be due to a cytotoxic reaction against the epidermal layer.

Animals↗

Demonstration of cell-mediated cytotoxicity to allogeneic and xenogeneic tissue in the American cockroach, Periplaneta americana, using a combination in vivo/in vitro assay.

Previous reports have claimed that insects lack the ability to reject integumentary allografts. However, when we followed the fate of the epidermal layer underneath the inert cuticle, we observed that the American cockroach not only rejected xenografts, but also demonstrated impressive reactivity against allografts. We have now developed a combination in vivo/in vitro assay that can quantitate the extent of allograft reactivity in the roach. Pieces of filter paper (3 X 4-mm) were implanted under the second and fourth tergites of donor animals. After 7 days, implants seeded with host hemocytes were removed, washed, and incubated in culture medium containing 3H-thymidine for 24 hr. Labeled grafts were reimplanted into paired animals to detect cytotoxicity as follows: grafts removed from under the second tergite were placed back into their original positions to serve as autograft controls; grafts removed from under the fourth tergite were reciprocally transferred between paired animals; and grafts were recovered after various time intervals and processed for scintillation counting. There was no significant difference in counts between allografts and autografts at day 1. Autografts sampled on days 3, 5, 7, and 10 had significantly higher counts than allografts, with peak reactivity occurring between days 3 and 5. This indicates that allogeneic cells were selectively destroyed, and confirms our recent data from conventional grafting studies that insects have the ability to react to allografts.

Animals↗

The effects of microtubule dissociating agents on the physiology and cytology of the sensory neuron in the femoral tactile spine of the cockroach, periplaneta americana L.

Microtubules are prominent cellular components of the mechanosensory and chemosensory sensilla associated with the insect cuticle, and a range of hypotheses have been proposed to account for their role in sensory transduction. Chemical agents such as colchicine and vinblastine, which dissociate microtubules, also interfere with transduction in these sensilla, and this has been attributed to their anti-microtubule activity. We have now examined the dynamic properties of sensory transduction in the mechanosensitive neuron of the cockroach femoral tactile spine, after the application of colchicine, vinblastine and lumicolchicine. Concurrently we have examined the ultrastructure of the same sensory ending by transmission electron microscopy. All of the drugs reduced the mechanical sensitivity o the receptor. Colchicine and vinblastine achieved this reduction without altering the dynamic properties of the receptor but lumicolchicine changed the dynamic response, and increased the relative sensitivity to rapid movements. Conduction velocity, another measure of neuronal function, which relies upon ionic currents flowing through the membrane, was reduced by all three drugs. The effects of the drugs upon the ultrastructure of the sensory ending were also disparate. In the case of colchicine there was complete dissociation of microtubules in the tubular body and distal dendrite before a total loss of mechanical sensitivity. Vinblastine was less effective in dissociating microtubules, although more effective in the reduction of mechanical sensitivity. With lumicolchicine the dominant morphological effect was a severe disruption of the dendritic membrane. We conclude from these experiments that microtubules are not essential in the transduction of mechanical stimuli by cuticular receptors and that the effects of these drugs upon mechanosensitivity are not directly related to their dissociation of the microtubules in the tubular body, but are more likely to arise from actions upon the cell membrane. These actions could include effects upon tubulin in the membrane or upon other membrane components.

Animals↗

Immunocytochemical localization of nicotinic acetylcholine receptors in the terminal abdominal ganglion of the cockroach (Periplaneta americana).

A polyclonal, monospecific antiserum raised against a nicotinic acetylcholine receptor protein affinity-purified from insect nervous tissue, was employed to demonstrate the localization of antigenic sites in the neuropile of the terminal (sixth) abdominal ganglion of the cockroach Periplaneta americana. In agreement with previously published autoradiographic mapping of specific [125I]alpha-bungarotoxin binding sites, specific areas of the central neuropile of this ganglion were densely stained, but not the cercal afferent axons. No staining was detected corresponding to the dense, peripheral, partly non-specific binding of alpha-bungarotoxin seen in autoradiographs of the same tissue. Certain peripherally located neuronal cell bodies, including the cell body of giant interneuron 2, contained intracellularly located antigenic sites.

Animals↗

A case for ancestral transfer of symbionts between cockroaches and termites.

Living species of the cockroach family Cryptocercidae have intestinal symbionts that are congeneric with some of the gut protozoa found in Isoptera. Presence of such closely related symbionts in cryptocercids and in termites has been frequently interpreted as a uniquely derived homologous character shared between the two xylophagous groups. This may not be the most parsimonious interpretation. Cryptocercus nymphs placed into Zootermopsis (dampwood termite) colonies were killed and eaten by the termites. Termites placed into a Cryptocercus nest box were also fully consumed. Modern Cryptocercus punctulatus and Zootermopsis are often found in the same decaying logs in the Pacific Northwest of the U.S.A., and it is likely that their ancestors also cohabited in at least a portion of their ranges. By occasionally killing and consuming an intruder from the other group, gut protozoa could have been acquired and exchanged between termites and Cryptocercus or their ancestors, under natural conditions and before the life histories of the protozoa became specialized within the host orders. Implications for assessing the phylogeny of the two dictyopteroid groups are also discussed.

Animals↗

Abundant distribution of locustatachykinin-like peptide in the nervous system and intestine of the cockroach Leucophaea maderae.

An antiserum raised to the locust neuropeptide locustatachykinin I (LomTK I) was used for analysis of the distribution of tachykinin-related peptide in the cockroach Leucophaea maderae. Extracts of dissected brains, suboesophageal ganglia, thoracic ganglia and midguts were separated by high performance liquid chromatography and the fractions analysed in enzyme-linked immunosorbent assay with use of the LomTK antiserum. Each of the tissues was found to contain LomTK-like immunoreactive (LomTK-LI) components with retention times corresponding approximately to synthetic LomTK I and II and callitachykinins I and II. The LomTK antiserum was also used for immunocytochemical mapping of peptide in the nervous system and intestine of L. maderae. A large number of LomTK-LI interneurons were detected in the proto-, deuto- and tritocerebrum of the brain and in the suboesophaegeal ganglion. The immunoreactive neurons supply processes to most parts of the brain: the central body, protocerebral bridge, mushroom body calyces, antennal lobes, optic lobe and most regions of the non-glomerular neuropil. A few protocerebral neurons send LomTK-LI processes to the glandular lobe of the corpora cardiaca. In each of the thoracic ganglia there are six LomTK-LI interneurons and in each of the unfused abdominal ones there are two interneurons. The fused terminal ganglion contains some additional cell bodies in the posterior neuromers. LomTK-LI cell bodies were detected in the frontal ganglion and fibres were seen in this ganglion as well as in the hypocerebral ganglion. The frontal ganglion supplies LomTK-LI processes to the muscle layer of the pharynx. The muscle layer of the midgut is innervated by LomTK-LI fibres from the stomatogastric system (oesophageal nerve and associated ganglia). Additionally the midgut contains numerous LomTK-LI endocrine cells. A number of the pharyngeal dilator muscles were also found to be innervated by LomTK-LI fibres, probably derived from cell bodies in the suboesophageal ganglion. All the LomTK-LI neurons of the central nervous system appear to be interneurons, suggesting a neuromodulatory role of the endogenous tachykinins. The tachykinin-like peptides from peripheral ganglia may be involved in the control of foregut and midgut contractility and possibly the peptide of the endocrine cells in the midgut has additional actions related to intestinal function.

Animals↗

Morphology as a basis for taxonomy of large spirochetes symbiotic in wood-eating cockroaches and termites: Pillotina gen. nov., nom. rev.; Pillotina calotermitidis sp. nov., nom. rev.; Diplocalyx gen. nov., nom. rev.; Diplocalyx calotermitidis sp. nov., nom. rev.; Hollandina gen. nov., nom.[TRUNCATED].

The purposes of this paper are (i) to present a framework for the morphometric analysis of large uncultivable spirochetes that are symbiotic in wood-eating cockroaches and termites; (ii) to revive, in accordance with the rules of the International Code of Nomenclature of Bacteria, the names of three genera (Pillotina, Diplocalyx, and Hollandina) and three species (Pillotina calotermitidis, Diplocalyx calotermitidis, and Hollandina pterotermitidis) for the same organisms to which the names were originally applied, because these names were not included on the 1980 Approved Lists of Bacterial Names; and (iii) to formally propose the name Clevelandina reticulitermitidis for a new genus and species of spirochetes from the termite Reticulitermes tibialis. None of these genera and species has been cultivated either axenically or in mixed culture; hence, all are based on type-descriptive material.

Animals↗

Crystallization and preliminary crystallographic study of a pheromone-binding protein from the cockroach Leucophaea maderae.

Pheromone-binding proteins (PBPs) are small helical proteins (13-18 kDa) present in various sensory organs of moths and other insect species. An antennal protein from the cockroach Leucophaea maderae (LmaPBP) has been found to share all the hallmarks of the PBP family and is expressed specifically in the female adult antennae, the gender that perceives the sex pheromone. Here, the crystallization of LmaPBP expressed as a recombinant protein in Escherichia coli periplasm is reported. Crystals of LmaPBP were obtained by the sitting-drop vapour-diffusion method using a nanodrop-dispensing robot. The protein crystallizes in two different crystal forms. Form 1 belongs to space group P1, with unit-cell parameters a = 43.2, b = 45.1, c = 45.7 A, alpha = 118.6, beta = 93.0, gamma = 106.9 degrees. With two molecules in the asymmetric unit, V(M) is 2.7 A(3) Da(-1) and the solvent content is 47%. A complete data set has been collected at 1.6 A resolution on beamline ID14-2 (ESRF, Grenoble). Form 2 was obtained in the presence of the pheromone (3-hydroxy-butan-2-one) and belongs to space group P2(1), with unit-cell parameters a = 38.2, b = 62.2, c = 45.1 A, beta = 93.0 degrees. With two molecules in the asymmetric unit, V(M) is 2.0 A(3) Da(-1) and the solvent content is 39%. A complete data set has been collected at 1.7 A resolution on beamline BM14 (ESRF, Grenoble). SeMet expression has been performed with a view to solving the structure by MAD data collection using the Se absorption edge.

Amino Acid Sequence↗

Olfactory receptor cells on the cockroach antennae: responses to the direction and rate of change in food odour concentration.

In insects, information about food odour is encoded by olfactory receptor cells with characteristic response spectra, located in several types of cuticular sensilla. Within short, hair-like sensilla on the cockroach's antenna, antagonistic pairs of olfactory receptor cells shape information inflow to the CNS by providing excitatory responses for both increases and decreases in food odour concentration. The segregation of food odour information into parallel ON and OFF responses suggests that temporal concentration changes become enhanced in the sensory output. When food odour concentration changes slowly and continuously up and down with smooth transition from one direction to another, the ON and OFF olfactory cells not only signal a succession of odour concentrations but also the rate with which odour concentration happens to be changing. Access to the values of such cues is of great use to an insect orientating to an odour source. With them they may extract concentration gradients from odour plumes.

Action Potentials↗