Studies on the cellular defense reactions of the Madeira cockroach, Leucophaea maderae: nodule formation in response to injected bacteria

Nodules were formed in the Madeira cockroach, Leucophaea maderae, in response to injections of low doses (3 x 10(4) bacteria/insect) of three strains of Bacillus cereus and Escherichia coli K12 D31. The most pathogenic strain of bacteria used, B. cereus B1, produced the greatest cellular response, while the least pathogenic, E. coli K12 D31, injected at the same dose, caused little nodule formation. Similarly, nodules were generally found to be larger following injection of pathogenic bacteria such as B. cereus B1 than to the weak pathogen, E. coli K12 D31. There was, however, no difference in the extent of nodule formation with the four bacterial strains/species if they were heat killed prior to injection. Histologically, the nodules formed in response to all bacterial species employed were similar, with a central necrotic core enclosing cell debris and occasional bacteria, and an outer, thin sheath of plasmatocyte-like hemocytes. Possible reasons for the enhanced cellular reactivity observed in L. maderae to pathogenic bacteria are discussed.     

Circadian rhythms in the mating behavior of the cockroach, Leucophaea maderae

Mating behavior of small populations of virgin males and females of the cockroach Leucophaea maderae were continuously monitored via time-lapse video recording in controlled laboratory conditions. The time of onset of copulation was found to be rhythmic in a light cycle of 12 h light alternated with 12 h of darkness, with the peak of mating behavior occurring near the light to dark transition. This rhythm persisted in constant dim red illumination and constant temperature. In constant conditions, the period of the rhythm was slightly less than 24 h, with a peak of copulation during the late subjective day. These data demonstrated that mating behavior is gated by a circadian clock. When males and females were taken from light cycles that were 12 h out of phase, a bimodal rhythm was observed with one peak in the males' late subjective day and a second peak of equal amplitude in the late subjective day of females. The results indicated that circadian systems in both males and females contribute to the circadian rhythm in copulation. Bilateral section of the optic tracts (OTX) of both males and females abolished the rhythm, but the rhythm persisted when OTX females were paired with intact males or when OTX males were paired with intact females. Furthermore, when OTX males or OTX females were paired with intact animals that were 12 h out of phase, a bimodal rhythm was still observed. These results suggested that the circadian pacemaker in the optic lobes of both male and female cockroaches participates in the control of mating, but that a pacemaker outside the optic lobes is also likely involved. Finally, it was shown that the female's olfactory response (measured by electroantennogram) to components of the male sex pheromone exhibited a circadian rhythm, but the data suggested the peripheral olfactory rhythm is not likely to be involved in the rhythm of mating behavior.     

Bursicon release and activity in haemolymph during metamorphosis of the cockroach,Leucophaea maderae

Bursicon activity first appears in the haemolymph of the cockroach, Leucophaea maderae, early in ecdysis as the old cuticle splits and separates over the thorax. Hormonal activity reaches high levels in the haemolymph before ecdysis is complete and remains so for about 1·5 hr, with a gradual decline and disappearance by 3 hr. The sensory mechanism controlling bursicon release is located in the thorax and appears to be stimulated as the ecdysial split widens for emergence of the thorax. If the abdomen is isolated before this time no tanning of abdominal cuticle occurs, while the isolated thorax proceeds to tan. Therefore the thoracic ganglia seem to be a site of release for bursicon. Release of the hormone from abdominal and head ganglia may also occur after neural stimulation from the thoracic system. Bursicon activity was found in all ganglia of the central nervous system and the corpora cardiaca-allata complex. Removal of the old cuticle prior to the start of ecdysial behaviour does not result in tanning of the new cuticle. However, if the old cuticle is removed after the insect begins to swallow air in preparation for ecdysis, then the new cuticle tans. Mechanical prevention of ecdysis and later removal of the old cuticle also does not result in tanning of the new cuticle. Therefore, shedding of the old cuticle only activates the release of bursicon in conjunction with other normal ecdysial events.     

Visceral muscles and myogenic activity in the hindgut of the cockroach,Leucophaea maderae

Summary The hindgut of the Madeira cockroach contains an intricate network of longitudinal and circular muscles that are distinctive for each region. In the rectum, the longitudinal muscles are symmetrically arranged in 6 distinct bands, while the circular muscles appear as a uniform layer over the rectal pads. In the colon, the muscle fibers are arranged in an irregular lattice with the longitudinal fibers generally superimposed on the circular ones but with an evident weaving between the layers. In addition to these muscle layers, a delicate, superficial network of muscle-like fibers covers many portions of the colon and rectum.In spite of the bewilderingly complex motile activity of deganglionated hindguts, all activity could be classified under 4 basic types after cinematographic analysis: segmentation, compression, peristalsis, or reverse peristalsis or a combination thereof. Although much of the activity that occurred was seemingly random, there was an evident rhythmicity that spontaneously arose and ended in several types of motility during the course of observations. The defined modes of activity seemed to be completely myogenic in nature, as all 4 categories were readily observed in hindguts 30 min after treatment with tetrodotoxin (10^–6 g/ml). Each region of the hindgut seemed to have its own particular rhythm.Action potentials were recorded both intracellularly and extracellularly from all regions of the hindgut; amplitude usually ranged between 10 and 20 mV for intracellular recordings, and such spike potentials were often preceded by a slow depolarizing pre-potential. Generally, however, the depolarization was abrupt. Transmembrane potentials from the visceral muscle fibers were never truly at rest. Slow, continuous fluctuations (3–8 mV) were common. At times, plateau-type action potentials were recorded, but generally the repolarization contour was almost linear with time. Contractions were evoked by action potentials but not by the slow, rhythmic fluctuations in the membrane potential.No particular region or structure in the hindgut showed an exclusive pacemaker function. However, there was an evident gradient of increased excitability progressing in an caudal direction from the ileum.In a sodium-free saline, the amplitude of action potentials was remarkable enhanced from 5 to 10 min after the initial change. Even after a 20 min exposure, action potentials were still often present although their frequency and amplitude dropped. Tetrodotoxin (10^–6g/ml) had no. pronounced effect on frequency or amplitude of action potentials. However, spike potentials ceased within 1.5 min after exposure to a sodium and calcium-free saline. When such preparations were re-exposed to a sodium-free saline containing normal calcium, the action potentials reappeared, suggesting that calcium might be a current-carrying ion. Although action potentials in a calcium-free medium showed variability, we generally saw a marked reduction in amplitude of potentials within 5 min. We further observed that 2 mM manganous ion completely abolished action potentials within 2 min. Thus, it seems likely that sodium is not the sole current-carrying ion in cockroach hindgut muscle.The authors express their indebtedness to Ms. Susan Swann, Mr. Gerald Holt, Mr. David Owens, and Ms. Mary Strand for their competent technical assistance.     

Immunocytochemical localization of leucomyosuppressin-like peptides in the CNS of the cockroach,Leucophaea maderae

Immunocytochemistry was used to determine sites of synthesis and pathways for the transport of the neuropeptide, Leucomyosuppressin (pQDVDHVFLRFamide) in the cockroach,Leucophaea maderae. This study led to identification of neurons in the brain and thoracic ganglia reactive to polyclonal antibodies raised against this peptide. No immunoreactive cells were found in the subsophageal or abdominal ganglia. Although the corpus cardiacum contained no intrinsic cells immunoreactive to LMS antibodies, the periphery of this organ and that of the nervi corporis allati contain an abundance of LMS-reactive terminals.     

The secretion and metabolism of alpha-ecdysone by cockroach (Leucophaea maderae) tissues in vitro

Hemolymph β-ecdysone levels are high (～1.6 μg/ml) in late last instar cockroach ($\text{Leucophaea}$$\text{maderae}$) nymphs; the level of α-ecdysone (～0.1 μg/ml) is evidently subphysiological. Cultured leg regenerates, target organs of ecdysone, are capable of slowly converting α- to β-ecdysone. Cultured prothoracic glands secrete α-ecdysone, which was identified by complete mass spectrometry. These results are consistent with the view that α-ecdysone, secreted by the prothoracic gland, functions as a prohormone which is converted into the active moulting hormone, β-ecdysone, in other tissues.     

Three shapes of mitochondria in femoral muscle of the cockroach, Leucophaea maderae Fabricius

Three distinct types of mitochondria as related to shape, position, and size in the femoral muscle of the cockroach, Leucophae maderae, are described. The elongated mitochondria are interposed between the myofibrils and are oriented parallel to the long axis of the fiber. Surface indentations on these mitochondria for surrounding structures indicate their permanent position. The oval mitochondria are situated under the sarcolemma. These organelles have a parallel orientation to the underlying muscle fiber but no alignment with respect to the “sarcomeric repeat” and thereby suggest that this type is mobile. The Y-shaped mitochondria are observed in the intermyofibrillar sarcoplasm. Their paired processes are on each side of the Z-disc and imply that the Y-shaped mitochondria are a sessile type. The cristae in all three categories of mitochondria display a tightly packed and complex arrangement.     

Primary structure and synthesis of a blocked myotropic neuropeptide isolated from the cockroach, Leucophaea maderae

A peptide which stimulates the contractile activity of the cockroach hindgut was isolated from head extracts of the cockroach, Leucophaea maderae. The inability of aminopeptidase M to degrade the peptide and the presence of glutamic acid in the hydrolysate suggested N-terminal blocking by pyroglutamic acid. The N-terminal pGlu was removed enzymatically and the unblocked fragment was sequenced with an automated gas-phase peptide sequencer. The structure determined (pGlu-Thr-Ser-Phe-Thr-Pro-Arg-Leu-NH2) was synthesized and shown to be both chemically and biologically identical with the natural product.     

Effect of lysozyme on the lectin-mediated phagocytosis of Bacillus cereus by haemocytes of the cockroach, Blaberus discoidalis

Lysozyme-like activity has been demonstrated in both cell-free haemolymph and, more abundantly, in haemocyte-lysate supernatants of Blaberus discoidalis. This activity was non-inducible, but heat-stable, with a maximum activity at pH 6.2. When B. cereus was pre-incubated in a concentration of chicken egg-white lysozyme equivalent to the concentration of lysozyme-like activity in cell-free haemolymph, the phagocytosis of B. cereus opsonized with GlcNAc-specific lectins, i.e. BDL2, WGA and HPA, was significantly reduced by up to 50%, while phagocytosis of B. cereus opsonized with mannose-specific lectins, such as BDL1 and Con A, was significantly increased. Pre-incubation of B. cereus in a higher concentration of lysozyme resulted in a smaller, shorter lived increase in the phagocytic rate of bacteria opsonized with these mannose-specific lectins. The action of lysozyme on the peptidoglycan in the cell wall of B. cereus probably resulted in a reduction in the number of binding sites for the GlcNAc-specific lectins, and, therefore, reduced the phagocytic rate of BDL2, HPA and WGA-opsonized B. cereus. Concomitantly, the breakdown of peptidoglycan probably exposed mannose-containing polysaccharides, previously embedded in the peptidoglycan layer, resulting in an increase in the phagocytic rate of the BDL1- and Con A-opsonized B. cereus. These results are discussed in relation to the immune-potential of B. discoidalis.     

Synthesis, transport, and release of a neurohormone by cultured neuroendocrine glands from the cockroach, Leucophaea maderae

A hindgut-stimulating neurohormone synthesized in vitro by the neurosecretory cells of cultured brains of Leucophaea maderae passes through the nervi corporis cardiaci I into the corpora cardiaca and is released into the culture medium. As much as 90 per cent of the hormone breaks down in the medium during a 3-day incubation period, and the amount recovered represents only a small fraction of the amount actually released.     

Action of taurine, 3-aminopropanesulfonic acid, and GABA on the hindgut and heart of the cockroach, Leucophaea maderae.

Taurine, glycine, glutamate, and gamma-aminobutyric acid (GABA) were all present in concentrations of greater than 1% of the total free amino acid content in the brain, thoracic, and abdominal ganglia of Leucophaea maderae. Hemolymph, subesophageal ganglia, and hindgut had substantial amounts of glutamate and glycine, but less than 0.3% taurine or GABA. Taurine, 3-aminopropanesulfonic acid (3-APS), cysteine-sulfinic acid (CSA), and GABA each had myotropic activity on the isolated cockroach hindgut, with 3-APS having the most consistent effect (ED50 = 0.63 mM), while taurine and CSA activities were similar to that of GABA on the hindgut. Both taurine and 3-APS had anti-arrhythmic effects on semi-isolated heart preparations of L. maderae, while GABA was inhibitory and induced arrhythmia. Bicuculline was antagonistic to the effects of GABA, taurine, and 3-APS on the hindgut, and induced arrhythmia in heart preparations; this arrhythmia was reversible by taurine, but not by GABA or 3-APS.     

Vitellogenin of the cockroach,Leucophaea maderae: nucleotide sequence,structure and analysis of processing in the fat body and oocytes

A cDNA encoding vitellogenin (Vg) of the cockroach, Leucophaea maderae was cloned and sequenced. The deduced amino acid sequence consisting of 1913 residues (including 15 residues for a putative signal peptide) was obtained. Amino-terminal sequence analysis demonstrated that the pro-Vg was cleaved into four polypeptide 'subunits' following the three consensus RXXR cleavage site sequences, which were secreted as four Vg polypeptides (apparent molecular weights = 112-, 100-, 92- and 55-kD), sequestered, and deposited in the egg as four respective vitellin (Vn) polypeptides. There was, however, an additional 90-kD Vn polypeptide existed in the egg. We show that this polypeptide is a processed product from 92-kD Vn polypeptide. Northern blot analysis of poly (A)+ RNA reveals that mRNA coding for Vg is present only in the female fat body cells but neither in the ovary nor in the male fat body cells. The deduced amino acid sequence contained a serine-rich stretch at the C-terminal region. This stretch occurred also in Vgs of Periplaneta americana (Vg1 and Vg2) and Blattella germanica. The Vg of L. maderae had 26% and 31% homology with those of P. americana (Vg1 and Vg2) and B. germanica, respectively. Phylogenetic analysis (neighbour-joining) was made using four cockroach Vgs and the tree was compared with other molecular and conventional phylogenetic trees.