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Identity of a second type of allatostatin from cockroach brains: an octadecapeptide amide with a tyrosine-rich address sequence.

An octadecapeptide that inhibits juvenile hormone synthesis has been isolated by HPLC from brain-retrocerebral complexes of the cockroach Diploptera punctata. The primary structure of this allatostatin has been elucidated by tandem mass spectrometry: Ala-Tyr-Ser-Tyr-Val-Ser-Glu-Tyr-Lys-Arg-Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu- NH2 (ASB2). The amidated three-residue C terminus of this type B allatostatin is identical to that of four known type A allatostatins, and the preceding three residues show close structural homology. ASB2 has over twice the activity of the type A tridecapeptide Ala-Pro-Ser-Gly-Ala-Gln-Arg-Leu-Tyr-Gly-Phe-Gly-Leu-NH2 (ASA1) in inhibiting juvenile hormone biosynthesis in corpora allata from females in early vitellogenesis (day 2), and its efficacy persists during pregnancy, but it is equally effective as ASA1 on glands from day-10 females (IC50 = 0.31 nM). The octadecapeptide is characterized by a potential dibasic cleavage site, Lys9-Arg10, the integrity of which is needed for high potency. The ASB2-(11-18)-octapeptide amide gives a full response at high concentrations at day 10 (IC50 = 48 nM), but the C-truncated (1-9)-, (1-11)-, and (1-17)-amide fragments of ASB2 are inactive. Thus, the endocrine message is located at the C terminus. N alpha-acetylation of the N-truncated (9-18), (10-18), and (11-18) fragments of ASB2 increases activity relative to the nonacetylated peptides. The site of action of type A and type B allatostatins is located before mevalonate kinase in the biosynthetic pathway for juvenile hormone.

Amino Acid Sequence↗

Molecular cloning of the gene for the allatostatin family of neuropeptides from the cockroach Diploptera punctata.

Allatostatins (ASTs) are insect neuropeptides that inhibit juvenile hormone biosynthesis by the corpora allata. We have isolated a cDNA from the cockroach Diploptera punctata that encodes a 41.5-kDa precursor polypeptide containing the AST family of peptides. Translation of the cDNA revealed a 370-amino acid pre-pro-peptide consisting of 13 AST-type peptides and appropriate processing sites for endoproteolytic cleavage and amidation. The 13 potential AST sequences are characterized by the C-terminal AST corestructure Phe-Gly-Leu-NH2, with only one exception. Separating the clustered ASTs in the precursor, three acidic spacer regions are found. Contained within the largest of these are two potentially related peptides that may also be processed. Southern blot analysis revealed the presence of a single copy of the AST gene per haploid genome, as well as the probability that the gene may be present in at least two allelic forms. In situ hybridization indicated the AST-encoding gene is expressed in neurosecretory cells of D. punctata brain.

Amino Acid Sequence↗

Application of chiral gas chromatography with electroantennographic detection to the determination of the stereochemistry of a cockroach sex pheromone.

The coupling of an electroantennographic detector to a chiral capillary gas chromatographic system provides a highly sensitive technique for the determination of pheromone stereochemistry. Electronic modification of the usual electroantennographic detector enables the detector to respond to the relatively broad peaks produced by chiral gas chromatographic systems. By using this methodology, supellapyrone, a female sex pheromone of the brownbanded cockroach Supella longipalpa (Orthoptera: Blattellidae), is shown to be 5-(2'R,4'R-dimethylheptanyl)-3-methyl-2H-pyran-2-one.

Animals↗

Cockroach diuretic hormones: characterization of a calcitonin-like peptide in insects.

Insect diuretic hormones are crucial for control of water balance. We isolated from the cockroach Diploptera punctata two diuretic hormones (DH), Dippu-DH(31) and Dippu-DH(46), which increase cAMP production and fluid secretion in Malpighian tubules of several insect species. Dippu-DH(31) and -DH(46) contain 31 and 46 amino acids, respectively. Dippu-DH(46) belongs to the corticotropin-releasing factor (CRF)-like insect DH family, whereas Dippu-DH(31) has little sequence similarity to the CRF-like DH, but is similar to the calcitonin family. Dippu-DH(46) and -DH(31) have synergistic effects in D. punctata but have only additive effects in Locusta migratoria. Dippu-DH(31) represents a distinct type of insect DH with actions that differ from those of previously identified insect peptides with diuretic activity.

Amino Acid Sequence↗

The crystal structure of a cockroach pheromone-binding protein suggests a new ligand binding and release mechanism.

Pheromone-binding proteins (PBPs) are small helical proteins found in sensorial organs, particularly in the antennae, of moth and other insect species. They were proposed to solubilize and carry the hydrophobic pheromonal compounds through the antennal lymph to receptors, participating thus in the peri-receptor events of signal transduction. The x-ray structure of Bombyx mori PBP (BmorPBP), from male antennae, revealed a six-helix fold forming a cavity that contains the pheromone bombykol. We have identified a PBP (LmaPBP) from the cockroach Leucophaea maderae in the antennae of the females, the gender attracted by pheromones in this species. Here we report the crystal structure of LmaPBP alone or in complex with a fluorescent reporter (amino-naphthalen sulfonate, ANS) or with a component of the pheromonal blend, 3-hydroxy-butan-2-one. Both compounds bind in the internal cavity of LmaPBP, which is more hydrophilic than BmorPBP cavity. LmaPBP structure ends just after the sixth helix (helix F). BmorPBP structure extends beyond the sixth helix with a stretch of residues elongated at neutral pH and folding as a seventh internalized helix at low pH. These differences between LmaPBP and BmorPBP structures suggest that different binding and release mechanism may be adapted to the hydrophilicity or hydrophobicity of the pheromonal ligand.

Amino Acid Sequence↗

Ion movements in the nervous system of the American cockroach, Periplaneta americana (L.), influenced by toxaphene.

The effect of toxaphene upon ion fluxes of the central nervous system (CNS) of the cockroach, Periplaneta americana (L.), was studied in vitro. The CNS, divided into three sections (brain, thoracic, and abdominal), was exposed to 10(-5) M toxaphene in saline containing 24Na+, 42K+, 36CL-, or 45Ca++. Uptake and efflux of these ion were evaluated. Toxaphene was responsible for significant increases in the internal levels of 42K+ and 45Ca++, but had little effect upon 24Na+ or 36Cl- movements. The resulting concentration changes of the ions may be involved in the neural activity observed with toxaphene poisoning. Of the three CNS sections, significant changes were more numerous and occurred earlier in abdominal sections.

Animals↗

MAP: microwave-assisted extraction of cockroaches.

A Microwave-Assisted Process (MAP is a Trade-Mark of Her Majesty the Queen in Right of Canada as represented by the Minister of the Environment) solvent extraction procedure was used in conjunction with GC-MS analysis to investigate the chemical composition of dried and live cockroaches. The main components extracted were classified into four groups: sterols. fatty acids and their esters, long chain alkanes and fused aromatic hydrocarbons.

Alkanes↗

The effects of dieldrin and chlordimeform on learning and memory in the cockroach, Periplaneta americana: a study in behavioral toxicology.

A one-session T-maze training procedure for cockroaches in which animals were trained to turn right or left to avoid a shock was utilized to investigate the effects of dieldrin and chlordimeform (two neurotoxic pesticides) on learning and memory. These animals were trained and then tested for retention 5 h later. Three behavioral measures were recorded: choice behavior or direction turned, the time taken to proceed down the runway (runway time), and the time taken to proceed from the runway to either choice arm (choice point time). In control animals the number of correct choices, the runway time, and the choice point time increased with succeeding trials during training. Furthermore, control animals showed retention of correct choice behavior from training to testing. A nontoxic dose (no overt behavioral signs) of dieldrin was injected 2 h before training or 15 min after training. Pretraining injections of dieldrin eliminated correct-choice learning but did not alter the increase with training in runway or choice point times. Posttraining dieldrin administration did not interfere with retention of correct-choice behavior upon testing. These findings indicate that dieldrin induces behavioral alterations in a low acute dose and that detrimental effects are more likely with more complex behavior. Furthermore, behavioral disruption is more likely the closer exposure is to the initial learning of a task. Nontoxic doses of chlordimeform injected 1 h before training eliminated correct-choice learning and facilitated an increase in runway times during training.

Amidines↗

Adjustability of the circadian clock in the cockroaches: a comparative study of two closely related species, Blattella germanica and Blattella bisignata.

A single 2h light pulse (250 lux) was given at various times to phase shift the locomotor circadian rhythm of two species of closely related cockroaches, Blattella bisignata and Blatella germanica. The phase-response curve (PRC) of both species showed a similar pattern. Phase delays and advances were induced by light pulse during the early and late subjective night, respectively, while no clear phase shifting was elicited during the subjective day. However, the magnitude of the phase delay (1.89h +/- 0.66h) and advance (0.69h +/- 0.36h) of B. bisignata was significantly larger than that of B. germanica (0.78h +/- 0.38h and 0.35h +/- 0.18h, respectively). This result indicates the superior adjustability of the circadian clock in B. bisignata. The period-response curve (PdRC) was also constructed for both species. Although both species did not show great flexibility in circadian period changes, the phase shifts were significantly correlated with the period changes in the advance zone of B. bisignata (r = 0.72, P < .1). This allowed the circadian clock of B. bisignata to display better entrainability since the phase advance adjustment was significantly more difficult than that of phase delay. The results indicate the overall adjustability of the circadian clock of B. germanica is inferior to that of B. bisignata. The significance of this finding is discussed from an ecological perspective.

Adaptation, Physiological↗

The period gene of the German cockroach and its novel linking power between vertebrate and invertebrate.

Circadian clock protein PERIOD (PER) is essential for the endogenous clockworks in diverse lineages within Metazoa, but the protein sequences, the clock protein interactions, and the photic responses are variant and different between vertebrate and invertebrate PER homologs. Here we identified the German cockroach PER homologs and found it could bridge the huge phylogenetic gap and make possible a more precise protein sequence comparison between vertebrate and invertebrate PER homologs. Seven blocks of conserved regions (c1-c7) interspersed within PER proteins were defined, and two new significant homologies were found in the upstream portion of c3 region and in the c7 region, respectively. In addition, we found all dipteran insects PER homologs lack the c7 region and its following amino acid residues. Our results not only reveal the homology and divergence, but also imply the constraint and plasticity of divergent PER proteins during the course of evolution. These findings lay a solid foundation for understanding the general and divergent properties of circadian clockworks in diverse lineages within Metazoa.

Amino Acid Sequence↗

The electron microscopy of the left colleterial gland of the cockroach.

A study has been made of the cells of the left colleterial gland of the cockroach, Periplaneta americana (L.), using the electron microscope, and the results compared with previous histological and histochemical studies. The colleterial gland consists of an arborescent bunch of long tubules composed mainly of the cells which secrete the structural protein of the egg case ("type 4 cells"). Other types of cells: chitinogenic cells and "type 2 and 3 cells" each with a different cytology are described. The type 4 cells, which form the structural protein, reveal a cytological pattern very similar to that described for mammalian cells in a state of active protein synthesis. There is an elaborate development of particle-studded membranes in the cytoplasm. Smaller, rounded agranular vesicles also occur. The free secretory surface of the secreting cells forms the "end-apparatus" of the light microscopists. The invaginated surface is cast into numerous long narrow processes usually radially arranged and directed into a funnel-like formation derived from the thin intima lining the lumen of the gland (Text-fig. 2). The secretion in the form of small balls may be seen in the cavity of the end-apparatus and sometimes in the narrow processes. The small chitinogenic cells, lying between the protein-forming cells and the thin intima which they secrete, have a different cytology perhaps related to the fact that they form a polysaccharide rather than a protein. There is a very poor development of the particle-studded membranes of the type found in protein-forming cells. The type 2 cells, supposed to form an oxidase, have an end-apparatus that is similar to, but more complex than, those of the type 4 cells and their cytoplasm is almost completely filled with mitochondria. There is some evidence that mitochondria play a part in forming the oxidase and pass into the tubules of the end-apparatus. Type 3 cells resemble both types 2 and 4 and are probably a transient intermediate form.

Animals↗

Absence of rDNA amplification in the uninucleolate oocyte of the cockroach Blattella germanica (Oorthoptera: Blattidae).

Amplification of the genes coding for ribosomal RNA oocurs in the oocytes of a wide variety of organisms. In oocytes of various species of crickets (Orthoptera: Gryllidae) the amplified DNA is contained in a large extrachromosomal DNA body. Multiple nucleoli form about the periphery of the DNA body during the diplotene stage of meiosis I. In contrast to the general pattern of orthopteran oocytes, oocytes of the cockroach Blattella germanica demonstrate a single large nucleolus instead of many nucleoli. In order to determine whether the genes coding for rRNA are amplified in the oocytes of B. germanica, the relative amount of rDNA in oocytes was compared with the rDNA content of spermatocytes and somatic cells. An extrachromosomal DNA body similar to that present in crickets is not present in B. germanica. A satellite DNA band which contains nucleotide sequences complementary to rRNA accounts for approximately 3-5% of the total DNA in somatic and in male and female gametogenic tissues. Female cells contain approximately twice as much rDNA as do male cells. An XX-XO sex-determining mechanism is operative in B. germanica. In situ hybridization with rRNA indicates that the nucleolar organizer is located on one end of the X chromosome and that oocytes do not contain more than twice the amount of rDNA found in spermato cytes. The data indicate that rDNA is not amplified in the uninucleolate oocyte of B germanica.

Animals↗

Gain control of synaptic transfer from second- to third-order neurons of cockroach ocelli.

Synaptic transmission from second- to third-order neurons of cockroach ocelli occurs in an exponentially rising part of the overall sigmoidal characteristic curve relating pre- and postsynaptic voltage. Because of the nonlinear nature of the synapse, linear responses of second-order neurons to changes in ligh intensity are half-wave rectified, i.e., the response to a decrement in light is amplified whereas that to an increment in light is compressed. Here I report that the gain of synaptic transmission from second- to third-order neurons changes by ambient light levels and by wind stimulation applied to the cerci. Transfer characteristics of the synapse were studied by simultaneous intracellular recordings of second- and third-order neurons. Potential changes were evoked in second-order neurons by a sinusoidally modulated light with various mean luminances. With a decrease in the mean luminance (a) the mean membrane potential of second-order neurons was depolarized, (b) the synapse between the second- and third-order neurons operated in a steeper range of the exponential characteristic curve, where the gain to transmit modulatory signals was higher, and (c) the gain of third-order neurons to detect a decrement in light increased. Second-order neurons were depolarized when a wind or tactile stimulus was applied to various parts of the body including the cerci. During a wind-evoked depolarization, the synapse operated in a steeper range of the characteristic curve, which resulted in an increased gain of third-order neurons to detect light decrements. I conclude that the nonlinear nature of the synapse between the second- and third-order neurons provides an opportunity for an adjustment of gain to transmit signals of intensity change. The possibility that a similar gain control occurs in other visual systems and underlies a more advanced visual function, i.e., detection of motion, is discussed.

Animals↗

The spectral sensitivities of the dorsal ocelli of cockroaches and honeybees; an electrophysiological study.

The spectral sensitivities of the dorsal ocelli of cockroaches (Periplaneta americana, Blaberus craniifer) and worker honeybees (Apis mellifera) have been measured by electrophysiological methods. The relative numbers of quanta necessary to produce a constant size electrical response in the ocellus were measured at various wave lengths between 302 and 623 mmicro. The wave form of the electrical response (ERG) of the dark-adapted roach ocellus depends on the intensity but not the wave length of the stimulating light. The roach ocellus appears to possess a single photoreceptor type, maximally sensitive about 500 mmicro. The ERG's of bee ocelli are qualitatively different in the ultraviolet and visible regions of the spectrum. The bee ocellus has two types of photoreceptor, maximally sensitive at 490 mmicro and at about 335 to 340 mmicro. The spectral absorption of the ocellar cornea of Blaberus craniifer was measured. There is no significant absorption between 350 and 700 mmicro.

Animals↗

Nonlinear cable properties of the giant axon of the cockroach Periplaneta americana.

The steady state nonlinear properties of the giant axon membrane of the cockroach Periplaneta americana were studied by means of intracellular electrodes. The resistivity of this membrane markedly decreases in response to small subthreshold depolarizations. The specific slope resistance is reduced by twofold at 5 mV depolarization and by a factor of 14 at 20 mV depolarization. As a result, the spatial decay, V(X), of depolarizing potentials is enhanced when compared with the passive (exponential) decay. This enhancement is maximal at a distance of 1-1.5 mm from a point of subthreshold (0-20 mV) depolarizing perturbation. At that distance, the difference between the actual potential and the potential expected in the passive axon is approximately 30%. The effects of membrane rectification on V(X) were analyzed quantitatively with a novel derivation based on Cole's theorem, which enables one to calculate V(X) directly from the input current-voltage (I0-V) relation of a long axon. It is shown that when the experimental I0-V curve is replotted as (I0Rin)-1 against V (where Rin is the input resistance at the resting potential), the integral between any two potentials (V1 greater than V2) on this curve is the distance, in units of the resting space constant, over which V1 attenuates to V2. Excellent agreement was found between the experimental V(X) and the predicted value based solely on the input I0-V relation. The results demonstrate that the rectifying properties of the giant axon membrane must be taken into account when the electrotonic spread of even small subthreshold potentials is studied, and that, in the steady state, this behavior can be extracted from measurements at a single point. The effect of rectification on synaptic efficacy is also discussed.

Animals↗

Monitoring for insecticide resistance in field-collected strains of the German cockroach (Dictyoptera: Blattellidae).

Forty-five field-collected strains of German cockroaches, Blattella germanica were tested for resistance to 12 different insecticides by the time-mortality response method in comparison with a known susceptible strain. Only low to moderate resistance to diazinon, chlorpyrifos, and acephate was detected. Resistance to malathion was widespread; about half of the strains tested showed high resistance. High resistance to the carbamates propoxur and bendiocarb also occurred. High resistance was uncommon with propoxur, but about 35 strains were highly resistant to bendiocarb. High resistance to pyrethrins was observed in about of the strains tested. Resistance to the pyrethroids allethrin, permethrin, phenothrin, fenvalerate, and cyfluthrin was detected in some of the strains examined. All of the strains tested were susceptible to one or more of the insecticides used. These results indicate that, although resistance is a serious problem in this species, satisfactory control should be possible by selection of an appropriate insecticide.

Animals↗

Effect of sublethal propoxur exposure on male German cockroaches (Orthoptera: Blattellidae) and their feeding behavior.

Exposure of male German cockroaches, Blattella germanica L., to propoxur produced significant water loss at 2 and 24 h after treatment. This effect was dependent upon the state of hydration before insecticide exposure. Hydrated males lost as much as 10% of their total body mass in the form of water; dehydrated males lost about 4.3%; and untreated controls lost approximately 1.8% during the same time period. No significant compensatory feeding responses were observed 1-4 d after treatment. Water depletion associated with propoxur exposure resulted in increased water consumption for the first 2 d after treatment and then reached a plateau for the duration of the study. Males consumed water daily regardless of insecticide exposure.

Animals↗

Topical and oral toxicity of sulfluramid, a delayed-action insecticide, against the German cockroach (Dictyoptera: Blattellidae).

The LD50 of sulfluramid topically applied to 2-d-old, fifth instars of the German cockroach, Blattella germanica (L.), was estimated at 14.5 micrograms/g (95% FL = 13.7-15.4 micrograms/g). Sulfluramid was significantly more toxic than topically applied hydramethylnon (LD50 = 29.2 [19.0-46.5] micrograms/g). Sulfluramid had delayed toxicity but caused mortality significantly faster than hydramethylnon after topical application. The oral LD50 against newly enclosed, fifth instars was estimated to be 4.1 (3.9-4.4) micrograms/g; this toxicity was significantly greater than when sulfluramid was topically applied. Mortality caused by sulfluramid occurred significantly more slowly in the dietary exposures than in the topical applications. Sulfluramid at 1,000 ppm in diets was not a feeding deterrent to nymphal B. germanica.

Animals↗