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PubMed1994

The preparation of trophocytes from disaggregated fat body of the cockroach (Periplaneta americana).

A method has been developed for disaggregating the fat body of the adult American cockroach, Periplaneta americana, using collagenase. The yield of cells is sensitive to the osmolarity of the dispersing medium and to the age of the cockroaches from which the fat bodies are taken. Trophocytes uncontaminated with other cells were obtained by taking advantage of the low density of these cells which causes them to float to the top of the dispersion medium. In contrast, the mycetocytes and urocytes being denser than the medium sink to the bottom. The trophocytes retain the ability to respond to the synthetic hyperglycaemic hormones, CCI and CCII, as shown by the activation of phosphorylase and the stimulation of trehalose efflux. The trophocytes incorporated leucine into protein secreted by the cells in a time dependent manner.

Aging↗
PubMed2006

Cloning and characterization of the adipokinetic hormone receptor from the cockroach Periplaneta americana.

Cockroaches have long been used as insect models to investigate the actions of biologically active neuropeptides. Here, we describe the cloning and functional expression in Chinese hamster ovary cells of an adipokinetic hormone (AKH) G protein-coupled receptor from the cockroach Periplaneta americana. This receptor is only activated by various insect AKHs (we tested eight) and not by a library of 29 other insect or invertebrate neuropeptides and nine biogenic amines. Periplaneta has two intrinsic AKHs, Pea-AKH-1, and Pea-AKH-2. The Periplaneta AKH receptor is activated by low concentrations of both Pea-AKH-1 (EC50, 5 x 10(-9)M), and Pea-AKH-2 (EC50, 2 x 10(-9)M). Insects can be subdivided into two evolutionary lineages, holometabola (insects with a complete metamorphosis during development) and hemimetabola (incomplete metamorphosis). This paper describes the first AKH receptor from a hemimetabolous insect.

Amino Acid Sequence↗
PubMed2005

Day/night fluctuations in melatonin content, arylalkylamine N-acetyltransferase activity and NAT mRNA expression in the CNS, peripheral tissues and hemolymph of the cockroach, Periplaneta americana.

Melatonin content measured by a radioenzymatic assay in the brain of the American cockroach (Periplaneta americana) showed a day/night fluctuation with higher levels at night under LD 12:12. The activity of arylalkylamine N-acetyltransferase (NAT) in brain was also higher at night and this pattern continued in constant darkness. The results suggest that the rhythmicity in melatonin content can be caused by NAT. Melatonin content in hemolymph showed an even greater day/night difference, more than 12 times that in brain under LD 12:12. Melatonin levels in retina were also higher at night while NAT activity was not significantly higher at night than at daytime. Using a probe designed from NAT cloned from testes we performed Northern blot analysis of total RNA, which revealed that the level of NAT mRNA was higher in midgut, ovary and female accessory glands than in fat body and brain. The level of transcript in midgut was higher at night, but the levels in ovary and female accessory reproductive gland showed the opposite pattern. We also used the antibody to whole Drosophila melanogaster aaNAT1 protein, seeking a homologous antigen in the cephalic ganglia. NAT-like antigen was detected in several restricted populations of cells in the brain that were partially co-localized with PER-like antigen. The results suggest that NAT exists in multiple forms in various tissues of the cockroach and that its functions and regulations can vary among tissues. The results in the brain led to the conclusion that NAT could be a clock-controlled gene functioning as an output regulator of the circadian clock.

Animals↗
PubMed2005

Discontinuous carbon dioxide release in the German cockroach, Blattella germanica (Dictyoptera: Blattellidae), and its effect on respiratory transpiration.

The discontinuous gas exchange cycle (DGC) was described in the German cockroach, Blattella germanica (L.) (Dictyoptera: Blattellidae) for the first time. Also, the effect of the DGC on water loss was investigated. The CO(2) emission pattern in both insecticide resistant and susceptible B. germanica varied with temperature. At 10, 15, and 20 degrees C the pattern was discontinuous. Cycle frequency increased at 25 and 30 degrees C, and at 35 degrees C the pattern became cyclic. In most DGCs, there was no clear distinction between the closed and flutter phases in both strains thus data for these phases were combined and analyzed as the interburst phase. The probability that B. germanica would breath discontinuously varied with temperature. Most cockroaches (62.8%) displayed DGCs at 10 degrees C, therefore measurement of metabolic rate and water loss was carried out at this temperature. Using repeated measures of analysis of variance, the interburst and burst V(.)(CO(2))(ml h(-1)) were not significantly different between the two strains. The variability in CO(2) emission during the interburst and burst phases over time was not significantly different from cycle to cycle or between strains. Overall metabolic rate during the entire recording was not significantly different between both strains. There was a significant difference in the duration of the interburst and burst phases between the strains. The susceptible strain had significantly longer interburst and burst phase durations during a complete DGC than the resistant strain. The interburst and burst phase durations were 5.01+/-0.19 and 6.21+/-0.13 min, respectively, for the resistant strain, whereas the durations were 7.16+/-0.37 and 6.73+/-0.17 min, respectively, for the susceptible strain. This resulted in a DGC of significantly longer duration (13.89+/-0.44 min) in the susceptible strain compared with the resistant strain (11.23+/-0.26 min). The duration of the interburst phase was significantly different from the open phase duration in the resistant strain such that during a single DGC lasting approximately 11.23 min, 43.5% consisted of the interburst phase while the burst phase made up 56.5% of the cycle. The cuticular permeability at 10 degrees C and 0% RH was 2.26 microg cm(-2) h(-1) mmHg(-1) for the resistant strain and 3.42 microg cm(-2) h(-1) mmHg(-1) for the susceptible strain. In both strains, cuticular transpiration accounted for approximately 95% of total water loss. The significantly longer duration of the interburst phase of the susceptible strain was not important in reducing water loss.

Analysis of Variance↗
PubMed2004

Kinetic and pharmacological characterization of desensitizing and non-desensitizing glutamate-gated chloride channels in cockroach neurons.

Glutamate-gated chloride channels (GluCls) are found only in invertebrate nerve and muscle, where they mediate inhibitory synaptic transmission, and are important target sites of insecticides. Two GluCl subtypes have previously been distinguished in isolated cockroach CNS neurons based on differential pharmacology. The present study characterizes the kinetics and pharmacological properties of desensitizing and non-desensitizing GluCls. Both types of GluCls were sensitive to glutamate and ibotenic acid. The non-desensitizing GluCl subtype was elicited by glutamate with an EC(50) of 115.8 microM and a Hill coefficient of 2.6 and was also sensitive to the agonist ibotenic acid with an EC(50) of 42 microM and a Hill coefficient of 1.7. The desensitizing and non-desensitizing currents were carried by chloride ions, and occurred either separately or in combination in individual neurons. The GluCls were also found to coexist with and function independently of the GABA-activated chloride channels. The desensitizing and non-desensitizing GluCls exhibited different sensitivities to the ligand-gated channel blocker picrotoxinin. The desensitizing GluCls were blocked only 8% by 30 microM picrotoxinin, whereas the non-desensitizing GluCls were potently blocked by picrotoxinin with an IC(50) of 4.1 microM. The insecticides fipronil and dieldrin at 1 microM inhibited the desensitizing currents by 56 and 13%, respectively, and the non-desensitizing currents by 98 and 43%, respectively. It is concluded that the two types of GluCls found in cockroach neurons exhibit significantly different electrophysiological and pharmacological characteristics.

Animals↗
PubMed2001

Myostimulatory neuropeptides in cockroaches: structures, distribution, pharmacological activities, and mimetic analogs.

In this brief overview we give the historical background on the discovery of myostimulatory neuropeptides in cockroaches. Related peptides were later found in other insect groups as well. We summarize the current knowledge on primary structures, localization, physiological and pharmacological effects of the different cockroach neuropeptides, including kinins, sulfakinins, pyrokinins, tachykinin-related peptides, periviscerokinins, corazonin, and proctolin. In addition, we briefly comment on the development of mimetic pseudopeptide analogs in the context of their possible use in insect pest management.

Journal Article↗
PubMed2001

History dependence of circadian pacemaker period in the cockroach.

The freerunning period of circadian clocks in constant environmental conditions can be history-dependent, and one effect of entrainment of circadian clocks by light cycles is to cause long-lasting changes in the freerunning period that are termed after-effects. We have studied after-effects of entrainment to 22-h (LD 8:14) and 26-h (LD 8:18) light cycles in the cockroach Leucophaea maderae. We find that in cockroaches, the freerunning period of the locomotor activity rhythm, measured in constant darkness (DD), is 0.7h less after entrainment to T22 than after entrainment to T26. Induction of after-effects requires several days (>1 week) entrainment, and after induction, after-effects will persist in DD for over 40 days. Further after-effects are unaltered by phase-resetting of up to 12h caused by exposure to low-temperature pulses (7 degrees C) of 24 or 48h duration. After-effects also persist through re-entrainment for 2 weeks to 24-h light cycles. These results indicate that after-effects arise from stable changes in the circadian system that are likely to be independent of phase relationships among oscillators within the circadian system. We also show that entrainment to temperature cycles does not generate after-effects indicating that light may be unique in its ability to generate lasting changes in pacemaker period.

Journal Article↗
PubMed2002

Temperature acclimation in overwintering nymphs of a cockroach, Periplaneta japonica: walking on ice.

Nymphs of the Yamato cockroach, Periplaneta japonica, showed a seasonal change in the ability to move at low temperature. Laboratory-reared nymphs buried in ice for 15 min-3 h took about 600 s to get up at 25 degrees C after being placed with their dorsal surface on the floor of a Petri dish. The mean time to get up at 25 degrees C (recovering time) after 15 or 60-min burial in ice was relatively long in early autumn, decreased rapidly in autumn, reached a minimal level in winter (<100 s) and increased in spring. Temperature was responsible for this seasonal change in physiology, and neither photoperiod nor diapause status was important. Recovering time depended on the acclimation temperature and its duration. For a 10-day exposure in a range of 0-25 degrees C, the lower the temperature the shorter the recovering. Longer periods of acclimation at these temperatures tended to shorten recovering time. The acquisition of the ability to move on ice is a seasonal phenomenon unique to P. japonica. No comparable response was found in three other cockroaches including two sub-tropical species (Periplaneta americana and Opisthoplatia orientalis) and a temperate species (Blattela nipponica).

Journal Article↗
PubMed2000

Sex-specific differences in nitrogen intake and investment by feral and laboratory-cultured cockroaches.

We compared nutrient selection in a laboratory culture of the American cockroach (Periplaneta americana: Insecta, Blattodea) and a feral population which was founded by escapees from the culture approximately 14 generations prior to the study. In a first experiment, adult male and female cockroaches were provided with two nutritionally complementary synthetic foods, and thus allowed to select the protein and carbohydrate components of their diet independently. There were no differences in the amounts of carbohydrate eaten by the two populations. However, feral males ingested more protein than cultured males. In a second experiment, the construction of nitrogen budgets showed that the additional nitrogen ingested by feral males was allocated preferentially to accessory sex glands, rather than somatic tissue or excretion via the faeces. This suggests a possible role for sexual selection in the dietary difference between the strains. By contrast with males, there was no statistically significant difference in the amount of protein eaten by females of the two populations. However, feral females were found to have a higher density of bacterial endosymbionts than cultured females. Since these symbionts are involved in the synthesis of essential amino acids, this might account for greater reproductive output observed in a previous study in the feral compared with the culture females.

Journal Article↗
PubMed2000

Biological activities of the allatostatin family of peptides in the cockroach, Diploptera punctata, and potential interactions with receptors.

Allatostatins are a family of peptides that inhibit the production of juvenile hormone in the cockroach, Diploptera punctata. It is likely that the allatostatin prohormone precursor is processed to give rise to all 13 members of the family simultaneously. All members of the family show potency and efficacy, in terms of their ability to inhibit juvenile hormone production, albeit with dramatically different IC(50) and ED(50) values, ranging from a maximum of 0.014 nM for Dippu-AST 2 to 107 nM for Dippu-AST 1 (ED(50)). The likely occurrence of all 13 peptides in tissues and in haemolymph suggests that they may act in concert to produce physiological effects. We have employed combinations of the allatostatins, including a cocktail of all 13, 12 (minus Dippu-AST 2) and 11 (minus Dippu-AST 2 and 5) as well as mixtures of high and low activity allatostatins (Dippu-AST 5 plus either Dippu-AST 1 or 13) in dose-response studies to examine the possibility of synergistic or additive effects of the peptides on biological activity. None of the peptide combinations yielded evidence of synergistic interactions between allatostatins. However, the data do provide insight into receptor-ligand interactions in cockroaches and suggest the allatostatins regulate JH biosynthesis through a complex mix of differing affinity interactions with receptors in the corpora allata.

Journal Article↗
PubMed2002

A stimulus generating system for studying wind sensation in the American cockroach.

A novel system for the generation and measurement of a two dimensional wind stimulus is proposed and described. This system was used to investigate the wind sensation of the American cockroach. The new aspects of this system are (a) a pair of computer driven wind tunnels that are shown to produce non-turbulent flows and (b) a novel fiber optic wind detector that measures both amplitude and direction of the wind. Winds can be produced and measured in behaviorally relevant frequency and amplitude ranges without perturbing the airflow. The combination of both the wind generation system and wind detector makes the system very flexible and allows the generation of stimuli with any given spectrum. The two dimensional wind stimulus is shown to be very reproducible. The wind detector is independent of the wind generation system so it can be used for measuring natural winds as well. Experimental data obtained on the cockroach are presented.

Air Movements↗
PubMed2003

Allatostatin gene expression in brain and midgut, and activity of synthetic allatostatins on feeding-related processes in the cockroach Blattella germanica.

Allatostatins of the YXFGLamide group were discovered in cockroaches through their capacity to inhibit juvenile hormone biosynthesis. Here, we assess the occurrence of preproallatostatin (preproAST) mRNA in the brain and midgut of adult females of the cockroach Blattella germanica, and estimate brain and midgut preproAST mRNA levels during the first reproductive cycle. Reverse transcription polymerase chain reaction (RT-PCR) shows that brain preproAST mRNA levels increase slightly during the gonadotrophic cycle, and remain high during ootheca transport. In the midgut, preproAST mRNA levels decline around the middle of the gonadotrophic cycle. The pattern of allatostatin expression in gut tissues suggests that these peptides play roles related to feeding and nutrition. Our results have shown that synthetic allatostatins inhibit hindgut motility and activate midgut alpha-amylase secretion. In addition, injected allatostatins inhibit food consumption, which might be connected to the above activities.

Animals↗
PubMed1997

Isolation and structural elucidation of two pyrokinins from the retrocerebral complex of the American cockroach.

By monitoring the contractile activity of the hyperneural muscle of the American cockroach in vitro two peptides were isolated from the retrocerebral complex of the American cockroach. Three purification steps using reversed-phase high performance liquid chromatography on C-18 columns containing trifluoroacetic acid or heptafluorobutyric acid as organic modifiers were sufficient to achieve homogeneous peptide preparations. The structures of both peptides were elucidated by a combination of Edman degradation and mass spectrometry which yielded the following structures: His-Thr-Ala-Gly Phe-Ile-Pro-Arg-Leu-NH2 (Pea-PK-1) and Ser-Pro-Pro-Phe-Ala-Pro-Arg-Leu-NH2 (Pea-PK-2). The C-terminal sequence Phe-X-Pro-Arg-Leu-NH2 characterized the peptides as members of the insect pyrokinin family. The synthetic peptides were shown to have the same retention times as the natural peptides. The occurrence of both peptides in the retrocerebral complex suggests a physiological role as neurohormones. The effects of the synthetic pyrokinis were clearly distinguishable in their actions on the hyperneural muscle. Regarding the threshold concentrations, Pea-PK-2 was only 0.3% as active as Pea-PK-1.

Amino Acid Sequence↗
PubMed2000

Novel point mutations in the German cockroach para sodium channel gene are associated with knockdown resistance (kdr) to pyrethroid insecticides.

Knockdown resistance (kdr) to pyrethroid insecticides has been attributed to point mutations in the para sodium channel gene in more than a half dozen insect pest species. In this study, we identified two novel para mutations in five highly resistant kdr-type German cockroach strains. The two mutations, from glutamic acid (E434) to lysine (K434) and from cysteine (C764) to arginine (R764), respectively, are located in the first intracellular linker connecting domains I and II. E434K is located near the beginning of the linker (closest to domain I), whereas C764R is found toward the end of the linker (closest to domain II). Two additional mutations from aspartic acid (D58) to glycine (G58), and from proline (P1880) to leucine (L1888), respectively, were found in one of the resistant strains. The four mutations coexist with the previously identified leucine to phenylalanine (L993F) kdr mutation in IIS6, and are present only in the highly resistant individuals of a given strain. These findings suggest that these mutations might be responsible for high levels of knockdown resistance toward pyrethroid insecticides in the German cockroach.

Amino Acid Sequence↗
PubMed2001

Cytochrome P450 CYP6L1 is specifically expressed in the reproductive tissues of adult male German cockroaches, Blattella germanica (L.).

A full-length cDNA encoding a new cytochrome P450, CYP6L1, was cloned from German cockroaches, Blattella germanica. CYP6L1 has an open reading frame of 1509 nucleotides with a deduced protein of 503 amino acids and molecular mass of 57 Kd. CYP6L1 is most similar to CYP6H1, a putative ecdysone 20-hydroxylase from Locusta migratoria. CYP6L1 mRNA was not detected in embryos nor nymphs, nor in adult females. CYP6L1 mRNA was detected only in the testes and accessory glands of male adult German cockroaches. Given that the testes and accessory glands are the most important components of the reproductive system in male insects, the expression of CYP6L1 mRNA exclusively in these tissues strongly suggests that CYP6L1 has a role in reproduction. Possible substrates for CYP6L1 are discussed.

Amino Acid Sequence↗
PubMed1997

Purification and characterization of arylalkylamine N-acetyltransferase from cockroach testicular organs.

Arylalkylamine N-acetyltransferase (NAT; EC2.3.1.87) catalyzes N-acetylation of various arylalkylamines using acetyl-CoA as a donor substrate. A type of NAT was purified 2700-fold from 451 pairs of cockroach testicular organs consisting of testis and its accessory gland. The NAT activity was recovered as a single peak on any column chromatography examined, suggesting that the testicular organ contained only one form of NAT. Five steps of successive column chromatographies gave a single protein band on SDS-polyacrylamide gel electrophoresis with estimated molecular mass of 28 kDa. The molecular mass of the native enzyme was also determined to be approximately 30 kDa by molecular sieve chromatography, indicating that the enzyme is a monomer protein. The enzyme acted on various arylalkylamines such as tryptamine, serotonin, dopamine, octopamine, norepinephrine, tyramine and methoxytryptamine, with K(m) values ranging from 20 to 50 microM. The optimum pH for these substrates was around 6.0. Internal amino acid sequences derived from two proteolytic fragments of the enzyme were determined as Leu-Leu-Gly-Glu-Asn-Gly-Asp-Glu and Phe-Phe-Phe-Leu-Glu-Glu-Pro-Leu-Asn-Ile-Ser-Leu-Gln, both of which exhibited significant homology to the C-terminal sequence of known vertebrate NATs; however, homology was less than 45%. These results suggest that a unique NAT is present in the cockroach testicular organ at high levels, and likely plays a role in the regulation of testicular function.

Amino Acid Sequence↗
PubMed1998

Molecular cloning of cDNA for p10, a novel protein that increases in the regenerating legs of Periplaneta americana (American cockroach).

Previously, we purified a protein with a molecular mass of 10 kDa (p10) that increases transiently during the regeneration of legs in the nymphal American cockroach Periplaneta americana, and showed that it is localized exclusively in the cytosol and on the external side of the newly formed epidermis of the regenerating legs [Nomura, A. et al. (1992) Int. J. Dev. Biol. 36, 391-389]. We isolated p10 cDNA and analyzed the expression of the p10 gene. The results indicated that p10 is synthesized as a precursor protein with a putative prosegment including a signal sequence at its N-terminal. The deduced amino acid sequence of p10 showed 53% and 47% identities with those of A10 (a Drosophila antennal protein) and CLP-1 (a moth, Cactoblastis cactorum labial palp protein), respectively. Expression of the p10 gene was shown to be significantly enhanced in regenerating Periplaneta legs. Immunoblotting analysis revealed that p10 was expressed not only in the regenerating legs, but also in the antennae and heads of nymphal and adult cockroaches.

Amino Acid Sequence↗
PubMed2001

Effect of carbon dioxide on the Oriental cockroach Blatta orientalis.

Exposure to high concentrations of carbon dioxide (CO2) killed adult and nymphal stages of the oriental cockroach Blatta orientalis L. (Dictyoptera: Blattidae) with LT50 values of 11.5-16.2 h for 60% CO2 in air and 5.7-7.1 h for 100% CO2 at 20 degrees C; corresponding LT50s at 28 degrees C were 2.8-4.6 h for 60% CO2 in air and 2.3-3.6 h for 100% CO2. Complete kill of mobile stages was obtained within 24 h using 60% CO2 at 20 degrees C. Survivors of treatments with 100% CO2 at 28 degrees C remained completely paralysed for up to 3 days post-treatment and took up to 5 days to regain normal movement, but adult females then resumed production of oothecae with no significant loss in fecundity. Oothecae 5 or 30 days after deposition required 60-84 h exposure to 60% CO2 at 20 degrees C to prevent emergence of nymphs but less time using 100% CO2 at 28 degrees C. At 28 degrees C, when adult females were treated with 100% CO2 and 52% r.h. for 6 h (giving 100% mortality) loss of weight was significantly greater than that following treatment with air at 52% r.h. for 6 h (giving no mortality). However, significantly greater weight loss also occurred when they were treated with dried air (< 10% r.h.) for 6 h, also with no mortality. The toxicity of CO2 to mobile stages of the oriental cockroach appeared to result from irreversible effects on the nervous system, rather than from water loss during exposure.

Animals↗