Cockroach Fat Body Research Articles

E93 is indispensable for reproduction in ametabolous and hemimetabolous insects.

Ecdysone-induced protein 93 (E93), known as the 'adult-specifier' transcription factor in insects, triggers metamorphosis in both hemimetabolous and holometabolous insects. Although E93 is conserved in ametabolous insects, its spatiotemporal expression and physiological function remain poorly understood. In this study, we first discover that, in the ametabolous firebrat Thermobia domestica, the previtellogenic ovary exhibits cyclically high E93 expression, and E93 mRNA is broadly distributed in previtellogenic ovarioles. E93 homozygous mutant females of T. domestica exhibit severe fecundity deficiency due to impaired previtellogenic development of the ovarian follicles, likely because E93 induces the expression of genes involved in ECM (extracellular matrix)-receptor interactions during previtellogenesis. Moreover, we reveal that in the hemimetabolous cockroach Blattella germanica, E93 similarly promotes previtellogenic ovarian development. In addition, E93 is also essential for vitellogenesis that is necessary to guarantee ovarian maturation and promotes the vitellogenesis-previtellogenesis switch in the fat body of adult female cockroaches. Our findings deepen the understanding of the roles of E93 in controlling reproduction in insects, and of E93 expression and functional evolution, which are proposed to have made crucial contributions to the origin of insect metamorphosis.

Comparative Transcriptomics of Fat Bodies between Symbiotic and Quasi-Aposymbiotic Adult Females of Blattella germanica with Emphasis on the Metabolic Integration with Its Endosymbiont Blattabacterium and Its Immune System.

We explored the metabolic integration of Blattella germanica and its obligate endosymbiont Blattabacterium cuenoti by the transcriptomic analysis of the fat body of quasi-aposymbiotic cockroaches, where the endosymbionts were almost entirely removed with rifampicin. Fat bodies from quasi-aposymbiotic insects displayed large differences in gene expression compared to controls. In quasi-aposymbionts, the metabolism of phenylalanine and tyrosine involved in cuticle sclerotization and pigmentation increased drastically to compensate for the deficiency in the biosynthesis of these amino acids by the endosymbionts. On the other hand, the uricolytic pathway and the biosynthesis of uric acid were severely decreased, probably because the reduced population of endosymbionts was unable to metabolize urea to ammonia. Metabolite transporters that could be involved in the endosymbiosis process were identified. Immune system and antimicrobial peptide (AMP) gene expression was also reduced in quasi-aposymbionts, genes encoding peptidoglycan-recognition proteins, which may provide clues for the maintenance of the symbiotic relationship, as well as three AMP genes whose involvement in the symbiotic relationship will require additional analysis. Finally, a search for AMP-like factors that could be involved in controlling the endosymbiont identified two orphan genes encoding proteins smaller than 200 amino acids underexpressed in quasi-aposymbionts, suggesting a role in the host-endosymbiont relationship.

Genome Sequence of a Blattabacterium Strain Isolated from the Viviparous Cockroach, Diploptera punctata.

Here, we report the genome sequence and characterization for a Blattabacterium strain isolated from the viviparous cockroach, Diploptera punctata, which provides amino acids critical for intrauterine embryo development. The genome was assembled by sequencing of the cockroach fat body, which is the location of this obligate symbiont.

Effect of certain entomopathogenic fungi on oxidative stress and mortality of Periplaneta americana

The present paper reports the effects of Metarhizium anisopliae, Isaria fumosoroseus and Hirsutella thompsonaii on Periplaneta americana. I. fumosoroseus and H. thompsonaii were cultured at 28±1°C on potato carrot agar and M. anisopliae was cultured at 28±1°C on potato dextrose agar for 14days. Conidial suspensions of fungi were given to cockroaches through different routes. M. anisopliae shows high virulence against adult cockroaches and mortality ranges from 38.65% to 78.36% after 48h. I. fumosoroseus and H. thompsonii show less virulence compared to M. anisopliae. We also investigated the effect of these three fungi on the activity of lactate dehydrogenase, lipid peroxidation and catalase in different tissues of the insect to gain an understanding of the different target site. The result suggested that the activity of lactate dehydrogenase, catalase and level of malondialdehyde varies in different organs and through different routes of exposure. Based on mortality percentages, all tested fungi had high potentials for biocontrol agents against P. americana. Our study reveals for the first time that I. fumosoroseus and H. thompsonaii fungal infections initiate oxidative stress in the midgut, fat body, whole body and hemolymph of cockroach thereby suggesting them to be the target organs for oxidative damage.

The cockroach Blattella germanica obtains nitrogen from uric acid through a metabolic pathway shared with its bacterial endosymbiont.

Uric acid stored in the fat body of cockroaches is a nitrogen reservoir mobilized in times of scarcity. The discovery of urease in Blattabacterium cuenoti, the primary endosymbiont of cockroaches, suggests that the endosymbiont may participate in cockroach nitrogen economy. However, bacterial urease may only be one piece in the entire nitrogen recycling process from insect uric acid. Thus, in addition to the uricolytic pathway to urea, there must be glutamine synthetase assimilating the released ammonia by the urease reaction to enable the stored nitrogen to be metabolically usable. None of the Blattabacterium genomes sequenced to date possess genes encoding for those enzymes. To test the host's contribution to the process, we have sequenced and analysed Blattella germanica transcriptomes from the fat body. We identified transcripts corresponding to all genes necessary for the synthesis of uric acid and its catabolism to urea, as well as for the synthesis of glutamine, asparagine, proline and glycine, i.e. the amino acids required by the endosymbiont. We also explored the changes in gene expression with different dietary protein levels. It appears that the ability to use uric acid as a nitrogen reservoir emerged in cockroaches after its age-old symbiotic association with bacteria.

Cloning of PaAtg8 and roles of autophagy in adaptation to starvation with respect to the fat body and midgut of the Americana cockroach, Periplaneta americana

Starvation, in particular amino acid deprivation, induces autophagy in trophocytes (adipocytes), the major component of the fat body cell types, in the larvae of Drosophila melanogaster. However, the fat body of cockroach has two additional cell types: urocytes depositing uric acid in urate vacuoles as a nitrogen resource and mycetocytes harboring an endosymbiont, Blattabacterium cuenoti, which can synthesize amino acids from the metabolites of the stored uric acid. These cells might complement the roles of autophagy in recycling amino acids in the fat body or other organs of cockroaches under starvation. We investigate the presence of autophagy in tissues such as the fat body and midgut of the American cockroach, Periplaneta americana, under starvation by immunoblotting with antibody against Atg8, a ubiquitin-like protein required for the formation of autophagosomes and by electron microscopy. Corresponding changes in acid phosphatase activity were also investigated as representing lysosome activity. Starvation increased the level of an autophagic marker, Atg8-II, in both the tissues, extensively stimulating the formation of autophagic compartments in trophocytes of the fat body and columnar cells of the midgut for over 2 weeks. Acid phosphatase showed no significant increase in the fat body of the starved cockroaches but was higher in the midgut of the continuously fed animals. Thus, a distinct autophagic mechanism operates in these tissues under starvation of 2 weeks and longer. The late induction of autophagy implies exhaustion of the stored uric acid in the fat body. High activity of acid phosphatase in the midgut of the fed cockroaches might represent enhanced assimilation and not an autophagy-related function.

Carbohydrate and lipid metabolism in cockroach ( Periplaneta americana) fat body are both activated by low and similar concentrations of Peram-AKH II

Injection of 0.1 pmol of the octapeptide Peram-AKH II (pGlu-Leu-Thr-Phe-Thr-Pro-Asn-TrpNH 2) elicits a significant hypertrehalosemic response in the American cockroach, Periplaneta americana; a maximal effect is obtained with 1 pmol. The latter amount also lowers the level of neutral lipid (NL) and phospholipid (PL) in the hemolymph. The evidence supports the idea that Peram-AKH II promotes the liberation of fatty acids from hemolymph phospholipid, and indirectly diacylglycerol in the same compartment. The fatty acids are then transported into the fat body where they are converted into triacylglycerol for storge. Because lipolysis and trehalose synthesis are initiated by a common concentration of Peram-AKH II it is reasonable to suggest that the physiological function of Peram-AKH II involves the participation of both metabolic pathways.

Lipid metabolism in the cockroach, Periplaneta americana, is activated by the hypertrehalosemic peptide, HTH-I

Phosphatidylcholine and phosphatidylethanolamine are the major constituents of the phospholipid pool in cockroach ( Periplaneta americana) fat body and hemolymph. Both species of phospholipid are significantly decreased 6 h after injecting hypertrehalosemic hormone I (HTH-I) into the hemocoel. Loss of phospholipid is accompanied by an accumulation of the phospholipid degradation products glycerophosphorylcholine and glycerol. HTH-I also increases phospholipase activity in the hemolymph and this is thought to be responsible for the depletion of hemolymph phospholipid. Phospholipase activity peaks approximately 2 h after injection of HTH-I and returns to normal at 6 h. In vitro, total phospholipid in the fat body is decreased by HTH-I whereas the concentration of diacylglycerol displays a corresponding increase. HTH-I elevates free fatty acid levels but has no effect on triacylglycerol. These effects of HTH-I are blocked by the phospholipase inhibitor mepacrine.

Stimulation of trehalose efflux from cockroach (Periplaneta americana) fat body by hypertrehalosemic hormone is dependent on protein kinase C and calmodulin.

Protein kinase C and calmodulin play key roles in cockroach fat body during activation of phosphorylase and trehalose efflux by HTH-II. The data support the view that an increase in cytosolic Ca2+ is prerequisite for enhanced activity of protein kinase C and calmodulin. Chelation of Ca2+ (i) with BAPTA blocks HTH-II-induced trehalose efflux from the fat body whereas thapsigargin, which raises [Ca2+]i to the same level as HTH-II, produces only a small, yet significant increase in trehalose efflux. Sphingosine, an inhibitor of protein kinase C, inhibits HTH-II-induced trehalose efflux in a concentration-dependent manner. Trehalose efflux is not activated by the protein kinase C activators OAG or PMA alone but in the presence of thapsigargin both agents increase trehalose efflux to a level comparable to that obtained with HTH-II. Thapsigargin has only a moderate activating effect on phosphorylase but in combination with OAG produces an activation indistinguishable from that provoked by HTH-II. Each of the structurally different calmodulin inhibitors, trifluoperazine, W-7, and calmidazolium, blocks completely the action of HTH-II on trehalose efflux, thus confirming the importance of calmodulin in HTH-II initiated trehalose efflux.

Regulation of phospholipase A 2 activity in cockroach ( Periplaneta americana) fat body by hypertrehalosemic hormone: evidence for the participation of protein kinase C

Phospholipase A 2 (PLA 2) associated with the membrane fraction of trophocytes from Periplaneta americana fat body increases by as much as 100% when the cells are incubated with hypertrehalosemic hormone (HTH-II). Activation with HTH-II is approximately halved by inclusion of the PKC inhibitor sphingosine in the incubation medium. Because activation of PLA 2 by HTH-II is blocked by the GDP analogue GDP-β-S, and the unactivated enzyme is activated by the GTP analogue GTP-γ-S it is likely that a G protein is involved in activation of the enzyme. Activation of PLA 2 was also achieved by treating the trophocytes with the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol in the presence of thapsigargin. This supports the view that protein kinase C is also involved in the activation process.

Control of phospholipase A 2 activity in cockroach ( Periplaneta americana) fat body trophocytes by hypertrehalosemic hormone: the role of calcium

Recently, synthetic HTH-I and HTH-II have been shown to increase the formation of free fatty acids in cockroach ( Periplaneta americana) fat body. In this study we show that HTH-II increases PLA 2 activity in dispersed trophocytes, thus implying that phospholipid is a potential source of the fatty acids. The increase in HTH-induced PLA 2 activity is triggered by an increase in [Ca 2+] i but extracellular Ca 2+ is also required for a maximal Ca 2+ signal: an effect that can be blocked by the introduction of BAPTA into the trophocytes. Treating trophocytes with ryanodine blocks the increase in PLA 2 activity that follows treatment of the cells with HTH-II. This indicates that the Ca 2+ release channels are distinct from those that respond to inositol trisphosphate. Thapsigargin, which releases Ca 2+ to the cytosol from an intracellular store, increases PLA 2 activity. The data show that the enzyme is translocated from the cytosol to the plasma membrane.

Inositol trisphosphate mediates the action of hypertrehalosemic hormone on fat body of the American cockroach, Periplaneta americana

The rate of synthesis of inositol trisphosphate (InsP 3) in trophocytes derived from disaggregated cockroach ( Periplaneta americana) fat body increases following treatment of the cells with hypertrehalosemic hormone I or II (HTH-I, -II) in vitro. Trophocytes preloaded with [ 3H]inositol display a significant increase in InsP 3 synthesis as early as 15 s after addition of the hormone. When the trophocytes are pre-incubated with LiCl and subsequently incubated with HTH the [ 3H] content of the InsP 3 fraction is greater than that found with HTH alone. This is taken as evidence that inositol monophosphate phosphatase is part of the mechanism for clearing InsP 3 from the cytosol. In contrast to HTH, octopamine, which is also capable of exerting a hypertrehalosemic effect in the cockroach, does not increase the synthesis of InsP 3. 1-Octadecyl-2-methyl- rac-glycero-3-phosphocholine (ET-18-OCH 3), a potent and selective inhibitor of phosphatidylinositol phospholipase C, blocks the activation of phosphorylase by HTH-I as well as the hypertrehalosemic effect induced by the hormone.

Antagonistic effects of hypertrehalosemic neuropeptide on the activities of 6-phosphofructo-1-kinase and fructose-1,6-bisphosphatase in cockroach fat body

Hypertrehalosemic neuropeptides from the corpora cardiaca such as the decapeptide Bld HrTH bring about a profound switch in the metabolic activity of cockroach fat body during which production of the blood sugar trehalose is stimulated while the catabolism of carbohydrate (glycolysis) is inhibited. The mechanisms of the metabolic switch are not fully understood. Incubation of isolated fat body from the cockroach Blaptica dubia with 10 −8 M Bld HrTH, for 10–60 min, stimulated glycogen breakdown and increased the content of the substrates of both the glycolytic enzyme 6-phosphofructo-1-kinase (PFK, EC 2.7.1.11) and the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase, EC 3.1.3.11) in the tissue. The glycolytic signal fructose 2,6-bisphosphate was markedly decreased in fat body on incubation with Bld HrTH. The content of ATP was slightly reduced, while the contents of ADP and AMP were increased after incubation with the hormone. Fructose 2,6-bisphosphate is a potent activator of PFK and a strong inhibitor of FBPase purified from fat body. The activity of PFK was decreased by about 90% when the hormone-dependent changes in effectors and substrates in fat body were simulated in vitro. FBPase, in contrast, was activated about 25-fold under these conditions, suggesting the hormone to stimulate gluconeogenesis in fat body. The data support the view that fructose 2,6-bisphosphate is a pivotal intracellular messenger in the hormone-induced metabolic switch from carbohydrate degradation to trehalose production in cockroach fat body.

Hormonal activation of phosphorylase in cockroach fat body trophocytes: A correlation with trans‐membrane calcium flux

AbstractThis study is an investigation of the temporal relationship between transmembrane Ca2+ fluxes, and glycogen phosphorylase activation in dispersed trophocytes from the fat body of the cockroach, Periplaneta americana. Phosphorylase is maximally activated within 5 min after treating the trophocytes with either of the hypertrehalosemic hormones, Pea‐HTH‐I and Pea‐HTH‐II. Activation caused by Pea‐HTH‐II is sustained for a longer period than that produced by Pea‐HTH‐I. Chelation of extracellular Ca2+ with EGTA blocks the activation of phosphorylase by HTH. Similarly, chelation of intracellular Ca2+ with Quin 2 greatly diminishes the phosphorylase activating effect of both HTHs. The data support the view that an increase in the intracellular Ca2+ concentration is required for the activation of phosphorylase and that extracellular Ca2+ is an essential, although not necessarily sole, source of Ca2+ for this purpose. Using 45Ca2+ to trace the movement of Ca2+ following a challenge with either Pea‐HTH‐I or ‐II, it was shown that 45Ca2+influx nearly doubled during the first 30 s. At this time, the trophocytes begin to expel Ca2+ at a rate higher than that of untreated cells and this state persists for approximately 4 min. The Ca2+ fluxes are consistent with its postulated role in the activation of phosphorylase. Arch. Insect Biochem. Physiol. 42:233–244, 1999. © 1999 Wiley‐Liss, Inc.

Hormonal activation of phosphorylase in cockroach fat body trophocytes: A correlation with trans‐membrane calcium flux

This study is an investigation of the temporal relationship between transmembrane Ca2+ fluxes, and glycogen phosphorylase activation in dispersed trophocytes from the fat body of the cockroach, Periplaneta americana. Phosphorylase is maximally activated within 5 min after treating the trophocytes with either of the hypertrehalosemic hormones, Pea-HTH-I and Pea-HTH-II. Activation caused by Pea-HTH-II is sustained for a longer period than that produced by Pea-HTH-I. Chelation of extracellular Ca2+ with EGTA blocks the activation of phosphorylase by HTH. Similarly, chelation of intracellular Ca2+ with Quin 2 greatly diminishes the phosphorylase activating effect of both HTHs. The data support the view that an increase in the intracellular Ca2+ concentration is required for the activation of phosphorylase and that extracellular Ca2+ is an essential, although not necessarily sole, source of Ca2+ for this purpose. Using 45Ca2+ to trace the movement of Ca2+ following a challenge with either Pea-HTH-I or -II, it was shown that 45Ca2+influx nearly doubled during the first 30 s. At this time, the trophocytes begin to expel Ca2+ at a rate higher than that of untreated cells and this state persists for approximately 4 min. The Ca2+ fluxes are consistent with its postulated role in the activation of phosphorylase. Arch. Insect Biochem. Physiol. 42:233–244, 1999.© 1999 Wiley-Liss, Inc.

Hormonal activation of phosphorylase in cockroach fat body trophocytes: A correlation with trans-membrane calcium flux.

This study is an investigation of the temporal relationship between transmembrane Ca(2+) fluxes, and glycogen phosphorylase activation in dispersed trophocytes from the fat body of the cockroach, Periplaneta americana. Phosphorylase is maximally activated within 5 min after treating the trophocytes with either of the hypertrehalosemic hormones, Pea-HTH-I and Pea-HTH-II. Activation caused by Pea-HTH-II is sustained for a longer period than that produced by Pea-HTH-I. Chelation of extracellular Ca(2+) with EGTA blocks the activation of phosphorylase by HTH. Similarly, chelation of intracellular Ca(2+) with Quin 2 greatly diminishes the phosphorylase activating effect of both HTHs. The data support the view that an increase in the intracellular Ca(2+ )concentration is required for the activation of phosphorylase and that extracellular Ca(2+) is an essential, although not necessarily sole, source of Ca(2+) for this purpose. Using (45)Ca(2+) to trace the movement of Ca(2+) following a challenge with either Pea-HTH-I or -II, it was shown that (45)Ca(2+)influx nearly doubled during the first 30 s. At this time, the trophocytes begin to expel Ca(2+) at a rate higher than that of untreated cells and this state persists for approximately 4 min. The Ca(2+) fluxes are consistent with its postulated role in the activation of phosphorylase. Arch.

Hormonal activation of cockroach fat body phospholipase A 2 is an inositol trisphosphate mediated event

Hormonal activation of cockroach fat body phospholipase A 2 is an inositol trisphosphate mediated event

Hypertrehalosaemic neuropeptides decrease levels of the glycolytic signal fructose 2,6-bisphosphate in cockroach fat body

In cockroach fat body, trehalogenesis and glycolysis compete for glucose phosphates as common substrates. During trehalogenesis, glycolysis is inhibited, although the mechanism responsible for this is not known. Incubation of the isolated fat body from the Argentine cockroach Blaptica dubia with an extract of the corpora cardiaca containing as little as 0.005 gland equivalents ml-1 of incubation medium increased the release of trehalose (anthrone-positive material) from the tissue by more than 100 %. The content of the glycolytic activator fructose 2,6-bisphosphate was decreased by up to 50 %. A decapeptide was isolated from the corpora cardiaca of B. dubia and shown to be identical to the naturally occurring Blaberus discoidalis hypertrehalosaemic peptide (Bld HrTH), which was also found in the corpora cardiaca. Synthetic Bld HrTH at 2 nmol l-1 and above increased trehalose production and decreased the content of fructose 2,6-bisphosphate to the same extent as did corpus cardiacum extract. The octapeptides Periplaneta americana cardioacceleratory hormones I and II (Pea CAH-I and Pea CAH-II) also had a significant effect on both parameters. Fructose 2,6-bisphosphate is a potent activator of phosphofructokinase from cockroach fat body if the enzyme is assayed at near-physiological concentrations of substrates and effectors. It is suggested that, because of the decrease in fructose 2,6-bisphosphate levels in the fat body, the activity of the key glycolytic enzyme phosphofructokinase is diminished. This can explain the inhibition of glycolytic flux by hypertrehalosaemic peptides which alters the balance of glucose metabolism in favour of trehalose formation.

Signal transduction in isolated fat body from the cockroach Blaptica dubia exposed to hypertrehalosaemic neuropeptide

Hypertrehalosaemic hormones stimulate trehalogenesis while inhibiting glycolysis in cockroach fat body. Signal transduction of the hypertrehalosaemic peptide Bld HrTH was examined in isolated fat body of the Argentine cockroach Blaptica dubia with respect to its effects on the increase in trehalose production and decrease in the content of the glycolytic activator fructose 2,6-bisphosphate in the tissue. Cyclic AMP does not seem to be involved in these processes as the cAMP analogue cpt-cAMP and the phosphodiesterase inhibitor IBMX, which both permeate cell membranes, had no effect on either parameter. Octopamine at physiological concentrations (10−7 mol · l−1) was also ineffective, but at 10−5 mol · l−1 or above, octopamine stimulated trehalose production although the content of fructose 2,6-bisphosphate in fat body was not affected. Both calcium entry and the release of Ca2+ from intracellular stores seem to be involved in the action of the hormone. If Ca2+ was omitted from the incubation medium, the hormone stimulated trehalose production less, though still significantly, whereas the hormone effect on fructose 2,6-bisphosphate was completely abolished in the absence of extracellular Ca2+. With Ca2+ present in the medium, the effect of the hormone on fructose 2,6-bisphosphate could be fully mimicked by the calcium ionophore A23187, suggesting that calcium entry is a␣decisive step in this signalling pathway. Trehalose production, on the other hand, was increased by thimerosal and thapsigargin which increase cytosolic Ca2+ from intracellular stores, whereas thimerosal in the absence of extracellular Ca2+ increased rather than decreased the content of fructose 2,6-bisphosphate, thus dissociating the two effects, which are normally coordinated by the hormone. Trehalose production and the content of fructose 2,6-bisphosphate were not significantly affected by mepacrine and mellitin, which are known to inhibit, respectively stimulate, phospholipase A2. Our data suggest that the effects of Bld HrTH on the stimulation of trehalose production and reduction of fructose 2,6-bisphosphate content in fat body are mediated by Ca2+, but that different signalling pathways are involved, suggesting that the two processes, although they are functionally linked, could be regulated separately.

^<13>C ラベル前駆体とトレハラーゼ阻害剤を用いた, In vitro でのゴキブリ脂肪体のトレハロース合成能の NMR による測定

^<13>C ラベル前駆体とトレハラーゼ阻害剤を用いた, In vitro でのゴキブリ脂肪体のトレハロース合成能の NMR による測定