Innate immune signaling in Drosophila shifts anabolic lipid metabolism from triglyceride storage to phospholipid synthesis to support immune function.

During infection, cellular resources are allocated toward the metabolically-demanding processes of synthesizing and secreting effector proteins that neutralize and kill invading pathogens. In Drosophila, these effectors are antimicrobial peptides (AMPs) that are produced in the fat body, an organ that also serves as a major lipid storage depot. Here we asked how activation of Toll signaling in the larval fat body perturbs lipid homeostasis to understand how cells meet the metabolic demands of the immune response. We find that genetic or physiological activation of fat body Toll signaling leads to a tissue-autonomous reduction in triglyceride storage that is paralleled by decreased transcript levels of the DGAT homolog midway, which carries out the final step of triglyceride synthesis. In contrast, Kennedy pathway enzymes that synthesize membrane phospholipids are induced. Mass spectrometry analysis revealed elevated levels of major phosphatidylcholine and phosphatidylethanolamine species in fat bodies with active Toll signaling. The ER stress mediator Xbp1 contributed to the Toll-dependent induction of Kennedy pathway enzymes, which was blunted by deleting AMP genes, thereby reducing secretory demand elicited by Toll activation. Consistent with ER stress induction, ER volume is expanded in fat body cells with active Toll signaling, as determined by transmission electron microscopy. A major functional consequence of reduced Kennedy pathway induction is an impaired immune response to bacterial infection. Our results establish that Toll signaling induces a shift in anabolic lipid metabolism to favor phospholipid synthesis and ER expansion that may serve the immediate demand for AMP synthesis and secretion but with the long-term consequence of insufficient nutrient storage.

JNK Signaling in Drosophila Aging and Longevity.

The evolutionarily conserved c-Jun N-terminal kinase (JNK) signaling pathway is a critical genetic determinant in the control of longevity. In response to extrinsic and intrinsic stresses, JNK signaling is activated to protect cells from stress damage and promote survival. In Drosophila, global JNK upregulation can delay aging and extend lifespan, whereas tissue/organ-specific manipulation of JNK signaling impacts lifespan in a context-dependent manner. In this review, focusing on several tissues/organs that are highly associated with age-related diseases-including metabolic organs (intestine and fat body), neurons, and muscles-we summarize the distinct effects of tissue/organ-specific JNK signaling on aging and lifespan. We also highlight recent progress in elucidating the molecular mechanisms underlying the tissue-specific effects of JNK activity. Together, these studies highlight an important and comprehensive role for JNK signaling in the regulation of longevity in Drosophila.

Luciferase isozymes from the Brazilian Aspisoma lineatum (Lampyridae) firefly: origin of efficient pH-sensitive lantern luciferases from fat body pH-insensitive ancestors.

Firefly luciferases usually emit green-yellow bioluminescence at physiological pH values. However, under acidic conditions, in the presence of heavy metals and, at high temperatures they emit red bioluminescence. To understand the structural origin of bioluminescence colors and pH-sensitivity, about 20 firefly luciferases have been cloned, sequenced and investigated. The proton and metal-binding site responsible for pH- and metal sensitivity in firefly luciferases was shown to involve the residues H310, E311 and E354 in firefly luciferases. However, it is still unclear how and why pH-sensitivity arose and evolved in firefly luciferases. Here, we cloned and characterized two novel luciferase cDNAs from the fat body and lanterns of the Brazilian firefly Aspisoma lineatum. The larval fat body isozyme (AL2) has 545 residues, and displays very slow luminescence kinetics and a pH-insensitive spectrum. The adult lantern isozyme (AL1) has 548 residues, displays flash-like kinetics and pH and metal sensitive bioluminescence spectra, and is at least 10 times catalytically more efficient than AL2. Thermostability and CD studies showed that AL2 is much more stable and rigid than the AL1 isozyme. Multialignment and modelling studies show that the E310Q substitution (E310 in AL2 and Q310 in AL1) may have been critical for the origin of pH-sensitivity in firefly luciferases. The results indicate that the lantern efficient flash-emitting pH-sensitive luciferases arose from less efficient glow-type pH-insensitive luciferases found in the fat body of ancestral larval fireflies by enzyme structure flexibilization and substitution at position 310.

Global transcriptome profiling and functional analysis reveal that tissue-specific constitutive overexpression of cytochrome P450s confers tolerance to imidacloprid in palm weevils in date palm fields.

BACKGROUND: Cytochrome P450-dependent monooxygenases (P450s), constituting one of the largest and oldest gene superfamilies found in many organisms from bacteria to humans, play a vital role in the detoxification and inactivation of endogenous toxic compounds. The use of various insecticides has increased over the last two decades, and insects have developed resistance to most of these compounds through the detoxifying function of P450s. In this study, we focused on the red palm weevil (RPW), Rhynchophorus ferrugineus, the most devastating pest of palm trees worldwide, and demonstrated through functional analysis that upregulation of P450 gene expression has evolved as an adaptation to insecticide stress arising from exposure to the neonicotinoid-class systematic insecticide imidacloprid. RESULTS: Based on the RPW global transcriptome analysis, we identified 101 putative P450 genes, including 77 likely encoding protein coding genes with ubiquitous expression. A phylogenetic analysis revealed extensive functional and species-specific diversification of RPW P450s, indicating that multiple CYPs actively participated in the detoxification process. We identified highly conserved paralogs of insect P450s that likely play a role in the development of resistance to imidacloprid: Drosophila Cyp6g1 (CYP6345J1) and Bemisia tabaci CYP4C64 (CYP4LE1). We performed a toxicity bioassay and evaluated the induction of P450s, followed by the identification of overexpressed P450s, including CYP9Z82, CYP6fra5, CYP6NR1, CYP6345J1 and CYP4BD4, which confer cross-resistance to imidacloprid. In addition, under imidacloprid insecticide stress in a date palm field, we observed increased expression of various P450 genes, with CYP9Z82, CYP4BD4, CYP6NR1 and CYP6345J1 being the most upregulated detoxification genes in RPWs. Expression profiling and cluster analysis revealed P450 genes with multiple patterns of induction and differential expression. Furthermore, we used RNA interference to knock down the overexpressed P450s, after which a toxicity bioassay and quantitative expression analysis revealed likely candidates involved in metabolic resistance against imidacloprid in RPW. Ingestion of double-stranded RNA (dsRNA) successfully knocked down the expression of CYP9Z82, CYP6NR1 and CYP345J1 and demonstrated that silencing of CYP345J1 and CYP6NR1 significantly decreased the survival rate of adult RPWs treated with imidacloprid, indicating that overexpression of these two P450s may play an important role in developing tolerance to imidacloprid in a date palm field. CONCLUSION: Our study provides useful background information on imidacloprid-specific induction and overexpression of P450s, which may enable the development of diagnostic tools/markers for monitoring the spread of insecticide resistant RPWs. The observed trend of increasing tolerance to imidacloprid in the date palm field therefore indicated that strategies for resistance management are urgently needed.

Comparison of Lactate Dehydrogenase Activity in Hive and Forager Honeybees May Indicate Delayed Onset Muscle Soreness - Preliminary Studies.

Active skeletal muscles produce lactate. H+ is generated during lactate neutralization in the Cori cycle, which leads to muscle acidosis and soreness (the so-called Delayed Onset Muscle Soreness, DOMS) in vertebrates. The aim of the study was to determine the activities/concentrations of compounds involved in the Cori cycle in worker and forager bees. Muscles, fat bodies, and hemolymph from 1- and 14-day-old workers and foragers were collected and assayed for the protein, lactate, glucose, NAD+, and NADH concentrations and lactate dehydrogenase (LDH) activity. Both lactate concentration and LDH activity in the hemolymph, muscles, and fat bodies increased with age. The concentrations of NAD+ and NADH in the tissues decreased with ageing/senescence, whereas protein concentrations increased until day 14 of bee's life and then decreased in foragers. The concentration of glucose decreased in the hemolymph and muscles and increased in the fat bodies. Elevated lactate concentrations in foragers may indicate transition from the aerobic to the anaerobic phase and development of metabolic acidosis that may eventually lead to muscle damage/soreness and shorter lifespan. When analyzing flight dynamics, load mass, and bee behavior, changes in the concentrations of Cori cycle compounds should be taken into account.

The Fat Body of the Hematophagous Insect, Panstrongylus megistus (Hemiptera: Reduviidae): Histological Features and Participation of the ß-Chain of ATP Synthase in the Lipophorin-Mediated Lipid Transfer.

In insects, lipid transfer to the tissues is mediated by lipophorin, the major circulating lipoprotein, mainly through a nonendocytic pathway involving docking receptors. Currently, the role of such receptors in lipid metabolism remains poorly understood. In this work, we performed a histological characterization of the fat body of the Chagas' disease vector, Panstrongylus megistus (Burmeister), subjected to different nutritional conditions. In addition, we addressed the role of the ß-chain of ATP synthase (ß-ATPase) in the process of lipid transfer from lipophorin to the fat body. Fifth-instar nymphs in either fasting or fed condition were employed in the assays. Histological examination revealed that the fat body was composed by diverse trophocyte phenotypes. In the fasting condition, the cells were smaller and presented a homogeneous cytoplasmic content. The fat body of fed insects increased in size mainly due to the enlargement of lipid stores. In this condition, trophocytes contained abundant lipid droplets, and the rough endoplasmic reticulum was highly developed and mitochondria appeared elongated. Immunofluorescence assays showed that the ß-ATPase, a putative lipophorin receptor, was located on the surface of fat body cells colocalizing partially with lipophorin, which suggests their interaction. No changes in ß-ATPase expression were found in fasting and fed insects. Blocking the lipophorin-ß-ATPase interaction impaired the lipophorin-mediated lipid transfer to the fat body. The results showed that the nutritional status of the insect influenced the morphohistological features of the tissue. Besides, these findings suggest that ß-ATPase functions as a lipophorin docking receptor in the fat body.

The dipteran parasitoid Exorista bombycis induces pro- and anti-oxidative reactions in the silkworm Bombyx mori: Enzymatic and genetic analysis.

Hymenopteran parasitoids inject various factors including polydnaviruses along with their eggs into their host insects that suppress host immunity reactions to the eggs and larvae. Less is known about the mechanisms evolved in dipteran parasitoids that suppress host immunity. Here we report that the dipteran, Exorista bombycis, parasitization leads to pro-oxidative reactions and activation of anti-oxidative enzymes in the silkworm Bombyx mori larva. We recorded increased activity of oxidase, superoxide dismutase, thioredoxin peroxidase, catalase, glutathione-S-transferase (GST), and peroxidases in the hemolymph plasma, hemocytes, and fat body collected from B. mori after E. bombycis parasitization. Microarray and qPCR showed differential expression of genes encoding pro- and anti-oxidant enzymes in the hemocytes. The significance of this work lies in increased understanding of dipteran parasitoid biology.

AMP-activated protein kinase protects against anoxia in Drosophila melanogaster.

During anoxia, proper energy maintenance is essential in order to maintain neural operation. Starvation activates AMP-activated protein kinase (AMPK), an evolutionarily conserved indicator of cellular energy status, in a cascade which modulates ATP production and consumption. We investigated the role of energetic status on anoxia tolerance in Drosophila and discovered that starvation or AMPK activation increases the speed of locomotor recovery from an anoxic coma. Using temporal and spatial genetic targeting we found that AMPK in the fat body contributes to starvation-induced fast locomotor recovery, whereas, under fed conditions, disrupting AMPK in oenocytes prolongs recovery. By evaluating spreading depolarization in the fly brain during anoxia we show that AMPK activation reduces the severity of ionic disruption and prolongs recovery of electrical activity. Further genetic targeting indicates that glial, but not neuronal, AMPK affects locomotor recovery. Together, these findings support a model in which AMPK is neuroprotective in Drosophila.

NONSULFATED SULFAKININ CHANGES METABOLIC PARAMETERS OF INSECT FAT BODY MITOCHONDRIA.

We investigated the effect of neuropeptide, the nonsulfated sulfakinin (SK) Zopat-SK-1 (pETSDDYGHLRFa) on the mitochondrial oxidative metabolism in the Zophobas atratus larval fat body. Mitochondria were isolated from beetle fat bodies 2 and 24 h after hormone injection. The administration of 20 pmol of Zopat-SK-1 to feeding larvae led to decreased mitochondrial oxidative activities in larval fat body. Diminished activities of citrate synthase and the cytochrome pathway, that is, nonphosphorylating and phosphorylating respiration during succinate oxidation, were observed. However, the effect of Zopat-SK-1 was more pronounced in fat body of insects after 24 h since hormone application. In hormone-treated larval fat bodies, mitochondrial respiration was decreased at the level of respiratory chain and the TCA cycle as well as at the level of mitochondrial biogenesis, as indicated by decreased activities of mitochondrial marker enzymes in fat body homogenates. The inhibition of succinate oxidation may indicate the role of Zopat-SK-1 in the regulation of mitochondrial complex II activity. Moreover, decreased respiratory chain activity was accompanied by the reduced activity of mitochondrial energy-dissipating pathway, uncoupling protein 4. The observed decrease in mitochondrial oxidative metabolism may reflect the Zopat-SK-1-induced reduction in the metabolic rate of larval fat body linked to actual energetic demands of animal.

Cytochrome P450s from the fall armyworm (Spodoptera frugiperda): responses to plant allelochemicals and pesticides.

Spodoptera frugiperda is a polyphagous lepidopteran pest that encounters a wide range of toxic plant metabolites in its diet. The ability of this insect to adapt to its chemical environment might be explained by the action of major detoxification enzymes such as cytochrome P450s (or CYP). Forty-two sequences coding for P450s were identified and most of the transcripts were found to be expressed in the midgut, Malpighian tubules and fat body of S. frugiperda larvae. Relatively few P450s were expressed in the established cell line Sf9. In order to gain information on how these genes respond to different chemical compounds, larvae and Sf9 cells were exposed to plant secondary metabolites (indole, indole-3-carbinol, quercetin, 2-tridecanone and xanthotoxin), insecticides (deltamethrin, fipronil, methoprene, methoxyfenozide) or model inducers (clofibrate and phenobarbital). Several genes were induced by plant chemicals such as P450s from the 6B, 321A and 9A subfamilies. Only a few genes responded to insecticides, belonging principally to the CYP9A family. There was little overlap between the response in vivo measured in the midgut and the response in vitro in Sf9 cells. In addition, regulatory elements were detected in the promoter region of these genes. In conclusion, several P450s were identified that could potentially be involved in the adaptation of S. frugiperda to its chemical environment.

dMyc expression in the fat body affects DILP2 release and increases the expression of the fat desaturase Desat1 resulting in organismal growth.

Drosophila dMyc (dMyc) is known for its role in cell-autonomous regulation of growth. Here we address its role in the fat body (FB), a metabolic tissue that functions as a sensor of circulating nutrients to control the release of Drosophila Insulin-like peptides (Dilps) from the brain influencing growth and development. Our results show that expression of dMyc in the FB affects development and animal size. Expression of dMyc, but not of CycD/cdk4 or Rheb, in the FB diminishes the ability to retain Drosophila Insulin-like peptide-2 (DILP2) in the brain during starvation, suggesting that expression of dMyc mimics the signal that remotely controls the release of Dilps into the hemolymph. dMyc also affects glucose metabolism and increases the transcription of Glucose-transporter-1 mRNA, and of Hexokinase and Pyruvate-Kinase mRNAs, key regulators of glycolysis. These animals are able to counteract the increased levels of circulating trehalose induced by a high sugar diet leading to the conclusion that dMyc activity in the FB promotes glucose disposal. dMyc expression induces cell autonomous accumulation of triglycerides, which correlates with increased levels of Fatty Acid Synthase and Acetyl CoA Carboxylase mRNAs, enzymes responsible for lipid synthesis. We also found the expression of Stearoyl-CoA desaturase, Desat1 mRNA significantly higher in FB overexpressing dMyc. Desat1 is an enzyme that is necessary for monosaturation and production of fatty acids, and its reduction affects dMyc's ability to induce fat storage and resistance to animal survival. In conclusion, here we present novel evidences for dMyc function in the Drosophila FB in controlling systemic growth. We discovered that dMyc expression triggers cell autonomous mechanisms that control glucose and lipid metabolism to favor the storage of nutrients (lipids and sugars). In addition, the regulation of Desat1 controls the synthesis of triglycerides in FB and this may affect the humoral signal that controls DILP2 release in the brain.

Parasitization by Scleroderma guani influences expression of superoxide dismutase genes in Tenebrio molitor.

Superoxide dismutase (SOD) is an antioxidant enzyme involved in detoxifying reactive oxygen species. In this study, we identified genes encoding the extracellular and intracellular copper-zinc SODs (ecCuZnSOD and icCuZnSOD) and a manganese SOD (MnSOD) in the yellow mealworm beetle, Tenebrio molitor. The cDNAs for ecCuZnSOD, icCuZnSOD, and MnSOD, respectively, encode 24.55, 15.81, and 23.14 kDa polypeptides, which possess structural features typical of other insect SODs. They showed 20-94% identity to other known SOD sequences from Bombyx mori, Musca domestica, Nasonia vitripennis, Pediculus humanus corporis, and Tribolium castaneum. Expression of these genes was analyzed in selected tissues and developmental stages, and following exposure to Escherichia coli and parasitization by Scleroderma guani. We recorded expression of all three SODs in cuticle, fat body, and hemocytes and in the major developmental stages. Relatively higher expressions were detected in late-instar larvae and pupae, compared to other developmental stages. Transcriptional levels were upregulated following bacterial infection. Analysis of pupae parasitized by S. guani revealed that expression of T. molitor SOD genes was significantly induced following parasitization. We infer that these genes act in immune response and in host-parasitoid interactions.

Differential gene expression in male and female fat body in the oriental fruit fly, Bactrocera dorsalis.

The sexual difference in gene expression in fat body between 8- and 10-day-old male and female Bactrocera dorsalis was examined using suppression subtractive hybridization. A total of 952 clones were sequenced and searched using BLAST from the subtracted cDNA library. About 22% of these clones showed homology with detoxification enzymes including cytochrome P450 monooxygenases (CYPs) and glutathione S-transferase. NADH dehydrogenases, distributed to energy metabolism, constituted about 9% of these clones. About 10% of these clones were cecropin, an antimicrobial peptide. Real-time quantitative polymerase chain reaction (qPCR) analysis showed that four transcripts were expressed at a higher level in fat body of males, compared to females. Bactrocera dorsalis cyp6g2 (Bdcyp6g2) was cloned (accession number KF469179) and the temporal profile of transcriptional expression showed that Bdcyp6g2 mRNA increased with age in males from day 3 after eclosion, but only on days 0-3 in females. Compared to females, the susceptibility of 9-day-old males to three insecticides was significantly less. These results suggested the genes expressed at a higher level in male act in its survival.

Inhibition of insect glutathione S-transferase (GST) by conifer extracts.

Insecticide synergists biochemically inhibit insect metabolic enzyme activity and are used both to increase the effectiveness of insecticides and as a diagnostic tool for resistance mechanisms. Considerable attention has been focused on identifying new synergists from phytochemicals with recognized biological activities, specifically enzyme inhibition. Jack pine (Pinus banksiana Lamb.), black spruce (Picea mariana (Mill.) BSP.), balsam fir (Abies balsamea (L.) Mill.), and tamarack larch (Larix laricina (Du Roi) Koch) have been used by native Canadians as traditional medicine, specifically for the anti-inflammatory and antioxidant properties based on enzyme inhibitory activity. To identify the potential allelochemicals with synergistic activity, ethanol crude extracts and methanol/water fractions were separated by Sephadex LH-20 chromatographic column and tested for in vitro glutathione S-transferase (GST) inhibition activity using insecticide-resistant Colorado potato beetle, Leptinotarsa decemlineata (Say) midgut and fat-body homogenate. The fractions showing similar activity were combined and analyzed by ultra pressure liquid chromatography-mass spectrometry. A lignan, (+)-lariciresinol 9'-p-coumarate, was identified from P. mariana cone extracts, and L. laricina and A. balsamea bark extracts. A flavonoid, taxifolin, was identified from P. mariana and P. banksiana cone extracts and L. laricina bark extracts. Both compounds inhibit GST activity with taxifolin showing greater activity compared to (+)-lariciresinol 9'-p-coumarate and the standard GST inhibitor, diethyl maleate. The results suggested that these compounds can be considered as potential new insecticide synergists.

A Farnesyl Pyrophosphate Synthase Gene Is Expressed in Fat Body Regulates Cantharidin Synthesis in Male Epicauta impressicornis Blister Beetle.

Blister beetles of Epicauta impressicornis have attracted attention because they contain a large amount of cantharidin (CTD). To date, however, the synthesis and transfer of CTD in adults of E. impressicornis are largely unknown. Here, we showed that the larvae E. impressicornis are capable of synthesizing CTD and they consume CTD during pupation. Before sexual maturity, both male and female adults synthesized a small amount of CTD, while after sexual maturity, males produced larger amounts of CTD, but females did not. The newly synthesized CTD in males first appeared in the hemolymph and then accumulated in the reproductive system. During the mating, the males transferred CTD to the reproductive system of females. In addition, a farnesyl pyrophosphate synthase (FPPS) gene was identified in male E. impressicornis. RNA-seq analysis, quantitative RT-PCR, and RNA interference analyses were conducted to investigate expression patterns and the functional roles of E. impressicornis FPPS (EiFPPS). Our results indicate that EiFPPS is highly expressed in the fat body of males. Moreover, the knock-down of EiFPPS led to a significant decrease in CTD synthesis. The current study indicates that EiFPPS is expressed in the fat body to regulate CTD synthesis in male E. impressicornis blister beetles.

The Fat Body-Specific GST Gene SlGSTe11 Enhances the Tolerance of Spodoptera litura to Cyantraniliprole and Nicotine.

Spodoptera litura is a significant agricultural pest, and its glutathione S-transferase (GST) plays a crucial role in insecticide resistance. This study aimed to investigate the relationship between the SlGSTe11 gene of S. litura and resistance to cyantraniliprole and nicotine. Transcriptome analysis revealed that SlGSTe11 is highly expressed mainly in fat bodies, with a significant increase in SlGSTe11 gene expression under induction by cyantraniliprole and nicotine. The ectopic expression of the SlGSTe11 gene in transgenic fruit flies resulted in a 5.22-fold increase in the tolerance to cyantraniliprole. Moreover, compared to the UAS-SlGSTe11 line, the Act5C-UAS>SlGSTe11 line laid more eggs and had a lower mortality after nicotine exposure. RNAi-mediated inhibition of SlGSTe11 gene expression led to a significant increase in the mortality of S. litura under cyantraniliprole exposure. In vitro metabolism experiments demonstrated that the recombinant SlGSTe11 protein efficiently metabolizes cyantraniliprole. Molecular docking results indicated that SlGSTe11 has a strong affinity for both cyantraniliprole and nicotine. These findings suggest that SlGSTe11 is involved in the development of resistance to cyantraniliprole and nicotine in S. litura.

De novo biosynthesis of sexual pheromone in the labial gland of bumblebee males.

De novo biosynthesis of male sex pheromone from two bumblebee species (Bombus terrestris and Bombus lucorum) was studied by using in vitro incubations of labial glands (LGs) with radioactive [1,2-(14)C]acetate and deuterated [D(3)]acetate. The labeled substrate was incorporated into several types of compounds, such as terpenic alcohols, fatty acids, esters, and hydrocarbons. A similar incubation of [1,2-(14)C]acetate with fat bodies (FB) led to the formation of fatty acids, triacylglycerols (TAG), and hydrocarbons. To support the results from in vitro incubations, PCR analysis of fatty acid synthase (FAS) transcripts in LG and FB was performed. Relative quantification of FAS transcription levels revealed that the abundance of mRNA from the FAS gene is a function of the age of B. terrestris males. A comparison of the relative FAS mRNA gene transcription level in FB and LGs of B. terrestris and B. lucorum males proved that high biosynthetic activity takes place in the LGs of both species. Together, these results indicate that pheromone components are synthesized de novo in the LG.

Effect of boric acid on antioxidant enzyme activity, lipid peroxidation, and ultrastructure of midgut and fat body of Galleria mellonella.

Boric acid is widely used as an insecticide, acaricide, herbicide, and fungicide and also during various industrial processings. Hence, numerous populations are subjects to this toxic compound. Its action on animals is still not fully known and understood. We examined the effect of boric acid on larvae of greater wax moth (Galleria mellonella). The chemical appeared to be toxic for larvae, usually in a concentration-dependent manner. Exposed groups revealed increased lipid peroxidation and altered activity of catalase, superoxide dismutase, glutathione S-transferase, and glutathione peroxidase. We also observed changes of ultrastructure, which were in tune with biochemical assays. We suggest that boric acid has a broad mode of action, which may affect exposed larvae, and even if sublethal, they may lead to disturbances within exposed populations.

Gene cloning and expression patterns of two prophenoloxidases from Catantops pinguis (Orthoptera: Catantopidae).

In insect, fat body plays major roles in insect innate immunity. Phenoloxidase (PO) is an important component in insect innate immunity and is necessary for acclimatization. In our study, two prophenoloxidase (PPO) subunits were obtained from fat body of Catantops pinguis (Stål). The full-length cDNA sequence of one PPO (CpPPO1) consisted of 2347 bp with an open reading frame (ORF) of 2187 bp encoding 728 amino acids, while the other subunit (CpPPO2) had a full length of 2445 bp, encoding 691 amino acids. Both the PPO gene products are predicted to possess all the structural features of other PPO members, including two putative tyrosinase copper-binding motifs with six highly conserved histidine residues and a thiolester-like motif. Tissue distribution analysis showed that both PPO mRNAs were abundantly expressed in the fat body among 11 tissues examined, and they were transiently up-regulated after Escherichia coli infection, consistent with them being immune-responsive genes. Total levels of CpPPO1 and CpPPO2 mRNA transcripts were much higher in first instar larvae and adults. A much higher transcript level of CpPPO1 was detected in several months, while there were extremely high mRNA expression levels of CpPPO2 in January, July, October, and December. The above results suggested that PPO from fat body might also bring significant function during the processes of development and acclimatization for C. pinguis.

UCP4 expression changes in larval and pupal fat bodies of the beetle Zophobas atratus under adipokinetic hormone treatment.

We investigated the influence of adipokinetic hormone (AKH), an insect neurohormone, on uncoupling protein 4 (ZaUCP4) expression and activity in larval and pupal fat body mitochondria of the beetle Zophobas atratus in relation to intermediary metabolism. Homologous Tenmo-AKH was administered to the beetle larvae and pupae as either a single dose or as two doses of 20pmol during a 24h interval. In the larval and pupal fat bodies, downregulation of ZaUCP4 expression at the mRNA and protein levels was observed 24h and 48h after AKH treatment, respectively. In both developmental stages, ZaUCP4 activity was lowered in fat body mitochondria 48h after AKH treatment. In the AKH-injected larvae, changes in ZaUCP4 expression were accompanied by the mobilization of carbohydrate reserves, no change in the concentration of total lipids and an increase in the free fatty acid level. In contrast, AKH had no effect on carbohydrate metabolism in the pupal fat body but induced lipid mobilization. It seems that AKH influences ZaUCP4 expression by triggering multiple events and that it has different physiological roles in controlling intermediary metabolism in the fat body of the beetle larvae and pupae.