Epoxide hydrolase activities and epoxy fatty acids in the mosquito Culex quinquefasciatus

Culex mosquitoes have emerged as important model organisms for mosquito biology, and are disease vectors for multiple mosquito-borne pathogens, including West Nile virus. We characterized epoxide hydrolase activities in the mosquito Culex quinquefasciatus, which suggested multiple forms of epoxide hydrolases were present. We found EH activities on epoxy eicosatrienoic acids (EETs). EETs and other eicosanoids are well-established lipid signaling molecules in vertebrates. We showed EETs can be synthesized in vitro from arachidonic acids by mosquito lysate, and EETs were also detected in vivo both in larvae and adult mosquitoes by LC-MS/MS. The EH activities on EETs can be induced by blood feeding, and the highest activity was observed in the midgut of female mosquitoes. The enzyme activities on EETs can be inhibited by urea-based inhibitors designed for mammalian soluble epoxide hydrolases (sEH). The sEH inhibitors have been shown to play diverse biological roles in mammalian systems, and they can be useful tools to study the function of EETs in mosquitoes. Besides juvenile hormone metabolism and detoxification, insect epoxide hydrolases may also play a role in regulating lipid signaling molecules, such as EETs and other epoxy fatty acids, synthesized in vivo or obtained from blood feeding by female mosquitoes.     

Ingestion of the epoxide hydrolase inhibitor AUDA modulates immune responses of the mosquito,Culex quinquefasciatus during blood feeding

Epoxide hydrolases (EHs) are enzymes that play roles in metabolizing xenobiotic epoxides from the environment, and in regulating lipid signaling molecules, such as juvenile hormones in insects and epoxy fatty acids in mammals. In this study we fed mosquitoes with an epoxide hydrolase inhibitor AUDA during artificial blood feeding, and we found the inhibitor increased the concentration of epoxy fatty acids in the midgut of female mosquitoes. We also observed ingestion of AUDA triggered early expression of defensin A, cecropin A and cecropin B2 at 6 h after blood feeding. The expression of cecropin B1 and gambicin were not changed more than two fold compared to controls. The changes in gene expression were transient possibly because more than 99% of the inhibitor was metabolized or excreted at 42 h after being ingested. The ingestion of AUDA also affected the growth of bacteria colonizing in the midgut, but did not affect mosquito longevity, fecundity and fertility in our laboratory conditions. When spiked into the blood, EpOMEs and DiHOMEs were as effective as the inhibitor AUDA in reducing the bacterial load in the midgut, while EETs rescued the effects of AUDA. Our data suggest that epoxy fatty acids from host blood are immune response regulators metabolized by epoxide hydrolases in the midgut of female mosquitoes, inhibition of which causes transient changes in immune responses, and affects growth of microbes in the midgut.     

The ACBP gene family in Rhodnius prolixus: Expression,characterization and function of RpACBP-1

The acyl-CoA-binding proteins (ACBP) constitute a family of conserved proteins that bind acyl-CoA with high affinity and protect it from hydrolysis. Thus, ACBPs may have essential roles in basal cellular lipid metabolism. The genome of the insect Rhodnius prolixus encodes five ACBP genes similar to those described for other insect species. The qPCR analysis revealed that these genes have characteristic expression profiles in insect organs, suggesting that they have specific roles in insect physiology. Recombinant RpACBP-1 was able to bind acyl-CoA in an in vitro gel-shift assay. Moreover, heterologous RpACBP-1 expression in acb1Δ mutant yeast rescued the multi-lobed vacuole phenotype, indicating that RpACBP-1 acts as a bona fide acyl-CoA-binding protein. RpACBP-1 knockdown using RNAi caused triacylglycerol accumulation in the insect posterior midgut and a reduction in the number of deposited eggs. The amount of stored triacylglycerol was reduced in flight muscle, and the incorporation of fatty acids in cholesteryl esters was increased in the fat body. These results showed that RpACBP-1 participates in several lipid metabolism steps in R. prolixus.     

An atypical residue in the pore of Varroa destructor GABA-activated RDL receptors affects picrotoxin block and thymol modulation

GABA-activated RDL receptors are the insect equivalent of mammalian GABA_A receptors, and play a vital role in neurotransmission and insecticide action. Here we clone the pore lining M2 region of the Varroa mite RDL receptor and show that it has 4 atypical residues when compared to M2 regions of most other insects, including bees, which are the major host of Varroa mites. We create mutant Drosophila RDL receptors containing these substitutions and characterise their effects on function. Using two electrode voltage clamp electrophysiology we show that one substitution (T6′M) ablates picrotoxin inhibition and increases the potency of GABA. This mutation also alters the effect of thymol, which enhances both insect and mammalian GABA responses, and is widely used as a miticide. Thymol decreases the GABA EC₅₀ of WT receptors, enhancing responses, but in T6′M-containing receptors it is inhibitory. The other 3 atypical residues have no major effects on either the GABA EC₅₀, the picrotoxin potency or the effect of thymol. In conclusion we show that the RDL 6′ residue is important for channel block, activation and modulation, and understanding its function also has the potential to prove useful in the design of Varroa-specific insecticidal agents.     

Alteration of plasma phospholipid fatty acid profile in patients with septic shock

In septic shock patients, alterations of plasma phospholipid fatty acid profile have never been described. The purpose of this monocentric, non-interventional, observational prospective study was to describe this fatty acid profile in the early phase of septic shock in intensive care unit. Thirty-seven adult patients with septic shock were included after the first day of stay in intensive care unit, before any form of artificial nutritional support. Plasma phospholipid fatty acid composition was determined by gas chromatography. All biological data from patients with septic shock were compared with laboratory reference values. Patients presented hypocholesterolemia and hypertriglyceridemia. They had low concentrations of phospholipid fatty acids specifically n-6 and n-3 polyunsaturated fatty acids (PUFAs) with a high n-6/n-3 ratio. Plasma phospholipid PUFA concentrations were strongly correlated with cholesterolemia. PUFAs/SFAs (saturated fatty acids) and PUFAs/MUFAs (monounsaturated fatty acids) ratios were low because of low percentage of n-6 and n-3 PUFAs and high percentage of SFAs and MUFAs. Low levels of plasma long chain PUFAs (≥20 carbons) were significantly associated with mortality at 28th day. In conclusion, plasma phospholipid FA profile of septic patients is very characteristic, close to that of acute respiratory distress syndrome and mortality is associated with long chain PUFA decrease. This profile could be explained by numerous non-exclusive physio-pathological processes 1) an activation of hepatic de novo lipogenesis that could contribute to hepatic steatosis, 2) an elevated adipose tissue lipolysis, 3) an increased free radical attack of FA by oxidative stress, 4) an over-production of inflammatory lipid mediators.     

A broadly tuned odorant receptor in neurons of trichoid sensilla in locust,Locusta migratoria

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. Odorant receptors (ORs) on the membrane of chemosensory neurons are believed to be key molecules in sensing exogenous chemical cues. ORs in different species of insects are diverse and should tune a species to its own specific semiochemicals relevant to their survival. The orthopteran insect, locust (Locusta migratoria), is a model hemimetabolous insect. There is very limited knowledge on the functions of locust ORs although many locust OR genes have been identified in genomic sequencing experiments. In this paper, a locust OR, LmigOR3 was localized to neurons housed in trichoid sensilla by in situ hybridization. LmigOR3 was expressed as a transgene in Drosophila trichoid olfactory neurons (aT1) lacking the endogenous receptor Or67d and the olfactory tuning curve and dose-response curves were established for this locust receptor. The results show that LmigOR3 sensitizes neurons to ketones, esters and heterocyclic compounds, indicating that LmigOR3 is a broadly tuned receptor. LmigOR3 is the first odorant receptor from Orthoptera that has been functionally analyzed in the Drosophila aT1 system. This work demonstrates the utility of the Drosophila aT1 system for functional analysis of locust odorant receptors and suggests that LmigOR3 may be involved in detecting food odorants, or perhaps locust body volatiles that may help us to develop new control methods for locusts.     

The immune signaling pathways of Manduca sexta

Signal transduction pathways and their coordination are critically important for proper functioning of animal immune systems. Our knowledge of the constituents of the intracellular signaling network in insects mainly comes from genetic analyses in Drosophila melanogaster. To facilitate future studies of similar systems in the tobacco hornworm and other lepidopteran insects, we have identified and examined the homologous genes in the genome of Manduca sexta. Based on 1:1 orthologous relationships in most cases, we hypothesize that the Toll, Imd, MAPK-JNK-p38 and JAK-STAT pathways are intact and operative in this species, as are most of the regulatory mechanisms. Similarly, cellular processes such as autophagy, apoptosis and RNA interference probably function in similar ways, because their mediators and modulators are mostly conserved in this lepidopteran species. We have annotated a total of 186 genes encoding 199 proteins, studied their domain structures and evolution, and examined their mRNA levels in tissues at different life stages. Such information provides a genomic perspective of the intricate signaling system in a non-drosophiline insect.     

Artificial miRNA-mediated silencing of ecdysone receptor (EcR) affects larval development and oogenesis in Helicoverpa armigera

The insect pests are real threat to farmers as they affect the crop yield to a great extent. The use of chemical pesticides for insect pest control has always been a matter of concern as they pollute the environment and are also harmful for human health. Bt (Bacillus thuringensis) technology helped the farmers to get rid of the insect pests, but experienced a major drawback due to the evolution of insects gaining resistance towards these toxins. Hence, alternative strategies are high on demand to control insect pests. RNA-based gene silencing is emerging as a potential tool to tackle with this problem. In this study, we have shown the use of artificial microRNA (amiRNA) to specifically target the ecdysone receptor (EcR) gene of Helicoverpa armigera (cotton bollworm), which attacks several important crops like cotton, tomato chickpea, pigeon pea, etc and causes huge yield losses. Insect let-7a precursor miRNA (pre-miRNA) backbone was used to replace the native miRNA with that of amiRNA. The precursor backbone carrying the 21 nucleotide amiRNA sequence targeting HaEcR was cloned in bacterial L4440 vector for in vitro insect feeding experiments. Larvae fed with Escherichia coli expressing amiRNA-HaEcR showed a reduction in the expression of target gene as well as genes involved in the ecdysone signaling pathway downstream to EcR and exhibited mortality and developmental defects. Stem-loop RT-PCR revealed the presence of amiRNA in the insect larvae after feeding bacteria expressing amiRNA-HaEcR, which was otherwise absent in controls. We also found a significant drop in the reproduction potential (oogenesis) of moths which emerged from treated larvae as compared to control. These results demonstrate the successful use of an insect pre-miRNA backbone to express amiRNA for gene silencing studies in insects. The method is cost effective and can be exploited as an efficient and alternative tool for insect pest management.     

Altered tyrosine metabolism and melanization complex formation underlie the developmental regulation of melanization in Manduca sexta

The study of hemolymph melanization in Lepidoptera has contributed greatly to our understanding of its role in insect immunity. Manduca sexta in particular has been an excellent model for identifying the myriad components of the phenoloxidase (PO) cascade and their activation through exposure to pathogen-associated molecular patterns (PAMPs). However, in a process that is not well characterized or understood, some insect species rapidly melanize upon wounding in the absence of added PAMPs. We sought to better understand this process by measuring wound-induced melanization in four insect species. Of these, only plasma from late 5th instar M. sexta was unable to melanize, even though each contained millimolar levels of the putative melanization substrate tyrosine (Tyr). Analysis of Tyr metabolism using substrate-free plasmas (SFPs) from late 5th instar larvae of each species showed that only M. sexta SFP failed to melanize with added Tyr. In contrast, early instar M. sexta larvae exhibited wound-induced melanization and Tyr metabolism, and SFPs prepared from these larvae melanized in the presence of Tyr. Early instar melanization in M. sexta was associated with the formation of a high mass protein complex that could be observed enzymatically in native gels or by PO-specific immunoblotting. Topical treatment of M. sexta larvae with the juvenile hormone (JH) analog methoprene delayed pupation and increased melanizing ability late in the instar, thus linking development with immunity. Our results demonstrate that melanization rates are highly variable in Lepidoptera, and that developmental stage can be an important factor for melanization within a species. More specifically, we show that the physiological substrate for melanization in M. sexta is Tyr, and that melanization is associated with the formation of a PO-containing protein complex.     

Biosynthetic pathway of the phytohormone auxin in insects and screening of its inhibitors

Insect galls are abnormal plant tissues induced by galling insects. The galls are used for food and habitation, and the phytohormone auxin, produced by the insects, may be involved in their formation. We found that the silkworm, a non-galling insect, also produces an active form of auxin, indole-3-acetic acid (IAA), by de novo synthesis from tryptophan (Trp). A detailed metabolic analysis of IAA using IAA synthetic enzymes from silkworms indicated an IAA biosynthetic pathway composed of a three-step conversion: Trp → indole-3-acetaldoxime → indole-3-acetaldehyde (IAAld) → IAA, of which the first step is limiting IAA production. This pathway was shown to also operate in gall-inducing sawfly. Screening of a chemical library identified two compounds that showed strong inhibitory activities on the conversion step IAAld → IAA. The inhibitors can be efficiently used to demonstrate the importance of insect-synthesized auxin in gall formation in the future.     

The steroid hormone 20-hydroxyecdysone promotes switching from autophagy to apoptosis by increasing intracellular calcium levels

Autophagy regulates cell survival (or cell death in several cases), whereas apoptosis regulates cell death. However, the relationship between autophagy and apoptosis and the regulative mechanism is unclear. We report that steroid hormone 20-hydroxyecdysone (20E) promotes switching from autophagy to apoptosis by increasing intracellular calcium levels in the midgut of the lepidopteran insect Helicoverpa armigera. Autophagy and apoptosis sequentially occurred during midgut programmed cell death under 20E regulation, in which lower concentrations of 20E induced microtubule-associated protein 1 light chain 3–phosphatidylethanolamine (LC3–II, also known as autophagy-related gene 8, ATG8) expression and autophagy. High concentrations of 20E induced cleavage of ATG5 to NtATG5 and pro-caspase-3 to active caspase-3, which led to a switch from autophagy to apoptosis. Blocking autophagy by knockdown of ATG5, ATG7, or ATG12, or with the autophagy inhibitor 3-methyladenine, inhibited 20E-induced autophagy and apoptosis. Blocking apoptosis by using the apoptosis inhibitor Ac-DEVD-CHO did not prevent 20E-induced autophagy, suggesting that apoptosis relies on autophagy. ATG5 knockdown resulted in abnormal pupation and delayed pupation time. High concentrations of 20E induced high levels of intracellular Ca²⁺, NtATG5, and active caspase-3, which mediated the switch from autophagy to apoptosis. Blocking 20E-mediated increase of cellular Ca²⁺ caused a decrease of NtATG5 and active caspase-3 and repressed the transformation from autophagy to apoptosis, thereby promoting cell survival. 20E induces an increase in the concentration of intracellular Ca²⁺, thereby switching autophagic cell survival to apoptotic cell death.     

Molecular characterization and functional expression of the Apis mellifera voltage-dependent Ca2+ channels

Voltage-gated Ca²⁺ channels allow the influx of Ca²⁺ ions from the extracellular space upon membrane depolarization and thus serve as a transducer between membrane potential and cellular events initiated by Ca²⁺ transients. Most insects are predicted to possess three genes encoding Cavα, the main subunit of Ca²⁺ channels, and several genes encoding the two auxiliary subunits, Cavβ and Cavα2δ; however very few of these genes have been cloned so far. Here, we cloned three full-length cDNAs encoding the three Cavα subunits (AmelCav1a, AmelCav2a and AmelCav3a), a cDNA encoding a novel variant of the Cavβ subunit (AmelCavβc), and three full-length cDNAs encoding three Cavα2δ subunits (AmelCavα2δ1 to 3) of the honeybee Apis mellifera. We identified several alternative or mutually exclusive exons in the sequence of the AmelCav2 and AmelCav3 genes. Moreover, we detected a stretch of glutamine residues in the C-terminus of the AmelCav1 subunit that is reminiscent of the motif found in the human Cav2.1 subunit of patients with Spinocerebellar Ataxia type 6. All these subunits contain structural domains that have been identified as functionally important in their mammalian homologues. For the first time, we could express three insect Cavα subunits in Xenopus oocytes and we show that AmelCav1a, 2a and 3a form Ca²⁺ channels with distinctive properties. Notably, the co-expression of AmelCav1a or AmelCav2a with AmelCavβc and AmCavα2δ1 produces High Voltage-Activated Ca²⁺ channels. On the other hand, expression of AmelCav3a alone leads to Low Voltage-Activated Ca²⁺ channels.     

TIL-type protease inhibitors may be used as targeted resistance factors to enhance silkworm defenses against invasive fungi

Entomopathogenic fungi penetrate the insect cuticle using their abundant hydrolases. These hydrolases, which include cuticle-degrading proteases and chitinases, are important virulence factors. Our recent findings suggest that many serine protease inhibitors, especially TIL-type protease inhibitors, are involved in insect resistance to pathogenic microorganisms. To clarify the molecular mechanism underlying this resistance to entomopathogenic fungi and identify novel genes to improve the silkworm antifungal capacity, we conducted an in-depth study of serine protease inhibitors. Here, we cloned and expressed a novel silkworm TIL-type protease inhibitor, BmSPI39. In activity assays, BmSPI39 potently inhibited the virulence protease CDEP-1 of Beauveria bassiana, suggesting that it might suppress the fungal penetration of the silkworm integument by inhibiting the cuticle-degrading proteases secreted by the fungus. Phenol oxidase activation studies showed that melanization is involved in the insect immune response to fungal invasion, and that fungus-induced excessive melanization is suppressed by BmSPI39 by inhibiting the fungal cuticle-degrading proteases. To better understand the mechanism involved in the inhibition of fungal virulence by protease inhibitors, their effects on the germination of B. bassiana conidia was examined. BmSPI38 and BmSPI39 significantly inhibited the germination of B. bassiana conidia. Survival assays showed that BmSPI38 and BmSPI39 markedly improved the survival rates of silkworms, and can therefore be used as targeted resistance proteins in the silkworm. These results provided new insight into the molecular mechanisms whereby insect protease inhibitors confer resistance against entomopathogenic fungi, suggesting their potential application in medicinal or agricultural fields.     

β-chain of ATP synthase as a lipophorin binding protein and its role in lipid transfer in the midgut of Panstrongylus megistus (Hemiptera: Reduviidae)

Lipophorin, the main lipoprotein in the circulation of the insects, cycles among peripheral tissues to exchange its lipid cargo at the plasma membrane of target cells, without synthesis or degradation of its apolipoprotein matrix. Currently, there are few characterized candidates supporting the functioning of the docking mechanism of lipophorin-mediated lipid transfer. In this work we combined ligand blotting assays and tandem mass spectrometry to characterize proteins with the property to bind lipophorin at the midgut membrane of Panstrongylus megistus, a vector of Chagas' disease. We further evaluated the role of lipophorin binding proteins in the transfer of lipids between the midgut and lipophorin. The β subunit of the ATP synthase complex (β-ATPase) was identified as a lipophorin binding protein. β-ATPase was detected in enriched midgut membrane preparations free of mitochondria. It was shown that β-ATPase partially co-localizes with lipophorin at the plasma membrane of isolated enterocytes and in the sub-epithelial region of the midgut tissue. The interaction of endogenous lipophorin and β-ATPase was also demonstrated by co-immunoprecipitation assays. Blocking of β-ATPase significantly diminished the binding of lipophorin to the isolated enterocytes and to the midgut tissue. In vivo assays injecting the β-ATPase antibody significantly reduced the transfer of [³H]-diacylglycerol from the midgut to the hemolymph in insects fed with [9,10-³H]-oleic acid, supporting the involvement of lipophorin-β-ATPase association in the transfer of lipids. In addition, the β-ATPase antibody partially impaired the transfer of fatty acids from lipophorin to the midgut, a less important route of lipid delivery to this tissue. Taken together, the findings strongly suggest that β-ATPase plays a role as a docking lipophorin receptor at the midgut of P. megistus.     

Mode of action of allatostatins in the regulation of juvenile hormone biosynthesis in the cockroach,Diploptera punctata

The FGLamide allatostatins (FGL/ASTs) are a family of neuropeptides with pleiotropic functions, including the inhibition of juvenile hormone (JH) biosynthesis, vitellogenesis and muscle contraction. In the cockroach, Diploptera punctata, thirteen FGLa/ASTs and one allatostatin receptor (AstR) have been identified. However, the mode of action of ASTs in regulation of JH biosynthesis remains unclear. Here, we determined the tissue distribution of Dippu-AstR. And we expressed Dippu-AstR in vertebrate cell lines, and activated the receptor with the Dippu-ASTs. Our results show that all thirteen ASTs activated Dippu-AstR in a dose dependent manner, albeit with different potencies. Functional analysis of AstR in multiple cell lines demonstrated that activation of the AstR receptor resulted in elevated levels of Ca²⁺ and cAMP, which suggests that Dippu-AstR can act through the Gα_q and Gα_s protein pathways. The study on the target of AST action reveals that FGL/AST affects JH biosynthesis prior to the entry of acetyl-CoA into the JH biosynthetic pathway.     

Acute fructose intake suppresses fasting-induced hepatic gluconeogenesis through the AKT-FoxO1 pathway

Excessive intake of fructose increases lipogenesis in the liver, leading to hepatic lipid accumulation and development of fatty liver disease. Metabolic alterations in the liver due to fructose intake have been reported in many studies, but the effect of fructose administration on hepatic gluconeogenesis is not fully understood. The aim of this study was to evaluate the acute effects of fructose administration on fasting-induced hepatic gluconeogenesis. C57BL/6J mice were administered fructose solution after 14 h of fasting and plasma insulin, glucose, free fatty acids, and ketone bodies were analysed. We also measured phosphorylated AKT and forkhead box O (FoxO) 1 protein levels and gene expression related to gluconeogenesis in the liver. Furthermore, we measured glucose production from pyruvate after fructose administration. Glucose-administered mice were used as controls. Fructose administration enhanced phosphorylation of AKT in the liver, without increase of blood insulin levels. Blood free fatty acids and ketone bodies concentrations were as high as those in the fasting group after fructose administration, suggesting that insulin-induced inhibition of lipolysis did not occur in mice administered with fructose. Fructose also enhanced phosphorylation of FoxO1 and suppressed gluconeogenic gene expression, glucose-6-phosphatase activity, and glucose production from pyruvate. The present study suggests that acute fructose administration suppresses fasting-induced hepatic gluconeogenesis in an insulin-independent manner.