Locust Fat Body Research Articles

Replication protein A is required for juvenile hormone-dependent vitellogenesis and oocyte maturation in locusts

Aside from inhibiting insect metamorphosis, juvenile hormone (JH) has a well-known role in stimulating various aspects of insect reproduction. Replication protein A (RPA), a heterotrimeric complex comprised of RPA1, RPA2 and RPA3 subunits plays an essential role in DNA replication and DNA repair. Here we report that RPAs are highly expressed in the fat body of adult female locust, Locusta migratoria. While RPA1 is upregulated by the JH receptor Methoprene-tolerant (Met), RPA2 and RPA3 expression appears to be primarily controlled by Forkhead box O transcription factor (FoxO). Knockdown of RPA1, RPA2 or RPA3 results in markedly reducd vitellogenin (Vg) expression in the fat body, accompanied by arrested ovarian growth and inhibited oocyte maturation. In addition, depletion of an RPA subunit leads to increased expression of other RPA subunits as well as a pro-apoptotic gene, Smac that is involved in DNA repair and apoptosis. The data indicate a crucial role of RPAs in JH-dependent vitellogenesis and oocyte maturation.

Juvenile hormone promotes locust fat body cell polyploidization and vitellogenesis by activating the transcription of Cdk6 and E2f1

Juvenile hormone (JH) is known to promote cell polyploidization for insect vitellogenesis and egg production, but the underlying mechanisms remain poorly understood. Using the migratory locust Locusta migratoria as a model system, we report here that the expression of cyclin-dependent kinase 6 (Cdk6) and adenovirus E2 factor-1 (E2f1), the core mediators in cell cycle progression is regulated by JH and its receptor Methoprene-tolerant (Met). JH acts through its receptor complex comprised of Met and Taiman to directly activate the transcription of Cdk6 and E2f1. Depletion of Cdk6 or E2f1 results in significantly decreased ploidy, precocious mitotic entry and increased cell numbers in the fat body, accompanied by substantial reduction of Vitellogenin gene expression, blocked ovarian growth and arrested oocyte maturation. These findings indicate a crucial role of Cdk6 and E2f1 in JH-regulated polyploidization and vitellogenesis as well as a novel regulatory machinery for endocycling in insects.

The creation of a recombinant single-chain antibody against α/β-hydrolase of microsporidium Paranosema locustae and immunolocalization of the enzyme in an infected host cell

Microsporidia are a group of widespread fungi-related obligate intracellular parasites. Direct contact of most microsporidia with the cytoplasm of an infected host cell entails possible secretion of various proteins from the parasite that allows control physiological processes of the host. Earlier, by means of polyclonal antibodies against α/β-hydrolase of microsporidium Paranosema locustae, the secretion of large amounts of the enzyme into the cytoplasm of fat body cells of infected migratory locust Locusta migratoria was demonstrated. However, yeast fungi Pichia pastoris did not recognize this enzyme as a secretory one during its heterologous expression. In the present study, a library of recombinant single-chain antibodies (scFv fragments) against proteins of the infected fat body of locust was constructed. The use of the phage display technology enabled choosing a miniantibody that specifically recognized the studied enzyme. Immunoblotting and immunolabeling of frozen sections of locust fat body with the selected scFv fragment confirmed the fact of secretion of P. locustae α/β-hydrolase (as two forms of different size) into the infected host cell. Prospects of using the selected scFv fragment for further studies of the secretion mechanism of the parasite’s protein and its role in host–parasite interactions are discussed.

A Novel Spore Wall Protein from Antonospora locustae (Microsporidia: Nosematidae) Contributes to Sporulation

Microsporidia are obligate intracellular parasites, existing in a wide variety of animal hosts. Here, we reported Aloc SWP2, a novel protein identified from the spore wall of Antonospora locustae (formerly, Nosema locustae, and synonym, Paranosema locustae), containing four cysteines that are conserved among the homologues of several Microspodian pathogens in insects and mammals. Aloc SWP2 was detected in the wall of mature spores via indirect immunofluorescence assay. In addition, immunocytochemistry localization experiments showed that the protein was observed in the wall of sporoblasts, sporonts, and meronts during sporulation within the host body, also in the wall of mature spores. AlocSWP2 was not detected in the fat body of infected locust until the 9th day after inoculating spores via RT‐PCR experiments. Furthermore, the survival percentage of infected locusts injected with dsRNA of AlocSWP2 on the 15th, 16th, and 17th days after inoculation with microsporidian were significantly higher than those of infected locusts without dsRNA treatment. Conversely, the amount of spores in locusts infected with A. locustae after treated with RNAi AlocSWP2 was significantly lower than those of infected locusts without RNAi of this gene. This novel spore wall protein from A. locustae may be involved in sporulation, thus contributing to host mortality.

Comparative transcriptomic analysis of immune responses of the migratory locust, Locusta migratoria, to challenge by the fungal insect pathogen, Metarhizium acridum.

BackgroundThe migratory locust, Locusta migratoria manilensis, is an immensely destructive agricultural pest that forms a devastating and voracious gregarious phase. The fungal insect pathogen, Metarhizium acridum, is a specialized locust pathogen that has been used as a potent mycoinsecticide for locust control. Little, however, is known about locust immune tissue, i.e. fat body and hemocyte, responses to challenge by this fungus.MethodsRNA-seq (RNA sequencing) technology were applied to comparatively examine the different roles of locust fat body and hemocytes, the two major contributors to the insect immune response, in defense against M. acridum. According to the sequence identity to homologies of other species explored immune response genes, immune related unigenes were screened in all transcriptome wide range from locust and the differential expressed genes were identified in these two tissues, respectively.ResultsAnalysis of differentially expressed locust genes revealed 4660 and 138 up-regulated, and 1647 and 23 down-regulated transcripts in the fat body and hemocytes, respectively after inoculation with M. acridum spores. GO (Gene Ontology) enrichment analysis showed membrane biogenesis related proteins and effector proteins significantly differentially expressed in hemocytes, while the expression of energy metabolism and development related transcripts were enriched in the fat body after fungal infection. A total of 470 immune related unigenes were identified, including members of the three major insect immune pathways, i.e. Toll, Imd (immune deficiency) and JAK/STAT (janus kinase/signal transduction and activator of transcription). Of these, 58 and three were differentially expressed in the insect fat body or hemocytes after infection, respectively. Of differential expressed transcripts post challenge, 43 were found in both the fat body and hemocytes, including the LmLys4 lysozyme, representing a microbial cell wall targeting enzyme.ConclusionsThese data indicate that locust fat body and hemocytes adopt different strategies in response to M. acridum infection. Fat body gene expression after M. acridum challenge appears to function mainly through activation of innate immune related genes, energy metabolism and development related genes. Hemocyte responses attempt to limit fungal infection primarily through regulation of membrane related genes and activation of cellular immune responses and release of humoral immune factors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2089-9) contains supplementary material, which is available to authorized users.

Endocrine control of TAG lipase in the fat body of the migratory locust, Locusta migratoria

Aspects of the role and activation of the enzyme triacylglycerol lipase (TAG lipase) in the fat body of the migratory locust Locusta migratoria were investigated. TAG lipase is under the hormonal control of the three endogenous adipokinetic peptides of the migratory locust, Locmi-AKH-I, Locmi-AKH-II and Locmi-AKH-III. Injection of low doses (5–10 pmol) of each peptide causes an increase in lipase activity. The activation of lipase is time dependent: an elevated activity was recorded 15 min after injection of 10 pmol Locmi-AKH-I and maximum activation was reached after 45–60 min. The activation of TAG lipase is also dose-dependent. Doses of 2 pmol of each Locmi-AKH had no effect, whereas 5 pmol caused a significant activation. Maximum activation is reached with a dose of 10 pmol. Analogues of the second messengers cAMP (cpt-cAMP) and IP 3 (F-IP 3) both activate the enzyme glycogen phosphorylase whereas only cpt-cAMP, but not F-IP 3, activates TAG lipase; cpt-cAMP elevates the lipid levels in the haemolymph. Activation of lipase is specific to the three endogenous AKH peptides: 5 pmol of the endogenous peptide Locmi-HrTH and 10 pmol of corazonin failed to activate lipase. High doses of octopamine did not activate lipase nor did they elevate the lipid concentration in the haemolymph. TAG lipase is stimulated by flight activity but activation is slower than that of glycogen phosphorylase: after 30 min of flight or after 5 min of flight plus 1 h of subsequent rest, activity of TAG lipase is increased, but not immediately after 5 min of flight. In contrast, glycogen phosphorylase is activated significantly after 5 min of flight. These activation patterns of the two enzymes mirror-image the concentration of their substrates in the haemolymph: there is a significant decrease in the concentration of carbohydrates after 5 min of flight, whereas no change of the concentration of lipids can be measured after such short time of flight activity; however, a subsequent rest period of 1 h is sufficient to increase the lipid concentration.

N-acylaziridines as potential proinsecticides of carboxylic acids☆Part VI. Direct HPLC monitoring of the metabolism in insect tissues

To determine the reversible masking potential of carboxylic acids afforded by the N-acyl structure in a proinsecticide perspective, the hydrolysis of monosubstituted N-acylaziridines and unsubstituted N-acylpyrrolidine was studied by reversed-phase high-performance liquid chromatography (HPLC) during in vitro assays conducted in the presence of insect tissues or of alpha-chymotrypsin. Chromatographic analysis of unextracted biological samples so-called "the direct injection approach" was simpler and more accurate than the "extraction approach" because it avoids problems associated with extraction. Thus, periodical injections of samples of biological insect tissues or of alpha-chymotrypsin incubated with N-acyl substrates were performed on packings allowing direct injection: a wide-pore column or a monolithic column. Moreover, to allow the simultaneous monitoring of the carboxylic acids and of the parent substrates, ion-pairing was used. In these conditions, it was shown that N-acylpyrrolidine is not hydrolyzed whatever the enzymatic conditions or the pH. On the other hand, the unmasking of the carboxylic acid is the preponderant mode of hydrolysis of N-acylaziridines in the presence of alpha-chymotrypsine and the exclusive one in the presence of locust fat-body, which establishes the convenience of this structure in our proinsecticide perspective. Due to the enzymatic character of the unmasking of the carboxylic acid during biological hydrolysis of N-acylaziridines, the research of possible chiral recognitions was undertaken. Thus, the enantiomeric composition of these substrates was analysed at the stage of their approximative half hydrolysis using a chiral alpha-AGP column. It appeared that locust fat-body hydrolyses preferentially the (R)-isomers of N-acylaziridines while the reverse is observed when alpha-chymotrypsine is used.

Biosynthesis and secretion of insect lipoprotein: involvement of furin in cleavage of the apoB homolog, apolipophorin-II/I

The biosynthesis of neutral fat-transporting lipoproteins involves the lipidation of their nonexchangeable apolipoprotein. In contrast to its mammalian homolog apolipoprotein B, however, insect apolipophorin-II/I (apoLp-II/I) is cleaved posttranslationally at a consensus substrate sequence for furin, resulting in the appearance of two apolipoproteins in insect lipoprotein. To characterize the cleavage process, a truncated cDNA encoding the N-terminal 38% of Locusta migratoria apoLp-II/I, including the cleavage site, was expressed in insect Sf9 cells. This resulted in the secretion of correctly processed apoLp-II and truncated apoLp-I. The cleavage could be impaired by the furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (decRVKRcmk) as well as by mutagenesis of the consensus substrate sequence for furin, as indicated by the secretion of uncleaved apoLp-II/I-38. Treatment of L. migratoria fat body, the physiological site of lipoprotein biosynthesis, with decRVKRcmk similarly resulted in the secretion of uncleaved apoLp-II/I, which was integrated in lipoprotein particles of buoyant density identical to wild-type high density lipophorin (HDLp). These results show that apoLp-II/I is posttranslationally cleaved by an insect furin and that biosynthesis and secretion of HDLp can occur independent of this processing step. Structure modeling indicates that the cleavage of apoLp-II/I represents a molecular adaptation in homologous apolipoprotein structures. We propose that cleavage enables specific features of insect lipoproteins, such as low density lipoprotein formation, endocytic recycling, and involvement in coagulation.

Identification of a glycogenolysis-inhibiting peptide from the corpora cardiaca of locusts.

A mass spectrometric study of the peptidome of the neurohemal part of the corpora cardiaca of Locusta migratoria and Schistocerca gregaria shows that it contains several unknown peptides. We were able to identify the sequence of one of these peptides as pQSDLFLLSPK. This sequence is identical to the part of the Locusta insulin-related peptide (IRP) precursor that is situated between the signal peptide and the B-chain. We designated this peptide as IRP copeptide. This IRP copeptide is also present in the pars intercerebralis, which is likely to be the site of synthesis. It is identical in both L. migratoria and S. gregaria. It shows no effect on the hemolymph lipid concentration in vivo or muscle contraction in vitro. The IRP copeptide is able to cause a decreased phosphorylase activity in locust fat body in vitro, opposite to the effect of the adipokinetic hormones and therefore possibly represents a glycogenolysis-inhibiting peptide.

Highly-resolving two-dimensional electrophoresis for the study of insect proteins.

In this paper, we describe methods for isolation, purification and solubilization of insect proteins from various tissues, including lipid-rich fat body. An Australian locust, Oedaleus australis, and its associated dipteran parasite, Trichopsidea oestracea, provided the protein samples. Protein samples of locust fat body, haemolymph and body wall as well as parasite whole-body extracts were isolated and purified of lipids and salts using chloroform-methanol extraction. Proteins were solubilized using two types of enhanced solubilizing solutions and arrayed using two-dimensional electrophoresis. We demonstrated substantial differences between the body wall protein spectra of normal locusts and those parasitized by T. oestracea. Proteins more abundant in parasitized locusts include two 70 kDa proteins with an isoelectric point (pI) of about 5.5, one approximately 55 kDa protein cluster with a pI of about 4.7 and three 40 kDa proteins with pI values of around 5.6. Proteins that decreased in parasitized locusts include a group of 45 kDa proteins with pI values between 6 and 6.8, and a cluster of 22 to 23 kDa proteins with pI values of approximately 5.4 and 5.6.

CDNA cloning of two different serine protease inhibitor precursors in the migratory locust, Locusta migratoria

Recently, a novel serine protease-inhibiting peptide family, designated as the 'pacifastin family', has been described in locusts and crayfish. All members of this family possess a characteristic cysteine-rich domain. The present study describes the cDNA cloning, sequencing and transcript distribution of two novel pacifastin-related peptide precursors in the migratory locust, Locusta migratoria. Only one of the encoded peptides (HI) was identified previously, whereas six others represent new members of the pacifastin family. Northern blot analysis showed that both precursor transcripts are present in adult locust fat body. These could not be detected in the midgut. Interestingly, an in silico data mining approach of the expressed sequence tags (EST) database revealed the existence of Manduca sexta and Bombyx mori cDNAs that display pronounced sequence similarities with these locust pacifastin-related transcripts.

Molecular cloning of a female-specific cDNA with unique repeat sequences from the fat body of the adult locust, Locusta migratoria

A cDNA clone encoding a 25-kDa protein (25K) was isolated from a cDNA library made from RNA isolated from the adult fat body and ovaries of the locust, Locusta migratoria. The longest open reading frame of this cDNA clone encodes a 225-amino acid polypeptide, the N-terminal end of which was similar to the 21-kDa and 19-kDa juvenile hormone induced proteins identified in the locust hemolymph, but the C-terminal end was different. The C-terminal end of the 25K cDNA contained seven unique repeat elements of 10 amino acids each, most of which are polar residues. Expression of the 25K mRNA was tissue-, development- and sex-specific. A 1.2-kb mRNA was detected using the 25K cDNA as a probe only in the fat body of adult females. The mRNA started to appear at day 4 after the insect molted to the adult and rapidly increased by day 6. The mRNA was absent in the ovarian follicle cells and fat body of adult males. In vitro transcription and translation of the 25K cDNA produced a protein that migrated around 32 kDa on sodium dodecyl sulfate polyacrylamide gels. The 25K cDNA was expressed in a baculovirus expression system and the protein produced also migrated around 32 kDa.

The phospholipase C signaling pathway in locust fat body is activated via G q and not affected by cAMP

Crosstalk between signal transduction pathways provides a complex intracellular avenue for fine tuning of hormone-induced signals. Over the last few years, we have studied the signaling mechanisms of three locust adipokinetic hormones (AKHs), which control mobilization of energy reserves from insect fat body as fuels for flight and transduce their signals via adenylyl cyclase- and phospholipase C- (PLC) dependent pathways. In this study, we examine possible crosstalk between these signaling routes. We show that cAMP does not affect basal and AKH-stimulated inositol phosphate (InsP n) production. Incubation of fat body with aluminium fluoride, an activator of G proteins, increased InsP n levels by 77%, whereas cholera toxin and pertussis toxin were ineffective. This implies that fat body PLC is not activated by Gβγ, but possibly by G qα. The involvement of this G protein in AKH signaling was demonstrated by our observation that the GPAntagonist-2A, which antagonizes G q, attenuated glycogen phosphorylase activation by AKH-I. As plasma membrane Ca 2+ channels may constitute another target for cAMP-mediated modulation, we studied the type of channels involved in AKH signaling using a variety of L-, N- and T-type Ca 2+ channel inhibitors. None of these blocked AKH-induced glycogen phosphorylase activation, suggesting that voltage-dependent Ca 2+ channels do not mediate AKH-induced Ca 2+ influx.

Chloromethyl ketones are insulin-like stimulators of lipid synthesis in locust fat body.

N alpha-tosyl-L-lysine chloromethyl ketone (TLCK) stimulates lipid synthesis in locust fat body in vitro, and is able to reverse the inhibitory effects of AKH-I on lipid synthesis. Effective stimulatory concentrations of TLCK were in the range of 0.2-1.0 mM. Similar stimulatory effects were also achieved with phenylalanine chloromethyl ketone (PheCK) and leucine chloromethyl ketone (LeuCK), but not with tosyl-phenylalanine chloromethyl ketone (TPCK), dansyl-glu-gly-arg-CK, chloroacetone, chloroacetic acid, chloroacetamide, chloroacetaldehyde, chloroacetyl-L-leucine or acetylated or fluorescamine-labelled TLCK, PheCK, and LeuCK. The level of stimulation caused by TLCK was dependent on incubation time, so that after a 5-h preincubation of fat body tissue with TLCK the stimulated rate was severalfold higher than the control. TLCK also increased the rate of uptake of trehalose and uridine, but not glucose, deoxyglucose or glycine. Increasing concentrations of bovine serum albumin (BSA) in the incubation medium caused a reduction in the rate of TLCK-stimulated acetate uptake, such that levels of uptake were no higher with 1% BSA than in the controls. A range of more specific protease and kinase inhibitors was tested, but none caused stimulation; thus the mode of action of TLCK on the stimulation of acetate uptake has yet to be identified. Elucidation of the mode of action of TLCK may facilitate the development of novel compounds for insect pest control.

Chloromethyl ketones are insulin‐like stimulators of lipid synthesis in locust fat body

Chloromethyl ketones are insulin‐like stimulators of lipid synthesis in locust fat body

The effect of analogues of adipokinetic hormone II on second messenger systems in the fat body of Schistocerca gregaria

Analogues of the locust adipokinetic hormone-2 from Schistocerca gregaria were tested for their ability to stimulate two signalling pathways in the locust fat body. A series of analogues in which successive amino acids were replaced by alanine was synthesized and their ability to stimulate the turnover of 3H-labelled inositol trisphosphates and the accumulation of cyclic AMP in vitro was investigated. Unmodified adipokinetic hormone-2 stimulated both pathways, but substitution of phenylalanine at position 4 or tryptophan at position 8 by alanine, produced peptides that were unable to stimulate either signalling pathway. Dose response curves are presented for the increase in inositol phosphates that occurred when fat body tissue was stimulated with the remaining analogues. The peptide in which pyroglutamate at position 1 was replaced gave a truncated inositol phosphate response whereas all other peptides produced full responses with varying sensitivities. The sensitivity of response to the analogue in which threonine at position 6 was replaced was considerably higher than that of the native hormone. None of the inactive or partially active peptides appeared to block the action of adipokinetic hormone-1 or -2. All peptides that were active in the inositol phosphate assay also stimulated the accumulation of cyclic AMP. Some similarities were found in the relative activities of the peptides in the two assay systems.

Metabolism of Stored Reserves in Insect Fat Body: Hormonal Signal Transduction Implicated in Glycogen Mobilization and Biosynthesis of the Lipophorin System*

The mobilization of carbohydrate and lipid reserves from the insect fat body as fuels for migratory flight activity is controlled by adipokinetic hormone (AKH), of which in Locusta migratoria three different forms occur: AKH-I, -II and -III. In fat body in vitro, each AKH is capable of activating glycogen phosphorylase and of stimulating cAMP production, but only in the presence of extracellular Ca2+. The hormones stimulate both the influx and the efflux of Ca2+, the higher influx probably causing an increase in intracellular [Ca2+]. AKH enhances the production of inositol phosphates among which inositol 1,4,5-triphosphate may mediate the mobilization of Ca2+ from intracellular stores. Evidence is presented in favor of the occurrence of a capacitative calcium entry mechanism. Results suggest that transduction of the AKH signal occurs through stimulatory G protein-coupled receptor(s). A tentative model is presented for the interactions between the AKH signaling pathways in the locust fat body cell. AKH-induced lipid mobilization during flight requires the presence in the insect blood of high-density lipophorin (HDLp) particles and apolipophorin III (apoLp-III). Both protein components are synthesized in the fat body. In the locust, the two integral, nonexchangeable HDLp apolipophorins (apoLp-I and -II) were shown to originate from a common precursor; an mRNA of 10.3 kb seems to code for this precursor protein. The models proposed for lipophorin assembly and secretion in a number of insects are not in agreement. The exchangeable apoLp-III may occur in two or more isoforms; locust apoLp-III is secreted from the fat body as one of the two isoforms and in the hemolymph converted into the truncated second one. The rationale for this process is as yet unknown.

Juvenile hormone and locust phase

Juvenile hormone (JH) is directly and indirectly involved in the determination of many phase characteristics of locusts. Temporal differences in JH titer and the biosynthetic capacity of excised corpora allata of solitary nymphs are correlated to some extent with the effects on them of treatment with JH or JH analogs. Juvenile hormone modulates cuticular melanization and the rate of reproductive maturation—specifically regulating vitellogenesis at the transcriptional level, and nonspecifically stimulating the translational capacity of the locust fat body. Juvenile hormone does not appear to be involved in behavioral phase transition of locusts. Long-term treatment of crowded nymphs with the JH analog methoprene does not lessen their gregarious behavior, and does not reduce hemolymph lipids or carbohydrates. Reducing endogenous JH levels of solitary nymphs by chemical allatectomy with precocene III does not induce gregarious behavior. However, methoprene does affect nymphal coloration. The relevance of these results to locust control is discussed. Arch. Insect Biochem. Physiol. 35:375–391, 1997. © 1997 Wiley-Liss, Inc.

Cloning and upstream sequence of a juvenile hormone-regulated gene from the migratory locust

As a step toward analyzing the molecular mechanism of action of juvenile hormone (JH), the gene encoding a JH-inducible 21-kDa protein (Jhp21) produced in the fat body of the migratory locust has been cloned. Four exons, representing 750 nucleotides of cDNA sequence, were found to be distributed through 13 kb of genomic DNA. Upstream 2 kb of DNA has been sequenced and three potential hormone-response elements have been identified.

Insect adipokinetic hormone stimulates inositol phosphate metabolism: roles for both Ins(1,4,5) P3 and Ins(1,3,4,5) P4 in signal transduction?

Adipokinetic hormones (AKHs) control the mobilization of energy reserves from the insect fat body as fuels for flight activity. As a part of our investigations on AKH signal transduction, we demonstrate in this study that the inositol lipid cycle may be involved in the action of AKH-I on fat body of the migratory locust. We show that [ 3H]inositol is incorporated into fat body phosphoinositides in vitro, whose hydrolysis leads to the formation of the following inositol phosphates (InsPs): Ins(1 and/or 3) P, Ins(4) P, Ins(1,3) P 2, Ins(1,4) P 2, Ins(3,4) P 2, Ins(1,3,4) P 3, Ins(1,4,5) P 3 and Ins(1,3,4,5) P 4. AKH stimulates the formation of these isomers, eliciting an increase in radioactivity of total Ins Ps already after 1 min. Mass measurements show that Ins(1,4,5)P 3 levels are substantially enhanced by AKH, which is indicative of hormonal activation of phospholipase C. In cell-free tissue preparations, Ins(1,4,5) P 3 is metabolized through dephosphorylation as well as further phosphorylation. Ins(1,3,4,5) P 4 is dephosphorylated primarily to Ins(1,3,4) P 3, although the a bility for its reconversion to Ins(1,4,5) P 3 suggests that in vivo Ins(1,3,4,5) P 4 may function as rapidly mobilizable pool for Ins(1,4,5) P 3 generation. Metabolic pathways for the conversion of InsPs to inositol in the locust fat body are proposed.