Locust Fat Body Research Articles

A new method for determining lipase activity in locust fat body

A new method for determining lipase activity in locust fat body is described. The procedure uses a micellar solution of triolein as substrate and demonstrates high and reproducible lipase activity in the fat body. The maximal activity is about 25-fold greater than that determined by using other substrates which have widely been used by many investigators. The activity depends on ionic strength with maximal activity observed at an ionic strength of 0.3. The optimal pH was 6.9. The V max was calculated to be about 2.6 μmol fatty acid/hr per g fat body and the apparent K m value was about 56 μM for triolein.

Juvenile hormone action on locust fat body

Production of vitellogenin (Vg) in fat body of adult female Locusta migratoria is abolished by removal or inactivation of the corpora allata and restored by administration of ( RS)-methoprene. Juvenile hormone III injection alone has little effect, but when it is injected together with the JH esterase inhibitor OTFP, active Vg synthesis is induced. This supports the assumption that methoprene acts in place of the natural hormone in this system. When fat body from vitellogenic females is maintained in synthetic medium for 48 hr, the proportion of Vg in the secreted protein drops greatly, but when methoprene is present in the medium the proportion of Vg is sustained. When fat body from JH-withdrawn locusts, in which Vg synthesis has declined to zero, is cultured with methoprene, Vg synthesis is re-induced. These results show that the JH analog can act directly on locust fat body to bring about expression of the Vg genes. Experiments to optimize JH action on fat body in vitro are continuing.

Juvenile hormone-regulated locust vitellogenin genes: Lack of expression after transfer intoDrosophila

In Locusta migratoria, vitellogenin (Vg) is normally produced only in adult female fat body under stimulation by juvenile hormone (JH). This permits study of (a) programming of genes for expression and (b) modulation of expression by JH. From L. migratoria genomic libraries, two Vg genes (A and B) have been cloned. Coding regions encompass 10-12 kbases of DNA, contain introns, and hybridize with 6,300 nucleotide mRNAs. Although the 5'-exons, coding for signal peptides, are similar in sequence, the remaining coding sequences have distinct restriction maps and show no crosshybridization. Upstream DNA from the two genes has some sequence similarity, corresponding to potential regulatory regions. Dot hybridization assays of mRNAs A and B in locust fat body after induction by methoprene show coordinate expression of the two Vg genes and also a third, unidentified JH-regulated gene. In the hope of providing a system for identification of control sequences, P element-mediated transformation has been used to transfer locust DNA into Drosophila. From Vg gene B, a central block was deleted, to give a mini-Vg gene comprising 5' and 3' terminal coding sequences plus upstream flanking DNA. This was incorporated into the P element vector Carnegie 20 and injected into Drosophila embryos. Three transformed fly lines were obtained in which the locust mini-Vg gene was integrated at different chromosomal sites. On Northern blots of fly RNA, however, no expression of the locust sequences could be detected, even though the accompanying rosy gene was expressed. This suggests that the locust regulatory sequences were not recognized in Drosophila, and current effort is directed toward developing a homologous locust transformation system for expression assay of cloned JH-regulated genes.

Nuclear development in locust fat body: The influence of juvenile hormone on inclusion bodies and the nuclear matrix

The hormonal induction of vitellogenesis in insects and in oviparous vertebrates are prime models of gene regulation in eukaryotes. In vertebrates the process is under estrogenic control and normally confined to females, although males can be artificially induced. In locust in contrast, juvenile hormone (JH) is central to fat body development in both males and females, yet the response is strongly sex limited not only for vitellogenin production but also in terms of total protein, DNA and RNA synthesis and nuclear ploidy levels. To differentiate further possible sex and/or JH related developmental aspects in locusts, large-scale nuclear events were examined during normal adult maturation and in animals treated with antiallatropins and JH analogs. Fat body nuclei undergo extensive restructuring during normal development in both sexes. This included progressive nuclear enlargement, accompanied by extensive proliferation of nuclear matrix components and elaboration of complex inclusion bodies (NB). The isolated protein matrix was unusually complex relative to similar structures from vertebrates and the NB were firmly anchored to it. Although matrix proteins were qualitatively similar to those from other sources, as assessed by SDS polyacrylamide gel electrophoresis, several major matrix polypeptides, including lamins A and B, and components >150 kD, fluctuated quantitatively during development and in concert with nuclear enlargement. The number and morphology of the NB were unrelated to sex, but increased in direct proportion to absolute nuclear volumes. All changes were more pronounced in females, where higher ploidy levels, larger nuclei and correspondingly more internal matrix elements occurred. Suppression of JH production by precocene prevented all foregoing nuclear changes, but re-exposure to methoprene rapidly induced normal development. The results are compared to analogous nuclear changes in steroid responsive vertebrate tissues.

The effects of synthetic locust adipokinetic hormone on dispersed locust fat body cell preparations: cAMP induction, lipid mobilization, and inhibition of protein synthesis

A procedure for the preparation of functional cells from adult locust fat bodies by collagenase treatment has been developed. The high variability of replicates encountered when whole fat bodies are incubated in vitro is greatly reduced in incubation of the dispersed cells. Synthetic locust adipokinetic hormone (AKH) (40 n M) stimulated release of lipids from the dispersed fat body cells at a rate comparable to that observed using whole fat bodies in vitro. Synthetic AKH elevated cAMP levels sixfold in dispersed cells. In addition, AKH inhibited protein synthesis to a maximum of 50–70% in a concentration-dependent manner. None of these actions of AKH required the presence of locust hemolymph components. These results demonstrate the utility of the isolated locust fat body cells for investigating hormonal action in vitro.

In vitro studies on hormone-stimulated lipid mobilization from fat body and interconversion of haemolymph lipoproteins of Locusta migratoria

Both adipokinetic hormone and octopamine have a stimulating effect on lipid release from locust fat body in vitro, when incubated in diluted haemolymph. The presence of adipokinetic hormone results in the formation of the flight-specific haemolymph lipoprotein A + accepting the increased amount of lipids released into the incubation medium. In contrast, interconversions of lipoproteins do not occur when octopamine is added to the incubation medium, which is in line with the expectations: the lipid-mobilizing effect of octopamine is a limited and short-term effect. When fat body tissue is incubated with isolated haemolymph protein fractions, the lipid-mobilizing effect of adipokinetic hormone only occurs when the incubation medium contains both lipoprotein, A y and protein fraction C, resulting in the formation of lipoprotein A +. In similar control incubations with the hormone omitted, some lipoprotein A + is also formed (concomitant with a slight amount of lipid released), though significantly less than in incubations with hormone. Besides a stimulating function on lipolytic processes in the fat body, adipokinetic hormone is suggested to influence haemolymph lipoprotein rearrangement. A possible counteracting function of another factor in the haemolymph is discussed.

Cyclic AMP in locust fat body: Correlation with octopamine and adipokinetic hormones during flight

The effects of flight upon the level of cyclic AMP in the fat body of Locusta migratoria have been examined. Flight induced two phases of cyclic AMP elevation; the first during the initial 10 min of flight, the second between 20–30 min of flight. Neck-ligated locusts have increased levels of cyclic AMP after 10 min of flight, indicating that the adipokinetic hormones are not necessary for this elevation. Injection of 6 mg trehalose, a procedure known to delay the release of adipokinetic hormones, prevented the increases seen at 10 and 30 min of flight. Injection of synthetic adipokinetic hormone I increased the levels of cyclic AMP within 5 min, and these were maintained for up to 15 min. The roles of octopamine and the adipokinetic hormones in increasing fat body cyclic AMP, and thereby regulating haemolymph lipid, during flight are discussed.

Substrate preferences of mitochondria isolated from locust (Locusta migratoria) and blowfly (Phormia regina) fat bodies

The preferred substrate of the fat body mitochondria of both the locust (Locusta migratoria) and the blowfly (Phormia regina) based on state three rates of oxidation is palmitoyl-L-carnitine. The fat body mitochondria of the locust do not oxidize proline plus pyruvate, pyruvate plus malate, or α-glycerophosphate under the conditions used. The mitochondria of the blowfly fat body do oxidize these substrates at high rates. The mitochondria isolated from the locust fat body do not oxidize fumarate or oxaloacetate at measurable rates. The mitochondria isolated from the blowfly fat body do not oxidize citrate, isocitrate, or fumarate at measurable rates.

Locust adipokinetic hormone stimulates lipid mobilization in Manduca sexta

Adipokinetic hormone, a decapeptide isolated from the locust, stimulates mobilization of diacylglycerols from the locust fat body and loading of the lipid transport protein, lipophorin. Injection of the synthetic locust adipokinetic hormone into a sphinx moth, Manduca sexta, causes lipid loading of lipophorin. The lipophorin decreases in density from 1.11 to 1.06 g/ml, and a soluble protein from the hemolymph (apolipophorin III) associates with the lipophorin particle. Administration of intermediate doses of hormone indicates that lipophorin is converted directly to the low density form; no appreciable amounts of intermediate density particles are formed.

Regulation of glycogen phosphorylase activity in fat body of Locusta migratoria and Periplaneta americana

Saline extracts of the corpus cardiacum (CC) of Locusta migratoria activate glycogen phosphorylase in locust fat body. The response of phosphorylase to CC extracts and to synthetic adipokinetic hormone (AKH) suggests that the factor responsible for the activating effect of the CC on phosphorylase is AKH, supplemented to a minor degree with Compound II. Octopamine does not influence fat body phosphorylase activity in locusts, however, it elicits a rapid short-term hyperlipemia. In cockroaches, Periplaneta americana, injection of octopamine results in a strong activation of fat body phosphorylase within 1 min. Cockroach CC extract exerts a more prolonged effect on phosphorylase activity than does octopamine.

Regulation of locust vitellogenesis

The reproduction of insects is frequently synchronized to the availability of specific host plants or to seasonal climatic condi- tions. Synchronization is achieved in most cases either by migration to favourable habitats or by diapause, a genetically determined state of suppressed develop- mental and metabolic rates, frequently induced by photoperiod, which enables the insect to over-winter (hibernate) or aesti- vate. In most cases, diapause involves the cessation of neuroendocrine activity. Some of the species of migratory locusts known from different parts of the world also diapause as adults and thus make use of both mechanisms for synchronization 1. Adult (or reproductive) diapause is charac- terized by the absence of vitellogenesis - the synthesis of female-specific egg-yolk protein which is produced in the fat body of locusts (a tissue analogous to the liver of vertebrates) and transported in the haemolymph to the developing oocyte. In conjunction, migration often commences during the previtellogenic phase of locust development. The vitellogenin molecule is a complex glycolipophosphoprotein that appears to have been highly conserved during evolu- tion. Perhaps this is related to the numerous constraints imposed on it subsequent to its primary translation and post-translational modification. These include: secretion and transport (and resistance at that stage to hydrolytic degradation), discriminatory recognition by the plasma membrane of the developing oocyte, specific uptake and packaging as yolk platelets. Some features of locust vitellogenesis and vitellogenin are similar to vertebrate systems with which we are more familiar. Other features, in particular, hormonal con- trol, but also structural features, such as low phosphate content and degree of proteolytic processing, differ remarkably from verte- brate systems. The extent to which such dif- ferences can be exploited to design agents for control of insect reproduction bears on the relevance of this approach to modern agricultural practice. Hormonal control of vitellogenesis Vitellogenin is induced in the vertebrate liver by estrogens 2. In locusts, no steroid hormones are known to participate in vitel- logenin induction. Instead, a class of hor-

The fate of Xenopus and locust vitellogenins made in Xenopus oocytes. An export-import processing model.

Xenopus liver or locust fat body RNA injected into Xenopus oocytes directs the formation of a high‐molecular‐weight species which is found transiently within the oocyte in a membranous vesicle fraction. The conversion of such internally generated Xenopus vitellogenin to yolk platelet proteins is reduced by the presence in the incubation medium of anti‐vitellogenin antibodies, or of tunicamycin, or of a mixture of colchicine and cytocholasin. but neither export nor conversion is blocked by removal of the follicular layers which surround the oocyte. Moreover, surrounding mRNA injected defolliculated oocytes with an excess of small defolliculated oocytes leads to a reduction in lipovitellin accumulation in the cells making vitellogenin and the appearance of lipovitellin in the platelets of the uninjected feeder cells.We propose that newly‐made locust and Xenopus vitellogenins are sequestered in vesicles and are then secreted by the oocyte, the locust species being further modified by cleavage just before export. Thus processing of heterologous yolk precursors follows the pathway characteristic of the cell type used to prepare the donor RNA. We suggest that the import mechanism discriminates between the exported locust and frog proteins, and that only the Xenopus vitellogenin subsequently enters the oocyte, where it is converted to lipovitellin and phosvitin by an enzyme present in the yolk platelets. Thus we explain the apparently paradoxical observations that internally generated and externally supplied frog vitellogenin are both converted to yolk platelet proteins, whilst injected vitellogenin is not.

In vitro studies on the effects of octopamine on locust fat body

The effects of octopamine on the fat body of locusts in vitro have been examined. The isolated fat body responded to octopamine by releasing lipid in a dose-dependent manner. Maximal stimulation was obtained with 5 × 10 −6 M octopamine. The octopamine stimulated release of lipid was blocked by α-adrenergic receptor antagonists but unaffected by β-adrenergic receptor antagonists. Octopamine was more potent than synephrine, dopamine, or tyramine in eliciting the response. Octopamine (5 × 10 −6 M) in the presence of IBMX, resulted in a six- to sevenfold elevation of cyclic AMP in the fat body; this response was also blocked by α- but not β-adrenergic receptor antagonists. Cyclic GMP levels were unaffected by octopamine. Adipokinetic hormone (AKH) in the presence of IBMX, also stimulated an elevation in cyclic AMP in the fat body, and stimulated the release of lipid. Unlike the octopamine stimulation, however, the AKH effect was not inhibited by the α-adrenergic receptor antagonists. Furthermore, AKH inhibited protein synthetic activity of fat body, while octopamine was without effect. It is concluded that a release of lipid from fat body of locusts is probably mediated by a rapid activation of an octopamine-sensitive adenylate cyclase. Furthermore, the mechanism of action and the receptors for octopamine and AKH are different.

Construction and partial characterization of a recombinant DNA probe for locust vitellogenin messenger RNA.

Double-stranded DNA complementary to poly(A)-containing RNA from the fat body of adult female locusts, Locusta migratoria, was synthesized. Hybrid molecules containing this cDNA was constructed in the PstI site of the plasmid pAT 153 by the technique of dC . dG tailing and amplified in Escherichia coli K-12 strain HB 101. Ten colonies of bacteria were identified as carrying recombinant plasmids containing DNA complementary to locust vitellogenin mRNA by (a) 'Northern' blot hybridization analysis and (b) hybrid selection of vitellogenin mRNA and immunological detection of the products of translation of the mRNA. Of the ten recombinant plasmids, one, termed plasmid 4E, containing a cDNA insert of about 650 nucleotides, was characterized in greater detail and a partial restriction map obtained. Using this hybrid plasmid it was possible to derive a value for the average content of vitellogenin mRNA in the adult female locust fat body as 1.5 x 10(5) molecules/cell, and to establish that the haploid genome of L. migratoria contains only one or two genes coding for vitellogenin.

Vitellogenin mRNA in locust fat body: identification, isolation, and quantitative changes induced by juvenile hormone

Change in RNA during the juvenile hormone (JH) stimulated synthesis of vitellogenin (Vg) in the fat body of adult female Locusta migratoria have been studied. Total RNA from mature females, but not that from males or prereproductive females, shows a 6300 nucleotide component, which has been isolated by binding to oligo(dT)-cellulose and sucrose gradient centrifugation, and identified as Vg mRNA by translation in Xenopus oocytes. It has been assayed quantitatively by photometric scanning after electrophoresis. During a gonotrophic cycle, Vg mRNA increased rapidly from 0 up to about 1% of the total fat body RNA, or more than 10(6) copies per cell. After destruction of the corpora allata (the source of JH) by treatment with ethoxyprecocene, Vg synthesis was stimulated by injection of 150 micrograms of the JH analog, methoprene. In primary stimulation, Vg mRNA was first detected at 24 h and showed a marked lag in accumulation; in secondary stimulation by methoprene after decay of the primary effect, Vg mRNA was detected after only 12 h and accumulation was much more rapid. Both in the natural cycle and in experimental stimulation, Vg mRNA did not disappear in correlation with declining Vg synthesis, which suggests conservation of mRNA in untranslated form. Total (chiefly ribosomal) RNA showed a different pattern, accumulating markedly during primary and only slightly during secondary stimulation. The data indicate that JH acts selectively (though not necessarily directly) on transcription of the Vg genes.

Action of formamidine pesticides on the octopamine receptors on locust fat body

1. The action of two formamidines, chlordimeform (CDM) and desmethylchlordimeform (DCDM), on isolated fat body of locusts has been examined. 2. Treatment with DCDM resulted in a dose-dependent stimulation of lipid release while CDM was ineffective at 10 −4M. 3. The action of DCDM was blocked by the α-adrenergic receptor blocker phentolamine, but not by the β-blocker propranolol. 4. DCDM (5 × 10 −6 M) in the presence of a phosphodiesterase inhibitor caused a 4-fold elevation of cyclic AMP in the fat body; this response was blocked by phentolamine but not by propranolol. CDM (10 −5 M) did not alter cyclic AMP levels in the fat body. 5. The effects of octopamine (10 −5 M) and DCDM (10 −5 M) on cyclic AMP level were not additive. 6. It is concluded that DCDM (or its metabolites) act upon octopamine receptors on locust fat body while CDM has no effect. In view of the positive action of CDM on other octopamine receptors this indicates there may be differences between octopamine receptors of different tissues.

Vitellogenin synthesis induced in locust fat body by juvenile hormone analog in vitro

Fat bodies from adult females ofLocusta migratoria continue to synthesize vitellogenin and other proteins when cultured in vitro. A strong secondary induction of vitellogenin synthesis was obtained in fat bodies cultured in the presence of methoprene, and a weaker but significant primary induction was also obtained using higher doses (>100 μg) of methoprene.

The influence of juvenile hormone on polyploidy and vitellogenesis in the fat body of Locusta migratoria.

Abstract Somatic polyploidy is a common developmental feature of endocrine target tissues, including vertebrate liver and insect fat body. Vitellogenin (Vg) synthesis in locust fat body is under strict control of juvenile hormone (JH) and is correlated with DNA synthesis and massive polyploidization of the tissue. We have used this system to concurrently monitor interactions between hormone, specific protein synthesis, and generation of cells with defined ploidy levels. Normal maturation of both males and females were compared, as were animals treated with precocene III and the JH analog ZR-515. Following are the main results and observations: Polyploidization occurs in both males and females but is much more pronounced in the latter. Peak DNA synthesis occurs in females just before onset of Vg synthesis and is coincident with predominant appearance of octoploid (8c) cells. In males DNA synthesis peaks earlier than in females, during transition from 2 c to 4 c cells, and then declines. Suppression of JH production by precocene prevents Vg synthesis completely and also inhibits DNA synthesis and generation of higher ploidy cells in both sexes. Reexposure of withdrawn animals to hormone reversed the foregoing conditions, but the disparity between sexes in timing of DNA synthesis and bulk tissue ploidy level was maintained. The developmental significance of these results is discussed.

Hormonal activation of protein kinase and lipid mobilization in the locust fat body in vitro

Cyclic nucleotide-activated protein kinase in locust fat body is elevated by extracts containing adipokinetic hormone (AKH) from locust corpora cardiaca or by synthetic redpigment-concentrating hormone (RPCH) which also exhibits lipid-mobilizing activity. Maximal activation is first attained in vitro 90 sec after RPCH is added to the incubation medium and 3.5 min after addition of AKH extract. This activation reflects the increase in endogenous cyclic nucleotides. Incubation of fat body in the presence of exogenous cyclic nucleotides activates lipase, and RPCH stimulation ultimately elevates the level of transported diacylglycerol 2.3-fold. RPCH stimulates fat body lipase in vivo.

Juvenile hormone-controlled vitellogenin synthesis in the fat body of the locust ( Locusta migratoria): Isolation and characterization of vitellogenin polysomes and their induction in vivo

With the aid of EGTA to control the endogenous RNase activity, a predominant population of heavy polysomes was isolated from the fat body of reproductively active female locusts. The identity of these as vitellogenin (Vg)-synthesizing polysomes was established by precipitation of the associated nascent polypeptides with anti-vitellin serum and by translation of the polysomal RNA in Xenopus laevis oöcytes. By EM observation these polysomes were found to contain 40–50 ribosome monomers. In normal development, accumulation of ribosomes in the fat body of female locusts began at about day 6 after adult ecdysis (terminal oöcyte = 2 mm or smaller), and was followed by the appearance of Vg-polysomes beginning at 3 mm terminal oöcyte length (about day 8–10 after adult ecdysis). The content of Vg-polysomes reached a maximum at 5–6 mm oöcyte length (about day 14), and then fell to an undetectable level as the oöcytes reached their maximum size (7 mm). In allatectomized females, Vg-polysomes were induced by treatment with an active juvenile hormone analogue, ZR-515. A single application with ZR-515 produced a massive accumulation of ribosomes and light polysomes in the first 48 hr which was followed by the rapid formation of Vg-polysomes to give a maximum at 72 hr. After the decay of this effect a second dose of ZR-515 resulted in the rapid appearance of Vg-polysomes without the initial generation of ribosomes.