Inhibited Juvenile Hormone Synthesis Research Articles

Inhibition of juvenile hormone synthesis in mosquitoes by the methylation inhibitor 3-deazaneplanocin A (DZNep)

Juvenile hormone (JH), synthesized by the corpora allata (CA), controls development and reproduction in mosquitoes through its action on thousands of JH-responsive genes. These JH-dependent processes can be studied using tools that increase or decrease JH titers in vitro and in vivo. Juvenile hormone acid methyl transferase (JHAMT) is a critical JH biosynthetic enzyme. JHAMT utilizes the methyl donor S-adenosyl-methionine (SAM) to methylate farnesoic acid (FA) into methyl farnesoate (MF), releasing the product S-adenosyl-L-homocysteine (AdoHcy), which inhibits JHAMT. S-adenosyl-homocysteine hydrolase (SAHH) catalyzes AdoHcy hydrolysis to adenosine and homocysteine, alleviating AdoHcy inhibition of JHAMT. 3-deazaneplanocin A (DZNep), an analog of adenosine, is an inhibitor of SAHH, and an epigenetic drug for cancer therapy. We tested the effect of DZNep on in vitro JH synthesis by CA of mosquitoes. DZNep inhibited JH synthesis in a dose-response fashion. Addition of MF, but not of FA relieved the inhibition, demonstrating a direct effect on JHAMT. In vivo experiments, with addition of DZNep to the sugar ingested by mosquitoes, resulted in a dose-response decrease in JH synthesis and JH hemolymphatic titers, as well as expression of early trypsin, a JH-dependent gene. Our studies suggest that DZNep can be employed to lower JH synthesis and titer in experiments evaluating JH-controlled processes in mosquitoes.

Evidence for a Phe-Gly-Leu-amide-like allatostatin in the beetle Tenebrio molitor

The allatostatins (ASTs) with Phe-Gly-Leu-amide C-terminal sequence are multifunctional neuropeptides discovered as inhibitors of juvenile hormone (JH) synthesis by corpora allata (CA) of cockroaches. Although these ASTs inhibit JH synthesis only in cockroaches, crickets, termites and locusts, isolation of peptides or of cDNA/genomic DNA or analysis of genomes indicates their occurrence in many orders of insects with the exception of coleopterans. The gene for these ASTs has not been found in the genome of the red flour beetle Tribolium castaneum (Family Tenebrionidae). Yet, in view of widespread occurrence of these peptides in insects, crustaceans and nematodes, they would be expected to occur in beetles. This study provides evidence for the presence of FGLa-like ASTs in the tenebrionid beetle, Tenebrio molitor, and scarabid beetle, Popillia japonica. Extract of brain from both beetles inhibited JH synthesis by cockroach CA dose dependently and reversibly. 20 brain equivalents of T. molitor and P. japonica extracts inhibited JH synthesis 64 ± 5 and 65 ± 0.6% respectively. Antibody against cockroach allatostatin ( Diploptera punctata AST-7) used in an enzyme-linked immunosorbent assay reacted with brain extract of these beetles. Antibody against D. punctata AST-5 localized FGLa-like ASTs in the brain and subesophageal ganglion of T. molitor and P. japonica. In addition, pretreatment of T. molitor brain extract with anti- D. punctata AST-5 reduced the inhibition of JH synthesis and pretreatment of anti- D. punctata AST-5 with D. punctata AST-5 diminished the immunoreactivity of the antibody. Thus we predict that FGLa-like allatostatins will be found in beetles.

Target of Rapamycin (TOR) Mediates the Transduction of Nutritional Signals into Juvenile Hormone Production

Anautogeny is a reproductive strategy by which females do not reproduce until they feed. Therefore, nutritional signals must inform the reproductive tissues, and cells that the organism has reached a nutritional status suitable for triggering reproductive processes. One of the possible pathways involved in anautogeny is the "target of rapamycin" (TOR) pathway, which has been described as connecting the nutritional status with growth, proliferation, and cancer. The German cockroach, Blattella germanica, is an anautogenous species whose vitellogenesis is governed by juvenile hormone. In the present report, we describe the cloning of TOR cDNA from B. germanica (BgTOR). Expression studies showed that BgTOR is expressed in adult female corpora allata and fat body. BgTOR knockdown using systemic RNAi in vivo produced a severe inhibition of juvenile hormone synthesis in adult female corpora allata, together with a reduction of mRNA levels corresponding to 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase-1, HMG-CoA synthase-2, and HMG-CoA reductase. In addition, there was a reduction of vitellogenin mRNA in the fat body, and ovaries did not grow. Analysis of TOR expression in corpora allata of fed and starved females suggested that TOR is not regulated at the transcriptional level. Nevertheless, there was a reduction in HMG-CoA synthases and reductase mRNA in corpora allata (but not in the fat body) of starved females, together with a dramatic reduction of juvenile hormone production and ovary development. Taken together, our results indicate that TOR knockdown mimics starvation in terms of corpora allata activity, and suggest that nutritional signals that activate juvenile hormone biosynthesis and vitellogenin production are mediated by the TOR pathway.

Isolation of the gene for the precursor of Phe-Gly-Leu-amide allatostatins in the termite Reticulitermes flavipes

Allatostatins (ASTs), with a C-terminal sequence Tyr/Phe-Xaa-Phe-Gly-Leu/Ile-amide, are multifunctional neuropeptides that were first discovered by their ability to inhibit juvenile hormone (JH) synthesis by the corpora allata (CA) in cockroaches. These A-type ASTs have since been demonstrated to inhibit JH synthesis in crickets, termites and more recently locusts. The gene for the precursor of A-type ASTs has been identified in several species of cockroaches, in crickets and in locusts, but not yet in termites, although 5 AST peptides were isolated from the lower termite Reticulitermes flavipes that are identical to known cockroach ASTs. In this study, primers designed from AST amino acid sequences of cockroaches are used to identify the gene for the preproAST peptides in R. flavipes. In addition, the expression of the gene in brain tissues is demonstrated for egg-laying and non-egg-laying neotenic reproductives. The gene codes for 14 individual peptides and its sequence is closer to that of cockroaches and the cricket than to that of other insect orders in which these peptides do not act as allatostatins. Among the known cockroach AST genes, the termite AST gene is most similar to that of Periplaneta americana, a species belonging to the primitive family Blattidae.

Identification of Phe-Gly-Leu-amide type allatostatin-7 in Reticulitermes flavipes: Its localization in tissues and relation to juvenile hormone synthesis

The allatostatins (ASTs), with a Tyr/Phe-Xaa-Phe-Gly-Leu/Ile-amide C-terminus, are neuropeptides that occur in many orders of insects, but are known to inhibit juvenile hormone (JH) synthesis by corpora allata (CA) only in cockroaches, crickets, and termites. 5 AST peptides with similar sequences to those of 6 species of cockroaches have been isolated and sequenced from extract of brain tissue of the termite Reticulitermes flavipes. The amino acid sequence of a 6th peptide, R. flavipes AST-7, determined by LC–MS/MS following HPLC fractionation of brain extract, is S-P-S-S-G-N-Q-R-L-Y-G-F-G-L-NH 2. The 8 terminal amino acids are identical to AST-7 of the cockroach Diploptera punctata. R. flavipes and D. punctata AST-7s inhibited JH synthesis by CA of both species equally and their affinity for antibody against D. punctata AST-7 is similar. Immunoreactivity of termite tissue with this antibody indicates neuro- and myomodulatory activity of the peptide in addition to its demonstrated allatostatic function. The density of AST immunostaining in axons within the CA of R. flavipes and the rate of JH synthesis by similar glands were negatively correlated. This is evidence that when AST is abundant in the glands it is being released in vivo to limit JH production.

Structure–activity studies with endogenous allatostatins from Periplaneta americana: Expressed receptor compared with functional bioassay

The A-allatostatins (F/YXFGLamides) are insect neuropeptides with inhibitory actions on juvenile hormone (JH) synthesis, muscular contraction and vitellogenesis. They exist in multiple forms within each species. In the cockroach, Periplaneta americana, only one receptor for A-allatostatin has been identified thus far. Here, we have characterised the receptor response to all 15 of the endogenous A-allatostatins encoded by the P. americana allatostatin prohormone gene, together with some analogues, using an indirect heterologous system involving co-expression of the receptor and a potassium channel subunit in Xenopus laevis oocytes and electrophysiological measurements. We have also determined the relative potency of the same peptides to inhibit JH synthesis in corpora allata. Our data reveal that the heterologously expressed receptor responds to all of the endogenous allatostatins and, although differences in potency are recorded, this cannot readily be related to particular differences in the primary structure of the peptides. Similarly, all allatostatins act on the corpora allata to inhibit the synthesis of JH, again with varying potency not readily related to peptide structure. Interestingly, some of the peptides did not perform consistently across the two assays. We show that the receptor is widely expressed in adult P. americana tissues (head, retrocerebral glands, fat body, ovary, male accessory gland, gut, leg muscle, Malpighian tubule and nerve cord) as well as in early larval instars. The spatial expression supports the known pleiotropic activity of allatostatins and role as a paracrine effector. This is the first report of such a detailed characterisation of an invertebrate receptor for allatostatin.

Allatoregulatory peptides in Lepidoptera, structures, distribution and functions

Allatoregulatory peptides either inhibit (allatostatins) or stimulate (allatotropins) juvenile hormone (JH) synthesis by the corpora allata (CA) of insects. However, these peptides are pleitropic, the regulation of JH biosynthesis is not their only function. There are currently three allatostatin families (A-, B-, and C-type allatostatins) that inhibit JH biosynthesis, and two structurally unrelated allatotropins. The C-type allatostatin, characterised by its blocked N-terminus and a disulphide bridge between its two cysteine residues, was originally isolated from Manduca sexta. This peptide exists only in a single from in Lepidoptera and is the only peptide that has been shown to inhibit JH synthesis by the CA in vitro in this group of insects. The C-type allatostatin also inhibits spontaneous contractions of the foregut. The A-type allatostatins, which exist in multiple forms in a single insect, have also been characterised from Lepidoptera. This family of peptides does not appear to have any regulatory effect on JH biosynthesis, but does inhibit foregut muscle contractions. Two structurally unrelated allatotropins stimulate JH biosynthesis in Lepidoptera. The first was identified in M. sexta (Manse-AT) and occurs in other moths. The second (Spofr AT2) has only been identified in Spodoptera frugiperda. Manduca sexta allatotropin also stimulates heart muscle contractions and gut peristalsis, and inhibits ion transport across the midgut of larval M. sexta. The C-terminal (amide) pentapeptide of Manse-AT is important for JH biosynthesis activity. The most active conformation of Manse-AS requires the disulphide bridge, although the aromatic residues also have a significant effect on biological activity. Both A- and C-type allatostatins and Manse-AT are localised in neurosecretory cells of the brain and are present in the corpora cardiaca, CA and ventral nerve cord, although variations in localisation exist in different moths and at different stages of development. The presence of Manse-AS and Manse-AT in the CA correlates with the biological activity of these peptides on JH biosynthesis. There is currently no explanation for the presence of A-type allatostatins in the CA. The three peptide types are also co-localised in neurosecretory cells of the frontal ganglion, and are present in the recurrent nerve that supplies the muscles of the gut, particularly the crop and stomodeal valve, in agreement with their role in the regulation of gut peristalsis. There is also evidence that they are expressed in the midgut and reproductive tissues.

Allatotropin regulation of juvenile hormone synthesis by the corpora allata from the lubber grasshopper, Romalea microptera

The in vitro synthesis of juvenile hormone (JH) by corpora allata (CA) from the lubber grasshopper ( Romalea microptera) was stimulated by low concentrations of brain extract and this effect was reduced at higher concentrations, suggesting the presence of allatotropin (AT) and allatostatin (AST) factors in the brain. The AT activity of brain extracts caused a rapid and reversible stimulation and appeared to be a peptide(s). Reversed phase (C18) HPLC analysis of brain extracts disclosed two peaks of AT activity but no significant AST activity. Manse-AT, Schgr-NPF, and Locmi-FLRF had no effect on JH synthesis by lubber CA, indicating that the Rommi-AT factors are distinct from these peptides. High concentrations of Dippu-AST-7 and Grybi-AST-1 inhibited JH synthesis, implying that AST factors might be present in lubber grasshoppers. CA response to AT activity of brain extracts varied during the oviposition cycle (∼35 days), with the maximum response occurring on days 16–18. AT activity of brain extracts also varied during the cycle, being highest on day 25. Our data suggest that the lubber CA is largely regulated by AT activity, and that JH synthesis reflects both CA response to AT activity and the level of AT activity in the brain.

Inhibition of juvenile hormone biosynthesis in mosquitoes: effect of allatostatic head factors, PISCF- and YXFGL-amide-allatostatins

We investigated the role of head factors and allatostatins (ASs) on the regulation of juvenile hormone (JH) synthesis in female adult mosquito. The biosynthetic activity of the Aedes aegypti corpora allata (CA) in vitro was inhibited by factors present in the head. Disconnecting the CA from the brain resulted in a significant increase in the rate of JH biosynthesis. Inhibition was not dependent on intact nervous connections; co-incubation of CA with brains or brain extracts resulted in a significant decrease of JH biosynthesis. This inhibitory effect of brain extracts was reversible and heat stable; extracts lost the inhibitory activity after proteinase K digestion suggesting a peptidic structure. In a first attempt to elucidate the nature of this inhibitory factor, we tested in our CA in vitro system the effect of members of two families of allatostatins already described in mosquitoes. Anopheles gambiae PISCF-allatostatin (homolog to Manduca PISCF-allatostatin) significantly inhibited JH synthesis, while Ae. aegypti YXFGL-amide-allatostatins (homologs to cockroach YXFGL-amide-allatostatins) did not affect JH synthesis. These results represent the first description of an allatostatic effect of PISCF-allatostatins outside the Lepidoptera.

Manipulation of intracellular calcium affects in vitro juvenile hormone synthesis by larval corpora allata of Manduca sexta

The effect of altering intracellular free Ca 2+ on juvenile hormone (JH) and acid synthesis by larval and pupally-committed corpora allata (CA) of fifth stadium Manduca sexta was investigated. Larval CA required extracellular Ca 2+ ≧ 0.1 mM for maximal JH synthesis, while JH acid synthesis by glands after pupal commitment was independent of extracellular Ca 2+. Free Ca 2+ in the hemolymph ranged from 1.4 to 2.1 mM during the fifth stadium. Both calcium ionophores and caffeine, which releases Ca 2+ from intracellular stores, inhibited JH synthesis by larval CA but stimulated JH acid synthesis by post-commitment CA. These results suggest that intracellular stores may be the principal source of Ca 2+ for the biosynthetic activity of the post-commitment gland. Calcium channel blockers (La 3+, Cd 2+) and antagonists (verapamil, isradipine and nitrendipine) decreased both JH and JH acid synthesis, indicating the existence of Ca 2+ channels in the CA cell membrane. Calmodulin (CaM) antagonists inhibited the activity of both larval and post-commitment CA, suggesting an integral relationship of CaM to the effects of Ca 2+ on gland activity. One of these effects is the demonstrated requirement of 0.1 mM extracellular Ca 2+ for allatostatin inhibition of JH I synthesis by larval CA.

Inhibition of juvenile hormone III biosynthesis in cockroach corpora allata by interference with the S‐adenosylmethionine‐dependent transmethylation

AbstractThe synthesis of juvenile hormone‐III by corpora allata of the cockroach Diploptera punctata is dependent under in vitro conditions upon a supply of exogenous methionine. Radiolabelled S‐adenosylmethionine was identified by HPLC in extracts of corpora allata incubated with either [methyl‐3H]methionine or [35S]methionine. Juvenile hormone (JH) synthesis by intact glands in vitro was inhibited by cycloleucine and selenomethionine, but this inhibition could be relieved by increasing the concentration of methionine. S‐adenosylhomocysteine or sinefungin had little or no inhibitory effect on JH synthesis by intact glands, but 5′‐deoxy‐5′‐methylthioadenosine was inhibitory. Adenosine and homocysteine synergistically inhibited JH synthesis. These results show that JH‐III synthesis by intact glands can be inhibited by interfering with the S‐adenosylmethionine‐dependent transmethylation, and suggest that the product and inhibitor of that reaction, S‐adenosyl‐homocysteine, is rapidly hydrolyzed to adenosine and homocysteine in the corpora allata.