The FGLamide-Allatostatins Influence Foraging Behavior in Drosophila melanogaster

Allatostatins (ASTs) are multifunctional neuropeptides that generally act in an inhibitory fashion. ASTs were identified as inhibitors of juvenile hormone biosynthesis. Juvenile hormone regulates insect metamorphosis, reproduction, food intake, growth, and development. Drosophila melanogaster RNAi lines of PheGlyLeu-amide-ASTs (FGLa/ASTs) and their cognate receptor, Dar-1, were used to characterize roles these neuropeptides and their respective receptor may play in behavior and physiology. Dar-1 and FGLa/AST RNAi lines showed a significant reduction in larval foraging in the presence of food. The larval foraging defect is not observed in the absence of food. These RNAi lines have decreased for transcript levels which encodes cGMP- dependent protein kinase. A reduction in the for transcript is known to be associated with a naturally occuring allelic variation that creates a sitter phenotype in contrast to the rover phenotype which is caused by a for allele associated with increased for activity. The sitting phenotype of FGLa/AST and Dar-1 RNAi lines is similar to the phenotype of a deletion mutant of an AST/galanin-like receptor (NPR-9) in Caenorhabditis elegans. Associated with the foraging defect in C. elegans npr-9 mutants is accumulation of intestinal lipid. Lipid accumulation was not a phenotype associated with the FGLa/AST and Dar-1 RNAi lines

Methyl Farnesoate Plays a Dual Role in Regulating \u3cem\u3eDrosophila\u3c/em\u3e Metamorphosis

Corpus allatum (CA) ablation results in juvenile hormone (JH) deficiency and pupal lethality in Drosophila. The fly CA produces and releases three sesquiterpenoid hormones: JH III bisepoxide (JHB3), JH III, and methyl farnesoate (MF). In the whole body extracts, MF is the most abundant sesquiterpenoid, followed by JHB3 and JH III. Knockout of JH acid methyl transferase (jhamt) did not result in lethality; it decreased biosynthesis of JHB3, but MF biosynthesis was not affected. RNAi-mediated reduction of 3-hydroxy-3-methylglutaryl CoA reductase (hmgcr) expression in the CA decreased biosynthesis and titers of the three sesquiterpenoids, resulting in partial lethality. Reducing hmgcr expression in the CA of the jhamt mutant further decreased MF titer to a very low level, and caused complete lethality. JH III, JHB3, and MF function through Met and Gce, the two JH receptors, and induce expression of Kr-h1, a JH primary-response gene. As well, a portion of MF is converted to JHB3 in the hemolymph or peripheral tissues. Topical application of JHB3, JH III, or MF precluded lethality in JH-deficient animals, but not in the Met gce double mutant. Taken together, these experiments show that MF is produced by the larval CA and released into the hemolymph, from where it exerts its anti-metamorphic effects indirectly after conversion to JHB3, as well as acting as a hormone itself through the two JH receptors, Met and Gce

Methyl Farnesoate Plays a Dual Role in Regulating \u3cem\u3eDrosophila\u3c/em\u3e Metamorphosis

Corpus allatum (CA) ablation results in juvenile hormone (JH) deficiency and pupal lethality in Drosophila. The fly CA produces and releases three sesquiterpenoid hormones: JH III bisepoxide (JHB3), JH III, and methyl farnesoate (MF). In the whole body extracts, MF is the most abundant sesquiterpenoid, followed by JHB3 and JH III. Knockout of JH acid methyl transferase (jhamt) did not result in lethality; it decreased biosynthesis of JHB3, but MF biosynthesis was not affected. RNAi-mediated reduction of 3-hydroxy-3-methylglutaryl CoA reductase (hmgcr) expression in the CA decreased biosynthesis and titers of the three sesquiterpenoids, resulting in partial lethality. Reducing hmgcr expression in the CA of the jhamt mutant further decreased MF titer to a very low level, and caused complete lethality. JH III, JHB3, and MF function through Met and Gce, the two JH receptors, and induce expression of Kr-h1, a JH primary-response gene. As well, a portion of MF is converted to JHB3 in the hemolymph or peripheral tissues. Topical application of JHB3, JH III, or MF precluded lethality in JH-deficient animals, but not in the Met gce double mutant. Taken together, these experiments show that MF is produced by the larval CA and released into the hemolymph, from where it exerts its anti-metamorphic effects indirectly after conversion to JHB3, as well as acting as a hormone itself through the two JH receptors, Met and Gce

MicroRNA clusters integrate evolutionary constraints on expression and target affinities : the miR-6/5/4/286/3/309 cluster in Drosophila

This research was supported by the Hong Kong Research Grant Council GRF Grant (14103516), The Chinese University of Hong Kong Direct Grant (4053248), and TUYF Charitable Trust (6903957) (JHLH).A striking feature of microRNAs is that they are often clustered in the genomes of animals. The functional and evolutionary consequences of this clustering remain obscure. Here, we investigated a microRNA cluster miR-6/5/4/286/3/309 that is conserved across drosophilid lineages. Small RNA sequencing revealed expression of this microRNA cluster in Drosophila melanogaster leg discs, and conditional overexpression of the whole cluster resulted in leg appendage shortening. Transgenic overexpression lines expressing different combinations of microRNA cluster members were also constructed. Expression of individual microRNAs from the cluster resulted in a normal wild-type phenotype, but either the expression of several ancient microRNAs together (miR-5/4/286/3/309) or more recently evolved clustered microRNAs (miR-6-1/2/3) can recapitulate the phenotypes generated by the whole-cluster overexpression. Screening of transgenic fly lines revealed down-regulation of leg patterning gene cassettes in generation of the leg-shortening phenotype. Furthermore, cell transfection with different combinations of microRNA cluster members revealed a suite of downstream genes targeted by all cluster members, as well as complements of targets that are unique for distinct microRNAs. Considered together, the microRNA targets and the evolutionary ages of each microRNA in the cluster demonstrates the importance of microRNA clustering, where new members can reinforce and modify the selection forces on both the cluster regulation and the gene regulatory network of existing microRNAs.PostprintPeer reviewe

Juvenile hormone counteracts the bHLH-PAS transcription factors MET and GCE to prevent caspase-dependent programmed cell death in Drosophila

Juvenile hormone (JH) regulates many developmental and physiological events in insects, but its molecular mechanism remains conjectural. Here we report that genetic ablation of the corpus allatum cells of th

A Compilation of the Diverse miRNA Functions in <i>Caenorhabditis elegans</i> and <i>Drosophila melanogaster</i> Development

MicroRNAs are critical regulators of post-transcriptional gene expression in a wide range of taxa, including invertebrates, mammals, and plants. Since their discovery in the nematode, Caenorhabditis elegans, miRNA research has exploded, and they are being identified in almost every facet of development. Invertebrate model organisms, particularly C. elegans, and Drosophila melanogaster, are ideal systems for studying miRNA function, and the roles of many miRNAs are known in these animals. In this review, we compiled the functions of many of the miRNAs that are involved in the development of these invertebrate model species. We examine how gene regulation by miRNAs shapes both embryonic and larval development and show that, although many different aspects of development are regulated, several trends are apparent in the nature of their regulation

The foraging distance of third instar larvae (path length) A. in the presence of food and B. absence of food for 5 mins was measured for controls 6896 x <i>w¹¹¹⁸</i>(black bar), <i>w¹¹¹⁸</i> self-cross (grey bar) and Dar-1and FGLa/AST RNAi lines crossed to 6896 (patterned bar) or <i>w¹¹¹⁸</i> (white bar); N = 30–34.

<p>The foraging distance of third instar larvae (path length) A. in the presence of food and B. absence of food for 5 mins was measured for controls 6896 x <i>w¹¹¹⁸</i>(black bar), <i>w¹¹¹⁸</i> self-cross (grey bar) and Dar-1and FGLa/AST RNAi lines crossed to 6896 (patterned bar) or <i>w¹¹¹⁸</i> (white bar); N = 30–34.</p

<i>for</i> mRNA expression levels of controls DaGal4 x <i>w¹¹¹⁸</i> (black bar), <i>w¹¹¹⁸</i> self-cross (grey bar), 6896 X <i>w¹¹¹⁸</i> (black bar with white diagonal stripes) and experimental Dar-1 and FGLa/AST RNAi lines x DaGal4 (patterned bars) or <i>w¹¹¹⁸</i>(white bars) and Dar1 and FGLa/AST RNAi lines x 6896 (white bars with black diagonal stripes).

<p>Each bar represents three independent RNA extractions that were each assayed by qPCR three times. Thirty third instar larvae were used for each extraction. Expression levels were normalized using RP49 as a standard. Asterisks indicate a significant difference * = p<0.05 and ** = p<0.001. Only the significance in comparison with <i>w¹¹¹⁸</i> self-cross is indicated although comparison with DaGal4 X <i>w¹¹¹⁸</i> was equivalent.</p

Relative mRNA expression levels of individual <i>D. melanogaster</i> stocks expressing RNAi to gene sequences for Dar-1 and FGLa/AST.

<p><b>A</b>. Dar-1 or FGLa/AST mRNA levels were measured in DaGal4 x <i>w¹¹¹⁸</i> and RNA levels in RNAi lines crossed to either DaGal4 (patterned bar) or <i>w¹¹¹⁸</i>(white bar) were measured relative to this control. <b>B</b>. The same comparisons as in A. with driver line 6896 X <i>w¹¹¹⁸</i> serving as the control. The number associated with each RNAi stock is the Transformation ID established by the Vienna <i>Drosophila</i> RNAi Center. Each bar represents two independent RNA extractions that were each assayed by qPCR three times. Thirty third instar larvae were used for each extraction. Expression levels were normalized using RP49 (ribosomal protein) as a standard. Asterisks indicate significant difference * = P<0.05; **P<0.001; ***P<0.0001.</p