Molecular genetic analysis of Drosophila melanogaster leucine-rich repeats containing GPCRs and sex peptide receptor.

In this study, several G protein-coupled receptors (GPCRs) of Drosophila melanogaster were investigated. This family constitutes the most extensive group of signal transducing receptor proteins and therefore, they are essential in intercellular communication. The larger part of the study was devoted to investigating the leucine-rich repeats containing GPCRs (LGRs), a family of rhodopsin-like GPCRs that are characterised by their exceptionally large extracellular domain. The genome of the fruit fly encodes four of these receptors of which only one (LGR2) has been extensively characterised. In a first approach, the transcript profile for the four LGRs was studied during the various developmental stages of the fruit fly and in different organs of the third larval and adult stage. Furthermore, the activity of the lgr1 gene promoter was characterised by means of a promoter reporter line. Analyses of the results showed that expression is highest in the adult salivary glands, the Malpighian tubules and the hindgut. Also, in the testes weak reporter signals could be detected. Using RNA interference to silence lgr1, itwas observed that pupariation was defective in these fruit flies. In association with this effect, it was shown that the titer of the moulting hormone 20-hydroxyecdysone (20E) remained low in comparison to control larvae and that the transcript levels of some genes known to be involved in the biosynthesis of 20E were significantly lowered. RNAi mediated knockdown of lgr1 in adults resulted in a quicker deathwhen the flies faced desiccation. With the objective of identifying the possible ligand(s) for LGR1, this receptor was exposed to ion transport peptide and prothoracicotropic hormone in a cell-based receptor activity assay. In an attempt to deorphanise LGR3 and/or 4, a peptide synthesis protocol was successfully developed for the production of D. melanogaster insulin-like peptide 2. However, in none of these tested cases was receptor activation witnessed.Another part of the presented work was focussed on the study of the D. melanogaster sex peptide receptor (SPR) which was characterised as such a few years ago. In this study, it was demonstrated that in addition to sex peptide (SP), the five myoinhibiting peptides (MIPs) from the fruit fly are potent agonists of SPR, as well as a chimeric peptide consisting of part of the N-terminal sequence of SP fused to MIP4. When SP or the chimeric peptide were injected into the haemolymph of virgin females, egg laying was induced. This was not the case when the flies were injected with MIPs.Transcripts of spr were demonstrated in all developmental stages. This is in contrast to sp, for which notable transcript levels can only be detected in male adults. On the other hand, mip transcript was present during all developmental stages. Furthermore, SPR homologues were identified in all insects with available genome data, except Hymenoptera. The same goes for MIP but not for SP, which can only be found in some Drosophila species. These observations have led to the conclusion that SPR is likely a MIP receptor that, during the course of evolution, was hijacked by SP for the function of post-mating responses in some Drosophila species. Other functionalities of SPR are likely still mediated by MIPactivation of the receptor.status: publishe

Sex peptides and MIPs can activate the same G protein-coupled receptor

In many animal species, copulation elicits a number of physiological and behavioral changes in the female partner. In Drosophila melanogaster, the main molecular effector of these physiological responses has been identified as sex peptide (SP). The sex peptide receptor (SPR) has been characterized and recently, its activation by Drosophila myoinhibiting peptides (MIPs)-in addition to SP-has been demonstrated. The myoinhibiting peptides are members of a conserved peptide family, also known as B-type allatostatins, which generally feature the C-terminal motif -WX6Wamide.status: publishe

B-Type Allatostatins and Sex peptides

edition: Secondstatus: publishe

Male reproduction is affected by RNA interference of period and timeless in the desert locust Schistocerca gregaria

In all living organisms, behavior, metabolism and physiology are under the regulation of a circadian clock. The molecular machinery of this clock has been conserved throughout the animal kingdom. Besides regulating the circadian timing of a variety of processes through a central oscillating mechanism in the brain, these circadian clock genes were found to have a function in peripheral tissues in different insects. Here, we provide evidence that the circadian clock genes period (per) and timeless (tim) have a role in the male locust reproduction. A knockdown of either of the two genes has no effect on male sexual maturation or behavior, but progeny output in their untreated female copulation partners is affected. Indeed, the fertilization rates of the eggs are lower for females with a per or tim RNAi copulation partner as compared to the eggs deposited by females that mated with a control male. As the sperm content of the seminal vesicles is higher in per or tim knockdown males, we suggest that this phenotype could be caused by a disturbance of the circadian regulated sperm transfer in the male reproductive organs, or an insufficient maturation of the sperm after release from the testes.status: publishe

The circadian clock genes affect reproductive capacity in the desert locust Schistocerca gregaria

The circadian clocks govern many metabolic and behavioral processes in an organism. In insects, these clocks and their molecular machinery have been found to influence reproduction in many different ways. Reproductive behavior including courtship, copulation and egg deposition, is under strong influence of the daily rhythm. At the molecular level, the individual clock components also have their role in normal progress of oogenesis and spermatogenesis. In this study on the desert locust Schistocerca gregaria, three circadian clock genes were identified and their expression profiles were determined. High expression was predominantly found in reproductive tissues. Similar daily expression profiles were found for period (per) and timeless (tim), while the clock (clk) mRNA level is higher 12h before the first per and tim peak. A knockdown of either per or tim resulted in a significant decrease in the progeny produced by dsRNA treated females confirming the role of clock genes in reproduction and providing evidence that both PER and TIM are needed in the ovaries for egg development. Since the knockdown of clk is lethal for the desert locust, its function remains yet to be elucidated.status: publishe

Age- and task-dependent foraging gene expression in the bumblebee Bombus terrestris

In eusocial insects, the division of labor within a colony, based on either age or size, is correlated with a differential foraging (for) gene expression and PKG activity. This paper presents in a first part a study on the for gene, encoding a cGMP-dependent protein kinase (PKG) in the bumblebee Bombus terrestris. Cloning of the ORF allowed phylogenetic tracing which showed conservation of PKGs among social insects. Our results confirm the proposed role for PKGs in division of labor. Btfor gene expression is significantly higher in the larger foragers compared to the smaller sized nurses. More importantly, we discovered an age-related decrease in Btfor expression in both nursing and foraging bumblebees. We therefore speculate that the presence of BtFOR is required for correct adaptation to new external stimuli and rapid learning for foraging. In a second series of experiments, worker bumblebee of B. terrestris were treated with two insecticides imidacloprid and kinoprene that have shown to cause impaired foraging behavior. Compared to controls, only the latter treatment resulted in a decreased Btfor expression which concurs with a stimulation of ovarian growth and a shift in labor towards nest related tasks. The data are discussed in relation to Btfor expression in the complex physiological event of foraging and side-effects by pesticides.status: publishe

Fruitless RNAi knockdown in the desert locust, Schistocerca gregaria, influences male fertility

In Drosophila melanogaster, the male-specific splice isoform of the fruitless gene (Fru(M)) encodes a set of transcription factors that are involved in the regulation of male courtship and copulation. Recent insights from non-drosophilid insects suggest a conserved evolutionary role for the transcription factor Fruitless. In the desert locust, Schistocerca gregaria and the German cockroach, Blatella germanica, both orthopteran insects, a conserved functional role for fruitless has been proposed. Fru specific RNAi knockdown in the third nymphal stage of male Schistocera gregaria delays copulation initiation and results in reduced progeny. In order to identify the origin of the observed phenotypic effects following a fruitless RNAi treatment in the male, we show that the fru knockdown has no detectable effect on spermio- or spermatogenesis and on the transfer of spermatozoa during copulation. Nevertheless, it is clear that the male seminal vesicles contain significantly less spermatozoa after fru RNAi as compared to gfp RNAi controls. We conclude that a lowered male fertility, caused by the fru knockdown in male desert locusts may be the direct cause for the reduction of the progeny numbers in their naïve female copulation partners.status: publishe

Cloning, constitutive activity and expression profiling of two receptors related to relaxin receptors in Drosophila melanogaster

Leucine-rich repeat containing G protein-coupled receptors (LGRs) comprise a cluster of transmembrane proteins, characterized by the presence of a large N-terminal extracellular domain. This receptor group can be classified into three subtypes. Belonging to the subtype C LGRs are the mammalian relaxin receptors LGR7 (RXFP1) and LGR8 (RXFP2), which mediate important reproductive and other processes. We identified two related receptors in the genome of the fruit fly and cloned their open reading frames into an expression vector. Interestingly, dLGR3 demonstrated constitutive activity at very low doses of transfected plasmid, whereas dLGR4 did not show any basal activity. Both receptors exhibited a similar expression pattern during development, with relatively high transcript levels during the first larval stage. In addition, both receptors displayed higher expression in male adult flies as compared to female flies. Analysis of the tissue distribution of both receptor transcripts revealed a high expression of dLGR3 in the female fat body, while the expression of dLGR4 peaked in the midgut of both the wandering and adult stage.publisher: Elsevier articletitle: Cloning, constitutive activity and expression profiling of two receptors related to relaxin receptors in Drosophila melanogaster journaltitle: Peptides articlelink: http://dx.doi.org/10.1016/j.peptides.2014.07.014 content_type: article copyright: Copyright © 2014 Elsevier Inc. All rights reserved.status: publishe

LGR1 signalling is required in the development of the fruit fly, Drosophila melanogaster

status: publishe