Julie Tobback

The cloning, phylogenetic relationship and distribution pattern of two new putative GPCR-type octopamine receptors in the desert locust (Schistocerca gregaria).

The biogenic amine octopamine functions as a neuromodulator, neurotransmitter and neurohormone in insect nervous systems. It plays a prominent role in modulating multiple physiological and behavioural processes in invertebrates. Octopamine exerts its effects by binding to specific receptor proteins that belong to the superfamily of G protein-coupled receptors. We found two partial sequences of putative octopamine receptors in the desert locust Schistocerca gregaria (SgOctalphaR and SgOctbetaR) and investigated their transcript levels in males and females of both phases and during the transition between long-term solitarious and gregarious locusts. The transcript levels of SgOctalphaR are the highest in the central nervous system, whereas those of SgOctbetaR are the highest in the flight muscles, followed by the central nervous system. Both SgOctalphaR and SgOctbetaR show higher transcript levels in long-term gregarious locusts as compared to solitarious ones. The rise of SgOctbetaR transcript levels already appears during the first 4h of gregarisation, during which also the behavioural changes take place.

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 article presents in the first part a study on the for gene, encoding a cGMP-dependent protein kinase (PKG) in the bumblebee Bombus terrestris. Cloning of the open reading frame 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 with 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 bumblebees of B. terrestris were treated with two insecticides imidacloprid and kinoprene, which have shown to cause impaired foraging behavior. Compared with controls, only the latter treatment resulted in a decreased Btfor expression, which concurs with a stimulation of ovarian growth and a shift in labor toward nest-related tasks. The data are discussed in relation to Btfor expression in the complex physiological event of foraging and side-effects by pesticides.

Locust phase polyphenism: Does epigenetic precede endocrine regulation?

The morphological, physiological and behavioural differences between solitarious and gregarious desert locusts are so pronounced that one could easily mistake the two phases as belonging to different species, if one has no knowledge of the phenomenon of phenotypic plasticity. A number of phase-specific features are hormonally controlled. Juvenile hormone promotes several solitarious features, the green cuticular colour being the most obvious one. The neuropeptide corazonin elicits the dark cuticular colour that is typical for the gregarious phase, as well as particular gregarious behavioural characteristics. However, it had to be concluded, for multiple reasons, that the endocrine system is not the primary phase-determining system. Our observation that longevity gets imprinted in very early life by crowding of the young hatchlings, and that it cannot be changed thereafter, made us consider the possibility that, perhaps, epigenetic control of gene expression might be, if not the missing, a primary phase-determining mechanism. Imprinting is likely to involve DNA methylation and histone modification. Analysis of a Schistocerca EST database of nervous tissue identified the presence of several candidate genes that may be involved in epigenetic control, including two DNA methyltransferases (Dnmts). Dnmt1 and Dnmt2 are phase-specifically expressed in certain tissues. In the metathoracic ganglion, important in the serotonin pathway for sensing mechanostimulation, their expression is clearly affected by crowding. Our data urge for reconsidering the role of the endocrine system as being sandwiched in between genetics and epigenetics, involving complementary modes of action.

Cloning and expression of PKG, a candidate foraging regulating gene in Vespula vulgaris

In honey bees, enhancement of cGMP-dependent protein kinase (PKG) expression accompanies a behavioural transition from in-hive working nursing bees towards outdoors foraging worker bees. Accordingly this gene was named amfor or Apis mellifera foraging gene. In the red harvester ant Pogonomyrmex barbatus a gene homologue aff ected food seeking behaviour as well, but in this species PKG expression decreased from the onset of foraging behaviour. Since the wasp Vespula vulgaris is phylogenetically positioned between ants and bees, in this paper we tried to elucidate whether the involvement of PKG in foraging behaviour can be extended to this species and if so, whether its expression is enhanced or decreased by the transition from nursing to foraging. To enable this candidate gene approach, we fi rst had to clone the PKG homologue from the common wasp. QPCR indicated a relevantly higher expression of Vvfor in nursing versus foraging wasps although interpretation of the results was hampered by a remarkable degree of variation as could be predicted from wasps captured in the wild as a source for mRNA extraction and quantifi cation.

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.

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.

Developmental‐ and food‐dependent foraging transcript levels in the desert locust

Drastic changes in the environment during a lifetime require developmental and physiological flexibility to ensure animal survival. Desert locusts, Schistocerca gregaria, live in an extremely changeable environment, which alternates between periods of rainfall and abundant food and periods of drought and starvation. In order to survive, locusts display an extreme form of phenotypic plasticity that allows them to rapidly cope with these changing conditions by converting from a cryptic solitarious phase to a swarming, voracious gregarious phase. To accomplish this, locusts possess different conserved mediators of phenotypic plasticity. Recently, attention has been drawn to the possible roles of protein kinases in this process. In addition to cyclic AMP‐dependent protein kinase (PKA), also cyclic GMP‐dependent protein kinase (PKG), which was shown to be involved in changes of food‐related behavior in a variety of insects, has been associated with locust phenotypic plasticity. In this article, we study the transcript levels of the S. gregaria orthologue of the foraging gene that encodes a PKG in different food‐related, developmental and crowding conditions. Transcript levels of the S. gregaria foraging orthologue are highest in different parts of the gut and differ between isolated and crowd‐reared locusts. They change when the availability of food is altered, display a distinct pattern with higher levels after a moult and decrease with age during postembryonic development.

RNA interference mortality points to noncircadian functions for the clock gene in the desert locust Schistocerca gregaria

One of the core genes in the circadian regulation network is clock (clk). By forming a heterodimer with CYCLE (CYC) that binds on an E‐box in the promoter region, it induces the transcription of other elements in the circadian transcriptional feedback loops and different clock output genes. In contrast to other insects, a clk double‐stranded RNA (dsRNA) treatment is lethal in adults and fifth instar nymphs of the desert locust, Schistocerca gregaria, in a dose‐dependent manner. Clk knock down fifth instar nymphs are able to undergo their imaginal moult but, depending on the amount of dsRNA, it takes them longer than the controls to reach adulthood. As adults, clk knock down animals do not develop their fat body and ovaries like the control animals. Therefore, we tested the expression of different genes involved in energy metabolism and reproduction to see the effect of the clk RNA interference knock down. Surprisingly, the expression of the vitellogenin gene was up‐regulated in the clk knock down females who did not appear to invest their energy in egg development. Taken together, our results point out that the clk gene in the desert locust has an additional function in development besides its established role in maintaining the circadian rhythms in the brain.