Cynthia Lenaerts

The pleiotropic allatoregulatory neuropeptides and their receptors: A mini-review

Juvenile hormones (JH) are highly pleiotropic insect hormones essential for post-embryonic development. The circulating JH titer in the hemolymph of insects is influenced by enzymatic degradation, binding to JH carrier proteins, uptake and storage in target organs, but evidently also by rates of production at its site of synthesis, the corpora allata (CA). The multiple processes in which JH is involved alongside the critical significance of JH in insect development emphasize the importance for elucidating the control of JH production. Production of JH in CA cells is regulated by different factors: by neurotransmitters, such as dopamine and glutamate, but also by allatoregulatory neuropeptides originating from the brain and axonally transported to the CA where they bind to their G protein-coupled receptors (GPCRs). Different classes of allatoregulatory peptides exist which have other functions aside from acting as influencers of JH production. These pleiotropic neuropeptides regulate different processes in different insect orders. In this mini-review, we will give an overview of allatotropins and allatostatins, and their recently characterized GPCRs with a view to better understand their modes of action and different action sites.

Receptors for Neuronal or Endocrine Signalling Molecules as Potential Targets for the Control of Insect Pests

Abstract In metazoans, neuronal and endocrine communication is based on the release of extracellular signalling molecules that are recognised in a physiological concentration range by specific receptor proteins present in the target cells. These receptors will elicit a cellular response upon activation by their physiological agonist. A highly diverse repertoire of naturally occurring receptor agonists has already been discovered. Peptides, proteins and biogenic amines constitute the most diverse agonist classes. Most of these interact with G protein-coupled receptors (GPCRs), the largest category of signal transducing receptors that controls virtually every physiological process in metazoans. For more than two decades, insect GPCRs have been hailed for their potentially excellent aptitude to serve as pharmacological targets for the development of novel products for insect pest control. In this review, we will address this issue and enumerate reasons why it would be worth investing more in these targets.

Pharmacological Characterization of a 5-HT1-Type Serotonin Receptor in the Red Flour Beetle, Tribolium castaneum

Serotonin (5-hydroxytryptamine, 5-HT) is known for its key role in modulating diverse physiological processes and behaviors by binding various 5-HT receptors. However, a lack of pharmacological knowledge impedes studies on invertebrate 5-HT receptors. Moreover, pharmacological information is urgently needed in order to establish a reliable classification system for invertebrate 5-HT receptors. In this study we report on the molecular cloning and pharmacological characterization of a 5-HT1 receptor from the red flour beetle, Tribolium castaneum (Trica5-HT1). The Trica5-HT1 receptor encoding cDNA shows considerable sequence similarity with members of the 5-HT1 receptor class. Real time PCR showed high expression in the brain (without optic lobes) and the optic lobes, consistent with the role of 5-HT as neurotransmitter. Activation of Trica5-HT1 in mammalian cells decreased NKH-477-stimulated cyclic AMP levels in a dose-dependent manner, but did not influence intracellular Ca2+ signaling. We studied the pharmacological profile of the 5-HT1 receptor and demonstrated that α-methylserotonin, 5-methoxytryptamine and 5-carboxamidotryptamine acted as agonists. Prazosin, methiothepin and methysergide were the most potent antagonists and showed competitive inhibition in presence of 5-HT. This study offers important information on a 5-HT1 receptor from T. castaneum facilitating functional research of 5-HT receptors in insects and other invertebrates. The pharmacological profiles may contribute to establish a reliable classification scheme for invertebrate 5-HT receptors.

Signalling properties and pharmacology of a 5‐HT7‐type serotonin receptor from Tribolium castaneum

In the last decade, genome sequence data and gene structure information on invertebrate receptors has been greatly expanded by large sequencing projects and cloning studies. This information is of great value for the identification of receptors; however, functional and pharmacological data are necessary for an accurate receptor classification and for practical applications. In insects, an important group of neurotransmitter and neurohormone receptors, for which ample sequence information is available but pharmacological information is missing, are the biogenic amine G protein‐coupled receptors (GPCRs). In the present study, we investigated the sequence information, pharmacology and signalling properties of a 5‐HT7‐type serotonin receptor from the red flour beetle, Tribolium castaneum (Trica5‐HT7). The receptor encoding cDNA shows considerable sequence similarity with cognate 5‐HT7 receptors and phylogenetic analysis also clusters the receptor within this 5‐HT receptor group. Real‐time reverse transcription PCR demonstrated high expression levels in the brain, indicating the possible importance of this receptor in neural processes. Trica5‐HT7 was dose‐dependently activated by 5‐HT, which induced elevated intracellular cyclic AMP levels but had no effect on calcium signalling. The synthetic agonists, α‐methyl 5‐HT, 5‐methoxytryptamine, 5‐carboxamidotryptamine and 8‐hydroxy‐2‐(dipropylamino)tetralin hydrobromide, showed a response, although with a much lower potency and efficacy than 5‐HT. Ketanserin and methiothepin were the most potent antagonists. Both showed characteristics of competitive inhibition on Trica5‐HT7. The signalling pathway and pharmacological profile offer important information that will facilitate functional and comparative studies of 5‐HT receptors in insects and other invertebrates. The pharmacology of invertebrate 5‐HT receptors differs considerably from that of vertebrates. The present study may therefore contribute to establishing a more reliable classification of invertebrate 5‐HT receptors.

The ecdysis triggering hormone system is essential for successful moulting of a major hemimetabolous pest insect, Schistocerca gregaria

Insects are enclosed in a rigid exoskeleton, providing protection from desiccation and mechanical injury. To allow growth, this armour needs to be replaced regularly in a process called moulting. Moulting entails the production of a new exoskeleton and shedding of the old one and is induced by a pulse in ecdysteroids, which activates a peptide-mediated signalling cascade. In Holometabola, ecdysis triggering hormone (ETH) is the key factor in this cascade. Very little functional information is available in Hemimetabola, which display a different kind of development characterized by gradual changes. This paper reports on the identification of the ETH precursor and the pharmacological and functional characterisation of the ETH receptor in a hemimetabolous pest species, the desert locust, Schistocerca gregaria. Activation of SchgrETHR by SchgrETH results in an increase of both Ca2+ and cyclic AMP, suggesting that SchgrETHR displays dual coupling properties in an in vitro cell-based assay. Using qRT-PCR, an in-depth profiling study of SchgrETH and SchgrETHR transcripts was performed. Silencing of SchgrETH and SchgrETHR resulted in lethality at the expected time of ecdysis, thereby showing their crucial role in moulting.

Ecdysteroid signalling components in metamorphosis and development of the desert locust, Schistocerca gregaria

The arthropod-specific hormone family of ecdysteroids plays an important role in regulating diverse physiological processes, such as moulting and metamorphosis, reproduction, diapause and innate immunity. Ecdysteroids mediate their response by binding to a heterodimeric complex of two nuclear receptors, the ecdysone receptor (EcR) and the retinoid-X-receptor/ultraspiracle (RXR/USP). In this study we investigated the role of EcR and RXR in metamorphosis and development of the desert locust, Schistocerca gregaria. The desert locust is a voracious, phytophagous, swarming pest that can ruin crops and harvests in some of the worlds poorest countries. A profound knowledge of the ecdysteroid signalling pathway can be used in the development of more target-specific insecticides to combat this harmful plague insect. Here we report an in-depth profiling study of the transcript levels of EcR and RXR, as well as its downstream response genes, in different tissues isolated throughout the last larval stage of a hemimetabolous insect, showing a clear correlation with circulating ecdysteroid titres. Using RNA interference (RNAi), the role of SgEcR/SgRXR in moulting and development was investigated. We have proven the importance of the receptor components for successful moulting of locust nymphs into the adult stage. Some SgEcR/SgRXR knockdown females were arrested in the last larval stage, and 65 % of them initiated vitellogenesis and oocyte maturation, which normally only occurs in adults. Furthermore, our results clearly indicate that at the peak of ecdysteroid synthesis, on day six of the last larval stage, knockdown of SgEcR/SgRXR is affecting the transcript levels of the Halloween genes, Spook, Shadow and Shade.

Molecular cloning and characterization of the allatotropin precursor and receptor in the desert locust, Schistocerca gregaria.

Allatotropins (ATs) are pleiotropic neuropeptides initially isolated from the tobacco hornworm, Manduca sexta. In 2008, the first receptor for AT-like peptides (ATR) was characterized in Bombyx mori. Since then, ATRs have also been characterized in M. sexta, Tribolium castaneum, Aedes aegypti and Bombus terrestris. These receptors show sequence similarity to vertebrate orexin (ORX) receptors. When generating an EST-database of the desert locust (Schistocerca gregaria) central nervous system, we found cDNA sequences encoding the Schgr-AT precursor and a fragment of its putative receptor. This receptor cDNA has now been completed and functionally expressed in mammalian cell lines. Activation of this receptor, designated as Schgr-ATR, by Schgr-AT caused an increase in intracellular calcium ions, as well as cyclic AMP (cAMP), with an EC50 value in the nanomolar range. In addition, the transcript distribution of both the Schgr-AT precursor and Schgr-ATR was investigated by means of quantitative real-time PCR. Moreover, we found more evidence for the myotropic and allatostimulatory actions of Schgr-AT in the desert locust. These data are discussed and situated in a broader context by comparison with literature data on AT and ATR in insects.

Role of the venus kinase receptor in the female reproductive physiology of the desert locust, Schistocerca gregaria

Venus kinase receptors (VKR) are a subfamily of invertebrate receptor tyrosine kinases, which have only recently been discovered. They contain an intracellular tyrosine kinase domain and an extracellular Venus FlyTrap domain. VKRs have been functionally and pharmacologically characterized in only two invertebrate species, namely the human parasite Schistosoma mansoni and the mosquito Aedes aegypti, where they play a crucial role in oogenesis. Here, we report the characterization of a VKR in the desert locust, Schistocerca gregaria. We performed an in-depth profiling study of the SgVKR transcript levels in different tissues throughout the female adult stage. Using the RNA interference technique, the possible role of SgVKR was investigated. SgVKR knockdown had significant effects on ovarian ecdysteroid levels and on the size of oocytes during the vitellogenic stage. SgVKR is probably involved in the complex cross-talk between several important pathways regulating female reproductive physiology. Contrary to A. aegypti and S. mansoni, we cannot conclude that this receptor is essential for reproduction, since silencing SgVKR did not affect fecundity or fertility. Considering the evolutionary distance between A. aegypti and S. gregaria, as well as the differences in regulation of their female reproductive physiology, this article constitutes a valuable asset in better understanding VKRs.

Puberty & Metamorphosis Related to Reproduction: Metamorphosis, Insects

Metamorphosis is a highly successful biological adaptation that has allowed the diversification of insects into the most abundant group of animals on earth. Depending on the degree of metamorphosis during their life cycle, insects can be divided in 3 major subgroups: Ametabola, Hemimetabola and Holometabola. Variation exists in the postembryonic development and reproductive physiology of these different subgroups. In this article, we first go into detail on the (hormonal) control of metamorphosis. Second, we discuss the maturation of the reproductive system in Hemimetabola and Holometabola.