Heleen Verlinden

Transcriptome analysis of the desert locust central nervous system: production and annotation of a Schistocerca gregaria EST database.

Background The desert locust (Schistocerca gregaria) displays a fascinating type of phenotypic plasticity, designated as ‘phase polyphenism’. Depending on environmental conditions, one genome can be translated into two highly divergent phenotypes, termed the solitarious and gregarious (swarming) phase. Although many of the underlying molecular events remain elusive, the central nervous system (CNS) is expected to play a crucial role in the phase transition process. Locusts have also proven to be interesting model organisms in a physiological and neurobiological research context. However, molecular studies in locusts are hampered by the fact that genome/transcriptome sequence information available for this branch of insects is still limited. Methodology We have generated 34,672 raw expressed sequence tags (EST) from the CNS of desert locusts in both phases. These ESTs were assembled in 12,709 unique transcript sequences and nearly 4,000 sequences were functionally annotated. Moreover, the obtained S. gregaria EST information is highly complementary to the existing orthopteran transcriptomic data. Since many novel transcripts encode neuronal signaling and signal transduction components, this paper includes an overview of these sequences. Furthermore, several transcripts being differentially represented in solitarious and gregarious locusts were retrieved from this EST database. The findings highlight the involvement of the CNS in the phase transition process and indicate that this novel annotated database may also add to the emerging knowledge of concomitant neuronal signaling and neuroplasticity events. Conclusions In summary, we met the need for novel sequence data from desert locust CNS. To our knowledge, we hereby also present the first insect EST database that is derived from the complete CNS. The obtained S. gregaria EST data constitute an important new source of information that will be instrumental in further unraveling the molecular principles of phase polyphenism, in further establishing locusts as valuable research model organisms and in molecular evolutionary and comparative entomology.

More than two decades of research on insect neuropeptide GPCRs: an overview

This review focuses on the state of the art on neuropeptide receptors in insects. Most of these receptors are G protein-coupled receptors (GPCRs) and are involved in the regulation of virtually all physiological processes during an insects life. More than 20 years ago a milestone in invertebrate endocrinology was achieved with the characterization of the first insect neuropeptide receptor, i.e., the Drosophila tachykinin-like receptor. However, it took until the release of the Drosophila genome in 2000 that research on neuropeptide receptors boosted. In the last decade a plethora of genomic information of other insect species also became available, leading to a better insight in the functions and evolution of the neuropeptide signaling systems and their intracellular pathways. It became clear that some of these systems are conserved among all insect species, indicating that they fulfill crucial roles in their physiological processes. Meanwhile, other signaling systems seem to be lost in several insect orders or species, suggesting that their actions were superfluous in those insects, or that other neuropeptides have taken over their functions. It is striking that the deorphanization of neuropeptide GPCRs gets much attention, but the subsequent unraveling of the intracellular pathways they elicit, or their physiological functions are often hardly examined. Especially in insects besides Drosophila this information is scarce if not absent. And although great progress made in characterizing neuropeptide signaling systems, even in Drosophila several predicted neuropeptide receptors remain orphan, awaiting for their endogenous ligand to be determined. The present review gives a précis of the insect neuropeptide receptor research of the last two decades. But it has to be emphasized that the work done so far is only the tip of the iceberg and our comprehensive understanding of these important signaling systems will still increase substantially in the coming years.

Endocrinology of reproduction and phase transition in locusts.

In the last decade, important progress has been made in the experimental analysis of the endocrine mechanisms controlling reproduction and phase transition in locusts. Phase transition is a very fascinating, but complex, phenomenon of phenotypic plasticity that is triggered by changes in population density and can lead to the formation of extremely devastating hopper bands and adult gregarious locust swarms. While some phase characteristics change within hours, others appear more gradually in the next stage(s), or even in the next generation(s). In adults, the phase status also has a major influence on the process of reproduction. A better understanding of how solitarious locusts become gregarious and how this switch affects reproductive physiology may result in novel strategies to fight locust plagues. In this paper, we will review the current knowledge concerning this close interaction between locust phase polyphenism and reproduction.

Tachykinin-related peptides and their receptors in invertebrates: A current view

Members of the tachykinin peptide family have been well conserved during evolution and are mainly expressed in the central nervous system and in the intestine of both vertebrates and invertebrates. In these animals, they act as multifunctional messengers that exert their biological effects by specifically interacting with a subfamily of structurally related G protein-coupled receptors. Despite the identification of multiple tachykinin-related peptides (TKRPs) in species belonging to the insects, crustaceans, mollusks and echiuroid worms, only five invertebrate receptors harboring profound sequence similarities to mammalian receptors for tachykinins have been functionally characterized to date. Three of these have been cloned from dipteran insect species, i.e. NKD (neurokinin receptor from Drosophila), DTKR (Drosophila tachykinin receptor) and STKR (tachykinin-related peptide receptor from the stable fly, Stomoxys calcitrans). In addition, two receptors from non-insect species, present in echiuroid worms and mollusks, respectively have been identified as well. In this brief review, we will survey some recent findings and insights into the signaling properties of invertebrate tachykinin-related peptides via their respective receptors. In this context, we will also point out the necessity to take into account differences in signaling mechanisms induced by distinct TKRP isoforms in insects.

Tissue-dependence and sensitivity of the systemic RNA interference response in the desert locust, Schistocerca gregaria

We report on a comprehensive study of the systemic RNAi-response in the desert locust, Schistocerca gregaria. Upon intra-abdominal injection of dsRNA for two housekeeping genes (alpha-tubulin 1a and gapdh) in the range of pg amounts of dsRNA per mg tissue, a potent reduction of their corresponding mRNA was obtained. Moreover, the observed transcript knockdown significantly increased in at least a 10 days period and eventually resulted in high mortality upon silencing of the alpha-tubulin 1a gene. A more moderate RNAi-response was however observed in the reproductive systems. Analysis of the tissue-dependent transcript level profile of several putative RNAi-genes indicated reduced levels of two genes, namely sg-dicer-2 and sg-argonaute-2, in the reproductive systems. By silencing these components, we confirmed their importance in the RNAi-process and suggest that their expression levels are determinant for tissue-dependent differences in the potency of RNAi in the desert locust.

Role of the Halloween genes, Spook and Phantom in ecdysteroidogenesis in the desert locust, Schistocerca gregaria

The functional characterization of the Halloween genes represented a major breakthrough in the elucidation of the ecdysteroid biosynthetic pathway. These genes encode cytochrome P450 enzymes catalyzing the final steps of ecdysteroid biosynthesis in the dipteran Drosophila melanogaster and the Lepidoptera Manduca sexta and Bombyx mori. This is the first report on the identification of two Halloween genes, spook (spo) and phantom (phm), from a hemimetabolous orthopteran insect, the desert locust Schistocerca gregaria. Using q-RT-PCR, their spatial and temporal transcript profiles were analyzed in both final larval stage and adult locusts. The circulating ecdysteroid titers in the hemolymph were measured and found to correlate well with changes in the temporal transcript profiles of spo and phm. Moreover, an RNA interference (RNAi)-based approach was employed to study knockdown effects upon silencing of both transcripts in the fifth larval stage. Circulating ecdysteroid levels were found to be significantly reduced upon dsRNA treatment.

Microarray-based transcriptomic analysis of differences between long-term gregarious and solitarious desert locusts

Desert locusts (Schistocerca gregaria) show an extreme form of phenotypic plasticity and can transform between a cryptic solitarious phase and a swarming gregarious phase. The two phases differ extensively in behavior, morphology and physiology but very little is known about the molecular basis of these differences. We used our recently generated Expressed Sequence Tag (EST) database derived from S. gregaria central nervous system (CNS) to design oligonucleotide microarrays and compare the expression of thousands of genes in the CNS of long-term gregarious and solitarious adult desert locusts. This identified 214 differentially expressed genes, of which 40% have been annotated to date. These include genes encoding proteins that are associated with CNS development and modeling, sensory perception, stress response and resistance, and fundamental cellular processes. Our microarray analysis has identified genes whose altered expression may enable locusts of either phase to deal with the different challenges they face. Genes for heat shock proteins and proteins which confer protection from infection were upregulated in gregarious locusts, which may allow them to respond to acute physiological challenges. By contrast the longer-lived solitarious locusts appear to be more strongly protected from the slowly accumulating effects of ageing by an upregulation of genes related to anti-oxidant systems, detoxification and anabolic renewal. Gregarious locusts also had a greater abundance of transcripts for proteins involved in sensory processing and in nervous system development and plasticity. Gregarious locusts live in a more complex sensory environment than solitarious locusts and may require a greater turnover of proteins involved in sensory transduction, and possibly greater neuronal plasticity.

Critical role for protein kinase A in the acquisition of gregarious behavior in the desert locust

The mechanisms that integrate genetic and environmental information to coordinate the expression of complex phenotypes are little understood. We investigated the role of two protein kinases (PKs) in the population density-dependent transition to gregarious behavior that underlies swarm formation in desert locusts: the foraging gene product, a cGMP-dependent PK (PKG) implicated in switching between alternative group-related behaviors in several animal species; and cAMP-dependent PK (PKA), a signal transduction protein with a preeminent role in different forms of learning. Solitarious locusts acquire key behavioral characters of the swarming gregarious phase within just 1 to 4 h of forced crowding. Injecting the PKA inhibitor KT5720 before crowding prevented this transition, whereas injecting KT5823, an inhibitor of PKG, did not. Neither drug altered the behavior of long-term gregarious locusts. RNAi against foraging effectively reduced its expression in the central nervous system, but this did not prevent gregarization upon crowding. By contrast, solitarious locusts with an RNAi-induced reduction in PKA catalytic subunit C1 expression behaved less gregariously after crowding, and RNAi against the inhibitory R1 subunit promoted more extensive gregarization following a brief crowding period. A central role of PKA is congruent with the recent discovery that serotonin mediates gregarization in locusts and with findings in vertebrates that similarly implicate PKA in the capacity to cope with adverse life events. Our results show that PKA has been coopted into effecting the wide-ranging transformation from solitarious to gregarious behavior, with PKA-mediated behavioral plasticity resulting in an environmentally driven reorganization of a complex phenotype.

Isolation and functional characterization of an allatotropin receptor from Manduca sexta.

Manduca sexta allatotropin (Manse-AT) is a multifunctional neuropeptide whose actions include the stimulation of juvenile hormone biosynthesis, myotropic stimulation, cardioacceleratory functions, and inhibition of active ion transport. Manse-AT is a member of a structurally related peptide family that is widely found in insects and also in other invertebrates. Its precise role depends on the insect species and developmental stage. In some lepidopteran insects including M. sexta, structurally-related AT-like (ATL) peptides can be derived from alternatively spliced mRNAs transcribed from the AT gene. We have isolated a cDNA for an AT receptor (ATR) from M. sexta by a PCR-based approach using the sequence of the ATR from Bombyx mori. The sequence of the M. sexta ATR is similar to several G protein-coupled receptors from other insect species and to the mammalian orexin receptor. We demonstrate that the M. sexta ATR expressed in vertebrate cell lines is activated in a dose-responsive manner by Manse-AT and each Manse-ATL peptide in the rank order ATL-I > ATL-II > ATL-III > AT, and functional analysis in multiple cell lines suggest that the receptor is coupled through elevated levels of Ca(2+) and cAMP. In feeding larvae, Manse-ATR mRNA is present at highest levels in the Malpighian tubules, followed by the midgut, hindgut, testes, and corpora allata, consistent with its action on multiple target tissues. In the adult corpora cardiaca--corpora allata complex, Manse-ATR mRNA is present at relatively low levels in both sexes.

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.