The macronuclear genomic landscape within Tetrahymena thermophila.

The extent of intraspecific genomic variation is key to understanding species evolutionary history, including recent adaptive shifts. Intraspecific genomic variation remains poorly explored in eukaryotic micro-organisms, especially in the nuclear dimorphic ciliates, despite their fundamental role as laboratory model systems and their ecological importance in many ecosystems. We sequenced the macronuclear genome of 22 laboratory strains of the oligohymenophoran Tetrahymena thermophila, a model species in both cellular biology and evolutionary ecology. We explored polymorphisms at the junctions of programmed eliminated sequences, and reveal their utility to barcode very closely related cells. As for other species of the genus Tetrahymena, we confirm micronuclear centromeres as gene diversification centres in T. thermophila, but also reveal a two-speed evolution in these regions. In the rest of the genome, we highlight recent diversification of genes coding for extracellular proteins and cell adhesion. We discuss all these findings in relation to this ciliate's ecology and cellular characteristics.

Post-feeding transcriptomics reveals essential genes expressed in the midgut of the desert locust.

The digestive tract constitutes an important interface between an animal's internal and external environment. In insects, available gut transcriptome studies are mostly exploratory or look at changes upon infection or upon exposure to xenobiotics, mainly performed in species belonging to holometabolan orders, such as Diptera, Lepidoptera or Coleoptera. By contrast, studies focusing on gene expression changes after food uptake and during digestion are underrepresented. We have therefore compared the gene expression profiles in the midgut of the desert locust, Schistocerca gregaria, between three different time points after feeding, i.e., 24 h (no active digestion), 10 min (the initial stage of feeding), and 2 h (active food digestion). The observed gene expression profiles were consistent with the polyphagous herbivorous lifestyle of this hemimetabolan (orthopteran) species. Our study reveals the upregulation of 576 genes 2 h post-feeding. These are mostly predicted to be associated with digestive physiology, such as genes encoding putative digestive enzymes or nutrient transporters, as well as genes putatively involved in immunity or in xenobiotic metabolism. The 10 min time point represented an intermediate condition, suggesting that the S. gregaria midgut can react rapidly at the transcriptional level to the presence of food. Additionally, our study demonstrated the critical importance of two transcripts that exhibited a significant upregulation 2 h post-feeding: the vacuolar-type H(+)-ATPase and the sterol transporter Niemann-Pick 1b protein, which upon RNAi-induced knockdown resulted in a marked increase in mortality. Their vital role and accessibility via the midgut lumen may make the encoded proteins promising insecticidal target candidates, considering that the desert locust is infamous for its huge migrating swarms that can devastate the agricultural production in large areas of Northern Africa, the Middle East, and South Asia. In conclusion, the transcriptome datasets presented here will provide a useful and promising resource for studying the midgut physiology of S. gregaria, a socio-economically important pest species.

Phenotypic plasticity through disposable genetic adaptation in ciliates.

Ciliates have an extraordinary genetic system in which each cell harbors two distinct kinds of nucleus, a transcriptionally active somatic nucleus and a quiescent germline nucleus. The latter undergoes classical, heritable genetic adaptation, while adaptation of the somatic nucleus is only short-term and thus disposable. The ecological and evolutionary relevance of this nuclear dimorphism have never been well formalized, which is surprising given the long history of using ciliates such as Tetrahymena and Paramecium as model organisms. We present a novel, alternative explanation for ciliate nuclear dimorphism which, we argue, should be considered an instrument of phenotypic plasticity by somatic selection on the level of the ciliate clone, as if it were a diffuse multicellular organism. This viewpoint helps to put some enigmatic aspects of ciliate biology into perspective and presents the diversity of ciliates as a large natural experiment that we can exploit to study phenotypic plasticity and organismality.

Schistocerca neuropeptides - An update.

We compiled a comprehensive list of 67 precursor genes encoding neuropeptides and neuropeptide-like peptides using the Schistocerca gregaria genome and several transcriptome datasets. 11 of these 67 precursor genes have alternative transcripts, bringing the total number of S. gregaria precursors identified in this study to 81. Based on this precursor information, we used different mass spectrometry approaches to identify the putative mature, bioactive peptides processed in the nervous system of S. gregaria. The thereby generated dataset for S. gregaria confirms significant conservation of the entire neuropeptidergic gene set typical of insects and also contains precursors typical of Polyneoptera only. This is in striking contrast to the substantial losses of peptidergic systems in some holometabolous species. The neuropeptidome of S. gregaria, apart from species-specific sequences within the known range of variation, is quite similar to that of Locusta migratoria and even to that of less closely related Polyneoptera. With the S. gregaria peptidomics data presented here, we have thus generated a very useful source of information that could also be relevant for the study of other polyneopteran species.

Can BRET-based biosensors be used to characterize G-protein mediated signaling pathways of an insect GPCR, the Schistocerca gregaria CRF-related diuretic hormone receptor?

G protein-coupled receptors (GPCRs) are membrane-bound receptors that are considered prime candidates for the development of novel insect pest management strategies. However, the molecular signaling properties of insect GPCRs remain poorly understood. In fact, most studies on insect GPCR signaling are limited to analysis of fluctuations in the secondary messenger molecules calcium (Ca2+) and/or cyclic adenosine monophosphate (cAMP). In the current study, we characterized a corticotropin-releasing factor-related diuretic hormone (CRF-DH) receptor of the desert locust, Schistocerca gregaria. This Schgr-CRF-DHR is mainly expressed in the nervous system and in brain-associated endocrine organs. The neuropeptide Schgr-CRF-DH induced Ca2+-dependent aequorin-based bioluminescent responses in CHO cells co-expressing this receptor with the promiscuous Gα16 protein. Furthermore, when co-expressed with the cAMP-dependent bioluminescence resonance energy transfer (BRET)-based CAMYEL biosensor in HEK293T cells, this receptor elicited dose-dependent agonist-induced responses with an EC50 in the nanomolar range (4.02 nM). In addition, we tested if vertebrate BRET-based G protein biosensors, can also be used to detect direct Gα protein subunit activation by an insect GPCR. Therefore, we analyzed ten different human BRET-based G protein biosensors, representing members of all four Gα protein subfamilies; Gαs, Gαi/o, Gαq/11 and Gα12/13. Our data demonstrate that stimulation of Schgr-CRF-DHR by Schgr-CRF-DH can dose-dependently activate Gαi/o and Gαs biosensors, while no significant effects were observed with the Gαq/11 and Gα12/13 biosensors. Our study paves the way for future biosensor-based studies to analyze the signaling properties of insect GPCRs in both fundamental science and applied research contexts.

First draft genome assembly of the desert locust, Schistocerca gregaria.

Background: At the time of publication, the most devastating desert locust crisis in decades is affecting East Africa, the Arabian Peninsula and South-West Asia. The situation is extremely alarming in East Africa, where Kenya, Ethiopia and Somalia face an unprecedented threat to food security and livelihoods. Most of the time, however, locusts do not occur in swarms, but live as relatively harmless solitary insects. The phenotypically distinct solitarious and gregarious locust phases differ markedly in many aspects of behaviour, physiology and morphology, making them an excellent model to study how environmental factors shape behaviour and development. A better understanding of the extreme phenotypic plasticity in desert locusts will offer new, more environmentally sustainable ways of fighting devastating swarms. Methods: High molecular weight DNA derived from two adult males was used for Mate Pair and Paired End Illumina sequencing and PacBio sequencing. A reliable reference genome of Schistocerca gregaria was assembled using the ABySS pipeline, scaffolding was improved using LINKS. Results: In total, 1,316 Gb Illumina reads and 112 Gb PacBio reads were produced and assembled. The resulting draft genome consists of 8,817,834,205 bp organised in 955,015 scaffolds with an N50 of 157,705 bp, making the desert locust genome the largest insect genome sequenced and assembled to date. In total, 18,815 protein-encoding genes are predicted in the desert locust genome, of which 13,646 (72.53%) obtained at least one functional assignment based on similarity to known proteins. Conclusions: The desert locust genome data will contribute greatly to studies of phenotypic plasticity, physiology, neurobiology, molecular ecology, evolutionary genetics and comparative genomics, and will promote the desert locust's use as a model system. The data will also facilitate the development of novel, more sustainable strategies for preventing or combating swarms of these infamous insects.

Quantitative Peptidomics with Isotopic and Isobaric Tags.

In differential peptidomics, peptide profiles are compared between biological samples and the resulting expression levels are correlated to a phenotype of interest. This, in turn, allows us insight into how peptides may affect the phenotype of interest. In quantitative differential peptidomics, both label-based and label-free techniques are often employed. Label-based techniques have several advantages over label-free methods, primarily that labels allow for various samples to be pooled prior to liquid chromatography-mass spectrometry (LC-MS) analysis, reducing between-run variation. Here, we detail a method for performing quantitative peptidomics using stable amine-binding isotopic and isobaric tags.

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.

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.

Fast and Reliable Quantitative Peptidomics with labelpepmatch.

The use of stable isotope tags in quantitative peptidomics offers many advantages, but the laborious identification of matching sets of labeled peptide peaks is still a major bottleneck. Here we present labelpepmatch, an R-package for fast and straightforward analysis of LC-MS spectra of labeled peptides. This open-source tool offers fast and accurate identification of peak pairs alongside an appropriate framework for statistical inference on quantitative peptidomics data, based on techniques from other -omics disciplines. A relevant case study on the desert locust Schistocerca gregaria proves our pipeline to be a reliable tool for quick but thorough explorative analyses.

Pharmacological and signalling properties of a D2-like dopamine receptor (Dop3) in Tribolium castaneum.

Dopamine is an important neurotransmitter in the central nervous system of vertebrates and invertebrates. Despite their evolutionary distance, striking parallels exist between deuterostomian and protostomian dopaminergic systems. In both, signalling is achieved via a complement of functionally distinct dopamine receptors. In this study, we investigated the sequence, pharmacology and tissue distribution of a D2-like dopamine receptor from the red flour beetle Tribolium castaneum (TricaDop3) and compared it with related G protein-coupled receptors in other invertebrate species. The TricaDop3 receptor-encoding cDNA shows considerable sequence similarity with members of the Dop3 receptor class. Real time qRT-PCR showed high expression in both the central brain and the optic lobes, consistent with the role of dopamine as neurotransmitter. Activation of TricaDop3 expressed in mammalian cells increased intracellular Ca(2+) signalling and decreased NKH-477 (a forskolin analogue)-stimulated cyclic AMP levels in a dose-dependent manner. We studied the pharmacological profile of the TricaDop3 receptor and demonstrated that the synthetic vertebrate dopamine receptor agonists, 2 - amino- 6,7 - dihydroxy - 1,2,3,4 - tetrahydronaphthalene hydrobromide (6,7-ADTN) and bromocriptine acted as agonists. Methysergide was the most potent of the antagonists tested and showed competitive inhibition in the presence of dopamine. This study offers important information on the Dop3 receptor from Tribolium castaneum that will facilitate functional analyses of dopamine receptors in insects and other invertebrates.

Characterization of a novel brain barrier ex vivo insect-based P-glycoprotein screening model.

In earlier studies insects were proposed as suitable models for vertebrate blood-brain barrier (BBB) permeability prediction and useful in early drug discovery. Here we provide transcriptome and functional data demonstrating the presence of a P-glycoprotein (Pgp) efflux transporter in the brain barrier of the desert locust (Schistocerca gregaria). In an in vivo study on the locust, we found an increased uptake of the two well-known Pgp substrates, rhodamine 123 and loperamide after co-administration with the Pgp inhibitors cyclosporine A or verapamil. Furthermore, ex vivo studies on isolated locust brains demonstrated differences in permeation of high and low permeability compounds. The vertebrate Pgp inhibitor verapamil did not affect the uptake of passively diffusing compounds but significantly increased the brain uptake of Pgp substrates in the ex vivo model. In addition, studies at 2°C and 30°C showed differences in brain uptake between Pgp-effluxed and passively diffusing compounds. The transcriptome data show a high degree of sequence identity of the locust Pgp transporter protein sequences to the human Pgp sequence (37%), as well as the presence of conserved domains. As in vertebrates, the locust brain-barrier function is morphologically confined to one specific cell layer and by using a whole-brain ex vivo drug exposure technique our locust model may retain the major cues that maintain and modulate the physiological function of the brain barrier. We show that the locust model has the potential to act as a robust and convenient model for assessing BBB permeability in early drug discovery.

Identification, functional characterization and phylogenetic analysis of double stranded RNA degrading enzymes present in the gut of the desert locust, Schistocerca gregaria.

RNA interference (RNAi) has become a widely used reverse genetics tool in eukaryotes and holds great potential to contribute to the development of novel strategies for insect pest control. While previous studies clearly demonstrated that injection of dsRNA into the body cavity of the desert locust, Schistocerca gregaria, is highly effective to induce gene silencing effects, we observed that the RNAi response is much less sensitive to orally delivered dsRNA. In line with this, we report on the presence of a potent dsRNA degrading activity in the midgut juice. Four different dsRNase sequences that belong to the DNA/RNA Non-specific Nuclease superfamily were retrieved from a transcriptome database of the desert locust. Surprisingly, we have found that, in the publicly available eukaryote nucleotide sequence databases, the presence of this group of enzymes is restricted to insects and crustaceans. Nonetheless, phylogenetic analyses predict a common origin of these enzymes with the Endonuclease G (EndoG) Non-specific Nucleases that display a widespread taxonomic distribution. Moreover, in contrast to the Sg-endoG transcript, the four Sg-dsRNase transcripts appear to be specifically expressed in the gut. Finally, by means of RNAi, we provide evidence for an important contribution of dsRNase2 to the dsRNA degrading activity that is present in the gut lumen of S. gregaria.

Identification of the short neuropeptide F precursor in the desert locust: evidence for an inhibitory role of sNPF in the control of feeding.

Peptides of the short neuropeptide F (sNPF) family have been shown to modulate feeding behavior in a wide variety of insect species. While these peptides stimulate feeding and food-searching behavior in Drosophila melanogaster and Apis mellifera, an opposite effect has recently been demonstrated in the desert locust, Schistocerca gregaria. In this study, we elaborate on these observations with the identification of the nucleotide sequence encoding the Schgr-sNPF precursor and the study of its role in the regulation of locust feeding behavior. We confirm that both Schgr-sNPF-like peptides, previously identified in mass spectrometric studies, are genuine precursor-encoded peptides. RNA interference mediated silencing of the Schgr-sNPF precursor transcript generates novel evidence for an inhibitory role of Schgr-sNPF in the regulation of feeding in S. gregaria. Furthermore, we show that starvation reduces the Schgr-sNPF precursor transcript level in the optic lobes, the primary visual centers of the locust brain. Our data indicate that Schgr-sNPF exerts an inhibitory effect on food uptake in the desert locust, which contrasts with effects of sNPF reported for several other insect species.

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.