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The SARS-CoV-2 pandemic has highlighted the need for improved technologies to help control the spread of contagious pathogens. While rapid point-of-need testing plays a key role in strategies to rapidly identify and isolate infectious patients, current test approaches have significant shortcomings related to assay limitations and sample type. Direct quantification of viral shedding in exhaled particles may offer a better rapid testing approach, since SARS-CoV-2 is believed to spread mainly by aerosols. It assesses contagiousness directly, the sample is easy and comfortable to obtain, sampling can be standardized, and the limited sample volume lends itself to a fast and sensitive analysis. In view of these benefits, we developed and tested an approach where exhaled particles are efficiently sampled using inertial impaction in a micromachined silicon chip, followed by an RT-qPCR molecular assay to detect SARS-CoV-2 shedding. Our portable, silicon impactor allowed for the efficient capture (>85%) of respiratory particles down to 300 nm without the need for additional equipment. We demonstrate using both conventional off-chip and in-situ PCR directly on the silicon chip that sampling subjects' breath in less than a minute yields sufficient viral RNA to detect infections as early as standard sampling methods. A longitudinal study revealed clear differences in the temporal dynamics of viral load for nasopharyngeal swab, saliva, breath, and antigen tests. Overall, after an infection, the breath-based test remains positive during the first week but is the first to consistently report a negative result, putatively signalling the end of contagiousness and further emphasizing the potential of this tool to help manage the spread of airborne respiratory infections. ispartof: BIOSENSORS & BIOELECTRONICS vol:217 ispartof: location:England status: published

Ecdysteroids are widely investigated for their role during the molting cascade in insects; however, they are also involved in the development of the female reproductive system. Ecdysteroids are synthesized from cholesterol, which is further converted via a series of enzymatic steps into the main molting hormone, 20-hydoxyecdysone. Most of these biosynthetic conversion steps involve the activity of cytochrome P450 (CYP) hydroxylases, which are encoded by the

Insects provide an unparalleled opportunity to link genomic changes with the rise of novel phenotypes, given tremendous variation in the numerous and complex adaptations displayed across the group. Among these numerous and complex adaptations, live-birth has arisen repeatedly and independently in insects and across the tree of life, suggesting this is one of the most common types of convergent evolution among animals. We sequenced the genome and transcriptome of the Pacific beetle-mimic cockroach, the only truly viviparous cockroach, and performed comparative analyses including two other viviparous insect lineages, the tsetse and aphids, to unravel the genomic basis underlying the transition to viviparity in insects. We identified pathways experiencing adaptive evolution, common in all viviparous insects surveyed, involved in uro-genital remodeling, maternal control of embryo development, tracheal system, and heart development. Our findings suggest the essential role of those pathways for the development of placenta-like structure enabling embryo development and nutrition. Viviparous transition seems also to be accompanied by the duplication of genes involved in eggshell formation. Our findings from the viviparous cockroach and tsetse reveal that genes involved in uterine remodeling are up-regulated and immune genes are down-regulated during the course of pregnancy. These changes may facilitate structural changes to accommodate developing young and protect them from the mothers immune system. Our results denote a convergent evolution of live-bearing in insects and suggest similar adaptive mechanisms occurred in vertebrates, targeting pathways involved in eggshell formation, uro-genital remodeling, enhanced tracheal and heart development, and reduced immunity.

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. The authors would like to thank Evelien Herinckx for taking care of the locust rearing facility at KU Leuven. We thank Christian Frese, Astrid Wilbrand-Hennes, Jan Krueger and Ursula Cullman (CECAD Cologne Proteomics Facility) for the Orbitrap MS analyses, Tobias Schulze (Biocenter Cologne) for IT support, and Marek Franitza and Janine Altmüller for transcriptome sequencing at Cologne Center for Genomics. This work was supported by grants from the Special Research Fund of KU Leuven [grant numbers C14/15/050, C14/19/069], the European Union’s Horizon 2020 Research and Innovation programme [grant number 634361 to RP and to JVB], and the Research Foundation of Flanders (FWO) [G0F2417N, G090919N] to JVB.

The SARS-CoV-2 pandemic has highlighted the need for improved technologies to help control the spread of contagious pathogens. While rapid point-of-need testing plays a key role in strategies to rapidly identify and isolate infectious patients, a cornerstone for any disease-control strategy, current test approaches have significant shortcomings related to assay limitations and sample type. Direct quantification of viral shedding in exhaled particles may offer a better rapid testing approach, since SARS-CoV-2 is believed to spread mainly by aerosols. It potentially measures contagiousness directly, the sample is easy to obtain, its production can be standardized between patients, and the limited sample volume lends itself to a fast and sensitive analysis. In view of these benefits, we developed and tested an approach where exhaled particles are efficiently sampled using inertial impaction in a micromachined silicon chip, followed by an in-situ RT-qPCR molecular assay to detect SARS-CoV-2 shedding. We demonstrate that sampling subjects using a one-minute breathing protocol, yields sufficient viral RNA to detect infections with a sensitivity comparable to standard sampling methods. A longitudinal study revealed clear differences in the temporal dynamics of viral load for nasopharyngeal swab, saliva, breath, and antigen tests. Overall, after an infection, the breath-based test is the first to consistently report a negative result, putatively signaling the end of contagiousness and further emphasizing the potential of this tool to help manage the spread of airborne respiratory infections.

Accurate control of innate behaviors associated with developmental transitions requires functional integration of hormonal and neural signals. Insect molting is regulated by a set of neuropeptides, which trigger periodic pulses in ecdysteroid hormone titers and coordinate shedding of the old cuticle during ecdysis. In the current study, we demonstrate that crustacean cardioactive peptide (CCAP), a structurally conserved neuropeptide described to induce the ecdysis motor program, also exhibits a previously unknown prothoracicostatic activity to regulate ecdysteroid production in the desert locust, Schistocerca gregaria. We identified the locust genes encoding the CCAP precursor and three G protein-coupled receptors that are activated by CCAP with EC50 values in the (sub)nanomolar range. Spatiotemporal expression profiles of the receptors revealed expression in the prothoracic glands, the endocrine organs where ecdysteroidogenesis occurs. RNAi-mediated knockdown of CCAP precursor or receptors resulted in significantly elevated transcript levels of several Halloween genes, which encode ecdysteroid biosynthesis enzymes, and in elevated ecdysteroid levels one day prior to ecdysis. Moreover, prothoracic gland explants exhibited decreased secretion of ecdysteroids in the presence of CCAP. Our results unequivocally identify CCAP as the first prothoracicostatic peptide discovered in a hemimetabolan species and reveal the existence of an intricate interplay between CCAP signaling and ecdysteroidogenesis. ispartof: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES vol:22 issue:24 ispartof: location:Switzerland status: published

ispartof: Physiological Entomology vol:46 issue:1 pages:70-81 status: published

Postembryonic development of insects is coordinated by juvenile hormone (JH) together with ecdysteroids. Whereas the JH early response gene kr&uuml, ppel-homolog 1 (Kr-h1) plays a crucial role in the maintenance of juvenile characteristics during consecutive larval stages, the ecdysteroid-inducible early gene E93 appears to be a key factor promoting metamorphosis and adult morphogenesis. Here, we report on the developmental and molecular consequences of an RNAi-mediated knockdown of SgE93 in the desert locust, Schistocerca gregaria, a hemimetabolan species. Our experimental data show that injection of gregarious locust nymphs with a double-stranded RNA construct targeting the SgE93 transcript inhibited the process of metamorphosis and instead led to supernumerary nymphal stages. These supernumerary nymphal instars still displayed juvenile morphological features, such as a nymphal color scheme and body shape, while they reached the physical body size of the adult locusts, or even surpassed it after the next supernumerary molt. Interestingly, when compared to control locusts, the total duration of the fifth and normally final nymphal (N5) stage was shorter than normal. This appeared to correspond with temporal and quantitative changes in hemolymph ecdysteroid levels, as well as with altered expression of the rate-limiting Halloween gene, Spook (SgSpo). In addition, the levels of the ecdysone receptor (SgEcR) and retino&iuml, d X receptor (SgRXR) transcripts were altered, indicating that silencing SgE93 affects both ecdysteroid synthesis and signaling. Upon knockdown of SgE93, a very potent upregulation of the SgKr-h1 transcript levels was observed in both head and fat body, while no significant changes were detected in the transcript levels of SgJHAMT and SgCYP15A1, the enzymes that catalyze the two final steps in JH biosynthesis. Moreover, the process of molting was disturbed in these supernumerary nymphs. While attempting ecdysis to the next stage, 50% of the N6 and all N7 nymphal instars eventually died. S. gregaria is a very harmful, swarm-forming pest species that destroys crops and threatens food security in many of the world&rsquo, s poorest countries. We believe that a better knowledge of the mechanisms of postembryonic development may contribute to the discovery of novel, more selective and sustainable strategies for controlling gregarious locust populations. In this context, identification of molecular target candidates that are capable of significantly reducing the fitness of this devastating swarming pest will be of crucial importance.

Neuropeptides belonging to the adipokinetic hormone (AKH) family elicit metabolic effects as their main function in insects, by mobilizing trehalose, diacylgycerol, or proline, which are released from the fat body into the hemolymph as energy sources for muscle contraction required for energy-intensive processes, such as locomotion. One of the AKHs produced in locusts is a decapeptide, Locmi-AKH-I (pELNFTPNWGT-NH2). A head-to-tail cyclic, octapeptide analog of Locmi-AKH-I, cycloAKH (cyclo[LNFTPNWG]) was synthesized to severely restrict the conformational freedom of the AKH structure. In vitro, cycloAKH selectively retains full efficacy on a pest insect (desert locust) AKH receptor, while showing little or no activation of the AKH receptor of a beneficial insect (honeybee). Molecular dynamic analysis incorporating NMR data indicate that cycloAKH preferentially adopts a type II β-turn under micelle conditions, whereas its linear counterpart and natural AKH adopts a type VI β-turn under similar conditions. CycloAKH, linear LNFTPNWG-NH2, and Locmi-AKH-I feature the same binding site during docking simulations with the desert locust AKH receptor (Schgr-AKHR), but differ in the details of the ligand/receptor interactions. However, cycloAKH failed to enter the binding pocket of the honeybee receptor 3D model during docking simulations. Since the locust AKH receptor has a greater tolerance than the honeybee receptor for the cyclic conformational constraint in vitro receptor assays, it could suggest a greater tolerance for a shift in the direction of the type II β turn exhibited by cycloAKH from the type VI β turn of the linear octapeptide and the native locust decapeptide AKH. Selectivity in biostable mimetic analogs could potentially be enhanced by incorporating conformational constraints that emphasize this shift. Biostable mimetic analogs of AKH offer the potential of selectively disrupting AKH-regulated processes, leading to novel, environmentally benign control strategies for pest insect populations. ispartof: Insect Biochemistry And Molecular Biology vol:125 ispartof: location:England status: published

Postembryonic development of insects is coordinated by juvenile hormone (JH) together with ecdysteroids. Whereas the JH early response gene krüppel-homolog 1 (Kr-h1) plays a crucial role in the maintenance of juvenile characteristics during consecutive larval stages, the ecdysteroid-inducible early gene E93 appears to be a key factor promoting metamorphosis and adult morphogenesis. Here, we report on the developmental and molecular consequences of an RNAi-mediated knockdown of SgE93 in the desert locust, Schistocerca gregaria, a hemimetabolan species. Our experimental data show that injection of gregarious locust nymphs with a double-stranded RNA construct targeting the SgE93 transcript inhibited the process of metamorphosis and instead led to supernumerary nymphal stages. These supernumerary nymphal instars still displayed juvenile morphological features, such as a nymphal color scheme and body shape, while they reached the physical body size of the adult locusts, or even surpassed it after the next supernumerary molt. Interestingly, when compared to control locusts, the total duration of the fifth and normally final nymphal (N5) stage was shorter than normal. This appeared to correspond with temporal and quantitative changes in hemolymph ecdysteroid levels, as well as with altered expression of the rate-limiting Halloween gene, Spook (SgSpo). In addition, the levels of the ecdysone receptor (SgEcR) and retinoïd X receptor (SgRXR) transcripts were altered, indicating that silencing SgE93 affects both ecdysteroid synthesis and signaling. Upon knockdown of SgE93, a very potent upregulation of the SgKr-h1 transcript levels was observed in both head and fat body, while no significant changes were detected in the transcript levels of SgJHAMT and SgCYP15A1, the enzymes that catalyze the two final steps in JH biosynthesis. Moreover, the process of molting was disturbed in these supernumerary nymphs. While attempting ecdysis to the next stage, 50% of the N6 and all N7 nymphal instars eventually died. S. gregaria is a very harmful, swarm-forming pest species that destroys crops and threatens food security in many of the world’s poorest countries. We believe that a better knowledge of the mechanisms of postembryonic development may contribute to the discovery of novel, more selective and sustainable strategies for controlling gregarious locust populations. In this context, identification of molecular target candidates that are capable of significantly reducing the fitness of this devastating swarming pest will be of crucial importance.

The desert locust, Schistocerca gregaria, is an infamous migrating insect, generally considered to be one of the most dangerous pest species worldwide. In an area that encompasses one fifth of the Earth’s land surface, desert locust swarms can devastate the agricultural production. Consequently, they pose a serious threat to food security and can have a dramatic socio-economic impact. Currently (in 2020), large parts of East Africa, Southwest and South Asia are experiencing the worst desert locust plague in many decades. Exceptionally high rainfall in different regions has caused the favorable environmental conditions for very successful reproduction and population growth. To better understand the molecular mechanisms responsible for this remarkable reproductive capacity, as well as to fill existing knowledge gaps regarding the regulation of male reproductive physiology, we decided to investigate the role of the methoprene-tolerant (Scg-Met) and taiman (Scg-Tai) genes in adult male desert locusts. Methoprene-tolerant and Taiman belong to the basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) family of transcription factors and form a complex that is responsible for transducing the Juvenile Hormone (JH) signal. So far, in vivo research has mainly focused on the role of these JH receptor components in post-embryonic development, as well as in the reproductive physiology of adult female insects. In the current study, we show that silencing these components by RNA interference strongly inhibits sexual maturation of gregarious desert locust males, thereby severely disrupting reproduction. This observation is evidenced by the absence of a yellow colored cuticle, the significant reduction in the relative weight of the testes, the inability to show mating behavior, and the drastically reduced phenylacetonitrile (PAN) pheromone levels of the treated males. In addition, we also observed significant reductions of the relative weight, as well as the relative protein content, of the male accessory glands in Scg-Met knockdown locusts. Interestingly, in these animals the size of the corpora allata (CA), the endocrine glands where JH is synthesized, was significantly increased, and a significant rise was observed in the relative transcript levels of JH acid methyltransferase (JHAMT), a rate-limiting enzyme in the JH biosynthesis pathway. Moreover, other endocrine pathways also appeared to be affected by the knockdown, as evidenced by significant changes in the levels of the transcripts coding for the insulin-related peptide and for two neuroparsins in the fat body. Our results demonstrate that JH signaling pathway components play a crucial role in male reproductive physiology. Since they are crucial for a successful reproduction, they may hold great potential as candidate targets for the design of novel strategies for locust population management.Significance statementDesert locusts can form devastating swarms that threaten the livelihood in many of the world’s poorest countries, as demonstrated by the plagues in East Africa, Southwest and South Asia that are currently covered in the news worldwide. Control of this migratory pest mainly relies on the large-scale use of neurotoxic insecticides, also affecting non-target organisms in their already fragile habitats. Therefore, the identification of novel molecular targets for the design of more sustainable and more selective pest management techniques remains of crucial importance. Potential candidate targets in this respect are pathway components that transduce signals of typical insect hormones, such as ecdysteroids and juvenile hormones. By focusing in our current study on the JH receptor components, we showed that the reproductive capacities of desert locust males can be severely inhibited in an early stage, resulting in individuals that display no mating behavior, do not produce an important pheromone and do not show the characteristic yellow color of sexually mature gregarious males. Specifically targeting molecular components of the JH signaling pathway in future, more biorational control strategies could therefore be a very successful way by which expansion of these devastating plagues might be prevented.

Kr&uuml, ppel-homolog 1 (Kr-h1) is a zinc finger transcription factor maintaining the status quo in immature insect stages and promoting reproduction in adult insects through the transduction of the Juvenile Hormone (JH) signal. Knockdown studies have shown that precocious silencing of Kr-h1 in the immature stages results in the premature development of adult features. However, the molecular characteristics and reproductive potential of these premature adult insect stages are still poorly understood. Here we report on an adult-like or &lsquo, adultoid&rsquo, phenotype of the migratory locust, Locusta migratoria, obtained after a premature metamorphosis induced by the silencing of LmKr-h1 in the penultimate instar. The freshly molted adultoid shows precocious development of adult features, corresponding with increased transcript levels of the adult specifier gene LmE93. Furthermore, accelerated ovarian maturation and vitellogenesis were observed in female adultoids, coinciding with elevated expression of LmCYP15A1 in corpora allata (CA) and LmKr-h1 and vitellogenin genes (LmVg) in fat body, whereas LmE93 and Methoprene-tolerant (LmMet) transcript levels decreased in fat body. In adultoid ovaries, expression of the Halloween genes, Spook (LmSpo) and Phantom (LmPhm), was elevated as well. In addition, the processes of mating and oviposition were severely disturbed in these females. L. migratoria is a well-known, swarm-forming pest insect that can destroy crops and harvests in some of the world&rsquo, s poorest countries. As such, a better understanding of factors that are capable of significantly reducing the reproductive potential of this pest may be of crucial importance for the development of novel locust control strategies.

Juvenile hormones (JH) are key endocrine regulators produced by the corpora allata (CA) of insects. Together with ecdysteroids, as well as nutritional cues, JH coordinates different aspects of insect postembryonic development and reproduction. The function of the recently characterized JH receptor, Methoprene-tolerant (Met), appears to be conserved in different processes regulated by JH. However, its functional interactions with other hormonal signalling pathways seem highly dependent on the feeding habits and on the developmental and reproductive strategies employed by the insect species investigated. Here we report on the effects of RNA interference (RNAi) mediated SgMet knockdown during the first gonadotrophic cycle in female desert locusts (Schistocerca gregaria). This voracious, phytophagous pest species can form migrating swarms that devastate field crops and harvests in several of the world’s poorest countries. A better knowledge of the JH signalling pathway may contribute to the development of novel, more target-specific insecticides to combat this very harmful swarming pest. Using RNAi, we show that the JH receptor Met is essential for ovarian maturation, vitellogenesis and associated ecdysteroid biosynthesis in adult female S. gregaria. Interestingly, knockdown of SgMet also resulted in a significant decrease of insulin-related peptide (SgIRP) and increase of neuroparsin (SgNP) 3 and 4 transcript levels in the fat body, illustrating the existence of an intricate regulatory interplay between different hormonal factors. In addition, SgMet knockdown in females resulted in delayed display of copulation behaviour with virgin males, when compared with dsGFP injected control animals. Moreover, we observed an incapacity of adult dsSgMet injected female locusts to oviposit during the time of the experimental setup. As such, SgMet is an essential gene playing crucial roles in the endocrine communication necessary for successful reproduction of the desert locust.

Ecdysteroid hormones influence the development and reproduction of arthropods by binding a heterodimeric complex of nuclear receptors, the ecdysone receptor (EcR) and the retinoid-X-receptor/ultraspiracle (RXR/USP). Here, we report on the in vivo role(s) of the ecdysone receptor complex, SchgrEcR/SchgrRXR, in the female reproductive physiology of a major phytophagous pest insect, i.e. the desert locust, Schistocerca gregaria. Tissue and temporal distribution profiles were analysed during the first gonadotrophic cycle of adult female locusts. RNA interference was used as a reverse genetics tool to investigate the in vivo role of the ecdysone receptor complex in ovarian maturation, oogenesis, fertility and fecundity. We discovered that silencing the ecdysone receptor complex in S. gregaria resulted in impaired ovulation and oviposition, indicative for a crucial role of this complex in chorion formation. We also found evidence for a feedback of SchgrEcR/SchgrRXR on juvenile hormone biosynthesis by the corpora allata. Furthermore, we observed a tissue-dependent effect of the SchgrEcR/SchgrRXR knockdown on the transcript levels of the insulin receptor and neuroparsin 3 and 4. The insulin receptor transcript levels were upregulated in the brain, but not the fat body and gonads. Neuroparsins 3 and 4 transcript levels were down regulated in the brain and fat body, but not in the gonads.

“One of the fundamental events during lifetime is founding the next generation” (Büning [1]). “…oogenesis is the necessary first step for development of the animal embryo since only in this unique cell does the nucleus exhibit its totipotent properties required for supporting normal embryogenesis.” (Mahowald [2]).

Currently (2020), Africa and Asia are experiencing the worst desert locust (Schistocerca gregaria) plague in decades. Exceptionally high rainfall in different regions caused favorable environmental conditions for very successful reproduction and population growth. To better understand the molecular mechanisms responsible for this remarkable reproductive capacity, as well as to fill existing knowledge gaps regarding the regulation of male reproductive physiology, we investigated the role of methoprene-tolerant (Scg-Met) and Taiman (Scg-Tai), responsible for transducing the juvenile hormone (JH) signal, in adult male locusts. We demonstrated that knockdown of these components by RNA interference strongly inhibits male sexual maturation, severely disrupting reproduction. This was evidenced by the inability to show mating behavior, the absence of a yellow-colored cuticle, the reduction of relative testes weight, and the drastically reduced phenylacetonitrile (PAN) pheromone levels of the treated males. We also observed a reduced relative weight, as well as relative protein content, of the male accessory glands in Scg-Met knockdown locusts. Interestingly, in these animals the size of the corpora allata (CA), the endocrine glands where JH is synthesized, was significantly increased, as well as the transcript level of JH acid methyltransferase (JHAMT), a rate-limiting enzyme in the JH biosynthesis pathway. Moreover, other endocrine pathways appeared to be affected by the knockdown, as evidenced by changes in the expression levels of the insulin-related peptide and two neuroparsins in the fat body. Our results demonstrate that JH signaling pathway components play a crucial role in male reproductive physiology, illustrating their potential as molecular targets for pest control. ispartof: Biomolecules vol:11 issue:2 ispartof: location:Switzerland status: published

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., info:eu-repo/semantics/published

Adipokinetic hormone (AKH) is a highly researched insect neuropeptide that induces the mobilization of carbohydrates and lipids from the fat body at times of high physical activity, such as flight and locomotion. As a naturally occurring ligand, AKH has undergone quite a number of amino acid changes throughout evolution, and in some insect species multiple AKHs are present. AKH acts by binding to a rhodopsin-like G protein-coupled receptor, which is related to the vertebrate gonadotropin-releasing hormone receptors. In the current study, we have cloned AKH receptors (AKHRs) from seven different species, covering a wide phylogenetic range of insect orders: the fruit fly, Drosophila melanogaster, and the yellow fever mosquito, Aedes aegypti (Diptera); the red flour beetle, Tribolium castaneum, and the large pine weevil, Hylobius abietis (Coleoptera); the honeybee, Apis mellifera (Hymenoptera); the pea aphid, Acyrthosiphon pisum (Hemiptera); and the desert locust, Schistocerca gregaria (Orthoptera). The agonistic activity of different insect AKHs, including the respective endogenous AKHs, at these receptors was tested with a bioluminescence-based assay in Chinese hamster ovary cells. All receptors were activated by their endogenous ligand in the nanomolar range. Based on our data, we can refute the previously formulated hypothesis that a functional AKH signaling system is absent in the beneficial species, Apis mellifera. Furthermore, our data also suggest that some of the investigated AKH receptors, such as the mosquito AKHR, are more selective for the endogenous (conspecific) ligand, while others, such as the locust AKHR, are more promiscuous and can be activated by AKHs from many other insects. This information will be of high importance when further analyzing the potential use of AKHRs as targets for developing novel pest control agents. ispartof: International Journal Of Molecular Sciences vol:19 issue:2 ispartof: location:Switzerland status: published

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.

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.

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 Pacific beetle cockroach, Diploptera punctata, has proven to be a valuable model insect in the study of the dynamics regulating juvenile hormone (JH) biosynthesis and metabolism, particularly during late nymphal development and reproduction. This stems in part from its unusual mode of reproduction, adenotrophic viviparity, in which females give birth to live young that have been nourished throughout embryonic development by a protein-rich 'milk' secreted by the wall of the brood sac or uterus. In this animal, as in most insects, JH regulates both vitellogenin production and its uptake by developing oocytes. However, JH has an antagonistic effect on embryonic development and following oviposition of the fertilized oocytes into the brood sac, JH production halts, in part through the action of a peptide family, the FGLa allatostatins. JH production remains at a low level throughout pregnancy and is only reinstated at the end of gestation, at which time, the next wave of oocytes begins to develop and enter vitellogenesis. Thus, JH production in this species is precisely regulated, since the appearance of JH at inappropriate times would result in abortion of the embryos. Numerous factors are responsible for the regulation of JH biosynthesis, including peptides, biogenic amines, neurotransmitters, ecdysteroids and second messenger effectors. In this review, we discuss these factors and highlight potentially fruitful areas of future research. Although several of the enzymes of the biosynthetic pathway have been cloned, the precise points of rate limitation remain uncertain. The dissection of the biosynthetic pathway and its control awaits the completion of the genome and transcriptome of this important model insect.

Neuropeptides are important regulatory factors that mediate key life processes, both in vertebrates and invertebrates. Many insect neuropeptides display pleiotropic activities, which means that they can influence multiple aspects of insect physiology. In the fruit fly, Drosophila melanogaster, Neuropeptide F (NPF) mediates diverse physiological processes, such as learning, stress responses, feeding and male courtship behavior. In locusts, only a truncated form of the predicted ‘‘full-length’’ NPF, the nonapeptide ‘‘trNPF’’, has been isolated. This nonapeptide previously proved to be biologically active, since it was shown to influence food intake and weight increase, as well as oocyte growth in adult female desert locusts (Schistocerca gregaria [Forskal]). In the present study, we have further analyzed the effect of trNPF on female reproductive physiology in S. gregaria. We confirmed that daily trNPF injections in adult females elicit an increase of oocyte size. In addition, an RNAi-mediated knockdown of the Schgr-NPF precursor transcript in adult female locusts resulted in the opposite effect, i.e. significantly smaller oocytes. Moreover, we discovered that daily injections of trNPF in adult female S. gregaria, caused higher ecdysteroid titers in the ovaries and accelerated the appearance of ecdysteroid peaks in the hemolymph of these animals. The RNAi-based knockdown of the Schgr-NPF precursor transcript clearly resulted in reduction of both hemolymph and ovarian ecdysteroid concentrations, confirming the stimulatory effects of trNPF injections on adult female ecdysteroid levels. The observed results are discussed in relation to previous reports on NPF activities in locusts and other insects.

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.

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 world's 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. publisher: Elsevier articletitle: Ecdysteroid signalling components in metamorphosis and development of the desert locust, Schistocerca gregaria journaltitle: Insect Biochemistry and Molecular Biology articlelink: http://dx.doi.org/10.1016/j.ibmb.2016.05.003 content_type: article copyright: © 2016 Elsevier Ltd. All rights reserved. ispartof: Insect Biochemistry and Molecular Biology vol:75 pages:10-23 ispartof: location:England status: published

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.

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.

Two genes coding for enzymes previously reported to be involved in the final steps of juvenile hormone (JH) biosynthesis in different insect species, were characterised in the desert locust, Schistocerca gregaria. Juvenile hormone acid O-methyltransferase (JHAMT) was previously described to catalyse the conversion of farnesoic acid (FA) and JH acid to their methyl esters, methyl farnesoate (MF) and JH respectively. A second gene, CYP15A1 was reported to encode a cytochrome P450 enzyme responsible for the epoxidation of MF to JH. Additionally, a third gene, FAMeT (originally reported to encode a farnesoic acid methyltransferase) was included in this study. Using q-RT-PCR, all three genes (JHAMT, CYP15A1 and FAMeT) were found to be primarily expressed in the CA of the desert locust, the main biosynthetic tissue of JH. An RNA interference approach was used to verify the orthologous function of these genes in S. gregaria. Knockdown of the three genes in adult animals followed by the radiochemical assay (RCA) for JH biosynthesis and release showed that SgJHAMT and SgCYP15A1 are responsible for synthesis of MF and JH respectively. Our experiments did not show any involvement of SgFAMeT in JH biosynthesis in the desert locust. Effective and selective inhibitors of SgJHAMT and SgCYP15A1 would likely represent selective biorational locust control agents.

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 (SgOct alpha R and SgOct beta R) 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 SgOctaR are the highest in the central nervous system, whereas those of SgOct beta R are the highest in the flight muscles, followed by the central nervous system. Both SgOct alpha R and SgOct beta R show higher transcript levels in long-term gregarious locusts as compared to solitarious ones. The rise of SgOct beta R transcript levels already appears during the first 4 h of gregarisation, during which also the behavioural changes take place.

The very first step in the study of the endocrine control of insect molting was taken in 1922. Stefan Kopec characterized a factor in the brain of the gypsy moth, Lymantria dispar which appeared to be essential for metamorphosis. This factor was later identified as the neuropeptide prothoracicotropic hormone (PTTH), the first discovery of a series of factors involved in the regulation of ecdysteroid biosynthesis in insects. It is now accepted that PTTH is the most important regulator of prothoracic gland (PG) ecdysteroidogenesis. The periodic increases in ecdysteroid titer necessary for insect development can basically be explained by the episodic activation of the PGs by PTTH. However, since the characterization of the prothoracicostatic hormone (PTSH), it has become clear that in addition to 'tropic factors', also 'static factors', which are responsible for the 'fine-tuning' of the hemolymph ecdysteroid titer, are at play. Many of these regulatory factors are peptides originating from the brain, but also other, extracerebral factors both of peptidic and non-peptidic nature are able to affect PG ecdysteroidogenesis, such as the 'classic' insect hormones, juvenile hormone (JH) and the molting hormone (20E) itself. The complex secretory pattern of ecdysteroids as observed in vivo is the result of the delicate balance and interplay between these ecdysiotropic and ecdysiostatic factors.

Literature on the question whether the juvenile stage of vertebrates is hormonally regulated is scarce. It seems to be intuitively assumed that this stage of development is automated, and does not require any specific hormone(s). Such reasoning mimics the state of affairs in insects until it was shown that surgical removal of a tiny pair of glands in the head, the corpora allata, ended larval life and initiated metamorphosis. Decades later, the responsible hormone was found and named "juvenile hormone" (JH) because when present, it makes a larva molt into another larval stage. JH is a simple ester of farnesol, a sesquiterpenoid present in all eukaryotes. Whereas vertebrates do not have an anatomical counterpart of the corpora allata, their tissues do contain farnesol-like sesquiterpenoids (FLS). Some display typical JH activity when tested in appropriate insect bioassays. Some FLS are intermediates in the biosynthetic pathway of cholesterol, a compound that insects and nematodes (=Ecdysozoa) cannot synthesize by themselves. They ingest it as a vitamin. Until a recent (2014) reexamination of the basic principle underlying insect metamorphosis, it had been completely overlooked that the Ca(2+)-pump (SERCA) blocker thapsigargin is a sesquiterpenoid that mimics the absence of JH in inducing apoptosis. In our opinion, being in the juvenile state is primarily controlled by endogenous FLS that participate in controlling the activity of Ca(2+)-ATPases in the sarco(endo)plasmic reticulum (SERCAs), not only in insects but in all eukaryotes. Understanding the control mechanisms of being in the juvenile state may boost research not only in developmental biology in general, but also in diseases that develop after the juvenile stage, e.g., Alzheimer's disease. It may also help to better understand some of the causes of obesity, a syndrome that holometabolous last larval insects severely suffer from, and for which they found a very drastic but efficient solution, namely metamorphosis. ispartof: Frontiers in Endocrinology vol:5 issue:DEC ispartof: location:Switzerland status: published

During development and reproduction the response to ecdysteroids is mediated by a heterodimeric receptor complex comprising the retinoid X receptor/ultraspiracle (RXR/USP) and the ecdysone receptor (EcR). Here, the role of these receptors in the endocrine control of reproduction is examined in the cockroach Diploptera punctata. We report the sequence of four DpRXR and three DpEcR splice variants, including the first description of a Drosophila EcRB2-like isoform in a hemimetabolous insect. DpRXR and DpEcR are broadly expressed in the tissues of adult females, with relatively high transcript levels in the corpora allata (CA), nervous tissue and ovary. Developmental profiling revealed an inverse correlation between DpRXR and DpEcR expression and the activity of the CA. RNAi-mediated depletion of DpRXR and DpEcR did not affect oocyte growth, but inhibited oviposition and impaired chorion formation. Retained oocytes exhibited a degenerating follicular epithelium and were slowly resorbed. Treated animals showed significantly higher rates of JH biosynthesis and a decrease in ecdysteroid titers at the end of vitellogenesis. Reduction of DpRXR and DpEcR expression resulted in an upregulation of genes involved in JH production and a downregulation of allatostatin receptor mRNA in the CA. Treatment with dsRNA also affected the expression of genes downstream of JH in target tissues including vitellogenin and Kruppel-homolog 1 as well as Broad-Complex, an early ecdysone response gene. Overall, results suggest that DpRXR and DpEcR are not required early in the reproductive cycle when events are JH-dependent, but do mediate critical ecdysteroid feedback to the CA late in the gonadotropic cycle.

The NMDA receptor (NMDAR) plays important roles in excitatory neurotransmission and in the regulation of reproduction in mammals. NMDAR in insects comprises two subunits, NR1 and NR2. In this study, we identified two NR1 paralogs and eleven NR2 alternatively spliced variants in the cockroach Diploptera punctata. This is the first report of NR1 paralogs in insects. The tissue distributions and expression profiles of DpNR1A, DpNR1B and DpNR2 in different tissues were also investigated. Previous studies have demonstrated NMDA-stimulated biosynthesis of juvenile hormone (JH) in the corpora allata through the influx of extracellular Ca2+ in Diploptera punctata. However, our data show that the transcript levels of DpNR1A, DpNR1B and DpNR2 were low in the corpora allata. MK-801, a high-affinity antagonist of NMDAR, did not show any effect on JH biosynthesis in vitro. In addition, neither partial knockdown of DpNR2 nor in vivo treatment with a physiologically relevant dose of MK-801 resulted in any significant change in JH biosynthesis or basal oocyte growth. Injection of animals with a high dose of MK-801 (30 µg per animal per injection), which paralyzed the animals for 4–5 h, resulted in a significant decrease in JH biosynthesis on days 4 and 5. However, the reproductive events during the first gonadotrophic cycle in female D. punctata were unaffected. Thus, NMDAR does not appear to play important roles in the regulation of JH biosynthesis or mediate reproduction of female D. punctata.

The FGLamide allatostatins (FGL/ASTs) are a family of neuropeptides with pleiotropic functions, including the inhibition of juvenile hormone (JH) biosynthesis, vitellogenesis and muscle contraction. In the cockroach, Diploptera punctata, thirteen FGLa/ASTs and one allatostatin receptor (AstR) have been identified. However, the mode of action of ASTs in regulation of JH biosynthesis remains unclear. Here, we determined the tissue distribution of Dippu-AstR. And we expressed Dippu-AstR in vertebrate cell lines, and activated the receptor with the Dippu-ASTs. Our results show that all thirteen ASTs activated Dippu-AstR in a dose dependent manner, albeit with different potencies. Functional analysis of AstR in multiple cell lines demonstrated that activation of the AstR receptor resulted in elevated levels of Ca(2+) and cAMP, which suggests that Dippu-AstR can act through the Gαq and Gαs protein pathways. The study on the target of AST action reveals that FGL/AST affects JH biosynthesis prior to the entry of acetyl-CoA into the JH biosynthetic pathway. publisher: Elsevier articletitle: Mode of action of allatostatins in the regulation of juvenile hormone biosynthesis in the cockroach, Diploptera punctata journaltitle: Insect Biochemistry and Molecular Biology articlelink: http://dx.doi.org/10.1016/j.ibmb.2014.09.001 content_type: article copyright: Copyright © 2014 Elsevier Ltd. All rights reserved. ispartof: Insect Biochemistry and Molecular Biology vol:54 pages:61-68 ispartof: location:England status: published

The paradigm saying that release of the brain neuropeptide big prothoracicotropic hormone (PTTH) initiates metamorphosis by activating the Torso-receptor/ERK pathway in larval prothoracic glands (PGs) is widely accepted nowadays. Upon ligand-receptor interaction Ca(2+) enters the PG cells and acts as a secondary messenger. Ecdysteroidogenesis results, later followed by apoptosis. Yet, some data do not fit in this model. In some species decapitated animals can still molt, even repeatedly, and metamorphose. PTTH does not universally occur in all insect species. PGs may also have other functions; PGs as counterpart of the vertebrate thymus? There are also small PTTHs. Finally, PTTH remains abundantly present in adults and plays a role in control of ecdysteroidogenesis (=sex steroid production) in gonads. This is currently documented only in males. This urges a rethinking of the PTTH-PG paradigm. The key question is: Why does PTTH-induced Ca(2+) entry only result in ecdysteroidogenesis and apoptosis in specific cells/tissues, namely the PGs and gonads? Indeed, numerous other neuropeptides also use Ca(2+) as secondary messenger. The recent rediscovery that in both invertebrates and vertebrates at least some isoforms of Ca(2+)-ATPase need the presence of an endogenous farnesol/juvenile hormone(JH)-like sesquiterpenoid for keeping cytosolic [Ca(2+)]i below the limit of apoptosis-induction, triggered the idea that it is not primarily PTTH, but rather the drop to zero of the JH titer that acts as the primordial initiator of metamorphosis by increasing [Ca(2+)]i. PTTH likely potentiates this effect but only in cells expressing Torso. PTTH: an evolutionarily ancient gonadotropin? publisher: Elsevier articletitle: Initiation of metamorphosis and control of ecdysteroid biosynthesis in insects: The interplay of absence of Juvenile hormone, PTTH, and Ca2+-homeostasis journaltitle: Peptides articlelink: http://dx.doi.org/10.1016/j.peptides.2014.07.025 content_type: article copyright: Copyright © 2014 Elsevier Inc. All rights reserved. ispartof: Peptides vol:68 pages:120-129 ispartof: location:United States status: published

Independent of the design of the life cycle of any insect, their growth and reproduction are highly choreographed through the action of two versatile hormones: ecdysteroids and juvenile hormones (JH). However, the means by which JH can target tissues and exert its pleiotropic physiological effects is currently still not completely elucidated. Although the identity of the one JH receptor is currently still elusive, recent evidence seems to point to the product of the Methoprene-tolerant gene (Met) as the most likely contender in transducing the action of JH. Studies on the role of this transcription factor have mostly been focused on immature insect stages. In this study we used the viviparous cockroach Diploptera punctata, a favorite model in studying JH endocrinology, to examine the role of Met during reproduction. A tissue distribution and developmental profile of transcript levels was determined for Met and its downstream partners during the first gonadotropic cycle of this cockroach. Using RNA interference, our study shows that silencing Met results in an arrest of basal oocyte development; vitellogenin is no longer transcribed in the fat body and no longer taken up by the ovary. Patency is not induced in these animals which fail to produce the characteristic profile of JH biosynthesis typical of the first gonadotropic cycle. Moreover, the ultrastructure of the follicle cells showed conspicuous whorls of rough endoplasmic reticulum and a failure to form chorion. Our study describes the role of Met on a cellular and physiological level during insect reproduction, and confirms the role of Met as a key factor in the JH signaling pathway.

A major breakthrough in elucidating the ecdysteroid biosynthetic pathway in insects was realized with the molecular identification and further functional characterization of the 'Halloween' genes. These genes were found to encode cytochrome P450 enzymes catalysing the final steps of ecdysteroid biosynthesis in the dipteran, Drosophila melanogaster, and in the Lepidoptera, Manduca sexta and Bombyx mori. A recent report focused on the identification of Halloween orthologs in the desert locust, Schistocerca gregaria, a member of the hemimetabolous insect order of the Orthoptera. In the present study, an additional Halloween gene Shade, is identified in the desert locust. In Diptera and Lepidoptera, this gene encodes a 20-hydroxylase, catalysing the conversion of ecdysone (E) to 20-hydroxyecdysone (20E). However, this enzymatic function has previously been suggested for CYP6H1 in another locust species, the migratory locust, Locusta migratoria. Using q-RT-PCR, the spatial and temporal transcript profiles of S. gregaria orthologs for Shade as well as CYP6H1 were analysed in last larval stage desert locusts. An RNA interference (RNAi)-based approach was employed to study whether these genes could possibly encode a functional 20-hydroxylase in the desert locust.

Diuretic hormones (DH) related to the vertebrate Corticotropin Releasing Factor (CRF) have been identified in diverse insect species. In the migratory locust, Locusta migratoria, the CRF-like DH (CRF/DH) is localized in the same neurosecretory cells as the Ovary Maturating Parsin (OMP), a neurohormone that stimulates oocyte growth, vitellogenesis and hemolymph ecdysteroid levels in adult female locusts. In this study, we investigated whether CRF-like DH can influence feeding and reproduction in the desert locust, Schistocerca gregaria. We identified two highly similar S. gregaria CRF-like DH precursor cDNAs, each of which also encodes an OMP isoform. Alignment with other insect CRF-like DH precursors shows relatively high conservation of the CRF/DH sequence while the precursor region corresponding to OMP is not well conserved. Quantitative real-time RT-PCR revealed that the precursor transcripts mainly occur in the central nervous system and their highest expression level was observed in the brain. Injection of locust CRF/DH caused a significantly reduced food intake, while RNAi knockdown stimulated food intake. Therefore, our data indicate that CRF-like DH induces satiety. Furthermore, injection of CRF/DH in adult females retarded oocyte growth and caused lower ecdysteroid titers in hemolymph and ovaries, while RNAi knockdown resulted in opposite effects. The observed effects of CRF/DH may be part of a wider repertoire of neurohormonal activities, constituting an integrating control system that affects food intake and excretion, as well as anabolic processes like oocyte growth and ecdysteroidogenesis, following a meal. Our discussion about the functional relationship between CRF/DH and OMP led to the hypothesis that OMP may possibly act as a monitoring peptide that can elicit negative feedback effects. ispartof: PLoS One vol:7 issue:2 pages:1-14 ispartof: location:United States status: published