Allatostatin A: Biological Overview | References
Gene name - Allatostatin A
Cytological map position - 96A20-96A20
Function - neuropeptide
Keywords - peptide secreted by the corpus cardiacum of the ring gland - a modulator of AKH and DILP signaling - a modulator of feeding choices between dietary carbohydrates and protein - adapts the fly to a digestive energy-saving state - conveys inhibitory input onto protocerebral dopamine neurons
Symbol - AstA
FlyBase ID: FBgn0015591
Genetic map position - chr3R:24,760,516-24,765,099
Classification - allatostatin preprohormone
Cellular location - secreted
|Recent literature||Christ, P., Hill, S. R., Schachtner, J., Hauser, F. and Ignell, R. (2017). Functional characterization of the dual allatostatin-A receptors in mosquitoes. Peptides 99: 44-55. PubMed ID: 29103918
The neuropeptide allatostatin-A (AstA) and its cognate receptors (AstARs) are involved in the modulation of feeding behavior, which in hematophagous insects includes the regulation of the disease vector-related behaviors, host seeking and blood feeding. In mosquitoes and other dipterans, there are two copies of AstAR, contrasting with the single copy found in other insects. This study identified and cloned the dual AstAR system of two important disease vectors Aedes aegypti and Culex quinquefasciatus, and compared them with those previously described, including those in Anopheles coluzzii and Drosophila melanogaster. Phylogenetic analysis of the AstARs revealed that the mosquito AstAR1s has retained a similar amino acid sequence as the AstARs from non-dipteran insect species. Intron analysis revealed that the number of introns accumulated in the AstAR2s is similar to that in other insects, and that introns are conserved within the receptor types, but that only the final two introns are conserved across AstAR1s and 2s. The dual AstARs were characterized in An. coluzzii, Ae. aegypti and Cx. quinquefasciatus by stably expressing the receptors in a Chinese hamster oocyte cell line (CHO) also stably expressing a promiscuous G-protein (G16), and challenged them with the endogenous isoforms of AstA from the three mosquito species. In the culicine mosquitoes, Ae. aegypti and Cx. quinquefasciatus, the AstARs demonstrated differential sensitivity to AstA, with the AstAR2s displaying a higher sensitivity than the AstAR1s, suggesting a divergence of functional roles for these AstARs. In contrast, both An. coluzzii AstARs demonstrated a similar sensitivity to the AstA ligands. These findings are discussed in the light of AstA acting as a regulator of blood feeding in mosquitoes. A better understanding of the regulation of host seeking and blood feeding in vector mosquitoes will lead to the rational development of novel approaches for vector control.
Coordinating metabolism and feeding is important to avoid obesity and metabolic diseases, yet the underlying mechanisms, balancing nutrient intake and metabolic expenditure, are poorly understood. Several mechanisms controlling these processes are conserved in Drosophila, where homeostasis and energy mobilization are regulated by the glucagon-related adipokinetic hormone (AKH) and the Drosophila insulin-like peptides (DILPs). This study provides evidence that the Drosophila neuropeptide Allatostatin A (AstA) regulates AKH and DILP signaling. The AstA receptor gene, Dar-2, is expressed in both the insulin and AKH producing cells. Silencing of Dar-2 in these cells results in changes in gene expression and physiology associated with reduced DILP and AKH signaling and animals lacking AstA accumulate high lipid levels. This suggests that AstA is regulating the balance between DILP and AKH, believed to be important for the maintenance of nutrient homeostasis in response to changing ratios of dietary sugar and protein. Furthermore, AstA and Dar-2 are regulated differentially by dietary carbohydrates and protein and AstA-neuronal activity modulates feeding choices between these types of nutrients. These results suggest that AstA is involved in assigning value to these nutrients to coordinate metabolic and feeding decisions, responses that are important to balance food intake according to metabolic needs (Hentze, 2015).
Imbalance between the amount and type of nutrients consumed and metabolized can cause obesity. It is therefore important to understand how animals maintain energy balancing, which is determined by mechanisms that guide feeding decisions according to the internal nutritional status. The fruit fly Drosophila melanogaster has become an important model for studies of feeding and metabolism, as the regulation of metabolic homeostasis is conserved from flies to mammals. In Drosophila, hormones similar to insulin and glucagon regulate metabolic programs and nutrient homeostasis. Adipokinetic hormone (AKH) is an important metabolic hormone and considered functionally related to human glucagon and a key regulator of sugar homeostasis (Lee, 2004). The release of AKH promotes mobilization of stored energy from the fat body, the equivalent of the mammalian liver and adipose tissues. Neuroendocrine cells in the corpus cardiacum (CC) express and release AKH3 that binds to the AKH receptor (AKHR), a G-protein coupled receptor (GPCR) expressed mainly in the fat body, and promotes mobilization of stored sugar and fat. Insulin and glucagon have opposing effects important to maintain balanced blood glucose levels. The Drosophila genome contains 7 genes coding for insulin-like peptides (DILPs), called dilp1-7, which are homologous to the mammalian insulin and insulin-like growth factors (IGFs). The seven DILPs are believed to act through one ortholog of the human insulin receptor that activates conserved intracellular signaling pathways. The DILPs are important regulators of metabolism, sugar homeostasis and cell growth. DILP2, 3 and 5 are produced in 14 neurosecretory cells in the brain; the insulin producing cells (IPCs). Genetic ablation of the IPCs results in a diabetic phenotype, increased lifespan and reduced growth. Because of the growth promoting effects, the activity of the DILPs is tightly linked to dietary amino acid concentrations (Hentze, 2015).
Although metabolism has been extensively studied, the mechanisms that coordinate metabolism and feeding decisions to maintain energy balancing are poorly understood. Neuropeptides are major regulators of behavior and metabolism in mammals and insects making them obvious candidates to coordinate these processes. Peptides with a FGL-amide carboxy terminus, called type A allatostatins, have previously been related to feeding and foraging behavior (Wang, 2012; Hergarden, 2012). Four Drosophila Allatostatin A (AstA) peptides have been identified (Lenz, 2000a) that are ligands for two GPCRs, the Drosophila Allatostatin A receptors DAR-1 and DAR-2. AstA peptides were originally identified as inhibitors of juvenile hormone (JH) synthesis from the corpora allata (CA) of the cockroach Diploptera punctata. However, recently it was shown that AstA does not regulate JH in Drosophila. Moreover, DAR-1 and DAR-2 are homologs of the mammalian galanin receptors, known to be involved in both feeding behavior and metabolic regulation (Hentze, 2015).
The function of AstA in Drosophila was examined in an effort to determine whether it is involved in the neuroendocrine mechanisms coupling feeding behavior to metabolic pathways that manage energy supplies. The data suggest that AstA is a modulator of AKH and DILP signaling. Dar-2 is expressed in both the IPCs and the AKH producing cells (APCs) of the CC. Silencing of AstA receptor gene Dar-2 in the APCs or IPCs resulted in changes in expression of genes associated with reduced AKH or DILP signaling, respectively. Moreover, loss of AstA is associated with increased fat body lipid levels, resembling the phenotype of mutants in the DILP and AKH pathways. The connection between nutrients and AstA signaling was also investigated, and AstA and Dar-2 were found to be regulated differently in response to dietary carbohydrates and protein, and activation of AstA-neurons was found to increase the preference for a protein rich diet, while AstA loss enhances sugar consumption. The results suggest that AstA is a key coordinator of metabolism and feeding behavior (Hentze, 2015).
In order to adjust energy homeostasis to different environmental conditions, feeding-related behavior needs to be coordinated with nutrient sensing and metabolism. The current data suggest that AstA is a modulator of AKH and DILP signaling that control metabolism and nutrient storage, but also affects feeding decisions. The positive effect of AstA on AKH signaling indicated by these observations is supported by the recent finding that expression of a presumably constitutive active mu opioid receptor, a mammalian GPCR which is also closely related to DAR-2, stimulates AKH release from the APCs in Drosophila. Moreover, AstA-type peptides have also been shown to stimulate AKH release in Locusta migratoria. AKH is primarily regulated at the level of secretion to allow a rapid response to metabolic needs. Considering that only a minor effect of Dar-2 silencing in the APCs on Akh transcription was detected, it is likely that AstA primarily acts at the level of AKH release in Drosophila (Hentze, 2015).
The data suggest regulation of both the DILPs and AKH by AstA indicating a close coupling between the activity of these two hormones. Consistent with this notion, the results also indicate a feedback relationship between the IPCs and APCs. The IPCs have processes that contact the corpora cardiaca (CC) cells of the ring gland and it is possible that DILPs released from these affect AKH release. The current findings are supported by a previous study that identifies a tight association between DILPs and AKH secretion in Drosophila. Furthermore, it was recently found that AKH regulates DILP3 release from the IPCs, and that sugar promotes DILP3 release, while DILP2 release is amino acid dependent. Interestingly, the data, which suggest that AstA is involved in the cross-talk between DILPs and AKH related specifically to sugar and protein, also indicate that AstA has a strong influence on dilp3 expression. Why is the relationship between the DILPs and AKH so tight? Even though insulin-like peptides reduce hemolymph sugar, they also reduce the content of stored glycogen and lipids, like AKH. Consistent with this, both AKH and the DILPs stimulate expression of tobi, which encodes a glycosidase believed to be involved in glycogen breakdown. However, since AKH and the DILPs have opposing effect on hemolymph sugar levels, a balance between these hormones is presumably required to maintain homeostasis. It is likely that different sources of AstA affect these two hormones, since the IPCs are located in the brain in proximity of AstA-positive neurites, while AstA-positive processes do not innervate the CC. Thus, it is likely that neuronal-derived AstA affects DILP secretion from the IPCs, while circulating AstA, which may be released from the endocrine cells of the gut, may be the source of AstA that acts on the APCs to regulate AKH. AstA regulation of DILP and AKH release may therefore not occur simultaneously and could also depend on the type of nutrient ingested, or be sequential. Since the data suggest feedback regulation between AKH and DILP, the overall outcome of simultaneous AstA induced activation of both cell types will not necessarily be a strong and equal increase in both hormones in the hemolymph. It is possible that AstA is involved in metabolic balancing, adjusting the ratio between AKH and DILPs in response to different dietary conditions. In mammals, glucagon and insulin are secreted simultaneously when the animal feeds on a protein-rich diet, to prevent hypoglycemia and promote cellular protein synthesis, since insulin is strongly induced after ingestion of amino acids. A similar mechanism has been proposed to explain the relationship between DILPs and AKH in Drosophila. The balance between DILP and AKH therefore may be important for resource allocation into growth and reproduction (Hentze, 2015).
Several differences in the expression of genes involved in energy mobilization were observed between males and females, which possibly reflects sex-specific strategies for energy mobilization and allocation of resources towards reproduction. Interestingly, 4EBP expression was significantly decreased in females with reduced AKH signaling, but upregulated in males. This suggests that in females AKH has a strong negative influence on DILP signaling that is not present in males. Why does the interaction between AKH and DILPs differ between sexes? An interesting possibility is that this sexually dimorphic interaction is related to the different preferences and requirements for sugar and protein in males and females. Males generally have a higher preference for sugar compared to females that prefer more dietary protein and show strong correlation between amino acid uptake, insulin and reproduction. In both mammals and Drosophila the balance between insulin and glucagon/AKH is important for nutrient homeostasis in response to high-protein versus high-sugar diets. This balance ensures that insulin promotes protein synthesis in response to dietary amino acids, while maintaining sugar levels stable, a function possibly important in females to allocate the high consumption of amino acids into reproduction. Thus, the sex-specific interplay between DILPs and AKH likely reflects difference in the metabolic wiring of males and females that underlie the sexually dimorphic reproductive requirements for dietary sugar and protein (Hentze, 2015).
Interestingly, AstA expression showed a general increase after feeding with a stronger transcriptional response of both AstA and Dar-2 to the carbohydrate rich diet compared to the protein rich diet. AstA may therefore be important for coordinating carbohydrate and protein dependent metabolic programs. The strong response to carbohydrates indicates that AstA may be involved in signaling related to carbohydrate feeding, although increased transcription may not necessarily result in elevated release of the mature AstA peptide. Nonetheless, the data indicate that feeding regulates AstA-signaling and that the response is influenced by the food composition. Consistent with the notion that AstA is involved in different responses to dietary carbohydrate and protein, this study found that flies with increased AstA neuronal activity increase their protein preference on the expense of their natural preference for sucrose. The AstA regulated circuitry may therefore be important for guiding the decision to feed on protein or sugar, a decision influenced by metabolic needs (Ribeiro, 2010). The AstA neurons have projections that may contact the Gr5a sugar sensing neurons and AstA>NaChBac flies with increased activity of the AstA neurons display reduced feeding and responsiveness to sucrose under starvation (Hergarden, 2012). Thus, the increased preference for dietary protein in AstA>NaChBac flies observed in this study may be caused by reduced sucrose responsiveness. If AstA signaling is high after feeding on carbohydrates as indicated by the data showing increased expression of AstA and Dar-2, then an increase in AstA signaling might mimic carbohydrate satiety. In line with this view, the data show that animals lacking AstA enhance their intake of dietary sugar. AstA signaling may therefore increase the animals preference for essential amino acids, as suggested by a recent study indicating that amino acid depleted flies increased their taste sensitivity for amino acids, even when they were replete with glucose. Based on the current data, it is therefore proposed that AstA plays a central role in a circuitry important for encoding nutritional value related to these distinct nutrients and the regulation of feeding decisions and metabolic programs. Excess dietary sugar is associated with obesity, and this study found that flies lacking AstA enhance intake of sugar and have increased lipid storage droplets in their fat bodies, like animals lacking AKH or its receptor. Thus, the data implicate AstA in regulation of appetite and food intake related to sugar, which is relevant for understanding obesity (Hentze, 2015).
This study suggests that AstA affects metabolism through its action on two key players, the DILPs and AKH. AstA expression is induced by feeding, but exhibits a differential nutritional response to dietary sugar and protein and influence metabolic programs and feeding choices associated with the intake of these nutrients. Interestingly, the homolog of AstA, galanin, regulates both feeding and metabolism in mammals and in Caenorhabditis elegans loss of the Allatostatin/galanin-like receptor npr9 affects foraging behavior and nutrient storage (Bendena, 2008). Altogether the data suggest that AstA is part of a conserved mechanism involved in coordinating nutrient sensing, feeding decisions and metabolism to ensure adequate intake of amino acids and sugar to maintain nutrient homeostasis under different feeding conditions (Hentze, 2015).
Feeding and sleep are fundamental behaviours with significant interconnections and cross-modulations. The circadian system and peptidergic signals are important components of this modulation, but still little is known about the mechanisms and networks by which they interact to regulate feeding and sleep. This study shows that specific thermogenetic activation of peptidergic Allatostatin A (AstA)-expressing posterior lateral protocerebrum (PLP) neurons and enteroendocrine cells reduces feeding and promotes sleep in the fruit fly Drosophila. The effects of AstA cell activation are mediated by AstA peptides with receptors homolog to galanin receptors subserving similar and apparently conserved functions in vertebrates. The PLP neurons are identified to be a downstream target of the neuropeptide pigment-dispersing factor (PDF), an output factor of the circadian clock. PLP neurons are contacted by PDF-expressing clock neurons, and express a functional PDF receptor demonstrated by cAMP imaging. Silencing of AstA signalling and continuous input to AstA cells by tethered PDF changes the sleep/activity ratio in opposite directions but does not affect rhythmicity. Taken together, these results suggest that pleiotropic AstA signalling by a distinct neuronal and enteroendocrine AstA cell subset adapts the fly to a digestive energy-saving state which can be modulated by PDF (Chen, 2016).
Neuropeptides and peptide hormones transfer a wide variety of neuronal or physiological information from one cell to the other by activating specific receptors on their target cells. Most if not all peptides are pleiotropic and can orchestrate diverse physiological, neuronal or behavioural processes. In vertebrates, such a pleiotropic effect is especially prominent in the regulation of feeding and sleep. Many different peptides (e.g. orexin/hypocretin, ghrelin, obestatin) modulate different aspects of both behaviours, which reciprocally influence each other. The temporal pattern of neuroendocrine activity and neuropeptide release is shaped by sleep homeostasis and the circadian clock which, in turn, reciprocally affects feeding and sleep-wake cycles. Significant progress has been made in this field during recent years. Still little characterised, however, is the neuronal architecture that enables the relevant peptidergic neurons to integrate energy status, circadian time and sleep-wake status in order to coordinate the timing of sleep, locomotor activity and feeding. Information about the output signals by which endogenous clocks provide time- and non-circadian information to relevant peptidergic cells is still limited (Chen, 2016).
During the last years, the fruit fly Drosophila has become an important model for research into the regulation of feeding and sleep. Drosophila offers advanced genetic tools, a small brain with only about 100.000 neurons and a quantifiable sleep- and feeding behaviour that shows characteristics very similar to that of mammals. These features greatly facilitate the analysis of the neuronal and endocrine underpinnings of feeding and sleep. Like in most animals, feeding and sleep follow a circadian pattern in the fruit fly with little characterised neuronal and hormonal pathways downstream of the central clock. Like in mammals, a number of neuropeptides have been shown to be involved in the regulation of feeding or sleep in Drosophila. Yet, so far, only sNPF and likely also NPF are implicated in the regulation of both feeding and sleep. Also Insulin-like peptide (DILP)-expressing neurons (IPCs) in the pars intercerebralis affect feeding and sleep, yet only feeding seems to be directly dependent on DILP signalling (Chen, 2016).
Recent work by Hergarden (2012) demonstrated that neurons expressing neuropeptides of the allatostatin A (AstA) family regulate feeding behaviour of the fruit fly. Constitutive activation of AstA cells contained in the AstA1-Gal4 expression pattern by ectopic expression of the bacterial low threshold voltage-gated NaChBac channel potently inhibited starvation-induced feeding (Nitabach, 2006). In contrast, constitutive inactivation of AstA1 cells by expression of the inwardly rectifying Kir2.1 potassium channel (Baines, 2001) increased feeding under restricted food availability. NaChBac activation of AstA1 cells also inhibited the starvation-induced increase of the proboscis extension reflex (PER), a behavioural indicator for glucose responsiveness (Hergarden, 2012). The AstA1 expression pattern includes a large number of brain neurons plus gut-innervating thoracico-abdominal ganglion (TAG) neurons and enteroendocrine cells (EECs) in the posterior midgut (Hergarden, 2012). This broad expression pattern is consistent with earlier described patterns of AstA-like immunoreactivity and suggests multiple functions for AstA. Earlier work had demonstrated an effect of AstA on gut motility (Vanderveken, 2014). Two AstA receptors, DAR-1 (= AlstR) and DAR-2 are characterised for Drosophila (Birgül, 1999; Lenz, 2000a; Lenz, 2000b; Larsen, 2001). Different genome-based phylogenetic GPCR analyses independently demonstrated their homology with the galanin receptor family of vertebrates (Chen, 2016).
Using anatomical subdivision and genetic manipulation of neuronal activity, this study aimed to identify AstA functions and assign them to subsets of AstA expressing cells. The results revealed new interconnected AstA functions that link feeding and sleep and identify AstA-expressing PLP neurons and EECs as a target of the central clock output factor PDF. Pleiotropic AstA signalling seems capable of coordinating multiple aspects of physiology and behaviour in a coherent manner to adapt the fly to a digestive energy-saving state. The functional range of AstA signalling in the fly is thus reminiscent of the pleiotropy found in mammalian galanin signalling (Chen, 2016).
This study shows that AstA cells via AstA signalling subserve an anorexigenic and sleep-promoting function in Drosophila. In mammals, a variety of neuropeptides and peptide hormones affect both sleep and feeding, and the results provide evidence that also further such peptides exist in the fly besides sNPF and possibly NPF. More specifically, the results with a new AstA34-Gal4 driver line show that activation of AstA-expressing PLP brain neurons or numerous EECs in the midgut strongly reduces food intake and promotes sleep. These behavioural effects are congruent with the anatomy of these cells. PLP interneurons are well positioned to modulate sleep as they widely arborise in the posterior superior protocerebrum, a projection area of sleep-relevant dopaminergic neurons, superior (dorsal) fan-shaped body neurons and neurons of the pars intercerebralis. AstA EECs in Drosophila are 'open type' EECs, possessing apical extensions that reach the gut lumen and likely express gustatory receptors. AstA-expressing EECs are thus potentially able to humorally signal nutritional information from the gut to brain centres regulating feeding and possibly also sleep and locomotor activity. If AstA is involved in inhibiting feeding and promoting sleep, one could expect AstA mutants to display decreased sleep and increased feeding in the absence of any other manipulation of AstA cells. It was observed, however, that a functional loss of the AstA gene did neither affect feeding nor locomotor activity under the experimental conditions with unrestricted access to a food source. This may suggest that AstA signalling is not part of a core feeding network, but represents an extrinsic modulator which becomes activated under specific yet so far uncharacterised conditions. Alternatively, as suggested by the observed difference in effect of constitutive vs. conditional electrical silencing of AstA cells, flies may be able to genetically or neuronally compensate for a constitutive loss of AstA signalling during development (Chen, 2016).
In larval Drosophila, AstA inhibits midgut peristalsis and affects K+ transport (Vanderveken, 2014) in order to concentrate ingested food. Together with the finding of a sleep-promoting and feeding-inhibiting effect of AstA, it is proposed that pleiotropic AstA signalling serves to coordinate behaviour and gut physiology to allow for efficient digestion. After food intake, AstA from the PLP neurons or EECs cause inhibition of further feeding, and -as the need for food search behaviour is relieved and nutrients need to be taken up- promotes sleep and inhibits gut peristalsis. Based on the gut content, enteroendocrine AstA is released and hormonally activates DAR-2 on key metabolic centers to tune adipokinetic hormone and insulin signalling (Hentze, 2015), and -at least in other insects- stimulates digestive enzyme activity in the midgut (Aguilar, 2003; Chen, 2016 and references therein).
The AstA receptors are homologues of the vertebrate galanin receptors that have pleiotropic functions. When activated in specific brain areas, galanin signalling has a strong orexigenic effect and has also been implicated in the control of arousal and sleep in mammals (Lang, 2015). In zebrafish, transgenic heat-shock induced expression of galanin decreased swimming activity, the latency to rest at night and decreased the responsiveness to various stimuli. Furthermore, the allatostatin/galanin-like receptor NPR-9 inhibits local search behaviour on food in the nematode C. elegans (Bendena, 2008). Similar to AstA in Drosophila (Vanderveken, 2014), galanin modulates intestinal motility and ion transport (Lange, 2007). Thus, in broad terms, the involvement of DARs/galanin receptors in modulating feeding, gut physiology and arousal/sleep appears to be evolutionarily conserved (Chen, 2016).
The neuronal clock network in Drosophila is intrinsically and extrinsically modulated by a variety of peptides (sNPF, NPF, calcitonin-gene related peptide/DH31, ion transport peptide, myoinhibiting peptides and PDF), which all affect sleep and locomotor activity and in part also act as clock output factors. Imaging results and constitutive activation of the PDF signalling pathway by t-PDF now suggest that the PLP neurons are modulated by PDF originating from the sLNv clock neurons. Unlike the peptides above, AstA from PLP neurons is outside and downstream of the central clock and seems not to modulate the clock network. Due to their anatomy and position, PLP neurons thus appear well-suited candidate cells by which clock neurons could modulate the complex cross-regulatory network regulating sleep, locomotor activity and perhaps also feeding. The rather mild effects on sleep and feeding of either t-PDF expression in AstA cells or thermogenetic activation of the sLNvs implies that this pathway is not the major output target of the central clock (if there is any) to modulate feeding and locomotor activity/sleep. This study found no shift in the circadian period or phase of feeding and locomotory activity/sleep upon AstA cell activation, suggesting that the main function of PDF-to-AstA cell signalling is not to time the respective behaviours but to modulate their amplitude. Similar non-timing functions of PDF have been demonstrated for other behaviours, including geotaxis and rival-induced mating duration (Chen, 2016).
At first sight, the current data suggesting that PDF activates PLP neurons to promote sleep seem to contradict earlier findings (Parisky, 2008). Since pdf01 mutants show increased sleep during the photophase, the arousal effect appears to be the dominant effect of PDF which is due to signalling between ventral lateral clock neurons (LNvs), with a major contribution of the PDF-expressing large LNvs. The PLP neurons are only contacted by the sLNvs, which upon activation induced a time-specific increase in sleep, but did not increase arousal. Thus, the sLNv-PLP pathway likely represents a sleep-promoting clock output branch. Besides PDF, the sLNvs but not the lLNvs also co-localise the sleep-promoting peptide sNPF. A recent report shows that hormonal PDF released from abdominal PDF neurons serves to couple the central clock with a peripheral clock in the oenocytes. Furthermore, the posterior midgut is innervated by the abdominal PDF neurons, and PDFR is expressed in the midgut. It is thus possible that the AstA-expressing EECs represent additional PDF targets and may contribute to the PDF-related effects of AstA cells (Chen, 2016).
In conclusion, the lack of effect on feeding upon AstA cell silencing under non-restricted food availability and an unaltered circadian locomotor rhythmicity after AstA cell silencing suggests that AstA signalling is neither a primary signal in feeding regulation nor in the clock output pathway timing rhythmic behaviour. Rather-like mammalian galanin signalling - it seems to be one out of several modulatory pathways that allow to adapt the intensity of feeding and locomotor activity/sleep to specific physiological or environmental conditions. For example, decreased locomotor activity to save energy and increased digestion efficiency to maximise energy uptake may be most important during restricted food conditions, at which AstA cell silencing leads to increased feeding (Hergarden, 2012). While our results allow now to raise such speculations, it is clear that more research is needed to reveal the conditions at which AstA signalling is functional and the modulatory PDF input is strongest (Chen, 2016).
Massive activation of dopamine neurons is critical for natural reward and drug abuse. In contrast, the significance of their spontaneous activity remains elusive. In Drosophila melanogaster, depolarization of the protocerebral anterior medial (PAM) cluster dopamine neurons en masse signals reward to the mushroom body (MB) and drives appetitive memory. Focusing on the functional heterogeneity of PAM cluster neurons, a single class of PAM neurons, PAM-γ3, mediates sugar reward by suppressing their own activity. PAM-γ3 is selectively required for appetitive olfactory learning, while activation of these neurons in turn induces aversive memory. Ongoing activity of PAM-γ3 gets suppressed upon sugar ingestion. Strikingly, transient inactivation of basal PAM-γ3 activity can substitute for reward and induces appetitive memory. Furthermore, the satiety-signaling neuropeptide Allatostatin A (AstA) was identified as a key mediator that conveys inhibitory input onto PAM-γ3. These results suggest the significance of basal dopamine release in reward signaling and reveal a circuit mechanism for negative regulation (Yamagata, 2016).
Sugar ingestion triggers multiple reward signals in the fly brain. This study has provided lines of evidence that part of the reward is signaled by inactivating dopamine neurons. The role of PAM-γ3 highlights the striking functional heterogeneity of PAM cluster dopamine neurons. The decrease and increase of dopamine can convey reward to the adjacent compartments of the same MB lobe-γ3 and γ4-. The reward signal by the transient decrease of dopamine is in stark contrast to the widely acknowledged role of dopamine. Midbrain dopamine neurons in mammals were shown to be suppressed upon the presentation of aversive stimuli or the omission of an expected reward, implying valence coding by the bidirectional activity. As depolarization of PAM-γ3 can signal aversive reinforcement, these neurons convey the opposite modulatory signals to the specific MB domain by the sign of their activity. Intriguingly, the presentation and cessation of electric shock act as punishment and reward, respectively. Such bidirectional activity of PAM-γ3 may represent the presentation and omission of reward. (Yamagata, 2016).
While thermoactivation of PAM-γ3 induced robust aversive memory, blocking their synaptic transmission did not affect shock learning, leaving a question regarding their role in endogenous aversive memory process. PAM-γ3 may only be involved in processing aversive reinforcement different from electric shock-like heat. However, two studies show that dopamine neurons mediating aversive reinforcement of high temperature and bitter N,N-Diethyl-3-methylbenzamide (DEET) are part of those for electric shock. Identification of such aversive stimuli that are signaled by PAM-γ3 activation is certainly interesting, as it is perceived as the opposite of sugar reward and thus provides the whole picture of the valence spectrum. Another scenario where sufficiency and necessity do not match is the compensation of the reinforcing effect by other dopamine cell types (e.g. MB-M3). The lack of PAM-γ3 requirements for electric shock memory may be explained by a similar mechanism. (Yamagata, 2016).
How can the suppression of PAM-γ3 modulate the downstream cell and drive appetitive memory? Optogenetic activation of the MB output neurons from the γ3 compartment induces approach behavior. This suggests that the suppression of the PAM-γ3 neurons upon reward leads to local potentiation of Kenyon cell output. This model is supported by recent studies showing the depression of MB output synapses during associative learning. A likely molecular mechanism is the de-repression of inhibitory D2-like dopamine receptors, DD2R. As D2R signaling is a widely conserved mechanism, it may be one of the most ancestral modes of neuromodulation. (Yamagata, 2016).
Furthermore, recent anatomical and physiological studies demonstrated that different MB-projecting dopamine neurons are connected to each other and act in coordination to respond to sugar or shock. Therefore, memories induced by activation or inhibition of PAM-γ3 may well involve the activity of other dopamine cell types (Yamagata, 2016).
The finding that appetitive reinforcement is encoded by both activation and suppression of dopamine neurons raises the question as to the complexity of reward processing circuits (see Reward signals by excitation and inhibition of dopamine neurons). It is, however, reasonable to implement a component like PAM-γ3 as a target of the satiety-signaling inhibitory neuropeptide AstA. Intriguingly, the visualization of AstA receptor distribution by DAR-1-GAL4 revealed expression in two types of MB-projecting dopamine neurons: PAM-γ3 and MB-MV1 (also named as PPL1- γ2α'1). Given the roles of MB-MV1 in aversive reinforcement and locomotion arrest, AstA/DAR-1 signaling may also inhibit a punishment pathway upon feeding. It is thus speculated that this complex dopamine reward circuit may be configured to make use of bidirectional appetitive signals in the brain (Yamagata, 2016).
The endocrine system employs peptide hormone signals to translate environmental changes into physiological responses. The diffuse endocrine system embedded in the gastrointestinal barrier epithelium is one of the largest and most diverse endocrine tissues. Furthermore, it is the only endocrine tissue in direct physical contact with the microbial environment of the gut lumen. However, it remains unclear how this sensory epithelium responds to specific pathogenic challenges in a dynamic and regulated manner.This study demonstrates that the enteroendocrine cells of the adult Drosophila melanogaster midgut display a transient, sensitive, and systemic induction of the pro-secretory factor dimmed (dimm) in response to the Gram-negative pathogen Pseudomonas entomophila (Pe). In enteroendocrine cells, dimm controls the levels of the targets phantom, cat-4 and the peptide hormone, Allatostatin A. Finally, dimm was identified as a host factor that protects against Pe infection and controls the expression of antimicrobial peptides. It is proposed that dimm provides "gain" in enteroendocrine output during the adaptive response to episodic pathogen exposure (Beebe, 2015).
Recent studies have identified paracrine and endocrine cells in the midgut of larval Drosophila melanogaster as well as midgut and hindgut receptors for multiple neuropeptides implicated in the control of fluid and ion balance. Although the effects of diuretic factors on fluid secretion by isolated Malpighian tubules of D. melanogaster have been examined extensively, relatively little is known about the effects of such factors on gut peristalsis or ion transport across the gut. The effects were measured of diuretic hormone 31 (DH31), drosokinin and allatostatin A (AST-A) on both K(+) transport and muscle contraction frequency in the isolated gut of larval D. melanogaster. K(+) absorption across the gut was measured using K(+) -selective microelectrodes and the scanning ion-selective electrode technique. Allatostatin A (AST-A; 1 muM) increased K(+) absorption across the anterior midgut but reduced K(+) absorption across the copper cells and large flat cells of the middle midgut. AST-A strongly inhibited gut contractions in the anterior midgut but had no effect on contractions of the pyloric sphincter induced by proctolin. DH31 (1 muM) increased the contraction frequency in the anterior midgut, but had no effect on K(+) flux across the anterior, middle, or posterior midgut or across the ileum. Drosokinin (1 μM) did not affect either contraction frequency or K(+) flux across any of the gut regions examined. Possible functions of AST-A, DH31, and drosokinin in regulating midgut physiology are discussed (Vanderveken, 2014).
How the brain translates changes in internal metabolic state or perceived food quality into alterations in feeding behavior remains poorly understood. Studies in Drosophila larvae have yielded information about neuropeptides and circuits that promote feeding, but a peptidergic neuron subset whose activation inhibits feeding in adult flies, without promoting metabolic changes that mimic the state of satiety, has not been identified. Using genetically based manipulations of neuronal activity, this study shows that activation of neurons (or neuroendocrine cells) expressing the neuropeptide allatostatin A (AstA) inhibits or limits several starvation-induced changes in feeding behavior in adult Drosophila, including increased food intake and enhanced behavioral responsiveness to sugar. Importantly, these effects on feeding behavior are observed in the absence of any measurable effects on metabolism or energy reserves, suggesting that AstA neuron activation is likely a consequence, not a cause, of metabolic changes that induce the state of satiety. These data suggest that activation of AstA-expressing neurons promotes food aversion and/or exerts an inhibitory influence on the motivation to feed and implicate these neurons and their associated circuitry in the mechanisms that translate the state of satiety into alterations in feeding behavior (Hergarden, 2012).
The problem of how neural circuits that control feeding are regulated by metabolism is a fundamental one, whose logic can be studied in model organisms independently of whether the particular gene products or neural circuits involved have direct mammalian homologs. To approach this question, it is necessary to define circuits that directly control feeding behavior, which may serve as targets of metabolic influence. This study demonstrates that AstA neurons exert an inhibitory influence on multiple aspects of feeding behavior in Drosophila. This influence can be observed in the absence of any detectable effects on metabolism or energy expenditure, arguing that AstA neurons do not simply promote metabolic changes that mimic or cause satiety. This study therefore provides evidence of a neural circuit that depresses food intake, which is distinct from circuits that promote feeding and acts downstream of metabolic influences (Hergarden, 2012).
The prevailing model for satiety in insects, based on studies in blowflies, is that proprioceptive feedback from foregut and crop distention is transmitted to the brain via the neck connective, thereby inhibiting central circuits that control feeding behavior. Although it is not known whether this mechanism operates in Drosophila, it raises the possibility that AstA neurons might inhibit feeding by regulating gut distention or proprioceptive feedback from the gut to the brain. However, this study found no evidence that activation of AstA neurons increases gut volume. No expression of AstA-GAL4 drivers, or of AstA itself, was seen in the foregut or crop. Nevertheless, both AstA and our AstA-GAL4 drivers are expressed in a subset of gut neuroendocrine cells. Because these neuroendocrine cells express 'panneuronal' drivers such as Elav, they cannot easily be manipulated independently of AstA neurons in the central brain and PNS. Therefore, a role for AstA-expressing gut neuroendocrine cells in the control of feeding behavior cannot be excluded (Hergarden, 2012).
AstA-expressing nerve fibers ramify within the SOG, where they exhibit varicosities in close proximity to the central projections of GR5a gustatory neurons, which detect sweet tastants. Given that GR5a neurons control proboscis extension reflex (PER) behavior, and that activating AstA neurons prevents the starvation-induced enhancement of sucrose-evoked PER behavior, it is possible that AstA neurons act in the SOG, either directly on GR5a fibers or on other neuronal populations that arborize in this structure, to regulate the PER. These observations, and the fact that AstA neuron activation inhibits feeding but not PER behavior in starved flies hand-fed 800 mM sucrose (as well as in unstarved flies), suggest that the PER and food intake may be controlled by different populations of AstA neurons (Hergarden, 2012).
The genetic manipulations of AstA neuronal activity performed in this study are likely to affect the release of both AstA itself, as well as other cotransmitters. Presently, there are no loss-of-function alleles of either AstA or its putative receptors, and, in the current study, expression of RNAi's for these genes was ineffective at reducing transcript levels. Furthermore, no feeding-related phenotype was observed upon injection of flies with AstA synthetic peptide. However, as noted earlier, injection of AstA peptide inhibits food intake in a number of other insect species. Furthermore, orthologs of AstA receptors have been shown to play a role in feeding in mammals and Caenorhabditis elegans. Given these data, it is likely that AstA itself plays a role to promote satiety or aversion to unpalatable food resources in Drosophila, but this remains to be demonstrated (Hergarden, 2012).
Genetic epistasis experiments were performed to examine interactions between AstA neurons and other classes of peptidergic neurons implicated in the control of feeding. Simultaneous activation of NPF neurons and AstA neurons largely relieved the inhibition of feeding caused by activation of AstA neurons on their own. This suggests that NPF and AstA neurons may act antagonistically to control feeding. In contrast, coactivation of neurons expressing Crz failed to rescue the reduced feeding caused by AstA neuron activation, despite the fact that activation of Crz neurons on its own enhanced food intake. Thus, although both NPF and Crz neurons promote food intake, they exhibit opposite epistatic interactions with AstA neurons (Hergarden, 2012).
In summary, these experiments identify a class of peptidergic cells whose activation suppresses feeding behavior in adult Drosophila, in a manner independent of any measurable effects on metabolism or energy reserves. It is suggested that AstA-expressing neurons and/or neuroendocrine cells exert an inhibitory influence on the motivation or drive to feed and/or promote aversion to unpalatable food resources. Further studies of these neurons, the circuitry they engage, and their regulation by food intake may shed light on mechanisms of satiety control and on the general question of how metabolic changes are translated into behavioral changes by the brain (Hergarden, 2012).
Allatostatins (ASTs) are multifunctional neuropeptides that generally act in an inhibitory fashion. ASTs were identified as inhibitors of juvenile hormone biosynthesis. Juvenile hormone regulates insect metamorphosis, reproduction, food intake, growth, and development. Drosophila melanogaster RNAi lines of PheGlyLeu-amide-ASTs (FGLa/ASTs) and their cognate receptor, Dar-1, were used to characterize roles these neuropeptides and their respective receptor may play in behavior and physiology. Dar-1 and FGLa/AST RNAi lines showed a significant reduction in larval foraging in the presence of food. The larval foraging defect is not observed in the absence of food. These RNAi lines have decreased for transcript levels which encodes cGMP- dependent protein kinase. A reduction in the for transcript is known to be associated with a naturally occurring allelic variation that creates a sitter phenotype in contrast to the rover phenotype which is caused by a for allele associated with increased for activity. The sitting phenotype of FGLa/AST and Dar-1 RNAi lines is similar to the phenotype of a deletion mutant of an AST/galanin-like receptor (NPR-9) in Caenorhabditis elegans. Associated with the foraging defect in C. elegans npr-9 mutants is accumulation of intestinal lipid. Lipid accumulation was not a phenotype associated with the FGLa/AST and Dar-1 RNAi lines (Wang, 2012).
Adipokinetic hormone (AKH) is a metabolic neuropeptide principally known for its mobilization of energy substrates, notably lipid and trehalose during energy-requiring activities, such as flight and locomotion. Drosophila melanogaster AKH cell localization in corpora cardiaca, as in other insect species, was confirmed by immunoreactivity and by a genetic approach using the UAS/GAL4 system. To assess AKH general physiological rules, AKH endocrine cells were ablated by specifically driving the expression of apoptosis transgenes in AKH cells. Trehalose levels were decreased in larvae and starved adults, when the stimulation by AKH of the production of trehalose from fat body glycogen is no longer possible. Moreover, these adults without AKH cells become progressively hypoactive. Finally, under starvation conditions, those hypoactive AKH-knockout cell flies survived approximately 50% longer than control wild-type flies, suggesting that the slower rate at which AKH-ablated flies mobilize their energy resources extends their survival (Isabel, 2005).
Adipokinetic hormones (AKHs) are metabolic neuropeptides, mediating mobilization of energy substrates from the fat body in many insects. In delving into the roles of the Drosophila Akh (dAkh) gene, its developmental expression patterns were examined and the physiological functions of the AKH-producing neurons were investigated using animals devoid of AKH neurons and ones with ectopically expressing dAkh. The dAkh gene is expressed exclusively in the corpora cardiaca from late embryos to adult stages. Projections emanating from the AKH neurons indicated that AKH has multiple target tissues as follows: the prothoracic gland and aorta in the larva and the crop and brain in the adult. Studies using transgenic manipulations of the dAkh gene demonstrated that AKH induced both hypertrehalosemia and hyperlipemia. Starved wild-type flies displayed prolonged hyperactivity prior to death; this novel behavioral pattern could be associated with food-searching activities in response to starvation. In contrast, flies devoid of AKH neurons not only lacked this type of hyperactivity, but also displayed strong resistance to starvation-induced death. From these findings, another role is proposed for AKH in the regulation of starvation-induced foraging behavior (Lee, 2004).
The type-A allatostatins A (AST-A) are a group of insect peptides with a common C-terminal motif Y/FXFGL-NH(2). The existence of at least four putative type A Drosophila melanogaster ASTs (called type A drostatins or DST-As) has been predicted from the sequence of a recently cloned DST-A preprohormone (Lenz, 2000a). SRPYSFGL-NH(2), (DST-3A), the only DST isolated from Drosophila so far, activated the first cloned DST-A GPCR (DAR-1) (Birgul,1999). A newly cloned orphan Dm GPCR, which shares 47% overall and 60% transmembrane region sequence identity with DAR-1, was classified as a second putative Dm DST-A receptor (DAR-2) (Lenz, 2000b). Although activation of DAR-2 by DSTs has been postulated, no experimental evidence for that has been presented to date. This study expressed both DAR-1 and DAR-2 in CHO cells and used a GTPγS and a Ca(2+) mobilization assay for pharmacological evaluation of the receptors. Synthetically prepared DST-As, as well as selected Diplotera punctata (cockroach) ASTs, activated DAR-1 and DAR-2 in both functional assays indicating ligand redundancy and cross species activity. Cell pretreatment with pertussis toxin led to some differences in the nature and magnitude of signaling pathways at the DAR-1 and DAR-2 receptors, suggesting possible differential coupling to cellular effector system(s) and distinct biological functions of each receptor in vivo (Larsen, 2001).
By using degenerate oligonucleotide primers deduced from the conserved regions of the mammalian somatostatin receptors, a novel G-protein-coupled receptor from Drosophila melanogaster has been isolated exhibiting structural similarities to mammalian somatostatin/galanin/opioid receptors. To identify the bioactive ligand, a 'reverse physiology' strategy was used whereby orphan Drosophila receptor-expressing frog oocytes were screened against potential ligands. Agonistic activity was electrophysiologically recorded as inward potassium currents mediated through co-expressed G-protein-gated inwardly rectifying potassium channels (GIRK). Using this approach a novel peptide was purified from Drosophila head extracts. Mass spectrometry revealed an octapeptide of 925 Da with a sequence Ser-Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH(2) reminiscent of insect allatostatin peptides known to control diverse functions such as juvenile hormone synthesis during metamorphosis or visceral muscle contractions. Picomolar concentrations of the synthesized octapeptide activated the cognate receptor response mediated through GIRK1, indicating that the 394-amino-acid Drosophila allatostatin receptor which is coupled to the Gi/Go class of G proteins was isolated (Birgü, 1999).
Search PubMed for articles about Drosophila Allatostatin A
Aguilar, R., Maestro, J. L., Vilaplana, L., Pascual, N., Piulachs, M. D. and Belles, X. (2003). Allatostatin gene expression in brain and midgut, and activity of synthetic allatostatins on feeding-related processes in the cockroach Blattella germanica. Regul Pept 115(3): 171-177. PubMed ID: 14556958
Baines, R. A., Uhler, J. P., Thompson, A., Sweeney, S. T. and Bate, M. (2001). Altered electrical properties in Drosophila neurons developing without synaptic transmission. J Neurosci 21(5): 1523-1531. PubMed ID: 11222642
Beebe, K., Park, D., Taghert, P. H. and Micchelli, C. A. (2015). The Drosophila pro-secretory transcription factor dimmed is dynamically regulated in adult enteroendocrine cells and protects against gram-negative infection. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 25999585
Bendena, W. G., Boudreau, J. R., Papanicolaou, T., Maltby, M., Tobe, S. S. and Chin-Sang, I. D. (2008). A Caenorhabditis elegans allatostatin/galanin-like receptor NPR-9 inhibits local search behavior in response to feeding cues. Proc Natl Acad Sci U S A 105(4): 1339-1342. PubMed ID: 18216257
Birgü, N., Weise, C., Kreienkamp, H. J. and Richter, D. (1999). Reverse physiology in Drosophila: identification of a novel allatostatin-like neuropeptide and its cognate receptor structurally related to the mammalian somatostatin/galanin/opioid receptor family. EMBO J 18(21): 5892-5900. PubMed ID: 10545101
Chen, J., Reiher, W., Hermann-Luibl, C., Sellami, A., Cognigni, P., Kondo, S., Helfrich-Förster, C., Veenstra, J.A. and Wegener, C. (2016). Allatostatin A signalling in Drosophila regulates feeding and sleep and is modulated by PDF. PLoS Genet 12: e1006346. PubMed ID: 27689358
Hentze, J. L., Carlsson, M. A., Kondo, S., Nassel, D. R. and Rewitz, K. F. (2015). The neuropeptide Allatostatin A regulates metabolism and feeding decisions in Drosophila. Sci Rep 5: 11680. PubMed ID: 26123697
Hergarden, A. C., Tayler, T. D. and Anderson, D. J. (2012). Allatostatin-A neurons inhibit feeding behavior in adult Drosophila. Proc Natl Acad Sci U S A 109(10): 3967-3972. PubMed ID: 22345563
Isabel, G., Martin, J. R., Chidami, S., Veenstra, J. A. and Rosay, P. (2005). AKH-producing neuroendocrine cell ablation decreases trehalose and induces behavioral changes in Drosophila. Am J Physiol Regul Integr Comp Physiol 288(2): R531-538. PubMed ID: 15374818
Lang, R., Gundlach, A. L. and Kofler, B. (2007). The galanin peptide family: receptor pharmacology, pleiotropic biological actions, and implications in health and disease. Pharmacol Ther 115(2): 177-207. PubMed ID: 17604107
Lang, R., Gundlach, A. L., Holmes, F. E., Hobson, S. A., Wynick, D., Hokfelt, T. and Kofler, B. (2015). Physiology, signaling, and pharmacology of galanin peptides and receptors: three decades of emerging diversity. Pharmacol Rev 67(1): 118-175. PubMed ID: 25428932
Larsen, M. J., Burton, K. J., Zantello, M. R., Smith, V. G., Lowery, D. L. and Kubiak, T. M. (2001). Type A allatostatins from Drosophila melanogaster and Diplotera puncata activate two Drosophila allatostatin receptors, DAR-1 and DAR-2, expressed in CHO cells. Biochem Biophys Res Commun 286(5): 895-901. PubMed ID: 11527383
Lee, G. and Park, J. H. (2004). Hemolymph sugar homeostasis and starvation-induced hyperactivity affected by genetic manipulations of the adipokinetic hormone-encoding gene in Drosophila melanogaster. Genetics 167(1): 311-323. PubMed ID: 15166157
Lenz, C., Sondergaard, L. and Grimmelikhuijzen, C. J. (2000a). Molecular cloning and genomic organization of a novel receptor from Drosophila melanogaster structurally related to mammalian galanin receptors. Biochem Biophys Res Commun 269(1): 91-96. PubMed ID: 10694483
Lenz, C., Williamson, M. and Grimmelikhuijzen, C. J. (2000b). Molecular cloning and genomic organization of a second probable allatostatin receptor from Drosophila melanogaster. Biochem Biophys Res Commun 273(2): 571-577. PubMed ID: 10873647
Nitabach, M. N., Wu, Y., Sheeba, V., Lemon, W. C., Strumbos, J., Zelensky, P. K., White, B. H. and Holmes, T. C. (2006). Electrical hyperexcitation of lateral ventral pacemaker neurons desynchronizes downstream circadian oscillators in the fly circadian circuit and induces multiple behavioral periods. J Neurosci 26(2): 479-489. PubMed ID: 16407545
Parisky, K. M., Agosto, J., Pulver, S. R., Shang, Y., Kuklin, E., Hodge, J. J., Kang, K., Liu, X., Garrity, P. A., Rosbash, M. and Griffith, L. C. (2008). PDF cells are a GABA-responsive wake-promoting component of the Drosophila sleep circuit. Neuron 60(4): 672-682. PubMed ID: 19038223
Ribeiro, C. and Dickson, B. J. (2010). Sex peptide receptor and neuronal TOR/S6K signaling modulate nutrient balancing in Drosophila. Curr Biol 20(11): 1000-1005. PubMed ID: 20471268
Vanderveken, M. and O'Donnell, M. J. (2014). Effects of diuretic hormone 31, drosokinin, and allatostatin A on transepithelial K(+) transport and contraction frequency in the midgut and hindgut of larval Drosophila melanogaster. Arch Insect Biochem Physiol 85(2): 76-93. PubMed ID: 24408875
Wang, C., Chin-Sang, I. and Bendena, W. G. (2012). The FGLamide-allatostatins influence foraging behavior in Drosophila melanogaster. PLoS One 7(4): e36059. PubMed ID: 22558326
Yamagata, N., Hiroi, M., Kondo, S., Abe, A. and Tanimoto, H. (2016). Suppression of Dopamine Neurons Mediates Reward. PLoS Biol 14(12): e1002586. PubMed ID: 27997541
date revised: 23 December 2016
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