InteractiveFly: GeneBrief

Capability: Biological Overview | References

BIOLOGICAL OVERVIEW

Environmental factors challenge the physiological homeostasis in animals, thereby evoking stress responses. Various mechanisms have evolved to counter stress at the organism level, including regulation by neuropeptides. In recent years, much progress has been made on the mechanisms and neuropeptides that regulate responses to metabolic/nutritional stress, as well as those involved in countering osmotic and ionic stresses. This study identified a peptidergic pathway that links these types of regulatory functions. The neuropeptide Corazonin (Crz), previously implicated in responses to metabolic stress, was uncovered as a neuroendocrine factor that inhibits the release of a diuretic hormone, CAPA, and thereby modulates the tolerance to osmotic and ionic stress. Both knockdown of Crz and acute injections of Crz peptide impact desiccation tolerance and recovery from chill-coma. Mapping of the Crz receptor (CrzR) expression identified three pairs of Capa-expressing neurons (Va neurons) in the ventral nerve cord that mediate these effects of Crz. Crz was shown to act to restore water/ion homeostasis by inhibiting release of CAPA neuropeptides via inhibition of cAMP production in Va neurons. Knockdown of CrzR in Va neurons affects CAPA signaling, and consequently increases tolerance for desiccation, ionic stress and starvation, but delays chill-coma recovery. Optogenetic activation of Va neurons stimulates excretion and simultaneous activation of Crz and CAPA-expressing neurons reduces this response, supporting the inhibitory action of Crz. Thus, Crz inhibits Va neurons to maintain osmotic and ionic homeostasis, which in turn affects stress tolerance. Earlier work demonstrated that systemic Crz signaling restores nutrient levels by promoting food search and feeding. It is additionally proposed that Crz signaling also ensures osmotic homeostasis by inhibiting release of CAPA neuropeptides and suppressing diuresis. Thus, Crz ameliorates stress-associated physiology through systemic modulation of both peptidergic neurosecretory cells and the fat body in Drosophila (Zandawala, 2021).

Environmental conditions continuously challenge the physiological homeostasis in animals, thereby evoking stress that can adversely affect the health and lifespan of an individual. For instance, lack of food and water, extreme temperatures, infection and predation can all evoke stress responses. In order to counter this stress and restore homeostasis, animals have evolved a multitude of physiological and behavioral mechanisms, which involve actions of multiple tissues and/or organs. The core of these mechanisms involves hormones and neuropeptides, which orchestrate the actions of various organs to counteract stress and maintain homeostasis. One well-studied mechanism counteracting water-deficit stress is the mammalian anti-diuretic system that involves hypothalamic osmoreceptors stimulating the sensation of thirst that leads to the release of the anti-diuretic hormone vasopressin, which targets multiple organs, including the kidney, to decrease urine output and conserve water. In insects such as the vinegar fly, Drosophila melanogaster, much progress has been made on the mechanisms and factors regulating metabolic homeostasis, nutritional stress and longevity. Several neuropeptides and peptide hormones have been shown to influence responses to nutrient stress via actions on peripheral tissues such as the liver-like fat body. Specifically, these peptide hormones include Drosophila insulin-like peptides (DILPs), adipokinetic hormone (AKH) and corazonin (Crz). In addition, mechanisms regulating the release of these hormones are being unraveled. Hence, the neural circuits and neuroendocrine pathways regulating metabolic homeostasis and nutritional stress are beginning to be understood. However, the circuits and/or pathways that regulate thermal, osmotic and ionic stresses remain largely unexplored (Zandawala, 2021).

Thus, this study asked what factors and cellular systems constitute the osmoregulatory axis in Drosophila (and other insects). Since nutrient and osmotic homeostasis are inter-dependent, it was hypothesized that regulation of osmotic stress may involve factors that also regulate nutritional stress. This study identified Crz signaling, a paralog of the AKH/Gonadotropin releasing hormone signaling system, as a candidate regulating these stresses. Based on previous research in Drosophila and other insects, it has been hypothesized that Crz modulates responses to stress, especially nutritional stress. Consistent with this, recent work has shown that neurosecretory cells co-expressing Crz and short neuropeptide F (sNPF) are nutrient sensing and modulate nutrient homeostasis through differential actions of the two co-expressed neuropeptides. Whereas sNPF acts on the insulin-producing cells (IPCs) and AKH-producing cells to stimulate DILP release and inhibit AKH release, respectively, systemic Crz signaling modulates feeding and nutritional stress through actions on the fat body (Zandawala, 2021).

Although the role of Crz in regulating responses to nutritional stress is now established, less is known about its role in cold tolerance and ion/water homeostasis. Hence, to address this, the role of Crz in modulating osmotic and ionic stresses was studied and the cellular systems constituting the Crz signaling axis is furthermore outlined. To this end, this study analyzed the effects of manipulating Crz signaling on desiccation tolerance and chill-coma recovery as these two assays are routinely used to assess responses to osmotic/ionic stress. Both knockdown of Crz and acute injections of Crz peptide impact desiccation tolerance and recovery from chill-coma. Comprehensive mapping of the Crz receptor (CrzR) expression revealed that these effects of Crz are not likely mediated by direct modulation of the osmoregulatory tissues but indirectly via three pairs of neurons (Va neurons) in the ventral nerve cord (VNC), which express diuretic CAPA neuropeptides. Knockdown of the CrzR in Va neurons affects CAPA release and ion/water balance, consequently influencing desiccation tolerance and chill-coma recovery. Crz acts via inhibition of cAMP production in Va neurons to inhibit release of CAPA and thereby reducing water loss during desiccation. These data, suggest that Crz is released into the hemolymph during nutritional stress and acts on the fat body to mobilize energy for food search to increase food intake. In summary, it is proposed that Crz acts upstream of CAPA signaling to regulate water and ion balance as well as restore nutrient levels caused by starvation. Thus, in addition to the hormonal actions of Crz on the fat body to maintain metabolic homeostasis and counter nutritional stress, this peptide also helps maintain osmotic homeostasis (Zandawala, 2021).

This study has found that a peptidergic neuroendocrine pathway in Drosophila, known to restore nutrient deficiency (utilizing Crz), integrates a further peptidergic component (CAPA) to maintain osmotic and ionic homeostasis. The Crz-CAPA signaling thereby also influences tolerance to osmotic and cold stress. An earlier study suggested that Crz is released during nutritional stress to mobilize energy stores from the fat body to fuel food search behavior (Kubrak, 2021). Furthermore, that study suggests that increased Crz signaling compromises resistance to starvation, desiccation and oxidative stress (Kubrak, 2021). This study has confirmed these findings, and also found that Crz inhibits a set of CrzR expressing Va neurons in the abdominal ganglia that produce CAPA peptides, which when released in vivo through optogenetic or thermogenetic control, leads to increased excretion and decreased whole body water content, respectively. Thus, the two peptidergic systems act together to maintain both energy and ion/water homeostasis (Zandawala, 2021).

The Capa gene-derived neuropeptides (CAPA1 and CAPA2) are well established as osmoregulatory factors that act on the Malpighian tubule principal cells (Davies, 2013; Paluzzi, 2012) and perhaps the hindgut, as indicated herein by receptor expression data. Previous ex vivo studies have largely showed that CAPA neuropeptides act as diuretic hormones in Drosophila and other dipterans while anti-diuretic actions have also been reported. This study's in vivo findings are more aligned with the observations supporting a diuretic role of CAPA peptides. Furthermore, this study shows that the CAPA-producing Va neurons are downstream of Crz signaling and it is proposed that under adverse conditions when flies are exposed to dry starvation (desiccation without food) the two signaling systems act in tandem to restore homeostasis. When the fly experiences starvation and nutrients diminish in the fly, nutrient sensors record this deficiency, which triggers release of hormones that act to restore metabolic homeostasis. As indicated above, one such hormone is Crz, known to provide energy for food search and induce feeding. The released Crz also inhibits CAPA release from Va cells during the nutrient shortage and results in diminished excretion (and defecation) during starvation and food search. Additionally, during the revision of this manuscript, a recent paper (Koyama, 2021) reported that CAPA from Va cells also acts on AKH-producing cells in the corpora cardiaca to inhibit AKH release and thereby decreasing lipolysis in adipocytes. Thus, when Crz inhibits the Va neurons, AKH signaling is likely to be elevated and results in increased energy mobilization from the fat body, further facilitating food search. In insects, increased food intake yields water, which requires post-feeding diuresis to be activated to restore water homeostasis. Therefore, the inhibition of CAPA release from Va cells needs to be lifted after successful food search and nutrient intake. This suggests that the Crz action occurs during the starvation/fasting, and thereafter postprandial CAPA action sets in to restore nutrient and water homeostasis. Unfortunately, it was not possible to provide direct evidence for this sequence of events since there is no method sensitive enough to monitor the timing of changes in hemolymph levels of Crz and CAPA in small organisms such as Drosophila. Indirect measurements, like monitoring levels of peptide-immunolabel in neurons of interest are not necessarily accurate, since these levels reflect the 'balance' between peptide release and production and therefore not useful for resolving onset and duration of release with accuracy (Zandawala, 2021).

The evidence for the Crz action on CAPA-producing Va neurons is based on the expression of CrzR in these cells, as well as functional imaging data which shows that Crz inhibits cAMP production in Va neurons (but has no impact on intracellular Ca2+ levels). This led to an investigation of the effects of manipulating Crz signaling and targeted CrzR knockdown in Va neurons on aspects of water and ion homeostasis and cold tolerance, which have been shown earlier to be affected by CAPA signaling.It was observed that global Crz or CrzR knockdown leads to delayed recovery from chill-coma (i.e. jeopardizing cold tolerance) and increased tolerance to ionic stress. Furthermore, experiments that target knockdown of CrzR to Va neurons also affected chill-coma recovery, and tolerance to starvation, desiccation, and ionic stresses. Injecting flies in vivo with Crz in the present study also resulted in effects on chill-coma recovery and survival during desiccation, further strengthening the model in which Crz modulates release of CAPA. However, the effect of CAPA injections on chill-coma recovery shown previously are not directly compatible with the current findings. This may be due to the difficulty in comparing the effects obtained from RNAi manipulations (chronic) and peptide injections (acute). Furthermore, Crz not only affects cold tolerance via its actions on CAPA signaling, it also regulates levels of trehalose, a cryoprotectant, via actions on the fat body. Thus, Crz signaling could modulate cold tolerance via two independent hormonal pathways (Zandawala, 2021).

Previous work in Drosophila showed that not only does global Crz knockdown result in increased resistance to starvation, desiccation and oxidative stress, but also found that CrzR knockdown in the fat body (and salivary glands) led to these same phenotypes. These findings suggest that Crz signaling to the fat body accounts for some of the stress tolerance phenotypes seen following Crz knockdown. Thus, it is possible that the effects observed on desiccation survival following Crz peptide injections in the present study are partly confounded by the actions of this peptide on the fat body and other tissues expressing the CrzR (Zandawala, 2021).

Earlier data show that after CrzR knockdown in the fat body, glucose, trehalose and glycogen levels are elevated in starved flies, but not in normally fed flies. Moreover, starved, but not normally fed flies with Crz knockdown, display increased triacyl glycerides and Crz transcript is upregulated in starved flies with CrzR knockdown in the fat body. This further emphasizes that systemic Crz signaling is critical under nutritional stress. The data on Crz immunolabeling intensity corroborate these earlier findings and suggest that Crz is released to restore metabolic homeostasis by mobilizing energy stores from the fat body to fuel food search behavior. After restoring nutritional and osmotic stress the Crz signaling decreases and thus the proposed inhibition of CAPA release from Va neurons is lifted (Zandawala, 2021).

The question as to how the Crz neurons sense nutrient deficiency or whether they can detect changes in osmolarity. However, it is known that Crz-producing DLPs express an aquaporin, Drip, and the carbohydrate-sensing gustatory receptors, Gr43a and Gr64a. A subset of the Crz neurons also express a glucose transporter (Glut1) that is involved in glucose-sensing. Thus, imbalances in internal nutritional and maybe even osmotic status could either be sensed cell autonomously by the Crz neurons or indirectly by signals relayed to them via other pathways (Zandawala, 2021).

A few other findings might support roles of Crz in ameliorating nutrient stress. This study found that Crz neurons innervate the antennal lobe (AL), and that the CrzR is strongly expressed in local interneurons of the AL. Possibly, Crz modulates odor sensitivity in hungry flies to increase food search, similar to peptides like short neuropeptide F (sNPF), tachykinin and SIFamide. Another peptide hormone, adipokinetic hormone (AKH), has been shown to be critical in initiating locomotor activity and food search in food deprived flies and AKH also affects sensitivity of gustatory neurons to glucose. The effect of AKH on increasing locomotor activity is evident only after 36h of starvation and may correlate with the proposed action of Crz during starvation. Possibly Crz acts in concert with AKH to allocate fuel during metabolic stress. Since it was found that the CrzR is not expressed in AKH-producing cells, it is likely that these two peptides act in parallel rather than in the same circuit/pathway. However, the DLPs also produce sNPF and this peptide is known to act on the AKH producing cells and thereby modulate glucose homeostasis and possibly, sensitivity of gustatory neurons. Thus, the DLPs may act systemically with Crz and by paracrine signaling with sNPF in the corpora cardiaca to act on AKH cells. Thereby the Crz and AKH systems could be linked by sNPF. In addition, while this manuscript was under revision, a paper (Koyama, 2021) was posted that added an interesting angle to the role of AKH signaling. That study revealed that the AKH producing cells (APCs) express the CAPA receptor and that CAPA acts on APCs to decrease AKH release, which diminishes lipolysis in adipocytes. Thus, in the fed fly, CAPA not only induces diuresis, it also diminishes energy mobilization. The same study also showed that the Va cells in flies are active after sucrose feeding or drinking, and that CAPA triggers nutrient uptake and peristalsis in the intestine. This interaction between CAPA-AKH signaling can also explain the direction of the phenotypes seen following CrzR knockdown in Va neurons. For instance, knockdown of CrzR in Va neurons results in increased CAPA release and one would predict decreased desiccation survival if CAPA is exclusively promoting excretion. However, increased CAPA signaling could also result in reduced AKH signaling that in turn promotes starvation survival. Thus, the direction of the phenotypes observed following CrzR knockdown in Va neurons can be explained by CAPA actions on excretion and AKH-release from APCs. Lastly, the possibility was not ruled out that other neuropeptides possibly coexpressed with CAPA could also contribute to the observed phenotypes (Zandawala, 2021).

In conclusion, it is suggested that Crz regulates acute metabolic stress-associated physiology and behavior via the fat body to ensure nutrient allocation to power food search and feeding during prolonged starvation. During food search and feeding, excretion is blocked by Crz acting on the Va cells to inhibit CAPA release. Following food intake and ensuing need for diuresis, Crz signaling ceases and CAPA can be released to ensure restoration of water and ion homeostasis. Taken together, these findings and those of previous studies indicate that Crz acts on multiple neuronal and peripheral targets to coordinate and sustain water, ion and metabolic homeostasis. It might even be possible that an ancient role of the common ancestor of Crz and AKH signaling systems was to modulate stress-associated physiology and that these paralogous signaling systems have sub-functionalized and neo-functionalized over evolution (Zandawala, 2021).

Anti-diuretic activity of a CAPA neuropeptide can compromise Drosophila chill tolerance

For insects, chilling injuries that occur in the absence of freezing are often related to a systemic loss of ion and water balance that leads to extracellular hyperkalemia, cell depolarization, and the triggering of apoptotic signalling cascades. The ability of insect ionoregulatory organs (e.g. the Malpighian tubules) to maintain ion balance in the cold has been linked to improved chill tolerance, and many neuroendocrine factors are known to influence ion transport rates of these organs. Injection of micromolar doses of Capability (CAPA) (an insect neuropeptide) have been previously demonstrated to improve Drosophila cold tolerance, but the mechanisms through which it impacts chill tolerance are unclear, and low doses of CAPA have been previously demonstrated to cause anti-diuresis in insects, including dipterans. This study provides evidence that low (fM) and high (microM) doses of CAPA impair and improve chill tolerance, respectively, via two different effects on Malpighian tubule ion and water transport. While low doses of CAPA are anti-diuretic, reduce tubule K(+) clearance rates and reduce chill tolerance, high doses facilitate K(+) clearance from the haemolymph and increase chill tolerance. By quantifying CAPA peptide levels in the central nervous system, the maximum achievable hormonal titres of CAPA was estimated, and evidence was further found that CAPA may function as an anti-diuretic hormone in Drosophila melanogaster. Evidence is provided of a neuropeptide that can negatively affect cold tolerance in an insect, and further evidence of CAPA functioning as an anti-diuretic peptide in this ubiquitous insect model (MacMillan, 2018).

This study is the first to report contrasting dose-dependent effects of CAPA peptides on fluid and ion secretion by the Malpighian tubules of Drosophila melanogaster, and the first to describe negative effects of a neuropeptide on the chill tolerance of any insect. These results support previous findings related to both high and low doses of CAPA peptides, and raise additional questions about the role of CAPA neuropeptides in insects in vivo. Similarly to prior evidence (Terhzaz, 2015), this study found that a micromolar dose of CAPA peptide led to faster recovery from chill coma in D. melanogaster. This study also reports, for the first time, a significant effect of CAPA administration on survival following prolonged chilling (MacMillan, 2018).

Notably, the effects of CAPA injection on chill CCRT were similar whether flies were injected early or late in the cold stress. Recovery from chill coma has been suggested to be dependent on the degree to which an insect has lost ion balance in the cold (dependent on the temperature and duration of cold stress), as well as the rate of ion and water homeostasis recovery following rewarming. If this is the case, the current result implies that the effects of CAPA peptide on ion and water balance are minimal during the cold stress and that the titre of CAPA in the haemolymph is instead primarily influencing rates of ion transport (and thus the recovery of ion balance) upon rewarming. Given these results, and the knowledge that CAPA receptors are located exclusively in the Malpighian tubule principal cells of D. melanogaster (Terhzaz, 2012), this study specifically considered the effects of CAPA peptide on Malpighian tubule function at room temperature, which is most relevant to its effects on chill coma recovery. It is possible that the peptide simply cannot bind at low temperatures or otherwise does not alter tubule function in the cold. Alternatively, this result may simply support the observation that rates of transport are strongly suppressed during chilling [∼20-fold between 25°C and 0°C in the same population of flies used in the present study, and as such, stimulation or further suppression is unlikely to have any measurable effect. To address these possibilities, careful analysis of the effects of temperature on neuropeptide signalling and renal function across a range of temperatures will be required, as the vast majority of neuropeptide effects in ectotherms have been documented at or near room temperature (MacMillan, 2018).

Injection of CAPA peptide was previously demonstrated to have no effect on the survival of D. melanogaster following 1 h at -6°C (Terhzaz, 2015). This approach in the present study differed in that the flies were instead subjected to a chronic exposure to a less extreme temperature (16 h at 0°C). Here, flies injected with a low dose of CAPA (10-15 mol l^-1) 30 min before they were removed from the cold suffered greater chilling injury, while those injected with a high dose ( 10^-6 mol l^-1) were significantly less injured than control flies 24 h following the cold stress. Injuries suffered from chilling in the absence of ice formation are often conceptually divided into direct chilling injury (resulting from severe acute cold stress) and indirect chilling injury (resulting from chronic, but milder cold stress). These two forms of injury have also been suggested to be associated with different underlying mechanisms; while direct chilling injury is thought to be a consequence of irreversible membrane phase changes and protein denaturation, indirect chilling injury is instead attributed to a more gradual loss of ion and water balance or oxidative stress. These results support the notion that direct and indirect chilling injury are influenced by independent physiological mechanisms, and that neuropeptide effects on ion and water balance may mitigate or exacerbate indirect chilling injury while having little effect on direct chilling injury. Regardless of the mechanisms at play, the effects of CAPA observed on survival following cold stress mirror the observations for CCRT; whereas high doses of CAPA improved chill tolerance in D. melanogaster, low doses had the opposite effect (MacMillan, 2018).

Receptors for CAPA peptides are found only in the Malpighian tubules of D. melanogaster (Terhzaz, 2012), so this study focused attention on the effects of low and high doses of CAPA on tubule fluid and ion secretion rates using Ramsay assays. Exposure of tubules to femtomolar (10-15 mol l^-1) doses of CAPA peptide reduced rates of ion and fluid secretion by the tubules. Since the initial description of CAPA peptides as modulators of Malpighian tubule secretion rates in D. melanogaster (Davies, 1995), no studies have tested the effects of this peptide on fluid secretion below concentrations of 1 nmol l^-1 (10-9 mol l^-1) in this species. Instead, most have described effects of higher concentrations (typically 10^-8 to 10^-6 mol l^-1), and this approach has led to the elucidation of the signalling pathway underlying this effect. Notably, Rodan and colleagues (Rodan et al., 2012) previously reported anti-diuretic effects of CAPA on the tubules of wild-type D. melanogaster. This antidiuretic effect was observed at 10-7 mol l^-1 CAPA, but only after a prolonged exposure of tubules to the peptide (greater than 30 min). The current results support an anti-diuretic role of CAPA, and further suggest that CAPA peptide is particularly anti-diuretic at lower (femtomolar to picomolar) concentrations in D. melanogaster. Very similar effects of low concentrations of CAPA peptide have been observed in both larval (Ionescu, 2012) and adult (Sajadi, 2018) mosquitoes (A. aegypti). The results suggest that low concentrations of CAPA impair cold tolerance by slowing rates of ion (particularly K+) and water flux through the Malpighian tubules upon rewarming, thereby reducing the speed at which flies can re-establish osmotic and ionic balance following cold exposure (MacMillan, 2018).

In contrast to several previous studies on D. melanogaster, this study found that exposing tubules to micromolar ( 10^-6 mol l^-1) doses of CAPA did not stimulate fluid secretion. It was not possible to account for this discrepancy. In the present study, however, despite failing to stimulate fluid secretion, 10^-6 mol l^-1 CAPA instead led to kaliuresis (higher [K+] in the secreted fluid). This finding is significant as it presents a plausible mechanism for increased chill tolerance following injection of high doses of CAPA peptide. In studies conducted on D. melanogaster to date, improvements in chill tolerance are associated with increased rates of K+ clearance by the tubules. Acclimation of flies to 10°C is associated with a compensatory increase in the rates of K+ secretion by the tubules. Similarly, cold-adapted Drosophila species maintain rates of tubule K+ secretion in the cold (3°C) that are higher than in species adapted to warmer climates. Either of these strategies would help flies to avoid hyperkalaemia in the cold and/or enable them to recover more rapidly from ionic imbalance upon rewarming, provided that rates of K+ reabsorption are simultaneously kept constant or reduced along the gut epithelia, as is the case for both cold acclimated and cold adapted Drosophila. Thus, although the direct effects of 10^-6 mol l^-1 CAPA on Malpighian tubule activity observed in this study are not in line with previous reports in Drosophila, they are internally consistent. It is therefore argued that micromolar doses of CAPA peptide improve chill tolerance via kaliuretic activity (i.e. by stimulating K+ secretion), with or without concurrent stimulation of fluid secretion (MacMillan, 2018).

If femtomolar doses of CAPA impair chill tolerance in Drosophila and do so by inhibiting fluid and ion secretion in the Malpighian tubules, it is predicted that they may do so via the NOS–cGMP–PKG pathway. In larval and adult mosquitoes (A. aegypti), low doses of cGMP (10^-9 to 10^-6 mol l^-1) mimic the anti-diuretic effects of low doses of CAPA (10^-15 mol l^-1), with maximal inhibition of secretion observed at 10^-8 mol l^-1 cGMP (Ionescu, 2012; Sajadi, 2018). In the case of larval mosquitoes, higher doses of cGMP (10-3 mol l^-1) induce a very modest (non-significant) increase in fluid secretion (Ionescu, 2012), while in adult mosquitoes no such stimulation could be induced with higher levels of cGMP (Sajadi, 2018). Accordingly, the present study tested whether similar low (10^-8 mol l^-1) and high (10^-3 mol l^-1) doses of cGMP could mimic these effects in Drosophila. Although the effects of 10^-8 mol l^-1 cGMP mirrored the effects of a low dose (10^-15 mol l^-1) of CAPA in Drosophila (reduced rates of fluid, Na+ and K+ secretion), 10^-3 mol l^-1 cGMP did not stimulate fluid secretion or induce kaliuresis. Indeed, exposure of D. melanogaster tubules to 10^-3 mol l^-1 cGMP had no significant effects on tubule secretion rates, ion concentrations in the secreted fluids or rates of ion flux by the tubules. Thus, the impact of cGMP on tubule secretion in adult Drosophila appears to mimic those observed in A. aegypti, as well as many other insects, including beetles and hemipterans. These results support the idea that low doses of CAPA peptide slow rates of fluid secretion through cGMP signalling, since this second messenger mimicked the anti-diuretic activity of this neuropeptide. In larval A. aegypti, stimulatory effects of high doses of AedesCAPA–PVK-1 or high doses of cGMP can be reversed by addition of specific inhibitors of protein kinase A (Ionescu, 2012), which suggests that high levels of CAPA peptide may be pharmacological, inadvertently activating the signalling cascade that drives diuresis and thereby overwhelming any effects of cGMP. As this study did not observe diuretic effects following application of high titres of CAPA peptide in the present study, it was not possible to test whether a similar effect can explain CAPA-induced diuresis in D. melanogaster (MacMillan, 2018).

Given the observations that CAPA peptide can both improve and hinder cold tolerance in D. melanogaster depending on the dose applied, it was of interest to ask whether flies are capable of reaching micromolar titres of CAPA peptide in the haemolymph. Accordingly, a D. melanogaster CAPA peptide-specific ELISA was developed. Despite pooling haemolymph of 60 flies per sample,it was not possible to detect CAPA peptides in the haemolymph of D. melanogaster, which suggests that total CAPA levels in these samples (collected from flies under control conditions, 23°C) are below the lowest standard (25 fmol). In order to detect CAPA in these pooled samples, each fly would have to contribute approximately 0.42 fmol of CAPA, which represents ∼1% of the CAPA peptide quantified in a single thoracicoabodominal ganglion, and the technique of haemolymph extraction typically obtains ∼56 nl of haemolymph from an adult female fly. Thus, in order to detect CAPA in the haemolymph, D. melanogaster would have to have a mean circulating titre of CAPA peptide ≥7.4 nmol l^-1. As CAPA was not detected in these samples, it is suggested that resting titres are below this concentration. Using the same ELISA, however, it was possible to detect CAPA peptide in pooled samples of the thoracicoabdominal ganglion, a region of the CNS that houses the Va neurons, where CAPA is produced and stored in Drosophila. Based on the abundance of CAPA neuropeptides in the entire nervous system where CAPA is produced, it is estimated that if all of this peptide was released at once, flies could reach a maximum of ∼500 nmol l^-1 CAPA peptide circulating in the haemolymph. This en masse release of all CAPA content is unlikely however, since neuropeptides are released as neurohormones from specialized neurohaemal organs, including the abdominal perivisceral organs where CAPA peptides have been localized and found to be most abundant in a variety of insects including D. melanogaster. Importantly, and consistent with earlier observations in the blowfly Calliphora erythrocephala, the D. melanogaster adult abdominal neurohaemal organs are directly incorporated into the fused ventral ganglion and localized to the dorsal neural sheath. In light of this, these results suggest that if flies are capable of reaching micromolar levels of CAPA in the haemolymph, it would likely require, at a minimum, doubling of CAPA peptide abundance above resting levels in the CNS and synchronous release of all CAPA peptides stored within the nervous system. However, this potential complete release en masse is unlikely, since in vitro induction of neuropeptide from neurohaemal organs has shown to release only fractional amounts compared with the total immunoreactive material present within the nervous system or neurohaemal organ. For example, in the cockroach Leucophaea maderae, leucokinin release from the retrocerebral complex induced by depolarization using high potassium saline accounted for only ∼2% of the total immunoreactive material present within the corpora cardiac–corpora allata complex. Similarly, in the house cricket Acheta domesticus, release of achetakinin following depolarization with high potassium saline from the retrocerebral complex, which is the richest source of this neuropeptide, represented <4% (i.e. ∼70 fmol released from ∼1800 fmol stored in each retrocerebral complex) of the total achetakinin immunoreactive material present within this neurohaemal organ. Finally, it is noted that both cold and desiccation stress have been demonstrated to cause upregulation of Capa mRNA, which may elevate CAPA levels in the CNS, and CAPA has been suggested to be released in D. melanogaster only upon removal from the desiccation or cold stress (Terhzaz, 2015). Further efforts are thus required to determine whether or not Drosophila and other dipterans are capable of reaching levels of CAPA that can stimulate diuresis, and if so, which abiotic conditions specifically lead to this strategy. Critical to this discussion is the direct detection and measurement of circulating levels of CAPA peptide in nanolitre scale haemolymph samples under a variety of highly dynamic conditions, and such an approach in future studies could involve matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MacMillan, 2018).

Chronic exposure to low temperatures suppresses the ability of insects to maintain ion and water homeostasis, causing progressive hyperkalemia and cell death. These results suggest that CAPA peptides can positively and negatively impact chill tolerance in D. melanogaster in a dose-responsive manner. Low (femtomolar) doses of CAPA cause anti-diuresis and limit clearance of K+ at the Malpighian tubules, limiting the ability of flies to recover ion and water balance upon rewarming and impairing chill tolerance. By contrast, high (micromolar) doses of CAPA cause kaliuresis (and based on previous reports also diuresis), facilitating K+ clearance from the haemolymph and improving chill tolerance. It is argued that the anti-diuretic effects of CAPA operate through cGMP, and question whether levels of CAPA peptide can reach micromolar levels and stimulate diuresis in vivo. Although a wide variety of other neuropeptides are known to influence insect ion and water balance through their effects on Malpighian tubule and gut epithelia, none other than CAPA have been tested in the context of chill tolerance (MacMillan, 2018).

Insect capa neuropeptides impact desiccation and cold tolerance

The success of insects is linked to their impressive tolerance to environmental stress, but little is known about how such responses are mediated by the neuroendocrine system. This study shows that the capability (capa) neuropeptide gene is a desiccation- and cold stress-responsive gene in diverse dipteran species. Using targeted in vivo gene silencing, physiological manipulations, stress-tolerance assays, and rationally designed neuropeptide analogs, it was demonstrated that the Drosophila melanogaster capa neuropeptide gene and its encoded peptides alter desiccation and cold tolerance. Knockdown of the capa gene increases desiccation tolerance but lengthens chill coma recovery time, and injection of Capa peptide analogs can reverse both phenotypes. Immunohistochemical staining suggests that Capa accumulates in the Capa-expressing Va neurons during desiccation and nonlethal cold stress but is not released until recovery from each stress. The results also suggest that regulation of cellular ion and water homeostasis mediated by capa peptide signaling in the insect Malpighian (renal) tubules is a key physiological mechanism during recovery from desiccation and cold stress. This work augments understanding of how stress tolerance is mediated by neuroendocrine signaling and illustrates the use of rationally designed peptide analogs as agents for disrupting protective stress tolerance (Terhzaz, 2015).

The Drosophila gene CG9918 codes for a pyrokinin-1 receptor

The database from the Drosophila Genome Project contains a gene, CG9918, annotated to code for a G protein-coupled receptor. The cDNA of this gene was cloned and functionally expressed in Chinese hamster ovary cells. A library of about 25 Drosophila and other insect neuropeptides, and seven insect biogenic amines were tested on the expressed receptor; it was activated by low concentrations of the Drosophila neuropeptide, pyrokinin-1 (TGPSASSGLWFGPRLamide; EC50, 5 x 10(-8) M). The receptor was also activated by other Drosophila neuropeptides, terminating with the sequence PRLamide (Hug-gamma, ecdysis-triggering-hormone-1, pyrokinin-2), but in these cases about six to eight times higher concentrations were needed. The receptor was not activated by Drosophila neuropeptides, containing a C-terminal PRIamide sequence (such as ecdysis-triggering-hormone-2), or PRVamide (such as capa-1 and -2), or other neuropeptides and biogenic amines not related to the pyrokinins. This paper is the first conclusive report that CG9918 is a Drosophila pyrokinin-1 receptor gene (Cazzamali, 2005).

Mechanism and function of Drosophila capa GPCR: a desiccation stress-responsive receptor with functional homology to human neuromedinU receptor

The capa peptide receptor, CapaR (CG14575), is a G-protein coupled receptor (GPCR) for the D. melanogaster capa neuropeptides, Drm-Capa-1 and -2 (Capa-1 and -2). To date, the capa peptide family constitutes the only known nitridergic peptides in insects, so the mechanisms and physiological function of ligand-receptor signalling of this peptide family are of interest. Capa peptide induces calcium signaling via CapaR with EC(5)(0) values for capa-1 = 3.06 nM and capa-2 = 4.32 nM. CapaR undergoes rapid desensitization, with internalization via a b-arrestin-2 mediated mechanism but is rapidly re-sensitized in the absence of Capa-1. Drosophila Capa peptides have a C-terminal -FPRXamide motif and insect-PRXamide peptides are evolutionarily related to vertebrate peptide neuromedinU (NMU). Potential agonist effects of human NMU-25 and the insect -PRLamides [Drosophila pyrokinins Drm-PK-1 (capa-3), Drm-PK-2 and Hugin-gamma [hugg]] against capaR were investigated. NMU-25, but not hugg nor Drm-PK-2, increases intracellular calcium ([Ca(2)(+)]i) levels via capaR. In vivo, NMU-25 increases [Ca(2)(+)]i and fluid transport by the Drosophila Malpighian (renal) tubule. Ectopic expression of human NMU receptor 2 in tubules of transgenic flies results in increased [Ca(2)(+)]i and fluid transport. Finally, anti-porcine NMU-8 staining of larval CNS shows that the most highly immunoreactive cells are capa-producing neurons. These structural and functional data suggest that vertebrate NMU is a putative functional homolog of Drm-capa-1 and -2. capaR is almost exclusively expressed in tubule principal cells; cell-specific targeted capaR RNAi significantly reduces capa-1 stimulated [Ca(2)(+)]i and fluid transport. Adult capaR RNAi transgenic flies also display resistance to desiccation. Thus, CapaR acts in the key fluid-transporting tissue to regulate responses to desiccation stress in the fly (Terhzaz, 2012).

Two nitridergic peptides are encoded by the gene capability in Drosophila melanogaster

A Drosophila gene (capability, capa) at 99D on chromosome 3R potentially encodes three neuropeptides: GANMGLYAFPRV-amide (capa-1), ASGLVAFPRV-amide (capa-2), and TGPSASSGLWGPRL-amide (capa-3). Capa-1 and capa-2 are related to the lepidopteran hormone cardioacceleratory peptide 2b, while capa-3 is a novel member of the pheromone biosynthesis-activating neuropeptide/diapause hormone/pyrokinin family. By immunocytochemistry, four pairs of neuroendocrine cells likely to release the capa peptides into the hemolymph were identified: one pair in the subesophageal ganglion and the other three in the abdominal neuromeres. In the Malpighian (renal) tubule, capa-1 and capa-2 increase fluid secretion rates, stimulate nitric oxide production, and elevate intracellular Ca(2+) and cGMP in principal cells. Capa-stimulated fluid secretion, but not intracellular Ca(2+) concentration rise, is inhibited by the guanylate cyclase inhibitor methylene blue. The actions of capa-1 and capa-2 are not synergistic, implying that both act on the same pathways in tubules. The capa gene is thus the first to be shown to encode neuropeptides that act on renal fluid production through nitric oxide (Kean, 2002).

Molecular cloning and functional expression of a Drosophila receptor for the neuropeptides capa-1 and -2

The Drosophila Genome Project website contains an annotated gene (CG14575) for a G protein-coupled receptor. This receptor was cloned, and it was found that the cloned cDNA did not correspond to the annotated gene; it partly contained different exons and additional exons located at the 5(')-end of the annotated gene. The coding part of the cloned cDNA was expressed in Chinese hamster ovary cells, and the receptor was found to be activated by two neuropeptides, Capa-1 and -2, encoded by the Drosophila capability gene. Database searches led to the identification of a similar receptor in the genome from the malaria mosquito Anopheles gambiae (58% amino acid residue identities; 76% conserved residues; and 5 introns at identical positions within the two insect genes). Because Capa-1 and -2 and related insect neuropeptides stimulate fluid secretion in insect Malpighian (renal) tubules, the identification of this first insect capa receptor will advance our knowledge on insect renal function (Iversen, 2002).

Search PubMed for articles about Drosophila Capability

Cazzamali, G., Torp, M., Hauser, F., Williamson, M. and Grimmelikhuijzen, C. J. (2005). The Drosophila gene CG9918 codes for a pyrokinin-1 receptor. Biochem Biophys Res Commun 335(1): 14-19. PubMed ID: 16054112

Davies, S. A., Huesmann, G. R., Maddrell, S. H., O'Donnell, M. J., Skaer, N. J., Dow, J. A. and Tublitz, N. J. (1995). CAP2b, a cardioacceleratory peptide, is present in Drosophila and stimulates tubule fluid secretion via cGMP. Am J Physiol 269(6 Pt 2): R1321-1326. PubMed ID: 8594932

Davies, S. A., Cabrero, P., Povsic, M., Johnston, N. R., Terhzaz, S. and Dow, J. A. (2013). Signaling by Drosophila capa neuropeptides. Gen Comp Endocrinol 188: 60-66. PubMed ID: 23557645

Ionescu, A. and Donini, A. (2012). AedesCAPA-PVK-1 displays diuretic and dose dependent antidiuretic potential in the larval mosquito Aedes aegypti (Liverpool). J Insect Physiol 58(10): 1299-1306. PubMed ID: 22820035

Iversen, A., Cazzamali, G., Williamson, M., Hauser, F. and Grimmelikhuijzen, C. J. (2002). Molecular cloning and functional expression of a Drosophila receptor for the neuropeptides capa-1 and -2. Biochem Biophys Res Commun 299(4): 628-633. PubMed ID: 12459185

Kean, L., Cazenave, W., Costes, L., Broderick, K. E., Graham, S., Pollock, V. P., Davies, S. A., Veenstra, J. A. and Dow, J. A. (2002). Two nitridergic peptides are encoded by the gene capability in Drosophila melanogaster. Am J Physiol Regul Integr Comp Physiol 282(5): R1297-1307. PubMed ID: 11959669

Koyama, T., Terhzaz, S., Naseem, M. T., Nagy, S., Rewitz, K., Dow, J. A. T., Davies, S. A. and Halberg, K. V. (2021). A nutrient-responsive hormonal circuit mediates an inter-tissue program regulating metabolic homeostasis in adult Drosophila. Nat Commun 12(1): 5178. PubMed ID: 34462441

Kubrak, O. I., Lushchak, O. V., Zandawala, M. and Nassel, D. R. (2016). Systemic corazonin signalling modulates stress responses and metabolism in Drosophila. Open Biol 6(11). PubMed ID: 27810969

Kubrak, O. I., Lushchak, O. V., Zandawala, M. and Nassel, D. R. (2016). Systemic corazonin signalling modulates stress responses and metabolism in Drosophila. Open Biol 6(11). PubMed ID: 27810969

MacMillan, H. A., Nazal, B., Wali, S., Yerushalmi, G. Y., Misyura, L., Donini, A. and Paluzzi, J. P. (2018). Anti-diuretic activity of a CAPA neuropeptide can compromise Drosophila chill tolerance. J Exp Biol. PubMed ID: 30104306

Paluzzi, J. P. (2012). Anti-diuretic factors in insects: the role of CAPA peptides. Gen Comp Endocrinol 176(3): 300-308. PubMed ID: 22226757

Rodan, A. R., Baum, M. and Huang, C. L. (2012). The Drosophila NKCC Ncc69 is required for normal renal tubule function. Am J Physiol Cell Physiol 303(8): C883-894. PubMed ID: 22914641

Sajadi, F., Curcuruto, C., Al Dhaheri, A. and Paluzzi, J. V. (2018). Anti-diuretic action of a CAPA neuropeptide against a subset of diuretic hormones in the disease vector Aedes aegypti. J Exp Biol 221(Pt 7). PubMed ID: 29496779

Terhzaz, S., Cabrero, P., Robben, J. H., Radford, J. C., Hudson, B. D., Milligan, G., Dow, J. A. and Davies, S. A. (2012). Mechanism and function of Drosophila capa GPCR: a desiccation stress-responsive receptor with functional homology to human neuromedinU receptor. PLoS One 7(1): e29897. PubMed ID: 22253819 Sajadi , F., Curcuruto , C., Al Dhaheri , A. and Paluzzi, J.-P. V. (2018). Anti-diuretic action of a CAPA neuropeptide against a subset of diuretic hormones in the disease vector, Aedes aegypti. J. Exp. Biol.  221, jeb177089

Terhzaz, S., Teets, N. M., Cabrero, P., Henderson, L., Ritchie, M. G., Nachman, R. J., Dow, J. A., Denlinger, D. L. and Davies, S. A. (2015). Insect capa neuropeptides impact desiccation and cold tolerance. Proc Natl Acad Sci U S A 112(9): 2882-2887. PubMed ID: 25730885

Terhzaz, S., Alford, L., Yeoh, J. G., Marley, R., Dornan, A. J., Dow, J. A. and Davies, S. A. (2018). Renal neuroendocrine control of desiccation and cold tolerance by Drosophila suzukii. Pest Manag Sci 74(4): 800-810. PubMed ID: 28714258

Watteyne, J., Peymen, K., Van der Auwera, P., Borghgraef, C., Vandewyer, E., Van Damme, S., Rutten, I., Lammertyn, J., Jelier, R., Schoofs, L. and Beets, I. (2020). Neuromedin U signaling regulates retrieval of learned salt avoidance in a C. elegans gustatory circuit. Nat Commun 11(1): 2076. PubMed ID: 32350283

Zandawala, M., Nguyen, T., Balanya Segura, M., Johard, H. A. D., Amcoff, M., Wegener, C., Paluzzi, J. P. and Nassel, D. R. (2021). A neuroendocrine pathway modulating osmotic stress in Drosophila. PLoS Genet 17(3): e1009425. PubMed ID: 33684132

date revised: 21 June 2024

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