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. 2016 Jun;80:96–107. doi: 10.1016/j.peptides.2016.02.004

The corticotropin-releasing factor-like diuretic hormone 44 (DH44) and kinin neuropeptides modulate desiccation and starvation tolerance in Drosophila melanogaster

Elizabeth Cannell a, Anthony J Dornan a, Kenneth A Halberg a,b, Selim Terhzaz a, Julian AT Dow a, Shireen-A Davies a,⁎
PMCID: PMC4889782  PMID: 26896569

Highlights

  • •

    CRF-like diuretic hormone 44 (DH44) signalling modulates desiccation tolerance in D. melanogaster.

  • •

    D. melanogaster kinin (Drome-kinin, DK) has a novel role in starvation stress tolerance.

  • •

    There are functional interactions between DH44 and kinin signalling pathways.

Keywords: DH44, Kinin, Desiccation, Starvation, Neuropeptide receptor, Drosophila melanogaster

Abstract

Malpighian tubules are critical organs for epithelial fluid transport and stress tolerance in insects, and are under neuroendocrine control by multiple neuropeptides secreted by identified neurons. Here, we demonstrate roles for CRF-like diuretic hormone 44 (DH44) and Drosophila melanogaster kinin (Drome-kinin, DK) in desiccation and starvation tolerance.

Gene expression and labelled DH44 ligand binding data, as well as highly selective knockdowns and/or neuronal ablations of DH44 in neurons of the pars intercerebralis and DH44 receptor (DH44-R2) in Malpighian tubule principal cells, indicate that suppression of DH44 signalling improves desiccation tolerance of the intact fly.

Drome-kinin receptor, encoded by the leucokinin receptor gene, LKR, is expressed in DH44 neurons as well as in stellate cells of the Malpighian tubules. LKR knockdown in DH44-expressing neurons reduces Malpighian tubule-specific LKR, suggesting interactions between DH44 and LK signalling pathways.

Finally, although a role for DK in desiccation tolerance was not defined, we demonstrate a novel role for Malpighian tubule cell-specific LKR in starvation tolerance. Starvation increases gene expression of epithelial LKR. Also, Malpighian tubule stellate cell-specific knockdown of LKR significantly reduced starvation tolerance, demonstrating a role for neuropeptide signalling during starvation stress.

1. Introduction

Diuretic and anti-diuretic hormones act on the insect excretory system [12] and are produced by neurosecretory cells in the brain and ventral ganglia. They are released into the haemolymph via neurohemal sites, where they activate their G protein-coupled receptors (GPCRs) located in the Malpighian tubules [2]. Several diuretic peptides have been identified and functionally characterized in Drosophila melanogaster, including CRF-like (DH44) and kinin (Drome-kinin, DK).

DH44 peptide is produced by neuroendocrine cells in the brain, specifically in three bilateral pairs of cells in the pars intercerebralis (PI) with axons extending to the retrocerebral complex of the corpus cardiacum [4]. DH44 neurons also receive inputs from the circadian-timing system, which is known to project to the PI [9], [24], [33], and the DH44 neurons are involved in rhythms of rest and activity in D. melanogaster [9]. DH44 neurons are also activated in response to nutritive sugars, a response that could underlie a coordinated response by the gut and Malpighian tubules to feeding [20].

DK is localised to both the brain and the ventral nerve cord (VNC) [7]. In adult Drosophila, the brain DK neurons are localized in the lateral horn of the procerebrum and in the subesophageal ganglia [17], [37]. In the VNC, DK neurons project to the heart and abdominal body wall [6].

DH44 acts through cyclic AMP to stimulate fluid secretion by Malpighian tubules [4], whereas DK increases fluid secretion by elevating intracellular Ca2+ levels and altering chloride shunt conductance [5], [44], [51]. DH44 acts on DH44 Receptor 2 (DH44-R2) localized to tubule principal cells. Another DH44 receptor DH44-R1 [30], is primarily expressed in the adult brain [10].

DK is encoded by the leucokinin (LK) gene (http://flybase.org/reports/FBgn0028418.html) and acts on the DK receptor encoded by the leucokinin receptor gene, LKR [44] (http://flybase.org/reports/FBgn0035610.html). LKR is expressed in tubule stellate cells [4], [26], [44] and also in DH44-expressing PI neurons [4] and in the adult gonads [44].

Consistent with the role of DH44 as a diuretic peptide [4], knockdown of DH44-R2 expression impairs osmotic stress survival [27]. Recently, DH44 has also been shown to increase gut contractions and to modulate waste excretion [20].

DK acts as a diuretic hormone in fluid homeostasis [5], [23], [44], [51] and DK signalling modulates desiccation stress tolerance [37]. Persistent inactivation of the LK neurons or ubiquitous knockdown of LKR results in bloating caused by increased haemolymph volume, a phenotype that is not recapitulated by neuronal knockdown of LKR [14], [37]. Thus, LK influences fluid homeostasis specifically through action on LKR in epithelial tissues. In addition to diuretic roles for DK, meal termination [1] and food intake [37] is also modulated by the DK neurons.

The co-localisation of LKR to the DH44 neurons suggests interaction between the two signalling pathways [4] and may represent a coordinated neuronal circuit regulating fluid homeostasis. Interplay and regulation is not unprecedented in terms of insect neuropeptides as synergistic effects on Malpighian tubule fluid secretion have been previously noted among diuretic hormones, for example between DK and calcitonin-like diuretic hormone [13], and multiple neuronal circuits have been identified as key moderators of tubule function [8]. Co-localisation is also observed between a number of other neuropeptides, including the presence of corazonin expression in DH44-R1 expression neurons in both adult and larval brains [31].

Here we have assessed the potential roles of DH44, DH44-R2 and LKR with respect to fluid homeostasis and stress responses. We demonstrate roles for DH44 signalling in desiccation stress; LKR in starvation responses; and interactions between DH44 and DK signalling pathways.

2. Methods

2.1. Drosophila stocks

Drosophila lines were reared on standard Drosophila diet at 22 °C, 45–55% relative humidity with a 12:12 h light:dark photoperiod. GAL4-UAS crosses were reared and maintained at 26 °C. Wild-type Canton-S (CS), ‘cantonised’ white honey (wh), UAS-mCD8:GFP, UAS-pStinger2, UAS-p35 and UAS-reaper fly lines were acquired from Bloomington Stock Center (Bloomington, IN). The DH44-GAL4 driver line (BL 39347) was created by the Janelia Farm FlyLight Project Team, which uses a short fragment of genomic DNA to control GAL4 expression [29], [43], while the UAS-DH44 RNAi line (BL 25804) was created by the Transgenic RNAi Project [39]. The UAS-LKR RNAi line (105155 KK) and UAS-DH44-R2 line (102292 KK) were acquired from Vienna Drosophila Resource RNAi Center. VDRC crosses were controlled using a VDRC control line gifted from Dr. Edward Green. The capaR-GAL4 line [50] and c724-GAL4 lines [48], [50] were generated in-house previously.

2.2. Immunocytochemistry

Immunocytochemistry against DH44 and LKR was performed as described elsewhere [34]. After anesthetizing flies on ice, brains were dissected from Drosophila in Schneider’s medium (Gibco Life Technologies), and then fixed in 4% paraformaldehyde. Brains were washed with PBTA (0.5% Triton X-100, 0.1% Azide in PBS), blocked with 10% normal goat serum (Sigma) in PBTA, and incubated overnight with DH44 antibody at a concentration of 1:4000 [4]. Following a second round of washing and blocking, brains were incubated with anti-rabbit Alexa Fluor 546 or 488 (Life Technologies) overnight at a dilution of 1:1000. After washing again, brains were mounted onto slides and analyzed using confocal microscopy. Labelling with LKR antibody was carried out at a dilution of 1:1000 [44].

2.3. Fluorescent-tagged DH44 peptide labelling

Ligand receptor assays were performed on live Malpighian tubules from 7–10 days old male wild-type flies using a Drosophila DH44 analogue conjugated to a high quantum yield fluorophore, BODIPY 543 (TMR)-C5-maleimide (DH44-F). The specificity and functional efficacy of DH44-F was tested with a ligand competition assay, using 10−5 M unlabelled DH44; and a tubule secretion assay using 10−7 M DH44-F, respectively, as detailed elsewhere [23]. Tubules were incubated in 1:1 of Schneider’s medium and Drosophila saline containing 500 ng/ml DAPI and 10−7 M DH44-F for 15 min, prior to being mounted on poly-l-lysine coated glass bottom dishes in PBS and then imaged using confocal microscopy using a Zeiss LSM 510 Meta inverted confocal microscope. Fluorescent signal analysis was performed as described previously [23], [40].

2.4. RNA isolation, cDNA synthesis and quantitative (Q)-RT-PCR

RNA was isolated from groups of 8 Drosophila (whole fly), 10 Drosophila bodies, or 20 heads from flies aged 5–10 days old using TRIzol Reagent (Life Technologies) following the manufacturer’s instructions. RNA levels were quantified using a NanoVue Plus spectrophotometer (GE Healthcare Life Sciences) and then samples were DNAse treated using the DNA-free DNA Removal kit (Life Technologies). Samples were quantified again and cDNA was synthesized from 500 ng RNA using SuperScript II RT (Thermo Fisher Scientific), following manufacturer’s instructions. Q-RT-PCR was performed using TaqMan Probe-Based Gene Expression Analysis (Life Technologies) in an ABI StepOnePlus Detection System (Applied Biosystems) using the following primers and probes: Dm02138400_m1 (DH44), Dm01824019_g1 (DH44-R1), Dm01793183_g1 (DH44-R2), Dm01843317_s1 (LK) and Dm01840198_m1 (LKR). TaqMan primers for alpha tubulin 84b was synthesised by Integrated DNA Technologies (forward-CCTCGAAATCGTAGCTCTACAC, reverse-ACCAGCCTGACCAACATG, probe-TCACACGCGACAAGGAAAATTCACAGA) using sequences similar to those published elsewhere [54]. RT-PCR data was analysed by the comparative CT method [46]. Fold change was compared to a normalized control using a two-tailed one-sample t-test with a null hypothesis of no change (i.e. fold change of 1) [36], [45]. Fold changes that were each normalized to a third shared sample were compared using a two-tailed two-sample t-test. These are reported on figures as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

2.5. Ramsay fluid secretion assay

Fluid secretion assays using Drosophila Malpighian tubules were performed as described previously [19]. Malpighian tubules were dissected in Schneider’s medium and transferred to a 9 μl drop of 1:1 of Schneider’s medium and Drosophila saline [4]. Baseline secretion was measured every 10 min for 30 min, after which 1 μl of peptide (DH44, DK from Genosphere Biotechnologies, Paris, France; or DH44-F from Cambridge Peptides, Birmingham, UK, all used at 10−6 M) was added to the drop. Stimulated secretion was measured every 10 min for a further 30 min. The percentage change of basal secretion rates were calculated as previously shown [38].

2.6. Stress tolerance assays

Desiccation survival assays were carried out on 5–10 day-old male flies at 22 °C, 45–55% relative humidity with a 12:12 h light:dark photoperiod and were performed by placing flies in empty vials and counting surviving flies until mortality reached 100% [32], [50], [53]. Starvation assays were conducted by placing male flies aged 5–10 days in vials with 1% low melting point agar (Roche), and counting surviving flies until mortality reached 100% [28], [53]. All experiments were run in triplicate with at least 30 flies in each run of specified genotype. Survival data were plotted as Kaplan–Meier curves. Statistical comparisons were made using the logrank test, with estimation of variance (SE) calculated using the Greenwood formula [15]. Hazard ratios were calculated using the Mantel Haenszel approach, as this test has been found to perform more accurately than the log-rank calculation of hazard when using large sample sizes [3]. Where hazard ratio is calculated against two control lines, the more conservative estimate is reported (i.e. closer to 1).

3. Results

3.1. Desiccation exposure suppresses non-neural DH44-R2 expression while starvation increases non-neural LKR and DH44 expression

Given that LKR is expressed in DH44 neurons, and that both DK and DH44 are diuretic peptides, putative roles for DH44 and DK signalling in desiccation stress were explored by measuring gene expression of DH44, the DK gene (LK) and brain-specific DH44-R1 in wild type flies, and non-neural LKR and DH44-R2 in bodies of wild-type flies, after exposure to 24 h of desiccation, or 24 h of starvation, and compared to a non-stressed control groups. Neither desiccation nor starvation had a significant effect on DH44-R1 or LK expression, while DH44-R2 expression was found to decrease significantly (p < 0.0001) following desiccation stress, and both DH44 (p < 0.05) and LKR (p < 0.0001) expression increased significantly following starvation stress (Fig. 1).

Fig. 1.

Fig. 1

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Desiccation and starvation stress impact DH44, DH44-R2 and LKR expression.

Quantitative RT-PCR analysis of RNA extracted from whole fly (DH44, DH44-R1, LK) or bodies (DH44-R2, LKR) of CS Drosophila exposed to 24 h of desiccation, 24 h of starvation, or no treatment. Data show no impact of either treatment on DH44-R1 or LK expression, but a 60% decrease in DH44-R2 expression following desiccation, and increases in DH44 (22%) and LKR (97%) expression following starvation.

The impact of desiccation on Malpighian tubule function was assessed using a secretion assay. The baseline and DH44-stimulated secretion rates of Drosophila exposed to 24 h of desiccation are significantly lower than that of control flies (Fig. 2A, B). However, the percentage change in secretion rate following stimulation with DH44 peptide is similar in tubules from both desiccated and non-desiccated flies (Fig. 2C).

Fig. 2.

Fig. 2

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Desiccation stress impacts fluid secretion rate of Malpighian tubules.

A, B. Baseline and DH44-stimluated secretion rates are significantly lower in desiccated wild-type flies compared to untreated controls. C. The percentage change in secretion rate following stimulation with 10−7 M DH44 peptide is similar in desiccated wild type flies and untreated controls.

Potential changes in DH44-R2 receptor abundance following desiccation exposure were assessed using fluorescently labelled DH44 peptide (DH44-F) binding to intact tubules. The specificity of DH44-F binding to tubule DH44-R2 receptors was verified by a ligand competition assay in which unlabelled peptide was able to displace DH44-F labelling (Fig. 3A) and by the ability of DH44-F to stimulate fluid secretion to a similar extent as unlabelled peptide during secretion assay (Fig. 3B). The intensity of fluorescent signal from DH44-F labelling of tubules from desiccated flies was found to be lower than that of the signal from unstressed controls (Fig. 3C).

Fig. 3.

Fig. 3

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DH44 binding to DH44-R2 in Malpighian tubules is reduced following desiccation exposure.

A. Unlabelled DH44 (10−5 M) displaces bound fluorescent-labelled DH44 (DH44-F; 10−7 M). B. Both DH44-F and DH44 significantly increase fluid secretion rate to a similar extent when applied to excised Malpighian tubules. C. DH44-F label intensity is reduced in Malpighian tubules of desiccated wild-type flies when compared to unstressed controls.

3.2. Manipulations of the DH44 neurons indicate a role for DH44 signalling in desiccation tolerance

As the data on desiccation-stressed wild-type flies indicated a role for the DH44 signalling pathway during desiccation exposure, manipulations of the DH44 neurons were performed and their impact on desiccation stress survival was assessed. In order to probe the function of these neurons, a DH44-GAL4 line in which GAL4 is expressed under the control of a known short fragment of genomic DNA containing the promoter sequence of the DH44 gene [29] was selected.

DH44 expression has previously been observed in a restricted number of neurons within the CNS, most notably in two bilateral clusters of 3 neurons localized to the pars intercerebralis (PI) [4], [9], [20], [35], [42]. The DH44-GAL4 transgene’s ability to reiterate endogenous gene expression was validated by co-expression with a DH44 antibody localizing to the DH44 neurons [4]. Expression of the GAL4-responsive mCD8:GFP (membrane-bound GFP) reporter in conjunction with the DH44 antibody demonstrated absolute co-localisation in the 6 DH44 neurons of the PI (Fig. 4A). In addition we were able to demonstrate, via co-expression of DH44 and DK receptor (LKR) antibodies, that these 6 neurons (Fig. 4B), are also positive for LKR expression.

Fig. 4.

Fig. 4

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Characterisation of DH44 expression pattern in 5–7 days adult CNS.

A. Co-expression of UAS-membrane-bound CD8:GFP (mGFP) driven by DH44-GAL4 and DH44 antibody in the adult brain. Co-localisation in the soma of 6 neurons of the pars intercerebralis indicated (arrows). B. Co-expression of LKR and DH44 in the adult brain. Co-localisation in the soma of 6 neurons of the pars intercerebralis indicated (arrows). C. UAS-pStingerII nuclear GFP (nGFP) driven by DH44-GAL4 in the adult brain. Two bilateral clusters of ∼2 smaller neurons in the suboseophageal ganglion indicated (arrows). D. UAS-pStingerII nuclear GFP (nGFP) driven by DH44-GAL4 in the adult ventral nerve cord (VNC), ventral view. Expression apparent in clusters in the prothoracic, mesothoracic and abdominal (Abg) ganglia. Pair of smaller neurons in the metathoracic ganglion indicated (arrows). E. UAS-pStingerII nuclear GFP (nGFP) driven by DH44-GAL4 in the adult ventral nerve cord (VNC), dorsal view. Pair of smaller neurons in the distal Abg indicated (arrows). Neuropil counterstained with anti-nC82 (nC82, magenta) where indicated. All patterns of expression are representative of both males and females. All views ventral unless otherwise indicated. Scale bars = 50 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

We performed a spatio-temporal assay of DH44 expression within the CNS using the DH44-GAL4 transgene driving nuclear (nGFP) as well as membrane-bound GFP (mGFP). In the adult DH44 expression is most notable in the two bilateral clusters of 3 neurons localized to the PI, with these clusters sending characteristic ipsilateral projections through the superior protocerebrum around the oesophageal foramen to form large dendritic arborisations on the prow and flange of the suboesophageal ganglion (Fig. 4C and Supplemental Fig. 1D). These arborisations obscure a further pair of bilateral clusters of smaller neurons that then send projections from the prow (Fig. 4C and Supplemental Fig. 1F), that have been identified as ramifying on the crop and midgut, and are associated with the detection and consumption of nutritive sugars [20].

In the adult VNC, expression is restricted to two sets of bilateral clusters of 3 neurons in the prothoracic and mesothoracic ganglions, an individual pair of smaller neurons in the metathoracic ganglion and finally a large grouping of interconnected neurons in the abdominal ganglion (Abg) (Fig. 4D, E and Supplemental Fig. 1E and G). The neurons in the metathoracic and abdominal ganglion appear to form an interconnected network of dendrites as well as projecting axons to peripheral (non-CNS) structures (Supplemental Fig. 1E and G). Again the most distal dendritic arborisation on the Abg occludes two smaller neurons that send axonal projections to the internal genitalia (Fig. 4E and Supplemental Fig. 1E and G), which, in the female, have been identified as modulating sperm-ejection and storage [35]. It has been shown previously that DH44 expression occurs in the embryo as well as the larva [4], [22], [57], we expanded this to show that expression is also present, though at reduced levels, in L1/L2 stages (data not shown) becoming overt by stage L3, and continuing on in an expanded pattern of expression in the pupal brain and VNC (Supplemental Fig. 1A–C). The more restricted numbers of neurons expressing DH44 in the adult, as compared to the larval and pupal, CNS is most likely a result of neuronal sculpting during metamorphosis, as expression of the GAL4 responsive anti-apoptotic transgene UAS-p35 [25] results in an expanded number of DH44-positive neurons in the CNS, most notably in the brain (Supplemental Fig. 1H).

A targeted RNAi knockdown approach was then used to test whether either DH44 or LKR within DH44 neurons modulates desiccation tolerance, starvation tolerance, or both. In order to reduce expression of DH44, DH44-GAL4 flies were crossed to a UAS-DH44 RNAi line. Immunochemistry using antibody against DH44 peptide showed a total loss of DH44 peptide in DH44-GAL4/UAS-DH44 RNAi progeny (Fig. 5A, B). Confirmation by Q-RT-PCR showed that DH44 mRNA expression in heads was reduced to approximately 42% of the levels found in parental controls crossed to wh (Fig. 5D). Immunostaining LKR knockdown in the DH44 neurons (65% decrease of LKR mRNA levels, data not shown) is also observed (Fig. 5E, F),

Fig. 5.

Fig. 5

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A–C. Elimination of DH44 peptide in pars intercerebralis achieved via RNAi knockdown and neuronal ablation.

A. Brains from control DH44-GAL4/+ progeny stained for DH44 show clear labelling in the pars intercerebralis (arrowed). B. DH44 staining in the pars intercerebralis is abolished in progeny from cross between DH44-GAL4 and UAS-DH44 RNAi (arrowed). C. Ablation of DH44 neurons via cross between DH44-GAL4 and UAS-reaper eliminates the distinctive DH44 staining pattern of six neurons in the pars intercerebralis (arrowed). D. Knockdown of DH44 gene expression in head upon either DH44 neuronal ablation or RNAi knockdown of DH44. E–G. Reduction or elimination of LKR expression in pars intercerebralis achieved via RNAi knockdown or neuronal ablation, respectively. E. Brains from control DH44-GAL4/+ progeny stained for LKR show clear labelling in the pars intercerebralis (arrowed). F. Decreased intensity of LKR staining in the pars intercerebralis in progeny from cross between DH44-GAL4 and UAS-LKR RNAi (arrowed). G. Ablation of DH44 neurons in progeny of cross between DH44-GAL4 and UAS-reaper eliminates LKR staining in the pars intercerebralis (arrowed).

Furthermore, in order to probe potential roles for DH44 neurons in desiccation and starvation tolerance, genetic ablation of DH44 neurons via GAL4-mediated expression of the reaper (apoptotic) transgene [55], [56] was performed, resulting in the complete loss of the DH44 neurons in the PI as demonstrated by absence of DH44 immunoreactivity (Fig. 5C and G) and reduction in overall gene expression (Fig. 5D).

DH44-GAL4/UAS-DH44 RNAi progeny were assayed for desiccation and starvation survival; the latter also controlled for any potential starvation effects during the desiccation stress experiments (Fig. 6). Knockdown of DH44 expression in the DH44 neurons was found to significantly extend survival time during desiccation exposure (p < 0.0001 against both controls; Logrank test; Fig. 6A). RNAi knockdown of DH44 peptide in DH44 neurons was associated with at least half the rate of death relative to control groups during desiccation stress (hazard ratio: 0.37, 95% confidence interval [CI]: 0.25–0.54) and an approximately 20% increase in median survival time. Survival time during starvation was not significantly impacted by DH44 knockdown when compared to both parental controls (Fig. 6B). Gravimetric analysis [5] to calculate water content [21] of the DH44-GAL4/UAS-DH44 RNAi flies and parental controls showed no significant difference in total body water content between GAL4/UAS-DH44 RNAi flies and parental controls for males and females (Supplementary Fig. 2). Thus, increased desiccation tolerance of DH44-GAL4/UAS-DH44 RNAi flies is not due to increased body water retention.

Fig. 6.

Fig. 6

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Consequence of targeted DH44 RNAi, LKR RNAi and reaper in the DH44 neurons on desiccation stress (left) and starvation stress (right).

A. RNAi knockdown of DH44 in the DH44 neurons increases survival time during desiccation stress exposure (p < 0.0001). B. RNAi knockdown of DH44 in the DH44 neurons did not significantly impact survival time during starvation stress exposure relative to both controls. C. Partial RNAi knockdown of LKR in the DH44 neurons resulted in decreased survival time during desiccation stress (p < 0.0001). D. Partial RNAi knockdown of LKR in the DH44 neurons did not significantly affect survival time during starvation stress. E. Ablation of DH44 neurons via targeted expression of reaper increased survival time during desiccation exposure (p < 0.0001). F. Ablation of DH44 neurons via targeted expression of reaper increased survival time during starvation exposure (p < 0.0001).

Partial knockdown of LKR in the DH44 neurons was found to have a different effect compared to knockdown of DH44 in the DH44 neurons. DH44-GAL4/UAS-LKR RNAi progeny exhibited significantly reduced survival time during desiccation exposure (p < 0.0001 against both controls; Logrank test), with a hazard ratio of 1.75 (95% CI: 1.40–2.18) and an 8% decrease in median survival time (Fig. 6C). Survival during starvation was not significantly impacted by the manipulation of LKR expression in DH44 neurons when compared to both parental controls (Fig. 6D).

Although knockdown of each DH44 and LKR expression in the DH44 neurons did not affect starvation tolerance, ablation of the DH44 neurons in DH44-GAL4/UAS-reaper progeny was found to significantly increase survival time during both desiccation stress exposure (p < 0.0001 against both controls; Logrank test) (Fig. 6E) and starvation exposure (p < 0.0001 against both controls; Logrank test) (Fig. 6F). Ablation of the DH44 neurons was associated with less than half the rate of death of controls during desiccation stress (Hazard ratio: 0.38; 95% CI: 0.25 to 0.58) and an approximately 16% increase in median survival time. During starvation stress, ablation of DH44 neurons resulted in a hazard ratio of 0.48 relative to parental controls (95% CI: 0.39–0.58) and an increase in median survival time of approximately 18%.

3.3. Malpighian tubule response to DH44 peptide is not affected by manipulation of DH44-producing neurons, although expression of DH44-R2 and LKR is altered

One way in which knockdown of DH44 in the DH44 neurons could potentially influence desiccation tolerance is by altering the abundance or functionality of the DH44 receptor, DH44-R2 in the Malpighian tubules [27]. As DH44-R2 invokes a diuretic effect, compromising its function could potentially promote fluid retention as is observed with the capa neuropeptide receptor, capaR [50]. In order to test this, basal and DH44-stimulated fluid secretion rates [4] were measured in intact tubules from flies in which the DH44 peptide was knocked down in the DH44 neurons and in flies with ablated DH44 neurons.

In the DH44-GAL4/UAS-reaper progeny tubules, both baseline secretion and stimulated secretion rates were similar to those of the control progeny, and the percentage change in the stimulated fluid transport rate compared to mean baseline secretion did not differ significantly between the groups (Fig. 7A). Similarly, knockdown of DH44 in the DH44 neurons using RNAi did not impact the baseline secretion rate of the tubules or the ability of the tubules to respond to DH44 stimulation (Fig. 7B). These results indicate that DH44-R2 remains functional in both DH44-GAL4/UAS-reaper and DH44-GAL4/UAS-DH44 RNAi tubules, and that the manipulation of the DH44 neurons does not have a feedback effect on DH44-R2 function in the Malpighian tubules.

Fig. 7.

Fig. 7

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DH44 neuron manipulation impacts mRNA expression of DH44-R2 and LKR in the Malpighian tubules, but not secretion response to DH44 peptide.

A. Baseline and DH44-stimulated secretion rates are not significantly different between flies with ablated DH44 neurons and parental controls. B. Baseline and DH44-stimulated secretion rates are similar between DH44 knockdown flies and parental controls. C. DH44-R2 expression in the Malpighian tubules is increased by RNAi knockdown of DH44 in DH44 neurons. D. LKR expression in the Malpighian tubules is decreased by ablation of the DH44 neurons (* = p < 0.05).

However, changes in mRNA expression of DH44-R2 and LKR were observed in Malpighian tubules of DH44-GAL4/UAS-reaper and DH44-GAL4/UAS-DH44 RNAi progeny. DH44-R2 mRNA expression was increased 2.5-fold in tubules of DH44-GAL4/UAS-DH44 RNAi flies compared to controls (Fig. 7C). DH44-R2 expression was also higher in tubules of DH44-GAL4/UAS-reaper progeny, but the difference was not statistically significant. By contrast, LKR mRNA expression was decreased by 2.2-fold in tubules of DH44-GAL4/UAS-reaper flies compared to controls (Fig. 7D). LKR expression was also decreased in DH44-GAL4/UAS-DH44 RNAi cross progeny tubules, although this was only significant relative to one parental control.

3.4. Knockdown of LKR and DH44-R2 in Malpighian tubules impacts fluid secretion, desiccation and starvation tolerance

Having demonstrated the impact of manipulation of neuronal DH44 signalling on desiccation survival (Fig. 6) but without effect on Malpighian tubule fluid secretion rates (Fig. 7), putative roles of Malpighian tubule DH44-R2 and LKR in desiccation tolerance were assessed by selective RNAi knockdown in either tubule principal or stellate cells, respectively. This was achieved using GAL4 drivers targeted to Malpighian tubule principal (capaR-GAL4) or stellate (c724-GAL4) cells.

c724-GAL4/UAS-LKR RNAi tubules were found to have a 91% reduction in LKR mRNA levels (Fig. 8A) compared to parental controls. A 60% reduction in tubule DH44-R2 mRNA levels in capaR-GAL4/UAS-DH44-R2 RNAi flies (Fig. 8B).

Fig. 8.

Fig. 8

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Knockdown of LKR in stellate cells of the Malpighian tubules suppresses response of tubules to DK peptide.

A. Expression of UAS-LKR RNAi in stellate cells of Malpighian tubules results in 91% knockdown of LKR mRNA levels in tubules. B. Expression of UAS-DH44-R2 RNAi in principal cells results in 60% knockdown of DH44-R2 mRNA levels in tubules. C. Knockdown of LKR in Malpighian tubule stellate cells impairs tubule response to 10−7 M DK. D. Knockdown of DH44-R2 in principal cells does not impact basal secretion rate or secretion rate in response to 10−7 M DH44.

The impact of reduced LKR and DH44-R2 expression on Malpighian tubule fluid secretion response to either DK or DH44, respectively, was assessed by secretion assay. c724-GAL4/UAS-LKR RNAi tubules were found to have a similar basal rate as parental controls, but a significantly reduced DK-stimulated secretion rate (Fig. 8C). By contrast, although DH44-R2 was also significantly reduced by targeted DH44-RNAi, the basal and DH44-stimulated secretion rates of capaR-GAL4/UAS-DH44-R2 RNAi tubules were similar to that of the parental control (Fig. 8D). It is likely that the 60% reduction in capaR-GAL4/UAS-DH44-R2 RNAi tubules is still sufficient for significant expression of DH44-R2. Efforts to obtain a more efficient RNAi knockdown via incorporation of dicer did not further reduce DH44-R2 gene expression (data not shown).

Having established tubule cell-specific LKR and DH44-R2 gene knockdowns, the role of Malpighian tubule LKR and DH44-R2 in desiccation and starvation survival was assessed by exposing c724-GAL4/UAS-LKR RNAi and capaR-GAL4/UAS-DH44-R2 RNAi flies to stress tolerance assays. Knockdown of LKR in tubule stellate cells did not significantly impact desiccation tolerance (Fig. 9A), but significantly impaired survival during starvation (Fig. 9B), resulting in a 3.7 fold rate of death relative to control (95% CI: 2.6–5.2) with a 26% lower median survival time. Inhibition of DK signalling pathways has previously been shown to result in a bloating phenotype of the abdomen [37] and an inflated crop in the gut [1]. The phenotype observed by Liu et al. is thought to be due to an increase in hemolymph volume, potentially due to the loss of DK diuretic action on the Malpighian tubule. Thus, it was expected that LKR knockdown in the Malpighian tubules may cause fluid retention that could be detected by gravimetric analysis of body water [21]. However, no difference in water content of c724-GAL4/UAS-LKR RNAi compared to parental controls was found. Also, unlike previous studies, these flies did not have a bloated phenotype. However, it may be that targeted LKR knockdown in only tubule stellate cells is not sufficient to impact fluid homeostasis, under conditions of normal LK secretion.

Fig. 9.

Fig. 9

Open in a new tab

Malpighian tubule diuretic receptors LKR and DH44-R2 are involved in desiccation and starvation survival.

A. Knockdown of LKR in tubule stellate cells does not significantly impact desiccation tolerance. B. Knockdown of LKR in tubule stellate cells significantly impairs survival during starvation stress (p < 0.0001), with a 26% decrease in median survival time. C. Knockdown of DH44-R2 in tubule principal cells significantly enhances desiccation tolerance (p < 0.001), with a 5% increase in median survival. D. Knockdown of DH44-R2 in tubule principal cells significantly impairs survival during starvation stress (p < 0.001), with a 9% decrease in median survival.

Knockdown of DH44-R2 in tubule principal cells significantly improved desiccation survival (Fig. 9C), resulting in a 0.6 fold rate of death (95% CI: 0.48–0.82) and a 5% increase in median survival time. DH44-R2 knockdown significantly impaired starvation tolerance (Fig. 9D), with a hazard ratio of 1.6 (95% CI: 1.2–2.1) and a 9% lower median survival time.

4. Discussion

We demonstrate that suppressing the DH44 signalling pathways, either by manipulating the DH44 neurons or by impacting the DH44-R2 in the tubules, improved desiccation survival. We show reduced DH44-R2 transcript levels and concomitantly reduced fluorescent-labelled DH44 binding in tubule principal cells upon desiccation. Targeted knockdown of DH44-R2 to Malpighian tubule principal cells also results in improved desiccation tolerance, which may also be modulated by re-absorption by the hindgut. Furthermore, ablation of DH44 neurons or DH44 knockdown in only DH44 neurons both have the effect of improving survival of flies during desiccation stress, possibly due to increased haemolymph volume. However, increased fluid retention was not detected in DH44-GAL4/UAS-DH44 RNAi flies.

Our data also imply a role for DH44 signalling in starvation tolerance. This is not unprecedented as the DH44 neurons also contain LKR, which is involved in feeding regulation [32]. Indeed, ablation of the DH44 neurons resulted in increased survival during starvation exposure, while knockdown of DH44 expression in the DH44 neurons via RNAi did not clearly impact starvation tolerance. However, in spite of the apparent lack of involvement of DH44 in the DH44 neurons in starvation tolerance, a decrease in survival during starvation exposure was observed following knockdown of DH44-R2 in Malpighian tubule principal cells. Consistent with these data was the finding that DH44 gene expression is increased after mild starvation exposure. Impairment of starvation survival by DH44-R2 knockdown could potentially be underpinned by a reduction in food consumption due to bloating, although tubule secretion rates in DH44-R2 knockdown flies are similar to control flies, and no bloating of these flies was observed.

The involvement of the DH44 neurons in starvation, however, is clearly indicated by the finding that ablation of these neurons greatly improves starvation survival. These neurons may be involved in circuitry that coordinates the physiological response to starvation, a finding that is perhaps consistent with the involvement of these neurons in nutrient sensing and the co-localization of LKR in these neurons, which may impact feeding behaviour [1], [20].

As with the DH44 peptide, a role for the diuretic hormone DK in desiccation tolerance can be hypothesized based on the finding that other diuretic hormones impact desiccation survival in Drosophila [32], [47], [50], [53]. Surprisingly, evidence for the involvement of DK signalling in desiccation tolerance from this study was limited. No changes in either whole fly LK expression or non-neural LKR expression (i.e. body samples) were found following 24 h of desiccation exposure. Consistent with these results was the finding that knockdown of LKR in the stellate cells of the Malpighian tubules does not impact desiccation survival. However, it may be that compensatory mechanisms occur via other neuropeptides which act through principal cells e.g. capa, DH31 and DH44, to maintain fluid secretion rates in stellate-cell LKR knockdown flies. Intriguingly, knockdown of LKR in DH44 neurons reduced desiccation survival. Also, manipulation of DH44 levels in the DH44 neurons via neuronal ablation or DH44 knockdown resulted in significantly reduced expression of the tubule-specific LKR. Thus, the DH44 and DK pathways interact, and could be co-regulated. Interactions between different neuropeptides and even classical neurotransmitters in the form of modulatory circuits have been proposed to occur elsewhere in the Drosophila brain [8], [49].

DK has demonstrated roles in feeding behaviour [1], [37], so a role in starvation tolerance is also plausible. Ablation of DH44 neurons (resulting in lack of neuronal LKR), but not RNAi knockdown of DH44 resulted in increased tolerance to starvation survival. By contrast, tubule stellate-cell specific knockdown of LKR results in reduced starvation survival; and expression of non-neuronal LKR is significantly increased under starvation conditions. These novel findings may be explained by the complex role of the Malpighian tubules, beyond osmoregulation. The Malpighian tubules are critical tissues not only for fluid homeostasis, but also for detoxification [11], [16], [18], [52]. Evidence indicates that lipid metabolism in the fat body is a particularly crucial source of energy during starvation [41]. Lipid mobilisation results in waste products being released into the hemolymph, which are then taken up by the Malpighian tubules for processing and excretion [41]. Interference with this process by reducing the ability of the Malpighian tubules to increase fluid secretion, potentially in response to changes in hemolymph osmolarity, could impact on the ability of the organism to mobilise energy resources. Thus, it could be interference with the role of the Malpighian tubule in detoxification, rather than in fluid homeostasis, that impacts starvation tolerance when LKR expression is reduced in the tubules. Moreover, the LKR gene has seven predicted binding sites for transcription factors [44], thereby providing several possible sites that could be used to modify gene expression during stress exposure.

Recently, insect diuretic neuropeptides that act on Malpighian tubules to modulate fluid homeostasis e.g. capa, kinin and DH44, have been found to modulate stress tolerance, metabolism and reproduction—and so are critical for organismal survival. The challenge will be to unravel the precise mechanisms of function of these neuropeptides, and to understand environmental ‘cues’ for potential co-regulation of neuropeptide gene expression, release, activation and signalling.

Author contributions

EC, AJD, KAH and ST performed the experiments, analysed the data and EC, SD, AJD wrote the manuscript. All work was conducted in the laboratory of SD/JATD, who designed experiments in conjunction with ST/EC/AJD/KAH.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

This work was funded by a Wellcome Trust studentship 096987/Z/11/Z (EC), European Commission H2020, Research and Innovation Grant 634361, (SD/JATD), Biotechnology and Biological Sciences Research Council (UK) grant BB/L002647/1 (SD/JATD/ST), and Fellowships from the Danish Council for Independent Research | Natural Sciences 0602-02523B and the Carlsberg Foundation CF14-0204 (KAH). The authors are grateful to the TRiP at Harvard Medical School (NIH/NIGMS R01-GM084947) for providing a transgenic RNAi fly stock, Professor Jan A. Veenstra, Bordeaux University, for providing antibody against DH44 peptide and Professor Dick Nässel, Stockholm University for helpful discussion and reagents.

Footnotes

Appendix A

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.peptides.2016.02.004.

Appendix A. Supplementary data

The following are Supplementary data to this article:

mmc1.docx (712.7KB, docx)

References

  • 1.Al-Anzi B., Armand E., Nagamei P., Olszewski M., Sapin V., Waters C. The leucokinin pathway and its neurons regulate meal size in Drosophila. Curr. Biol. 2010;20:969–978. doi: 10.1016/j.cub.2010.04.039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Altstein M., Nassel D.R. Neuropeptide signalling in insects. Adv. Exp. Med. biol. 2010;692:155–165. doi: 10.1007/978-1-4419-6902-6_8. [DOI] [PubMed] [Google Scholar]
  • 3.Bernstein L., Anderson J., Pike M.C. Estimation of the proportional hazard in two-treatment-group clinical trials. Biometrics. 1981;37:513–519. [PubMed] [Google Scholar]
  • 4.Cabrero P., Radford J.C., Broderick K.E., Costes L., Veenstra J.A., Spana E.P. The Dh gene of Drosophila melanogaster encodes a diuretic peptide that acts through cyclic AMP. J. Exp. Biol. 2002;205:3799–3807. doi: 10.1242/jeb.205.24.3799. [DOI] [PubMed] [Google Scholar]
  • 5.Cabrero P., Terhzaz S., Romero M.F., Davies S.A., Blumenthal E.M., Dow J.A. Chloride channels in stellate cells are essential for uniquely high secretion rates in neuropeptide-stimulated Drosophila diuresis. Proc. Natl. Acad. Sci. U. S. A. 2014;111:14301–14306. doi: 10.1073/pnas.1412706111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Cantera R., Hansson B.S., Hallberg E., Nassel D.R. Postembryonic development of leucokinin I-immunoreactive neurons innervating a neurohemal organ in the turnip moth Agrotis segetum. Cell Tissue Res. 1992;269:65–77. doi: 10.1007/BF00384727. [DOI] [PubMed] [Google Scholar]
  • 7.Cantera R., Nassel D.R. Segmental peptidergic innervation of abdominal targets in larval and adult dipteran insects revealed with an antiserum against leucokinin I. Cell Tissue Res. 1992;269:459–471. doi: 10.1007/BF00353901. [DOI] [PubMed] [Google Scholar]
  • 8.Carlsson M.A., Diesner M., Schachtner J., Nassel D.R. Multiple neuropeptides in the Drosophila antennal lobe suggest complex modulatory circuits. J. Comp. Neurol. 2010;518:3359–3380. doi: 10.1002/cne.22405. [DOI] [PubMed] [Google Scholar]
  • 9.Cavanaugh D.J., Geratowski J.D., Wooltorton J.R., Spaethling J.M., Hector C.E., Zheng X. Identification of a circadian output circuit for rest:activity rhythms in Drosophila. Cell. 2014;157:689–701. doi: 10.1016/j.cell.2014.02.024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Chintapalli V.R., Wang J., Dow J.A. Using FlyAtlas to identify better Drosophila melanogaster models of human disease. Nat. Genet. 2007;39:715–720. doi: 10.1038/ng2049. [DOI] [PubMed] [Google Scholar]
  • 11.Chung H., Sztal T., Pasricha S., Sridhar M., Batterham P., Daborn P.J. Characterization of Drosophila melanogaster cytochrome P450 genes. Proc. Natl. Acad. Sci. U. S. A. 2009;106:5731–5736. doi: 10.1073/pnas.0812141106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Coast G.M. Handbook of Biologically Active Peptides. Academic Press; California, USA: 2006. Insect diuretic and antidiuretic hormones; pp. 157–162. [Google Scholar]
  • 13.Coast G.M., Webster S.G., Schegg K.M., Tobe S.S., Schooley D.A. The Drosophila melanogaster homologue of an insect calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit fly Malpighian tubules. J. Exp. Biol. 2001;204:1795–1804. doi: 10.1242/jeb.204.10.1795. [DOI] [PubMed] [Google Scholar]
  • 14.Cognigni P., Bailey A.P., Miguel-Aliaga I. Enteric neurons and systemic signals couple nutritional and reproductive status with intestinal homeostasis. Cell. Metab. 2011;13:92–104. doi: 10.1016/j.cmet.2010.12.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Cox D., Oakes D. Chapman & Hall/CRC; USA: 1984. Analysis of Survival Data: Monograph on Statistics and Applied Probability. [Google Scholar]
  • 16.Davies S.A., Cabrero P., Overend G., Aitchison L., Sebastian S., Terhzaz S. Cell signalling mechanisms for insect stress tolerance. J. Exp. Biol. 2014;217:119–128. doi: 10.1242/jeb.090571. [DOI] [PubMed] [Google Scholar]
  • 17.de Haro M., Al-Ramahi I., Benito-Sipos J., Lopez-Arias B., Dorado B., Veenstra J.A. Detailed analysis of leucokinin-expressing neurons and their candidate functions in the Drosophila nervous system. Cell Tissue Res. 2010;339:321–336. doi: 10.1007/s00441-009-0890-y. [DOI] [PubMed] [Google Scholar]
  • 18.Dow J.A. Insights into the Malpighian tubule from functional genomics. J. Exp. Biol. 2009;212:435–445. doi: 10.1242/jeb.024224. [DOI] [PubMed] [Google Scholar]
  • 19.Dow J.A., Maddrell S.H., Gortz A., Skaer N.J., Brogan S., Kaiser K. The Malpighian tubules of Drosophila melanogaster: a novel phenotype for studies of fluid secretion and its control. J. Exp. Biol. 1994;197:421–428. doi: 10.1242/jeb.197.1.421. [DOI] [PubMed] [Google Scholar]
  • 20.Dus M., Lai J.S., Gunapala K.M., Min S., Tayler T.D., Hergarden A.C. Nutrient sensor in the brain directs the action of the brain-gut axis in Drosophila. Neuron. 2015;87:139–151. doi: 10.1016/j.neuron.2015.05.032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Folk D.G., Han C., Bradley T.J. Water acquisition and partitioning in Drosophila melanogaster: effects of selection for desiccation-resistance. J. Exp. Biol. 2001;204:3323–3331. doi: 10.1242/jeb.204.19.3323. [DOI] [PubMed] [Google Scholar]
  • 22.Fontana J.R., Crews S.T. Transcriptome analysis of Drosophila CNS midline cells reveals diverse peptidergic properties and a role for castor in neuronal differentiation. Dev. Biol. 2012;372:131–142. doi: 10.1016/j.ydbio.2012.09.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Halberg K.A., Terhzaz S., Cabrero P., Davies S.A., Dow J.A. Tracing the evolutionary origins of insect renal function. Nat. Commun. 2015;6:6800. doi: 10.1038/ncomms7800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Hall J.C. Genetics and molecular biology of rhythms in Drosophila and other insects. Adv. Genet. 2003;48:1–280. doi: 10.1016/s0065-2660(03)48000-0. [DOI] [PubMed] [Google Scholar]
  • 25.Hay B.A., Wolff T., Rubin G.M. Expression of baculovirus P35 prevents cell death in Drosophila. Development. 1994;120:2121–2129. doi: 10.1242/dev.120.8.2121. [DOI] [PubMed] [Google Scholar]
  • 26.Hector C.E., Bretz C.A., Zhao Y., Johnson E.C. Functional differences between two CRF-related diuretic hormone receptors in Drosophila. J. Exp. Biol. 2009;212:3142–3147. doi: 10.1242/jeb.033175. [DOI] [PubMed] [Google Scholar]
  • 27.Hector C.E., Bretz C.A., Zhao Y., Johnson E.C. Functional differences between two CRF-related diuretic hormone receptors in Drosophila. J. Exp. Biol. 2009;212:3142–3147. doi: 10.1242/jeb.033175. [DOI] [PubMed] [Google Scholar]
  • 28.Iijima K., Zhao L., Shenton C., Iijima-Ando K. Regulation of energy stores and feeding by neuronal and peripheral CREB activity in Drosophila. PLoS One. 2009;4:e8498. doi: 10.1371/journal.pone.0008498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Jenett A., Rubin G.M., Ngo T.T., Shepherd D., Murphy C., Dionne H. A GAL4-driver line resource for Drosophila neurobiology. Cell Rep. 2012;2:991–1001. doi: 10.1016/j.celrep.2012.09.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Johnson E.C., Bohn L.M., Taghert P.H. Drosophila CG8422 encodes a functional diuretic hormone receptor. J. Exp. Biol. 2004;207:743–748. doi: 10.1242/jeb.00818. [DOI] [PubMed] [Google Scholar]
  • 31.Johnson E.C., Shafer O.T., Trigg J.S., Park J., Schooley D.A., Dow J.A. A novel diuretic hormone receptor in Drosophila: evidence for conservation of CGRP signalling. J. Exp. Biol. 2005;208:1239–1246. doi: 10.1242/jeb.01529. [DOI] [PubMed] [Google Scholar]
  • 32.Kahsai L., Kapan N., Dircksen H., Winther A.M., Nassel D.R. Metabolic stress responses in Drosophila are modulated by brain neurosecretory cells that produce multiple neuropeptides. PLoS One. 2010;5:e11480. doi: 10.1371/journal.pone.0011480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Kaneko M., Hall J.C. Neuroanatomy of cells expressing clock genes in Drosophila: transgenic manipulation of the period and timeless genes to mark the perikarya of circadian pacemaker neurons and their projections. J. Comp. Neurol. 2000;422:66–94. doi: 10.1002/(sici)1096-9861(20000619)422:1<66::aid-cne5>3.0.co;2-2. [DOI] [PubMed] [Google Scholar]
  • 34.Kean L., Cazenave W., Costes L., Broderick K.E., Graham S., Pollock V.P. Two nitridergic peptides are encoded by the gene capability in Drosophila melanogaster. Am. J. Physiol.-Regul. Integr. Comp. Physiol. 2002;282:R1297–R1307. doi: 10.1152/ajpregu.00584.2001. [DOI] [PubMed] [Google Scholar]
  • 35.Lee K.-M., Daubnerová I., Isaac R.E., Zhang C., Choi S., Chung J. A neuronal pathway that controls sperm ejection and storage in female Drosophila. Curr. Biol. 2015;25:790–797. doi: 10.1016/j.cub.2015.01.050. [DOI] [PubMed] [Google Scholar]
  • 36.Levenson J.M., Roth T.L., Lubin F.D., Miller C.A., Huang I.C., Desai P. Evidence that DNA (cytosine-5) methyltransferase regulates synaptic plasticity in the hippocampus. J. Biol. Chem. 2006;281:15763–15773. doi: 10.1074/jbc.M511767200. [DOI] [PubMed] [Google Scholar]
  • 37.Liu Y., Luo J., Carlsson M.A., Nassel D.R. Serotonin and insulin-like peptides modulate leucokinin-producing neurons that affect feeding and water homeostasis in Drosophila. J. Comp. Neurol. 2015;523:1840–1863. doi: 10.1002/cne.23768. [DOI] [PubMed] [Google Scholar]
  • 38.MacPherson M.R., Lohmann S.M., Davies S.A. Analysis of Drosophila cGMP-dependent protein kinases and assessment of their in vivo roles by targeted expression in a renal transporting epithelium. J. Biol. Chem. 2004;279:40026–40034. doi: 10.1074/jbc.M405619200. [DOI] [PubMed] [Google Scholar]
  • 39.Ni J.Q., Liu L.P., Binari R., Hardy R., Shim H.S., Cavallaro A. A Drosophila resource of transgenic RNAi lines for neurogenetics. Genetics. 2009;182:1089–1100. doi: 10.1534/genetics.109.103630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Overend G., Cabrero P., Halberg K.A., Ranford-Cartwright L.C., Woods D.J., Davies S.A. A comprehensive transcriptomic view of renal function in the malaria vector, Anopheles gambiae. Insect Biochem. Mol. Biol. 2015;67:47–58. doi: 10.1016/j.ibmb.2015.05.007. [DOI] [PubMed] [Google Scholar]
  • 41.Palanker L., Tennessen J.M., Lam G., Thummel C.S. Drosophila HNF4 regulates lipid mobilization and beta-oxidation. Cell Metab. 2009;9:228–239. doi: 10.1016/j.cmet.2009.01.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Park D., Veenstra J.A., Park J.H., Taghert P.H. Mapping peptidergic cells in Drosophila: where DIMM fits in. PLoS One. 2008;3:e1896. doi: 10.1371/journal.pone.0001896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Pfeiffer B.D., Jenett A., Hammonds A.S., Ngo T.T., Misra S., Murphy C. Tools for neuroanatomy and neurogenetics in Drosophila. Proc. Natl. Acad. Sci. U. S. A. 2008;105:9715–9720. doi: 10.1073/pnas.0803697105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Radford J.C., Davies S.A., Dow J.A. Systematic G-protein-coupled receptor analysis in Drosophila melanogaster identifies a leucokinin receptor with novel roles. J. Biol. Chem. 2002;277:38810–38817. doi: 10.1074/jbc.M203694200. [DOI] [PubMed] [Google Scholar]
  • 45.Roth T.L., Lubin F.D., Funk A.J., Sweatt J.D. Lasting epigenetic influence of early-life adversity on the BDNF gene. Biol. Psychiatry. 2009;65:760–769. doi: 10.1016/j.biopsych.2008.11.028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Schmittgen T.D., Livak K.J. Analyzing real-time PCR data by the comparative C(T) method. Nat. Protoc. 2008;3:1101–1108. doi: 10.1038/nprot.2008.73. [DOI] [PubMed] [Google Scholar]
  • 47.Soderberg J.A., Birse R.T., Nassel D.R. Insulin production and signalling in renal tubules of Drosophila is under control of tachykinin-related peptide and regulates stress resistance. PLoS One. 2011;6:e19866. doi: 10.1371/journal.pone.0019866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Sozen M.A., Armstrong J.D., Yang M., Kaiser K., Dow J.A. Functional domains are specified to single-cell resolution in a Drosophila epithelium. Proc. Natl. Acad. Sci. U. S. A. 1997;94:5207–5212. doi: 10.1073/pnas.94.10.5207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Taghert P.H., Nitabach M.N. Peptide neuromodulation in invertebrate model systems. Neuron. 2012;76:82–97. doi: 10.1016/j.neuron.2012.08.035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Terhzaz S., Cabrero P., Robben J.H., Radford J.C., Hudson B.D., Milligan G. Mechanism and function of Drosophila capa GPCR: a desiccation stress-responsive receptor with functional homology to human neuromedinU receptor. PLoS One. 2012;7:e29897. doi: 10.1371/journal.pone.0029897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Terhzaz S., O'Connell F.C., Pollock V.P., Kean L., Davies S.A., Veenstra J.A. Isolation and characterization of a leucokinin-like peptide of Drosophila melanogaster. J. Exp. Biol. 1999;202:3667–3676. doi: 10.1242/jeb.202.24.3667. [DOI] [PubMed] [Google Scholar]
  • 52.Terhzaz S., Overend G., Sebastian S., Dow J.A., Davies S.A. The D. melanogaster capa-1 neuropeptide activates renal NF-kB signalling. Peptides. 2014;53:218–224. doi: 10.1016/j.peptides.2013.08.004. [DOI] [PubMed] [Google Scholar]
  • 53.Terhzaz S., Teets N.M., Cabrero P., Henderson L., Ritchie M.G., Nachman R.J. Insect capa neuropeptides impact desiccation and cold tolerance. Proc. Natl. Acad. Sci. U. S. A. 2015;112:2882–2887. doi: 10.1073/pnas.1501518112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Thomas A., Lee P.J., Dalton J.E., Nomie K.J., Stoica L., Costa-Mattioli M. A versatile method for cell-specific profiling of translated mRNAs in Drosophila. PLoS One. 2012:2012. doi: 10.1371/journal.pone.0040276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.White K., Grether M.E., Abrams J.M., Young L., Farrell K., Steller H. Genetic control of programmed cell death in Drosophila. Science. 1994;264:677–683. doi: 10.1126/science.8171319. [DOI] [PubMed] [Google Scholar]
  • 56.White K., Tahaoglu E., Steller H. Cell killing by the Drosophila gene reaper. Science. 1996;271:805–807. doi: 10.1126/science.271.5250.805. [DOI] [PubMed] [Google Scholar]
  • 57.Žitňan D., Sehnal F., Bryant P.J. Neurons producing specific neuropeptides in the central nervous system of normal and pupariation-delayed Drosophila. Dev. Biol. 1993;156:117–135. doi: 10.1006/dbio.1993.1063. [DOI] [PubMed] [Google Scholar]

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