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

Keywords: DH(44); Desiccation; Drosophila melanogaster; Kinin; Neuropeptide receptor; Starvation.

Fig. 1

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.

Fig. 2

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.

Fig. 3

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.

Fig. 4

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.)

Fig. 5

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).

Fig. 6

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).

Fig. 7

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).

Fig. 8

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.

Fig. 9

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.