Orchestration of Drosophila post-feeding physiology and behavior by the neuropeptide leucokinin

Behavior and physiology are orchestrated by neuropeptides acting as neuromodulators and/or circulating hormones. A central question is how these neuropeptides function to coordinate complex and competing behaviors. The neuropeptide leucokinin (LK) modulates diverse functions, including circadian rhythms, feeding, water homeostasis, and sleep, but the mechanisms underlying these complex interactions remain poorly understood. Here, we delineate the LK circuitry that governs homeostatic functions that are critical for survival. We found that impaired LK signaling affects diverse but coordinated processes, including regulation of stress, water homeostasis, locomotor activity, and metabolic rate. There are three different sets of LK neurons, which contribute to different aspects of this physiology. We show that the calcium activity of abdominal ganglia LK neurons (ABLKs) increases specifically following water consumption, but not under other conditions, suggesting that these neurons regulate water homeostasis and its associated physiology. To identify targets of LK peptide, we mapped the distribution of the LK receptor (Lkr), mined brain single-cell transcriptome dataset for genes coexpressed with Lkr, and utilized trans-synaptic labeling to identify synaptic partners of LK neurons. Lkr expression in the brain insulin-producing cells (IPCs), gut, renal tubules and sensory cells, and the post-synaptic signal in sensory neurons, correlates well with regulatory roles detected in the Lk and Lkr mutants. Furthermore, these mutants and flies with targeted knockdown of Lkr in IPCs displayed altered expression of insulin-like peptides (DILPs) in IPCs and modulated stress responses. Thus, some effects of LK signaling appear to occur via DILP action. Collectively, our data suggest that the three sets of LK neurons orchestrate the establishment of post-prandial homeostasis by regulating distinct physiological processes and behaviors such as diuresis, metabolism, organismal activity and insulin signaling. These findings provide a platform for investigating neuroendocrine regulation of behavior and brain-to-periphery communication.


Introduction
post-synaptic to SELKs. However, no colocalization is seen between the IPCs and post-242 synaptic signal of LKs. In addition, the post-synaptic signal is not coexpressed with 243 Hugin neurons (labeled with anti-CAPA antibody) although these have similar axonal 244 projections (Fig. S9). Hence, these anatomical data indicate that the IPCs express the 245 Lk receptor, but may receive non-synaptic (paracrine) inputs from LK neurons, or 246 possibly via the circulation from ABLKs. 247 Since Lkr is expressed in the IPCs we wanted to examine if the expression of 248 DILPs is altered in Lk and Lkr mutants. In Lk mutant flies, DILP3 immunolabeling is 249 increased and in Lkr mutants both DILP2 and DILP3 levels are significantly higher (Fig.  250 10C-F), indicating that LK could affect the release of DILP2 and DILP3 (as increased 251 immunolabeling has been proposed to reflect decreased peptide release [31]). No effect 252 on DILP5 levels was seen for any of the mutants, suggesting that LK selectively 253 modulates DILP function (Fig. S11). 254 Next, we examined DILP2, DILP3 and DILP5 transcript levels by qPCR after 255 targeted knockdown of the Lkr in the IPCs of flies using two different Lkr-RNAi lines and 256 a DILP2-GAL4 driver; also different diets were tested since DILP expression in IPCs are 257 influenced by carbohydrate and protein levels in the food [32]. The experimental flies 258 developed to pupation on normal diet and were transferred as adults to three different 259 diets, high sugar+high protein, low sugar+high protein and normal diet. UAS-Lkr-RNAi-260 #1 did not drive efficient knockdown and was thus excluded from the analysis; data 261 shown for UAS-Lkr-RNAi-#2. Significant effects on DILP transcripts were only seen for 262 DILP3, which was increased in flies after Lkr-RNAi under normal and high-sugar+high-263 protein diets, and DILP5, which was decreased in normal diet. Having noted an effect 264 on DILP/DILP levels in mutant flies and after Lkr knockdown in the IPCs we went on to 265 determine the effects of this manipulation on fly weight as well as survival during 266 starvation and desiccation. As seen in Fig. S12, there was a slight increase in survival 267 during desiccation and a small increase in dry weight of the flies with reduced Lkr in 268

IPCs. 269
Taken together, we identify roles for the Lkr within the CNS and in the 270 periphery that uniquely regulate physiological homeostasis. The Lkr expression in the 271 periphery suggests LK signaling to be associated with water balance, gut function and 272 chemosensation (Fig. 12). Within the CNS, LK signaling modulates specific 273 neurosecretory cells of the brain that are known to regulate stress responses, feeding, 274 metabolism, energy storage and activity patterns, including sleep ( Fig. 12)

277
In this study we established the role of LK signaling in orchestrating behavioral 278 and physiological homeostasis in Drosophila. More specifically, we determined a set of 279 effects caused by loss of LK signaling, which indicates that this neuropeptide regulates 280 physiology related to water homeostasis and metabolism, as well as associated stress, 281 locomotor activity and metabolic rate. We suggest that LK signaling regulates post-282 feeding physiology, metabolism and behavior, as this seems to link most of the observed 283 phenotypes observed after peptide and receptor knockdown. 284 In support of the physiological roles of LK signaling, we show distribution of the 285 Lkr expression in cells of the renal tubules and intestine, including the water-regulating 286 rectal pads, as well as in the IPCs, which are known to signal with DILPs to affect 287 feeding, metabolism, sleep, activity and stress responses [33][34][35][36]38]. Lkr is also 288 expressed by another set of brain neurosecretory cells (ipc-1/ipc-2a) known to regulate 289 stress responses by means of three different coexpressed neuropeptides [24]. 290 In the CNS of the adult fly, LK is produced at high levels by a small number of 291 neurons of three major types: two pairs of interneurons in the brain and about 20 292 neurosecretory cells, ABLKs, in the abdominal ganglia [6,7]. There is mounting evidence 293 that the ABLKs use LK as a hormonal signal that targets peripheral tissues, including the 294 renal tubules [17] and that the brain LK neurons act in neuronal circuits within the CNS 295 [18-20,39]. More specifically, the LHLK brain neurons are part of the output circuitry of 296 the circadian clock in regulation of locomotor activity and sleep suppression induced by 297 starvation [19,20,39] and the SELKs of the subesophageal zone may regulate feeding 298 [18]. In fact we show here that these SELKs have axons that exit through subesophageal 299 nerves known to innervate muscles of the feeding apparatus. We found in this study that 300 the ABLKs display increased calcium activity in response to drinking in desiccated flies, 301 but not during starvation, desiccation or regular feeding. This finding supports a role of 302 ABLKs and hormonal LK in regulation of water balance. These neurons have also been 303 implicated more broadly in control of water and ion homeostasis and in responses to 304 starvation, desiccation and ionic stress [17]. The LHLKs and SELKs did not display 305 changes in calcium signaling under the tested conditions, strengthening the unique 306 function of ABLKs in diuresis. 307 The regulation of metabolic rate, as determined by measurement of CO2 308 production, is a novel phenotype that we can link to LK signaling. This may be 309 associated with the overall activity of the flies, as suggested by the correlation between 310 activity and CO2 levels in our data. Thus, the regulation of activity and metabolic rate 311 might be coordinated by means of the LK neurons. 312 Using anatomical and experimental strategies, we identified a novel circuit 313 linking LK to insulin signaling. Lkr expression was detected in the brain IPCs using two 314 independently generated GAL4 lines plus single-cell transcriptome analysis. We also 315 observed that Lk and Lkr mutants displayed increased levels of DILP2 and DILP3 316 immunoreactivity in the brain IPCs and targeted knockdown of Lkr in IPCs increased 317 DILP3 expression. Associated with this we found that Lkr-RNAi targeted to IPCs 318 increased resistance to desiccation. However, using the trans-Tango method for 319 anterograde trans-synaptic labeling [30], we could not demonstrate direct synaptic 320 inputs to IPCs from LK neurons. The LHLKs did not yield any detectable signal; 321 however, the Lk-GAL4 line displayed very weak expression in the LHLKs. The SELKs 322 drove postsynaptic marker signal in sets of neurons in the SEG, some of which have 323 processes impinging on the IPCs. These findings suggest that SELKs form no 324 conventional synaptic contacts with IPCs, but paracrine LK signaling to these neurons is 325 not excluded since the two sets of neurons have processes in close proximity in the 326 tritocerebrum and the subesophageal zone. Nonsynaptic paracrine signaling with 327 neuropeptides has been well established in mammals (see [40][41][42]) and is likely to occur 328 also in insects. Alternatively, the LK input to IPCs could occur systemically at the 329 peripheral axon terminations of the IPCs after hormonal release from ABLKs. Whether  We suggest that LK signaling regulates post-feeding physiology and behavior 341 seen in the mutants as reduced metabolic rate and locomotor activity, diminished PER, 342 and reduced diuresis, as well as increased resistance to starvation and desiccation. Our 343 data also indicate that in wild type flies LK triggers release of IPC-derived DILPs that are 344 required for post-feeding metabolism and satiety, and it acts on other cells to induce 345 diuresis, and to increase activity (especially evening activity) and metabolic rate. An . This may suggest that the LK neurons receive nutrient-related 357 information from insulin-producing cells in the brain or elsewhere. 358 In conclusion, we found that LK signaling is likely to orchestrate postprandial 359 physiology and behavior in Drosophila. Food ingestion is followed by increased insulin 360 signaling, activation of diuresis, increased metabolic rate, and lowered locomotor activity For DILP2>Lkr-RNAi qPCR, crosses were established in normal food (NutriFly 378 Bloomington formulation) and eggs were laid for 24 hours. After adult eclosion, males 379 were transferred to alternative diets (normal diet described above; high-sugar high-380 protein: normal diet except with 20% sucrose and 10% yeast; low-sugar high-protein: 381 normal diet except 5% sucrose and 10% yeast). After 5-7 days on these media, heads 382 were dissected for qPCR. 383 384

Generation of GAL4 knock-in mutants and transgenic lines 387
Lk-/-and Lkr -/-were generated using the CRISPR/Cas9 system to induced homology-388 dependent repair (HDR) using one guide RNA (Lk-/-: GATCTTTGCCATCTTCTCCAG 389 and Lkr-/-: GTAGTGCAATACATCTTCAG). At gRNA target sites a donor plasmid was 390 inserted containing a GAL4::VP16 and floxed 3xP3-RFP cassette. For Lk -/-, the knockin 391 cassette was incorporated immediately following the ATG start site (4bp to 10bp, relative 392 to start site). For Lkr-/-, the knock in cassette was incorporated upstream of the ATG start 393 site (-111bp to -106bp, relative to start site). All lines were generated in the w 1118 394 background. Proper insertion loci for both mutations were validated by genomic PCR.

RT-qPCR 421
To quantify Lk and Lkr transcript levels in mutant flies, the following method was used. 422 Briefly, ten or more fed flies were flash frozen for each sample.  incubated for 16 hours. In addition, one set of flies were desiccated for 13 hours and then 478 transferred to a vial containing 1% agar (re-watered). Following this period, the flies were 479 fixed, dissected brains processed for immunohistochemistry and the GFP fluorescence 480 was quantified as described above.

Water-content measurements 492
For water content measurements, 15 flies per replicate (4 biological replicates) were 493 either frozen immediately on dry ice or desiccated as above for 9 hours and then frozen. 494 The samples were stored at -80C until use. To determine their wet weight, flies were 495 brought to room temperature and their weight was recorded using a Mettler Toledo MT5 496 microbalance (Columbus, USA). The flies were then dried for 24-48 hours at 60C before 497 recording their dry weight. The water content of the flies was determined by subtracting 498 dry weight from wet weight. 499 500

Blue dye feeding assay 501
Short-term food intake was measured as previously described [66]. Briefly, flies were 502 starved for 24 hours on 1% agar (Fisher Scientific) or maintained on standard fly food. At 503 ZT0, flies were transferred to food vials containing 1% agar, 5% sucrose, and 2. acclimation. After 24 hours acclimation in DAMS tubes with food, baseline activity was 552 measured for 24 hours. Tubes were maintained in a 25°C incubator with 12:12 LD cycles. 553

Mining public datasets for expression of genes 555
Lkr distribution in various tissues was determined by mining the FlyAtlas database [27]. 556 Lkr expression in the different regions of the gut and its cell types was obtained using 557 Flygut-seq [28]. A single-cell transcriptome atlas of the Drosophila brain was mined using 558 SCope ( http://scope.aertslab.org ) to identify genes coexpressed with Lkr [29]. 559 560

Statistical analyses 561
The experimental data are presented as means  s.e.m. Unless stated otherwise, one-562 way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test was 563 used for comparisons between three genotypes and an unpaired t test was used for 564 comparisons between two genotypes. All statistical analyses were performed using 565 GraphPad Prism with a 95% confidence limit (p < 0.05). Survival and stress curves were 566 compared using Mantel-Cox log-rank test.                ABLKs that could be detected is higher in rewatered flies compared to other conditions. 781 (assessed by one-way ANOVA followed by Tukey's multiple comparisons test). 782 short-term feeding compared to control flies as measured using a blue-dye feeding assay 792 (assessed by one-way ANOVA). (F) Expression of tetanus toxin in Lk neurons also has 793 no effect on short-term feeding. Average metabolic rate of Lkr mutants is lowered compared to control flies. (* p < 0.05, ** 805 p < 0.01, *** p < 0.001, **** p < 0.0001 as assessed by one-way ANOVA). 806  increase in dry weight. (* p < 0.05 as assessed by Log-rank (Mantel-Cox) test for (B), and 946 * p < 0.05 and ** p < 0.01 for (C) as assessed by one-way ANOVA followed by Tukey's 947 multiple comparisons test). 948